Bioactives and Traditional Herbal Medicine for the

Bioactives and Traditional Herbal Medicine for the Treatment of CardiovascularCerebrovascular Diseases 2015

Guest Editors Joen-Rong Sheu Pitchairaj Geraldine and Mao-Hsiung Yen

Evidence-Based Complementary and Alternative Medicine

Bioactives and Traditional Herbal Medicine forthe Treatment of CardiovascularCerebrovascularDiseases 2015



Guest Editors Joen-Rong Sheu Pitchairaj Geraldineand Mao-Hsiung Yen

Copyright copy 2015 Hindawi Publishing Corporation All rights reserved

This is a special issue published in ldquoEvidence-Based Complementary and Alternative Medicinerdquo All articles are open access articlesdistributed under the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in anymedium provided the original work is properly cited

Editorial Board

Mona Abdel-Tawab GermanyJon Adams AustraliaGabriel A Agbor CameroonUlysses P Albuquerque BrazilSamir Lutf Aleryani USAAther Ali USAGianni Allais ItalyTerje Alraek NorwayShrikant Anant USAIsabel Andujar SpainLetizia Angiolella ItalyVirginia A Aparicio SpainMakoto Arai JapanHyunsu Bae Republic of KoreaGiacinto Bagetta ItalyOnesmo B Balemba USAWinfried Banzer GermanyPanos Barlas UKVernon A Barnes USASamra Bashir PakistanPurusotam Basnet NorwayJairo Kennup Bastos BrazilSujit Basu USAArpita Basu USAGeorge D Baxter New ZealandAndre-Michael Beer GermanyAlvin J Beitz USALouise Bennett AustraliaMaria Camilla Bergonzi ItalyAnna R Bilia ItalyYong C Boo Republic of KoreaMonica Borgatti ItalyFrancesca Borrelli ItalyGloria Brusotti ItalyArndt Bussing GermanyRainer W Bussmann USAAndrew J Butler USAGioacchino Calapai ItalyGiuseppe Caminiti ItalyRaffaele Capasso ItalyFrancesco Cardini ItalyOpher Caspi IsraelSubrata Chakrabarti CanadaPierre Champy FranceShun-Wan Chan Hong Kong

Il-Moo Chang Republic of KoreaChun-Tao Che USAKevin Chen USAEvan P Cherniack USASalvatore Chirumbolo ItalyJae Youl Cho KoreaKathrine Christensen DenmarkShuang-En Chuang TaiwanY Clement Trinidad And TobagoPaolo Coghi ItalyMarisa Colone ItalyLisa A Conboy USAKieran Cooley CanadaEdwin L Cooper USAOlivia Corcoran UKMuriel Cuendet SwitzerlandRoberto K N Cuman BrazilVincenzo De Feo ItalyRocıo De la Puerta SpainLaura De Martino ItalyNunziatina De Tommasi ItalyAlexandra Deters GermanyFarzad Deyhim USAManuela Di Franco ItalyClaudia Di Giacomo ItalyAntonella Di Sotto ItalyM-G Dijoux-Franca FranceLuciana Dini ItalyTieraona L Dog USACaigan Du CanadaJeng-Ren Duann USANativ Dudai IsraelThomas Efferth GermanyAbir El-Alfy USATobias Esch USAGiuseppe Esposito ItalyKeturah R Faurot USAYibin Feng Hong KongNianping Feng ChinaPatricia D Fernandes BrazilJosue Fernandez-Carnero SpainAntonella Fioravanti ItalyFabio Firenzuoli ItalyPeter Fisher UKFilippo Fratini Italy

Brett Froeliger USAMaria pia Fuggetta ItalyJoel J Gagnier CanadaSiew Hua Gan MalaysiaJian-Li Gao ChinaMary K Garcia USASusana Garcia de Arriba GermanyDolores G Gimenez SpainGabino Garrido ChileIpek Goktepe QatarMichael Goldstein USAYuewen Gong CanadaSettimio Grimaldi ItalyGloria Gronowicz USAMaruti Ram Gudavalli USAAlessandra Guerrini ItalyNarcis Gusi SpainSvein Haavik NorwaySolomon Habtemariam UKAbid Hamid IndiaMichael G Hammes GermanyKuzhuvelil Harikumar IndiaCory S Harris CanadaJan Hartvigsen DenmarkThierry Hennebelle FranceLise Hestbaek DenmarkEleanor Holroyd AustraliaMarkus Horneber GermanyChing-Liang Hsieh TaiwanBenny T K Huat SingaporeRoman Huber GermanyHelmut Hugel AustraliaCiara Hughes UKAttila Hunyadi HungarySumiko Hyuga JapanH Stephen Injeyan CanadaChie Ishikawa JapanAngelo A Izzo ItalyChris J Branford-White UKSuresh Jadhav IndiaG K Jayaprakasha USAStefanie Joos GermanyZeev L Kain USAOsamu Kanauchi JapanWenyi Kang China

Shao-Hsuan Kao TaiwanJuntra Karbwang JapanKenji Kawakita JapanDeborah A Kennedy CanadaCheorl-Ho Kim Republic of KoreaYoun C Kim Republic of KoreaYoshiyuki Kimura JapanToshiaki Kogure JapanJian Kong USATetsuya Konishi JapanKarin Kraft GermanyOmer Kucuk USAVictor Kuete CameroonYiu W Kwan Hong KongKuang C Lai TaiwanIlaria Lampronti ItalyLixing Lao Hong KongChristian Lehmann CanadaMarco Leonti ItalyLawrence Leung CanadaShahar Lev-ari IsraelMin Li ChinaXiu-Min Li USAChun G Li AustraliaBi-Fong Lin TaiwanHo Lin TaiwanChristopher G Lis USAGerhard Litscher AustriaI-Min Liu TaiwanYijun Liu USAVıctor Lopez SpainThomas Lundeberg SwedenFilippo Maggi ItalyValentina Maggini ItalyGail B Mahady USAJamal Mahajna IsraelJuraj Majtan SlovakiaFrancesca Mancianti ItalyCarmen Mannucci ItalyArroyo-Morales Manuel SpainFulvio Marzatico ItalyMarta Marzotto ItalyJames H McAuley AustraliaKristine McGrath AustraliaJames S McLay UKLewis Mehl-Madrona USAPeter Meiser GermanyKarin Meissner Germany

Albert S Mellick AustraliaAyikoe Mensah-Nyagan FranceAndreas Michalsen GermanyOliver Micke GermanyRoberto Miniero ItalyGiovanni Mirabella ItalyDavid Mischoulon USAFrancesca Mondello ItalyAlbert Moraska USAGiuseppe Morgia ItalyMark Moss UKYoshiharu Motoo JapanKamal Moudgil USAYoshiki Mukudai JapanFrauke Musial GermanyMinKyun Na Republic of KoreaHajime Nakae JapanSrinivas Nammi AustraliaKrishnadas Nandakumar IndiaVitaly Napadow USAMichele Navarra ItalyIsabella Neri ItalyPratibha Nerurkar USAKaren Nieber GermanyMenachem Oberbaum IsraelMartin Offenbaecher GermanyJunetsu Ogasawara JapanKi-Wan Oh Republic of KoreaYoshiji Ohta JapanOlumayokun Olajide UKThomas Ostermann GermanySiyaram Pandey CanadaBhushan Patwardhan IndiaBerit S Paulsen NorwayPhilip Peplow New ZealandFlorian Pfab GermanySonia Piacente ItalyAndrea Pieroni ItalyRichard Pietras USAAndrew Pipingas AustraliaJose M Prieto UKHaifa Qiao USAWaris Qidwai PakistanXianqin Qu AustraliaEmerson Queiroz SwitzerlandRoja Rahimi IranKhalid Rahman UKCheppail Ramachandran USA

Elia Ranzato ItalyKe Ren USAMan H Rhee Republic of KoreaLuigi Ricciardiello ItalyDaniela Rigano ItalyJose L Rıos SpainPaolo di Sarsina ItalyMariangela Rondanelli ItalyOmar Said IsraelAvni Sali AustraliaMohd Z Salleh MalaysiaA Sandner-Kiesling AustriaManel Santafe SpainTadaaki Satou JapanMichael A Savka USAClaudia Scherr SwitzerlandG Schmeda-Hirschmann ChileAndrew Scholey AustraliaRoland Schoop SwitzerlandSven Schroder GermanyHerbert Schwabl SwitzerlandVeronique Seidel UKSenthamil Selvan USAFelice Senatore ItalyHongcai Shang ChinaKaren J Sherman USARonald Sherman USAKuniyoshi Shimizu JapanKan Shimpo JapanYukihiro Shoyama JapanMorry Silberstein AustraliaKuttulebbai Sirajudeen MalaysiaGraeme Smith UKChang-Gue Son KoreaRachid Soulimani FranceDidier Stien FranceCon Stough AustraliaAnnarita Stringaro ItalyShan-Yu Su TaiwanBarbara Swanson USAGiuseppe Tagarelli ItalyO Taglialatela-Scafati ItalyTakashi Takeda JapanGhee T Tan USAHirofumi Tanaka USALay Kek Teh MalaysiaNorman Temple CanadaMayankThakur Germany

Menaka C Thounaojam USAEvelin Tiralongo AustraliaStephanie Tjen-A-Looi USAMichał Tomczyk PolandLoren Toussaint USAYew-Min Tzeng TaiwanDawn M Upchurch USAKonrad Urech SwitzerlandTakuhiro Uto JapanSandy van Vuuren South AfricaAlfredo Vannacci ItalyS Vemulpad AustraliaCarlo Ventura ItalyGiuseppe Venturella Italy

Pradeep Visen CanadaAristo Vojdani USADawnWallerstedt USAShu-Ming Wang USAChong-Zhi Wang USAYong Wang USAJonathan Wardle AustraliaKenji Watanabe JapanJ Wattanathorn ThailandMichael Weber GermanySilvia Wein GermanyJanelle Wheat AustraliaJenny M Wilkinson AustraliaDarren Williams Republic of Korea

Christopher Worsnop AustraliaHaruki Yamada JapanNobuo Yamaguchi JapanJunqing Yang ChinaLing Yang ChinaEun Yang Republic of KoreaKen Yasukawa JapanAlbert S Yeung USAArmando Zarrelli ItalyC Zaslawski AustraliaRuixin Zhang USAM S Ali-Shtayeh Palestinian Authority

Contents

Bioactives and Traditional Herbal Medicine for the Treatment of CardiovascularCerebrovascularDiseases 2015 Joen-Rong Sheu Pitchairaj Geraldine and Mao-Hsiung YenVolume 2015 Article ID 320545 2 pages

Effects of Tetramethylpyrazine on Functional Recovery and Neuronal Dendritic Plasticity afterExperimental Stroke Jun-Bin Lin Chan-Juan Zheng Xuan Zhang Juan Chen Wei-Jing Liao and Qi WanVolume 2015 Article ID 394926 10 pages

Cardioprotective Potential of Polyphenolic Rich Green Combination in Catecholamine InducedMyocardial Necrosis in Rabbits Fatiqa Zafar Nazish Jahan Khalil-Ur-Rahman Ahrar Khanand Waseem AkramVolume 2015 Article ID 734903 9 pages

Hinokitiol Negatively Regulates Immune Responses through Cell Cycle Arrest in ConcanavalinA-Activated Lymphocytes Chi-Li Chung Kam-Wing Leung Wan-Jung Lu Ting-Lin Yen Chia-Fu HeJoen-Rong Sheu Kuan-Hung Lin and Li-Ming LienVolume 2015 Article ID 595824 8 pages

Effects of the Pinggan Qianyang Recipe on MicroRNA Gene Expression in the Aortic Tissue ofSpontaneously Hypertensive Rats Guangwei Zhong Xia Fang Dongsheng Wang Qiong Chenand Tao TangVolume 2015 Article ID 154691 10 pages

Antrodia camphorata Potentiates Neuroprotection against Cerebral Ischemia in Rats viaDownregulation of iNOSHO-1Bax and Activated Caspase-3 and Inhibition of Hydroxyl RadicalFormation Po-Sheng Yang Po-Yen Lin Chao-Chien Chang Meng-Che Yu Ting-Lin YenChang-Chou Lan Thanasekaran Jayakumar and Chih-Hao YangVolume 2015 Article ID 232789 8 pages

EditorialBioactives and Traditional Herbal Medicine for the Treatment ofCardiovascularCerebrovascular Diseases 2015

Joen-Rong Sheu1 Pitchairaj Geraldine2 and Mao-Hsiung Yen3

1Graduate Institute of Medical Sciences College of Medicine Taipei Medical University Taipei 110 Taiwan2Department of Animal Science Bharathidasan University Tiruchirappalli Tamil Nadu 620 024 India3Department of Pharmacology National Defense Medical Center Taipei Taiwan

Correspondence should be addressed to Joen-Rong Sheu sheujrtmuedutw

Received 8 June 2015 Accepted 8 June 2015

Copyright copy 2015 Joen-Rong Sheu et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cardiovascular diseases (CVDs) are still the principal causeof death worldwideWeakened endothelial function followedby inflammation of the vessel wall hints at atheroscle-rotic lesion formation that causes myocardial infarctionand stroke Heart failure can arise as consequence of largemyocardial infarctions In its more severe stages heartfailure patients have a life anticipation that is parallel todestructive cancers Accordingly the increase in risk factorload by metabolic diseases and age augments the incidencefor vascular and cardiac diseases and provides a challengefor developing efficient treatmentsThere is widespread proofto show that drug treatment of conventional risk factors iseffective in reducing cardiovascular events More effectivetreatment of CVD with various classes of antihypertensivedrugs has been associated with greater benefits but somerecent studies suggest wemay be reaching the optimal level oftreated blood pressure in some patient groups Apart from thetreatment of cardiovascular risk factorswith pharmacologicalagents and the use of antithrombotic drugs there is growingawareness of the role of dietary factors and herbal medicinesin the prevention of CVD and the possibility of their use intreatment Investigators from different places of the worldlike China Taiwan Bangladesh Pakistan and so forthcontributed to this special issue by presenting tremendouspapers These papers deliver an analysis in this field andcreate innovative contributions concerning themechanismofaction of bioactives and traditional herbal medicine for thetreatment of cardiovascularcerebrovascular diseases

Some interesting papers in this special issue addressthe cardioprotective effects of Chinese herbal medicine and

natural compounds For instance a paper summarized thesynergetic cardioprotective potential of herbal combinationof four plants namely Terminalia arjuna Cactus grandi-florous Crataegus oxyacantha and Piper nigrum throughcurative and preventive mode of treatment analysis and thispaper reported preadministration and postadministration ofherbal mixture restore the levels of biomarker of cardiotox-icity which includes cardiac marker enzymes lipids profileand antioxidant enzymes Similarly another paper in thisissue reports the cardioprotective effects of Sundarban honeyon cardiac troponin I cardiac marker enzymes the lipidprofile lipid peroxidation products and histoarchitecture ofthe myocardium against isoproterenol-induced myocardialinfarction in Wistar rats Pinggan Qianyang recipe (PQR) aChinese medicine recipe has long been used for calming theliver It has also been used to treat essential hypertension withsatisfactory results Consistent with this concern this specialissue published a paper that reports PQR exerts its antihyper-tensive effect through deterioration of the vascular remod-eling process The mechanism might be associated withregulating differentially expressed miRNAs in aorta tissue

Despite the fact that there are major developments intreating ischemic stroke over the last decade stroke is still aserious concern for which effective drug therapy is not yetavailable In the search for neuroprotective agents from nat-ural sources a number of plant extracts and several naturalproducts were isolated and reported to provide neuroprotec-tion against ischemic stroke A few papers in this special issuereport the neuroprotective effects of Chinese herbalmedicineand natural compounds For instance Antrodia camphorata

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 320545 2 pageshttpdxdoiorg1011552015320545

2 Evidence-Based Complementary and Alternative Medicine

(A camphorata) a fungus generally used in Chinese folkmedicine for the treatment of viral hepatitis and cancer hasshown neuroprotective effects in embolic rats This effectmay correlate with the downregulation of the iNOS HO-1 Bax and activated caspase-3 and the inhibition of OH∘signals Another study shows alpha-lipoic acid attenuatesmiddle cerebral artery occlusion-induced cerebral ischemiaand reperfusion injury via insulin receptor-dependent andPI3KAkt-dependent inhibition of NADPH oxidase More-over an interesting study in this special issue established theeffects of tetramethylpyrazine (TMP) on functional recoveryand neuronal dendritic plasticity after experimental stroke Inthis study the authors have shown that enhanced dendriticplasticity contributes to TMP-elicited functional recoveryafter ischemic stroke

Hinokitiol is a naturally occurring compound isolatedfrom the wood of Chamaecyparis taiwanensis It is involvedin multiple biological activities including antimicrobial andantitumorigenic activities Although hinokitiol has beenreported to inhibit inflammation its immunological regula-tion in lymphocytes remains inadequate With this context awell-designed study reported that hinokitiol downregulatedcyclin D3 E2F1 and Cdk4 expression and upregulated p21expression in concanavalinA- (ConA-) stimulatedT lympho-cytes It further demonstrated that hinokitiol upregulates p21expression and attenuates IFN-120574 secretion in T lymphocytesfrom the spleens ofmice thereby arresting the cell cycle in theG0G1 phase These authors concluded that hinokitiol pro-vides benefits in treating patients with autoimmune diseasesWe expect that this special issue grants inventive awarenessto increase the therapeutic value of herbal andor Chinesemedicines for treatment or prevention of cardiovascular andischemia-reperfusion injury-related disorders

Joen-Rong SheuPitchairaj Geraldine

Mao-Hsiung Yen

Research ArticleEffects of Tetramethylpyrazine on Functional Recovery andNeuronal Dendritic Plasticity after Experimental Stroke

Jun-Bin Lin1 Chan-Juan Zheng12 Xuan Zhang1 Juan Chen3 Wei-Jing Liao1 and Qi Wan3

1Department of Rehabilitation Medicine Zhongnan Hospital of Wuhan University Wuhan 430071 China2Department of Rehabilitation Medicine Center of Brain Department Hubei Xinhua Hospital Wuhan 430015 China3Department of Physiology School of Medicine Wuhan University Wuhan 430071 China

Correspondence should be addressed to Wei-Jing Liao weijingliaosinacom and Qi Wan qwanwhueducn

Received 28 September 2014 Revised 22 December 2014 Accepted 26 December 2014

Academic Editor Joen-Rong Sheu

Copyright copy 2015 Jun-Bin Lin et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The 2356-tetramethylpyrazine (TMP) has been widely used in the treatment of ischemic stroke by Chinese doctors Here wereport the effects of TMP on functional recovery and dendritic plasticity after ischemic stroke A classical model of middle cerebralartery occlusion (MCAO) was established in this study The rats were assigned into 3 groups sham group (sham operated ratstreated with saline) model group (MCAO rats treated with saline) and TMP group (MCAO rats treated with 20mgkgd TMP)The neurological function test of animals was evaluated using the modified neurological severity score (mNSS) at 3 d 7 d and14 d after MCAO Animals were euthanized for immunohistochemical labeling to measure MAP-2 levels in the peri-infarct areaGolgi-Cox staining was performed to test effect of TMP on dendritic plasticity at 14 d after MCAO TMP significantly improvedneurological function at 7 d and 14 d after ischemia increased MAP-2 level at 14 d after ischemia and enhanced spine density ofbasilar dendrites TMP failed to affect the spine density of apical dendrites and the total dendritic length Data analyses indicate thatthere was significant negative correlation between mNSS and plasticity measured at 14 d after MCAO Thus enhanced dendriticplasticity contributes to TMP-elicited functional recovery after ischemic stroke

1 Introduction

Stroke is the leading cause of long-term disability in thewestern world which is a severe disease characterized by itshighmorbidity mortality disability and recurrence [1] It hasbecome a heavy burden to patients families and societiesdue to the excessive costs of long hospitalizations nursingcare and rehabilitation [2] Ischemic stroke accounts forapproximately 87 of stroke [3]

2356-Tetramethylpyrazine (TMP Figure 1) is an activeingredient extracted from a traditional Chinese herbal med-icine Ligusticum chuanxiong Hort and has been widely usedin ischemic stroke by Chinese doctors [4] TMP exerts phar-macological effects in multiple ways with multiple targetsTMP is reported to protect ischemia reperfusion injuryof heart brain and kidney via reducing oxidative stressattenuating Ca2+ overload inhibiting apoptosis inhibiting

inflammatory reaction and so forth [5ndash7] Besides the above-mentioned effects it is also demonstrated that TMP caninhibit platelet aggregation depress blood viscosity and ame-liorate microcirculation [8] which could be another impor-tant mechanism to treat cardiovascular and cerebrovasculardiseases Recently it has been found that TMP could protecthepatic fibrosis by modulating multiple signal pathways [9ndash11] Furthermore TMP had a significant therapeutic effecton diabetic nephropathy [12] which could be mediated bydownregulated expression of vascular endothelial growthfactor in the kidney and reduction of lipoperoxidation [1314] Additionally TMP has been reported to have beneficialeffects in various types of cancer [15ndash17] Specific to ischemicstroke according to previous studies TMP can play a protec-tive role through the following mechanisms antiexcitotoxic-ity [18] inhibiting inflammatory reaction [19] anti-apoptosis[20] antioxidant activity [21] suppression of calcium [21]



N

N

Figure 1 The structure of TMP

Core

Penumbra

Figure 2 The schematic diagram of ischemic penumbra (IP)

thrombolytic effect [22] enhancing neurogenesis and celldifferentiation [23]

There are at least three processes during recovery afterstroke resolution of acute tissue damage behavioral compen-sation and plasticity [24] Based on the information abovemost studies focus on TMPrsquos inhibitory roles in postischemiccascade process in acute phase However the effects andmechanisms of TMP on neuroplasticity are still not clear upto nowThe plasticity of dendrites is an important componentof plasticity [25 26] When challenged by ischemic strokedendrites in ischemic penumbra (IP) show a series of changeswith morphological modifications [27] which suggest thatfacilitating or optimizing the plasticity of dendrites is likely tobe a promising therapeutic target Indeed dendritic changesafter ischemic injury could be induced by drugs and rehabil-itative trainings

Ischemic penumbra (IP) was first proposed by Astrup etal in 1981 [28] It was defined as a region of reduced cerebralblood flow (CBF) with absent spontaneous or induced elec-trical potentials that still maintained ionic homeostasis andtransmembrane electrical potentials It has the potential forfunctional recovery if local blood flow can be reestablishedwithin a limited period and is a key target for the treatmentof acute stroke [29] It is located in the peri-infarct area andFigure 2 shows schematic diagram of ischemic core and IP

In this study we tested the effects of TMP on func-tional recovery and dendritic plasticity after ischemic strokeA classical focal cerebral ischemia reperfusion model wasinduced by middle cerebral artery occlusion (MCAO) in therat and we conducted a TTC staining Firstly we measuredthe neurological function performance using the modifiedneurological severity score (mNSS) In order to measure thedendritic plasticity after behavioral testing immunohisto-chemistry was employed to evaluate the levels of microtubuleassociated protein-2 (MAP-2 marker of neuronal dendrites)

and a modified Golgi-Cox staining was conducted to exam-ine dendritic morphologic plasticity Finally correlationsanalyses between functional outcome and plasticity wereperformed

2 Materials and Methods

21 Animals A total of 78 eight-week-old male SpragueDawley (SD) rats weighing 200ndash250 g (purchased fromExperimental Animal Center of Wuhan University WuhanHubei China) were used for this experiment The ratswere acclimated for 3 or more days before the start of anyexperiments They were housed in a controlled environment(4 animals per cages 55plusmn5 relative humidity 22∘C 12 12 hlightdark cycle) and provided with free access to food andwater All experimental procedures involving animals wereapproved by the Animal Care and Use Committee of WuhanUniversity Medical School We made all efforts to minimizethe number of animals used and their suffering

22 Model MCAO was induced using the modified intralu-minal filament technique [30] Briefly rats were anesthetizedwith 10 chloral hydrate (400mgkg) intraperitoneally andafter a median incision of the neck skin the right carotidartery (CCA) external carotid artery (ECA) and internalcarotid artery (ICA) were carefully isolated The right MCAwas occluded with a monofilament nylon filament (BeijingCinontech Biotech Co Ltd Beijing China) by inserting itthrough the right CCA and gently advancing into the ICA upto a point approximately 17mmdistal to the bifurcation of thecarotid artery The filament was fixed in place and the animalwas allowed to recover fromanesthesia After 2 h the filamentwas withdrawn to permit reperfusion In sham group allsurgical procedures were the same as above without insertinga nylon filament A heating pad was used to maintain a rectaltemperature of 370 plusmn 05∘C during the surgical procedure

6 MCAO rats were anesthetized with an overdose ofchloral hydrate and sacrificed by decapitation at 3 d afterMCAO The brains were quickly removed and chilled atminus20∘C for 10min 2mm coronal slices were cut for eachbrain and immersed in a PBS solution (pH = 74) containing2 triphenyl tetrazolium chloride (TTC) (Sigma St LouisMO USA) at 37∘C in the dark for 30min The stainedsections were then fixed in 4 paraformaldehyde for 1 hAll stained sections were scanned and the infarct volumeswere analyzed by Image Pro Plus 60 (Media Cybernetics IncBethesda MD USA) To eliminate the effect of brain edemaand differential shrinkage resulting from tissue processingthe percentage of infarct volume was calculated as reportedpreviously [31]

23 Grouping and Administration In this study the animalswere randomly assigned into 3 groups sham group (shamoperated rats treated with saline) model group (MCAO ratstreatedwith saline) andTMPgroup (MCAOrats treatedwith20mgkgd TMP (Aladdin Chemistry Co Ltd ShanghaiChina))The first administrationwas conducted immediatelyafter reperfusion All injections were conducted through

Evidence-Based Complementary and Alternative Medicine 3

Neurological function

mNSS

ShammodelTMP

Biomarker

MAP-23 d7 d

14 d

14 d

Dendritic plasticity

Dendritic morphology

Total dendritic lengthspine density

Rats

Figure 3 A simple flow-chart of experimental design

intraperitoneal injection daily and in the volume of 5mLkguntil the day before they were sacrificed After neurologicalfunction test 54 rats were sacrificed at 3 d 7 d and 14 d afterMCAO for immunohistochemistry (119899 = 6 in each group ateach time point) and 18 rats for Golgi-Cox staining (119899 = 6in each group) at 14 d after MCAO A brief flow diagram isshown in Figure 3

24 Neurological Function Test Modified neurological sever-ity score (mNSS) test [32] was measured at 3 d 7 d and14 d after MCAO by an observer blinded to experimentalgroups The mNSS is a composite of motor sensory reflexand balance tests and is graded on a scale of 0ndash18 (normalscore 0 maximal deficit score 18) In the severity scores ofinjury 1 score point is awarded for the inability to performthe test or for the lack of a tested reflex thus the higher thescore is the more severe the injury is It is classified into threelevels 13 to 18 are graded as severe injury 7 to 12 as moderateinjury and 1 to 6 as mild injury

25 Immunohistochemistry At 3 d 7 d and 14 d after MCAOrats in each group at each time point (119899 = 6) were anes-thetized with an overdose of chloral hydrate and transcar-dially perfusedwith 150mL of 09 saline followed by 150mLof 4 paraformaldehydeThe brains were removed and post-fixed in 4 paraformaldehyde overnight Thereafter paraffinembedded blocks (bregma minus2 to +2mm) were obtained andsliced into sections of 6120583mandmounted onto the polylysine-coated slides Streptavidin-peroxidase (S-P) method [33]was adopted for immunostaining (1) tissue sections weredeparaffinized with xylene and rehydrated in ethanol (2)theywere incubated in endogenous peroxidase blocking solu-tion (Maixin Technology Co Ltd Fuzhou Fujian China)for 10min at room temperature (3) after being incubatedwith normal rabbit serum (Maixin Technology Co LtdFuzhou Fujian China) the brain sections were incubatedovernight with rabbit anti-MAP-2 antibody (1 200 BosterWuhanHubei China) at 4∘C (4) the sectionswere incubatedwith biotin-conjugated second antibody (Maixin TechnologyCo Ltd Fuzhou Fujian China) for 15min (5) they were

incubated with HRP-Streptavidin-Peroxidase (Maixin Tech-nology Co Ltd Fuzhou Fujian China) for 15min (6) thesections were stainedwith 3 31015840-diaminobenzidine andH

2O2

washed with tap water and counterstained with hematoxylinThe sections were rinsed with phosphate-buffered saline(PBS pH = 74) 3 times for 3min between every procedureof staining Finally the sections were dehydrated and cover-slipped To investigate the specificity of the reactions negativecontrols were established by replacing the primary antibodywith PBS and normal rabbit serum

For quantitative analysis three randomly selected sec-tions of each subject and five visual fields (400x) fromeach section in peri-infarct area were randomly capturedunder a microscope using a digital camera Integrated opticaldensity (IOD)wasmeasured using Image Pro Plus 60 (MediaCybernetics Inc Bethesda MD USA) for analysis Theanalysis procedure was conducted by an investigator in ablind fashion

26 Golgi-Cox Staining Procedure At 14 d after MCAO ratsin each group (119899 = 6) were injected intraperitoneally with alethal dose of chloral hydrate to induce anesthesia Removethe brains as soon as possible without perfusion and rinsetissue in double distilled water for 2-3 seconds to removeblood from the surface Hito Golgi-Cox OptimStain Kit(Hitobiotec Inc Wilmington DE USA) was applied fortissue preparation and staining procedure The whole Golgi-Cox staining procedure was conducted in strict accordancewith the manufacturerrsquos user manual and material safetydata sheet A series of 100120583m thick coronal sections wassliced from the caudal forelimb region of the motor cortex(approximately from bregma to +20mm from bregma) [34]using a microtome (Leica CM1950 cryostat Leica BiosystemsGmbH Wetzlar Germany)

27 Selection Criteria for Pyramidal Cells To be included foranalysis neurons should be selected according to specificcriteria [35] (1) the dendritic trees had to bewell impregnatedto facilitate accurate observation and analysis (2) the cellbodies and dendrites had to be in full view and not obscuredby other blood vessels astrocytes or clustering of dendritesfrom other pyramidal cells (3) they also had to appear intactand visible in the plane of section

28 Sholl Analysis To acquire images for analyzing layer Vpyramidal cells within peri-infarct area were traced at 200xmagnification Pyramidal neurons were readily identified bytheir characteristic triangular soma-shape apical dendritesextending toward the pial surface and numerous dendriticspines [36] In order to measure the length of dendritesSholl analysis [37] was conducted using a Sholl analysisplug-in (available at httpfijiscSholl Analysis) for Image Jsoftware (National Institutes of Health Bethesda MD USA)The number of intersections of dendrites with a series ofconcentric rings at 20120583m intervals from the centre of the cellbody was counted for each cell A reflection of total dendriticlength can be determined by multiplying the number of


Figure 4 A representative photograph of TTC staining of MCAOrat

intersections by 20 [38] Five cells per rat were measured forstatistical analysis

29 Measurement of Spine Density Dendritic spine densitywas analyzed from layer V pyramidal neurons within peri-infarct area For each cell at least 30 120583m long segments ofterminal basilar densities (third order or greater 119899 = 5) andapical densities (lower half of the apical segments 119899 = 5)on the same cell were traced at 1000x magnification [39]The number of spines was counted and the exact length ofthe dendritic segment was calculated to yield spines10120583mdata [39] We did not make any attempt to correct for spineshidden by the overlying dendrites Therefore the data may belikely to underestimate the actual density

210 Statistical Analysis All data was expressed as meanplusmn standard deviation (SD) and analyzed using SPSS 190software (SPSS Inc Chicago IL USA) Behavior data andimmunohistochemical data were analyzed using repeatedmeasures analysis of variance (rANOVA) and when theassumptions of sphericity were violated (Mauchlyrsquos test 119875 lt005) the Greenhouse-Geisser correction was applied Posthoc analyses used group designed 119905-test and Turkeyrsquos testOne-way analysis of variance (ANOVA) andTukeyrsquos test wereused for analyzing dendritic morphological data Correla-tions analysis between functional outcome andplasticitywereperformed using the Spearman correlation coefficients 119875 lt005 was considered statistically significant

3 Results

31 TTC forModel Rats Figure 4 shows a typical photographof coronal sections ofMCAO ratThe infarct region appearedwhite and the normal tissue was red Rats after MCAOexhibited obvious infarction which was located in cortex andstriatum The infarct volume was 3842 plusmn 442

32 Neurological Functional Assessment As shown inFigure 5 for model group and TMP group rats showedfunctional improvement with time going on Repeatedmeasures analysis of variance showed significant groupeffects (119865 = 11621 119875 = 0003) TMP treatment significantlyimproved functional recovery as evidenced by improvedmNSS at 7 d (model 1092 plusmn 168 versus TMP 933 plusmn 172119905 = 2281 119875 = 0033 decreased 1456) and 14 d (model842 plusmn 138 versus TMP 642 plusmn 116 119905 = 3839 119875 = 0001decreased 2375) compared with model group Howeverthere was no significant difference between the two groupsat 3 d after MCAO (model 1275 plusmn 166 versus TMP

18

16

14

12

10

8

6

4

2

0

mN

SS

lowast

lowastlowast

ModelTMPSham

3 d 7 d 14 d

Figure 5 Effect of TMP on neurological status in rats with ischemiccerebral injury The data were presented as mean plusmn standarddeviation (119899 = 12) lowast119875 lt 005betweenmodel group andTMPgrouplowastlowast

119875 lt 001 between model group and TMP group

1192 plusmn 124 119905 = 1394 119875 = 0177) All rats in sham groupperformed very well without any neurological deficit

33 MAP-2 Expression In this study IOD values wereapplied to indicate the expression of MAP-2 (Figure 6) Insham group obvious MAP-2 immunostaining was observedin the dendrites of the cells Repeated measures analysis ofvariance showed there was significant group effects (119865 =77753 119875 lt 0001) Post hoc analyses showed that there weresignificant differences between three groups at 3 d (sham3863539 plusmn 264921 versus model 1795893 plusmn 124488 versusTMP 1912820 plusmn 179569 119865 = 205913 119875 lt 0001) 7 d(sham 3800915 plusmn 271561 versus model 2263595 plusmn 210293versus TMP 2552122 plusmn 176414 119865 = 8061 119875 lt 0001)and 14 d (sham 3905986plusmn283129 versus model 3120385plusmn247853 versus TMP 3714730 plusmn 216838 119865 = 16017 119875 lt0001) Compared to shamgroup rats inmodel group showedsignificantly lower expression of MAP-2 (3 d 7 d and 14 dall 119875 lt 0001 decreased 5352 4045 and 2011 resp)although they exhibited an increasing trend from 3 d to 14 dafter MCAO TMP treatment resulted in upregulation inMAP-2 expression in peri-infarct area compared to modelgroup at 14 d (119875 = 0003 increased 1905) after MCAO

34 Dendritic Morphology The morphological analysis pre-sented here is based on a total of 180 neurons from 18animals Golgi-Cox staining clearly filled the dendritic shafts(Figure 7) and the spines of neurons from layer V pyramidalneurons The total dendritic length and dendritic spinedensity were obtained for analysis

341 Total Dendritic Length There was no significant differ-ence between three groups at 14 d after MCAO by a one-way


Sham

Model

TMP

3 d 7 d 14 d

(a)

50000

40000

30000

20000

10000

0

IOD

lowastlowastlowastlowastlowastlowastlowast

ShamModelTMP

3 d 7 d 14 d

(b)

Figure 6 The expression levels of MAP-2 within peri-infarct area of three groups in sham model and TMP groups at 3 d 7 d and 14 d afterMCAO (a) Immunohistochemical staining of three groups (400x) (b) MAP-2 levels of three groups through measuring the integral opticaldensity (IOD) Data were presented as mean plusmn standard deviation (119899 = 6) lowast119875 lt 001 and lowastlowast119875 lt 0001

ANOVA (sham 188567 plusmn 18073 versus model 178600 plusmn16602 versus TMP 181467 plusmn 14567 119865 = 0582 119875 = 0571)(Figure 8)

342 Spine Density of Basilar Dendrites For layer V pyra-midal neurons a one-way ANOVA of basilar dendrites spinedensity found difference between groups at 14 d after MCAO(sham 943plusmn085 versusmodel 770plusmn073 versus TMP 907plusmn084 119865 = 7642 119875 = 0005) (Figure 9) A following Tukeyrsquostest revealed that the dendritic spine density in model groupwas lower than that of sham group (119875 = 0006 decreased1835) and TMP treatment increased the dendritic spinedensity compared to model group (119875 = 0027 increased1779)

343 Spine Density of Apical Dendrites For apical dendritesa similar trend was observed (Figure 9) A one-way ANOVAof spine density also revealed difference between groups at14 d after MCAO (sham 973 plusmn 116 versus model 830 plusmn067 versus TMP 873 plusmn 085 119865 = 3870 119875 = 0044) Afollowing Tukeyrsquos test showed a decrease in spine density ofmodel group compared to sham group (119875 = 0040 decreased1470) while no significant increase of density was foundafter TMP treatment (119875 = 0175)

35 Correlations Analysis The Spearman correlation coef-ficients test showed that there were significant negativecorrelations between mNSS and plasticity measured at 14 dafter MCAO (mNSS and MAP-2 119903 = minus0619 119875 = 0032


Figure 7 A representative dendriticmorphology of layer V pyrami-dal cells of rats (Golgi-Cox staining) Photomicrograph was viewedat times200 magnification Bar = 50120583m

2200

2000

1800

1600

1400

1200

1000

800

600

400

200

0

Tota

l den

driti

c len

gth

(120583m

)

Sham Model TMP

Figure 8 Quantification analysis of effect of TMP on total dendriticlength using Sholl analysis Data were presented as mean plusmn standarddeviation (119899 = 6)

mNSS and total dendritic length 119903 = minus0640 119875 = 0025mNSS and spine density of basilar dendrites 119903 = minus0705119875 = 0010) But there was no significant correlation betweenmNSS and spine density of apical dendrites (119903 = minus0501119875 = 0097) (Figure 10)

4 Discussion

MCAO model is classical model and produces obviousinfarction induced by focal occlusion of middle cerebralartery [40] TTC staining is a traditional and widely usedmethod for the research of infarct size In our study relativelystable and large-sized infarction in cortex and striatum wasinduced by MCAO in rats in model group which showedsimilar results with previous studies [23 31]

Ischemic stroke often triggers a complex cascade of cel-lular and molecular events including excitotoxicity calciumoverload oxidative stress and the following apoptosis and

neuroinflammation [2] TMP could block multiple events ofthe injury cascade to provide protection [19ndash21] Up to nowmost studies focused on the inhibitory mechanisms of TMPin the early stage of cerebral ischemia injury and only a fewstudies analyzed the repair mechanisms of TMP [4 20 23]We reported the TMPrsquos effects on dendritic plasticity in arelative late stage whichmay provide a new target and awidertherapeutic window

In our study neurological score using mNSS showedobvious difference between sham and model group in alltime points which indicates that MCAO induced relativesevere neurological function deficits There must be a naturalrecovery process after cerebral ischemia reperfusion injury[41 42] which could be confirmed by our study TMP isa small molecular weight medicine and reported to haveappreciable blood-brain barrier penetrability [43] Accordingto our data TMP could improve functional outcome afterfocal stroke

MAP-2 is selectively concentrated in the neuron bodyand dendrites which plays a key role in maintaining neu-roarchitecture cellular differentiation and structural andfunctional plasticity [30] MAP-2 has an intimate relation-ship with ischemic cerebral injury and is considered to bean indication of compensatory dendrites reconstruction inremaining neurons [44 45] Several studies revealed that theexpression ofMAP-2 decreased after ischemic cerebral injury[46ndash48] In our study in sham groupMAP-2(+) cells showedstaining mainly in the dendrites of the cells in ischemicanimals we examined the expression of MAP-2 in peri-infarct area at 3 d 7 d and 14 d after MCAO the level ofMAP-2 markedly decreased compared to sham group andpersistently increased from 3 d to 14 d after stroke which wasconsistent with previous study [48] These results indicatedthat the expression ofMAP-2 showed a dynamic process afterstroke (decreasing in early stage and increasing gradually)which may represent degeneration and reconstruction ofdendritic structure Two studies [25 49] declared there were apeak point and following downtrend during dendrites recon-struction However we did not observe this process whichmay be due to the relatively short period of observation

Our data showed that treatment of TMP significantlyincreased MAP-2 expression level in peri-infarct area afterstroke and the neurological function was improved mean-while indicating that promotion of the reconstruction ofdendrites may contribute to the improvements of neuro-logical function The mechanism is not clear but may beassociated with inhibition of calpains Calpains could beactivated by elevated levels of intracellular calcium afterischemic injury [50 51] causing proteolysis of numerousneuronal cytoskeletal and regulatory proteinsThe increase incalpain expression in the ischemic area was accompanied by aloss of its substrate MAP-2 [52] TMP is a calcium antagonistand could markedly reverse the increased intercellular freecalcium concentration [21] This effect may contribute toupregulation of MAP-2 level Correlation analysis showedthat there was a significant negative correlation betweenmNSS and expression of MAP-2 indicating that TMPrsquoseffect on improvement of neurological function may be theassociation with upregulation of MAP-2


Sham Model TMP

Basilar

Apical

(a)

12

10

8

6

4

2

0

lowast

Num

ber o

f spi

nes (10120583

m)

ShamModelTMP

Basilar Apical

lowastlowastlowast

(b)

Figure 9 Quantification analyses of effect of TMP on dendritic spine density (basilar dendrites and apical dendrites resp) (a)The segmentswere acquired from layer V pyramidal cells and viewed at times1000 magnification Scale bar = 10 120583m for all segments (b) The dendritic spinedensity was expressed as spines10 120583m and the data were presented as mean plusmn standard deviation (119899 = 6) lowast119875 lt 005 and lowastlowast119875 lt 001

MAP-2 is an indirect marker which can be used forrepresenting dendritic plasticity However morphologicalstudy is more distinct and more direct for assessments ofdendrites Golgi-Cox staining method has been used broadlyfor studying morphology of neurites including quantitativeanalysis of dendritic length arborization and spine density[53] of which spine density is the most important parameterDendritic length reflected the total space for synapses andspine density represented the density of excitatory synapsesto some extent [54] Sholl analysis was a classical method formeasuring dendritic length which is an important parameterreflecting dendritic plasticity We found that the dendriticlength of layer V pyramidal cells within peri-infarct area didnot change compared to sham group In fact the evidenceabout changes of dendritic length after stroke is controversialsome studies found a shortening of dendrites after corticallesions [38 55] another study found no difference or exten-sion of dendrites in peri-infarct cortex afterMCAO[56] Suchparadoxical results are perhaps associated with the absence ofa peri-infarct baseline or absence of dynamic study Brown etal [57] conducted a longitudinal study and found there wasa balance between dendrites extension and retraction afterstroke which may be a mechanism to explain our resultsIn addition no obvious alternations of total dendritic lengthwere observed after being treated by TMP indicating that

TMP may fail to affect dendritic length totally at 14 d afterstroke Increasing of dendritic length is good for recovery ofstroke but the result is not good in this regard

Dendrites and dentritic spines are the primary postsynap-tic targets which receive the majority of excitatory synapses[58] Previous studies have shown that spine density couldbe enhanced by drugs [39] or rehabilitative training [59]after experimental stroke which was likely to play a key rolein mediating functional changes that occurred during andafter stroke [27] In our studies the dentritic spine densityof layer V pyramidal neurons decreased significantly in peri-infarct area at 14 d after MCAO indicating the degenerationof dendrites which is in accordance with previous study[60] After chronic treatment with TMP the spine densityof basilar dendrites increased compared to model group forapical dendrites there was no significant difference betweenmodel group and TMP group One explanation is that themodifications of basilar dendrites and apical dendrites didnot occur at the same time in the recovery period [61]The degeneration and reorganization of dendritic spines is acomplicated process and could be regulated throughmultiplemechanisms including receptors scaffolding proteins andregulators of the cytoskeleton [62 63] However the phys-iological mechanism responsible for TMP stimulating thisincrease is unclear in this experiment Correlation analysis


12

11

10

9

8

7

6

5

4

mN

SS

27000 30000 33000 36000 39000 42000

MAP-2 level (IOD value)

r = minus0619 P = 0032

(a)

12

11

10

9

8

7

6

5

4

mN

SS

1400 1600 1800 2000 2200

Total dendritic length

r = minus0640 P = 0025

(b)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11

Spine density of basilar dendrites

r = minus0705 P = 0010

(c)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11

Spine density of apical dendrites

r = minus0501 P = 0097

(d)

Figure 10 Scatterplots present correlations analysis ofmNSS and plasticitymeasured at 14 d afterMCAO (a) Scatterplots ofmNSS andMAP-2 level (b) Scatterplots of mNSS and total dendritic length (c) Scatterplots of mNSS and spine density of basilar dendrites (d) Scatterplotsof mNSS and spine density of apical dendrites

showed that there was a significant negative correlationbetween mNSS and spine density of basilar dendrites indi-cating that TMPrsquos effect on improvement of neurologicalfunction may be also the association with increase of spinedensity of basilar dendrites

There is a dynamic change of dendrites and dendriticspine after ischemic injury over time [27] We did not meas-ure the dendriticmorphology of other time points so it is oneof limitations that we could not revealmorphological changesduring ischemic stroke and recovery

5 Conclusion

TMP may increase MAP-2 level after cerebral ischemiareperfusion anddecrease the alterations of neuronal dendriticspines induced by ischemia suggesting that TMPmay have apotential and specific effect on the neuronal dendritic plastic-ity in rats with transient focal cerebral ischemia reperfusionMeanwhile TMP also improved functional outcome afterstroke Taken together after cerebral ischemia reperfusion

dendritic plasticity is one of themechanisms that contributedto functional recovery which might be regulated by TMP

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This study was supported by a research grant from theNational Natural Science Foundation of China (no81072917)

References

[1] Z-Q Lu Y-J Deng and J-X Lu ldquoEffect of aloe polysaccharideon caspase-3 expression following cerebral ischemia and reper-fusion injury in ratsrdquoMolecular Medicine Reports vol 6 no 2pp 371ndash374 2012


[2] E Candelario-Jalil ldquoInjury and repair mechanisms in ischemicstroke considerations for the development of novel neurother-apeuticsrdquo Current Opinion in Investigational Drugs vol 10 no7 pp 644ndash654 2009

[3] D Lloyd-Jones R J Adams T M Brown et al ldquoHeart diseaseand stroke statisticsmdash2010 update a report from the AmericanHeart Associationrdquo Circulation vol 121 no 7 pp e46ndashe2152010

[4] S-L Liao T-K Kao W-Y Chen et al ldquoTetramethylpyrazinereduces ischemic brain injury in ratsrdquo Neuroscience Letters vol372 no 1-2 pp 40ndash45 2004

[5] L Feng N Ke F Cheng et al ldquoThe protective mechanismof ligustrazine against renal ischemiareperfusion injuryrdquo TheJournal of Surgical Research vol 166 no 2 pp 298ndash305 2011

[6] W Qian X Xiong Z Fang H Lu and Z Wang ldquoPro-tective effect of tetramethylpyrazine on myocardial ischemia-reperfusion injuryrdquo Evidence-Based Complementary and Alter-native Medicine vol 2014 Article ID 107501 9 pages 2014

[7] Y Chang G Hsiao S H Chen et al ldquoTetramethylpyrazinesuppresses HIF-1alpha TNF-alpha and activated caspase-3expression in middle cerebral artery occlusion-induced brainischemia in ratsrdquo Acta Pharmacologica Sinica vol 28 no 3 pp327ndash333 2007

[8] X Cai Z Chen X Pan et al ldquoInhibition of angiogenesisfibrosis and thrombosis by tetramethylpyrazine mechanismscontributing to the SDF-1CXCR4 axisrdquo PLoS ONE vol 9 no2 Article ID e88176 2014

[9] X Zhang F Zhang D Kong et al ldquoTetramethylpyrazineinhibits angiotensin II-induced activation of hepatic stellatecells associated with interference of platelet-derived growthfactor 120573 receptor pathwaysrdquo FEBS Journal vol 281 no 12 pp2754ndash2768 2014

[10] F Zhang Z Zhang D Kong et al ldquoTetramethylpyrazinereduces glucose and insulin-induced activation of hepaticstellate cells by inhibiting insulin receptor-mediated PI3KAKTand ERK pathwaysrdquoMolecular and Cellular Endocrinology vol382 no 1 pp 197ndash204 2014

[11] F Zhang C Ni D Kong et al ldquoLigustrazine attenuates oxida-tive stress-induced activation of hepatic stellate cells by inter-rupting platelet-derived growth factor-120573 receptor-mediatedERK and p38 pathwaysrdquo Toxicology and Applied Pharmacologyvol 265 no 1 pp 51ndash60 2012

[12] B Wang Q Ni X Wang and L Lin ldquoMeta-analysis of theclinical effect of ligustrazine on diabetic nephropathyrdquo TheAmerican Journal of Chinese Medicine vol 40 no 1 pp 25ndash372012

[13] Q-H Yang Y Liang Q Xu Y Zhang L Xiao and L-Y SildquoProtective effect of tetramethylpyrazine isolated from Ligus-ticum chuanxiong on nephropathy in rats with streptozotocin-induced diabetesrdquo Phytomedicine vol 18 no 13 pp 1148ndash11522011

[14] L-M Lee C-F Liu and P-P Yang ldquoEffect of tetrameth-ylpyrazine on lipid peroxidation in streptozotocin-induceddiabetic micerdquo The American Journal of Chinese Medicine vol30 no 4 pp 601ndash608 2002

[15] K Yu Z Chen X Pan et al ldquoTetramethylpyrazine-mediatedsuppression of C6 gliomas involves inhibition of chemokinereceptor CXCR4 expressionrdquo Oncology Reports vol 28 no 3pp 955ndash960 2012

[16] Y Zhang X Liu T Zuo Y Liu and J H Zhang ldquoTetram-ethylpyrazine reverses multidrug resistance in breast cancer

cells through regulating the expression and function of P-glycoproteinrdquo Medical Oncology vol 29 no 2 pp 534ndash5382012

[17] X-B Wang S-S Wang Q-F Zhang et al ldquoInhibition oftetramethylpyrazine on P-gp MRP2 MRP3 and MRP5 inmultidrug resistant human hepatocellular carcinoma cellsrdquoOncology Reports vol 23 no 1 pp 211ndash215 2010

[18] Y-H Shih S-L Wu W-F Chiou H-H Ku T-L Ko andY-S Fu ldquoProtective effects of tetramethylpyrazine on kainateinduced excitotoxicity in hippocampal culturerdquo NeuroReportvol 13 no 4 pp 515ndash519 2002

[19] T-K Kao C-Y Chang Y-C Ou et al ldquoTetramethylpyrazinereduces cellular inflammatory response following permanentfocal cerebral ischemia in ratsrdquo Experimental Neurology vol247 pp 188ndash201 2013

[20] T-K Kao Y-C Ou J-S Kuo et al ldquoNeuroprotection bytetramethylpyrazine against ischemic brain injury in ratsrdquo Neu-rochemistry International vol 48 no 3 pp 166ndash176 2006

[21] Q Tang R Han H Xiao J Shen Q Luo and J Li ldquoNeuropro-tective effects of tanshinone IIA andor tetramethylpyrazine incerebral ischemic injury in vivo and in vitrordquo Brain Researchvol 1488 pp 81ndash91 2012

[22] Y Sun J Jiang Z Zhang et al ldquoAntioxidative and thrombolyticTMP nitrone for treatment of ischemic strokerdquo Bioorganic ampMedicinal Chemistry vol 16 no 19 pp 8868ndash8874 2008

[23] X Xiao Y Liu C Qi et al ldquoNeuroprotection and enhancedneurogenesis by tetramethylpyrazine in adult rat brain after focalischemiardquo Neurological Research vol 32 no 5 pp 547ndash5552010

[24] S T Carmichael ldquoPlasticity of cortical projections after strokerdquoThe Neuroscientist vol 9 no 1 pp 64ndash75 2003

[25] R J Nudo ldquoPlasticityrdquoNeuroRx vol 3 no 4 pp 420ndash427 2006[26] B B Johansson and P V Belichenko ldquoNeuronal plasticity and

dendritic spines effect of environmental enrichment on intactand postischemic rat brainrdquo Journal of Cerebral Blood Flow ampMetabolism vol 22 no 1 pp 89ndash96 2002

[27] C E Brown andTHMurphy ldquoLivinrsquo on the edge imaging den-dritic spine turnover in the peri-infarct zone during ischemicstroke and recoveryrdquo The Neuroscientist vol 14 no 2 pp 139ndash146 2008

[28] J Astrup B K Siesjo and L Symon ldquoThresholds in cerebralischemiamdashthe ischemic penumbrardquo Stroke vol 12 no 6 pp723ndash725 1981

[29] W-D Heiss ldquoThe ischemic penumbra how does tissue injuryevolverdquo Annals of the New York Academy of Sciences vol 1268no 1 pp 26ndash34 2012

[30] Q Zhou Q Zhang X Zhao et al ldquoCortical electrical stimu-lation alone enhances functional recovery and dendritic struc-tures after focal cerebral ischemia in ratsrdquo Brain Research vol1311 pp 148ndash157 2010

[31] Y M Zhang H Xu H Sun S H Chen and F M WangldquoElectroacupuncture treatment improves neurological functionassociated with regulation of tight junction proteins in ratswith cerebral ischemia reperfusion injuryrdquo Evidence-BasedComplementary and Alternative Medicine vol 2014 Article ID989340 10 pages 2014

[32] J Chen Y Li LWang et al ldquoTherapeutic benefit of intravenousadministration of bone marrow stromal cells after cerebralischemia in ratsrdquo Stroke vol 32 no 4 pp 1005ndash1011 2001

[33] X Bao X Tian X Hu Z Zhao Y Qu and C Song ldquoDiscoveryof specific tryptophan hydroxylase in the brain of the beetle


Harmonia axyridisrdquo Brain Research vol 1073-1074 no 1 pp202ndash208 2006

[34] G Paxinos and C WatsonThe Rat Brain in Stereotaxic Coordi-nates Elsevier London UK 2007

[35] C L R Gonzalez O A Gharbawie P T Williams J A KleimB Kolb and I Q Whishaw ldquoEvidence for bilateral control ofskilled movements ipsilateral skilled forelimb reaching deficitsand functional recovery in rats follow motor cortex and lateralfrontal cortex lesionsrdquoEuropean Journal of Neuroscience vol 20no 12 pp 3442ndash3452 2004

[36] F Alcantara-Gonzalez I Juarez O Solis et al ldquoEnhanceddendritic spine number of neurons of the prefrontal cortexhippocampus and nucleus accumbens in old rats after chronicdonepezil administrationrdquo Synapse vol 64 no 10 pp 786ndash7932010

[37] D A Sholl ldquoDendritic organization in the neurons of the visualand motor cortices of the catrdquo Journal of anatomy vol 87 no 4pp 378ndash406 1953

[38] R L Gibb C L R Gonzalez W Wegenast and B E KolbldquoTactile stimulation promotes motor recovery following corti-cal injury in adult ratsrdquo Behavioural Brain Research vol 214 no1 pp 102ndash107 2010

[39] O Hurtado A Cardenas J M Pradillo et al ldquoA chronictreatment with CDP-choline improves functional recoveryand increases neuronal plasticity after experimental strokerdquoNeurobiology of Disease vol 26 no 1 pp 105ndash111 2007

[40] F Liu and L D McCullough ldquoMiddle cerebral artery occlusionmodel in rodents methods and potential pitfallsrdquo Journal ofBiomedicine amp Biotechnology vol 2011 Article ID 464701 9pages 2011

[41] D C Morris M Chopp L Zhang M Lu and Z G ZhangldquoThymosin 1205734 improves functional neurological outcome in arat model of embolic strokerdquo Neuroscience vol 169 no 2 pp674ndash682 2010

[42] M Song Y-J KimY-HKim J Roh SUKim andB-WYoonldquoEffects of duplicate administration of human neural stem cellafter focal cerebral ischemia in the ratrdquo International Journal ofNeuroscience vol 121 no 8 pp 457ndash461 2011

[43] T-H Tsai and C-C Liang ldquoPharmacokinetics of tetram-ethylpyrazine in rat blood and brain using microdialysisrdquoInternational Journal of Pharmaceutics vol 216 no 1-2 pp 61ndash66 2001

[44] Y Li N Jiang C Powers and M Chopp ldquoNeuronal damageand plasticity identified by microtubule-associated protein 2growth-associated protein 43 and cyclin D1 immunoreactivityafter focal cerebral ischemia in ratsrdquo Stroke vol 29 no 9 pp1972ndash1980 1998

[45] P C Garcia C C Real A F B Ferreira S R Alouche L R GBritto and R S Pires ldquoDifferent protocols of physical exerciseproduce different effects on synaptic and structural proteins inmotor areas of the rat brainrdquo Brain Research vol 1456 pp 36ndash48 2012

[46] M Sun Y Zhao Y Gu and C Xu ldquoNeuroprotective actionsof aminoguanidine involve reduced the activation of calpainand caspase-3 in a rat model of strokerdquo Neurochemistry Inter-national vol 56 no 4 pp 634ndash641 2010

[47] M Sun Y Zhao Y Gu and C Xu ldquoInhibition of nNOSreduces ischemic cell death through down-regulating calpainand caspase-3 after experimental strokerdquo Neurochemistry Inter-national vol 54 no 5-6 pp 339ndash346 2009

[48] F Wang Z Liang Q Hou et al ldquoNogo-A is involved insecondary axonal degeneration of thalamus in hypertensive rats

with focal cortical infarctionrdquo Neuroscience Letters vol 417 no3 pp 255ndash260 2007

[49] T A Jones S D Bury D L Adkins-Muir L M Luke R PAllred and J T Sakata ldquoImportance of behavioral manipula-tions and measures in rat models of brain damage and brainrepairrdquo ILAR Journal vol 44 no 2 pp 144ndash152 2003

[50] B CWhite J M Sullivan D J DeGracia et al ldquoBrain ischemiaand reperfusion molecular mechanisms of neuronal injuryrdquoJournal of the Neurological Sciences vol 179 no 1-2 pp 1ndash332000

[51] R T Bartus R L Dean K Cavanaugh D Eveleth D L Car-riero and G Lynch ldquoTime-related neuronal changes followingmiddle cerebral artery occlusion implications for therapeuticintervention and the role of calpainrdquo Journal of Cerebral BloodFlow amp Metabolism vol 15 no 6 pp 969ndash979 1995

[52] M Liebetrau H Martens N Thomassen et al ldquoCalpaininhibitor A-558693 in experimental focal cerebral ischemia inratsrdquo Neurological Research vol 27 no 5 pp 466ndash470 2005

[53] R Gibb and B Kolb ldquoA method for vibratome sectioning ofGolgi-Cox stained whole rat brainrdquo Journal of NeuroscienceMethods vol 79 no 1 pp 1ndash4 1998

[54] B Kolb R Brown A Witt-Lajeunesse and R Gibb ldquoNeuralcompensations after lesion of the cerebral cortexrdquo NeuralPlasticity vol 8 no 1-2 pp 1ndash16 2001

[55] R Mostany and C Portera-Cailliau ldquoAbsence of large-scaledendritic plasticity of layer 5 pyramidal neurons in peri-infarctcortexrdquoThe Journal of Neuroscience vol 31 no 5 pp 1734ndash17382011

[56] C L R Gonzalez and B Kolb ldquoA comparison of differentmodels of stroke on behaviour and brain morphologyrdquo TheEuropean Journal of Neuroscience vol 18 no 7 pp 1950ndash19622003

[57] C E Brown J D Boyd and THMurphy ldquoLongitudinal in vivoimaging reveals balanced and branch-specific remodeling ofmature cortical pyramidal dendritic arbors after strokerdquo Journalof Cerebral Blood FlowampMetabolism vol 30 no 4 pp 783ndash7912010

[58] X Yu and Y Zuo ldquoSpine plasticity in the motor cortexrdquo CurrentOpinion in Neurobiology vol 21 no 1 pp 169ndash174 2011

[59] J Biernaskie and D Corbett ldquoEnriched rehabilitative trainingpromotes improved forelimb motor function and enhanceddendritic growth after focal ischemic injuryrdquo The Journal ofNeuroscience vol 21 no 14 pp 5272ndash5280 2001

[60] T Jiang R X Xu A W Zhang et al ldquoEffects of transcranialdirect current stimulation on hemichannel pannexin-1 and neu-ral plasticity in rat model of cerebral infarctionrdquo Neurosciencevol 226 pp 421ndash426 2012

[61] T A Jones and T Schallert ldquoOvergrowth and pruning ofdendrites in adult rats recovering from neocortical damagerdquoBrain Research vol 581 no 1 pp 156ndash160 1992

[62] J Lippman and A Dunaevsky ldquoDendritic spine morphogenesisand plasticityrdquo Journal of Neurobiology vol 64 no 1 pp 47ndash572005

[63] T Tada and M Sheng ldquoMolecular mechanisms of dendriticspinemorphogenesisrdquoCurrent Opinion in Neurobiology vol 16no 1 pp 95ndash101 2006

Research ArticleCardioprotective Potential of Polyphenolic RichGreen Combination in Catecholamine Induced MyocardialNecrosis in Rabbits

Fatiqa Zafar1 Nazish Jahan1 Khalil-Ur-Rahman2 Ahrar Khan3 and Waseem Akram4

1Department of Chemistry University of Agriculture Faisalabad 38000 Pakistan2Department of Biochemistry University of Agriculture Faisalabad 38000 Pakistan3Department of Pathology University of Agriculture Faisalabad 38000 Pakistan4Department of Entomology University of Agriculture Faisalabad 38000 Pakistan

Correspondence should be addressed to Nazish Jahan nazishjahanuafyahoocom

Received 5 February 2015 Revised 13 May 2015 Accepted 21 May 2015


Copyright copy 2015 Fatiqa Zafar et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The present study was designed to develop safer effective and viable cardioprotective herbal combination to control oxidative stressrelated cardiac ailments as new alternatives to synthetic drugs The synergetic cardioprotective potential of herbal combinationof four plants T arjuna (TA) P nigrum (PN) C grandiflorus (C) and C oxyacantha (Cr) was assessed through curative andpreventive mode of treatment In preventive mode of treatment the cardiac injury was induced with synthetic catecholamine(salbutamol) to pretreated rabbits with the proposed herbal combination for three weeks In curative mode of treatmentcardiotoxicityoxidative stress was induced in rabbits with salbutamol prior to treating them with plant mixture Cardiac markerenzymes lipids profile and antioxidant enzymes as biomarker of cardiotoxicity were determined in experimental animals Rabbitsadministrated with mere salbutamol showed a significant increase in cardiac marker enzymes and lipid profile and decrease inantioxidant enzymes as compared to normal control indicating cardiotoxicity and myocardial cell necrosis However pre- andpostadministration of plant mixture appreciably restored the levels of all biomarkers Histopathological examination confirmedthat the said combination was safer cardioprotective product

1 Introduction

Cardiovascular diseases have become a global threat to life[1] and are major reason of 171 million fatalities every yearIt is expected that death toll due to cardiac diseases willreach up to 20 million in 2020 [2] In Pakistan the conditionhas become really alarming as cardiac ailments contributeto about 25 of deaths in the country [3] Diverging to theconsistent efforts of medical and pharmaceutical scientiststo combat the heart diseases rather than to minimize theprevalence the numbers of cardiac patients are increasing[4] Currently available synthetic cardioprotective medicineshave not only been related to a number of side effects but arealso very costly [5] The easy availability comparatively lessside effects and low cost ofmedicinal plantsmake themmoreattractive therapeutic agents [6]

Medicinal plants enriched with polyphenols possess-ing free radical scavenging potential may reduce the riskof heart diseases because of inverse relationship betweencardiovascular diseases and intake of polyphenols [7] Freeradicals are reactive species generated in the body as a resultof many endogenous (metabolic pathways) and exogenous(environmental pollution pesticides and exposure to radi-ations) sources [8] Different environmental factors elevatethe level of free radicals and cells become unable to workefficiently against the free radicals leading to accumulationof radicals and oxidative stress which is involved in celldamage necrosis and apoptosis and has main causativerole in pathogenesis of cardiovascular diseases [9 10] Manyantioxidants like Vitamins C and E and plant polyphenols areefficient tools in oxidative stress and cardiovascular disordersas potential therapeutic agents [11]



Various medicinal plants possess certain preventiveeffects regarding heart diseases [12] Botanical therapeuticswith multicomponent has several advantages over singleplant extractisolated compound that may earn them a moreprominent place in the field of herbal medicines Multicom-ponent therapeutics offer bright prospects for the control ofmany diseases in a synergistic manner [13]

Mixtures of interacting bioactive compounds producedby plants may provide important combination therapiesthat simultaneously affect multiple pharmacological targetsand provide clinical efficacy beyond the reach of singlecompound-based drugs Therefore four medicinal plantswere selected to evaluate their combined cardioprotectivepotentialMedicinal plantsCrataegus oxyacantha (Cr) exhibithypotensive cardiotonic antispasmodic diuretic and seda-tive properties It helps to treat heart disease by dilatingperipheral and coronary blood vessels and improves thesupply of blood to the heart and extenuating symptoms inearly period of heart failure [14] Cactus grandiflorus (C) isparticularly useful in treating different ailments associatedwith the heart and is a very good source of polyphenolsIt has the ability to reduce the oxidative stress due to itspowerful antioxidant activity [15] Piper nigrum (PN) com-monly known asBlack Pepper is used to treat cardiac diseasesbeing a very good combination of antioxidants Terminaliaarjuna (TA) has significant antioxidant properties and is agood heart tonic [16] Gemmomodified extract of this plant(TA (g)) is a rich source of bioactive substances Gemmopreparations (freshly growing parts) of medicinal plants areimportant as these contain many active substances that startto disappear as plant reaches maturity [17]

Findingways to screen the synergistic combinations fromnumerous herbal pharmacological agents is still an ongoingchallenge In the present research work extracts of the abovefour medicinal plants being used by alternative practitionersand those have known folk medicinal background were usedin the ratio of (C Cr PN TA (g) = 2 1 2 2) for the assess-ment of synergetic cardioprotective activity These plantshave been previously analyzed by our research group fortheir individual antioxidant potential In the present researchsynergistic cardioprotective potential of the combinationwas evaluated in salbutamol induced cardiotoxicity throughanimal model

2 Methodology

21 Sample Collection Freshly growing leaves (gemmo parts)of medicinal plant Terminalia arjuna (Arjun) were col-lected from the Botanical garden University of AgricultureFaisalabad and got identified from plant taxonomist at theDepartment of Botany University of Agriculture FaisalabadPakistan Piper nigrum (Black pepper) was bought frommarket and ground into fine powder Ethanolic extracts ofmedicinal plants Cactus grandiflorus and Crataegus werepurchased from a branded company of Germany ldquoSchwaberdquofrom Homoeopathic Medical store

22 Sample Preparation Freshly growing leaves (gemmoparts) of Terminalia arjuna were washed with cold water to

remove dirt and were used in the form of gemmomodifiedextract Piper nigrum was purchased from herbal store andwas ground into fine powder whereas prepared ethanolicextracts of Cactus and Crataegus were used

23 Preparation of Plant Extracts Gemmomodified extractof Terminalia arjuna was prepared by maceration processThe fresh plant material was blended in a mixture of alcoholand glycerin having 2 1 ratio for 21 days [17] Aqueous extractof Piper nigrum was prepared by boiling the plant materialwith water for ten minutes and filtrate was used

24 Determination of Phenolics by HPLC For the determi-nation of phenolic contents by HPLC method of Pak-Dek etal [18] was followed Plant extract (50mg) was dissolved in24mL methanol and homogenized and then distilled water(16mL) and HCl (10mL 6M) were added This mixturewas thermostated for 2 h at 95∘C The final solution wasfiltered using a 045120583m nylon membrane filter and HighPerformance Liquid Chromatography (HPLC) analysis wascarried out The conditions used for the HPLC analysis aregiven in Table 1

25 Preparation of Herbal Combinations Herbal combina-tion was prepared by appropriately mixing the extracts ofCactus Crataegus Arjuna and Piper nigrum in the ratioof 2 1 2 2 These plant extracts were individually analyzedby our research group for their total polyphenolic contentsantioxidant activity and cardioprotective potential Presentstudy was planned to evaluate their synergistic cardioprotec-tive potential

26 Animals Male albino rabbits weighing 1ndash15 kg wereselected for this study Rabbits were kept under standardconditions of environment in the department of ClinicalMedicine and Surgery (CMS) University of AgricultureFaisalabad Pakistan andwere allowed free access to standarddiet and water All international ethical considerations aboutanimal studies were monitored during the experiment

27 Experimental Protocol Rabbits were kept for one weekacclimatization period and then randomly divided into dif-ferent groups Each group comprised three rabbits

Group I (Normal Controls) Rabbits were given standard dietonly

Group II (Salbutamol Control Group) Salbutamol was ingest-ed to the rabbits (60mgKg bwt) for two consecutive days toinduce oxidative stressmyocardial cell necrosis

Group III (Baseline Group) Herbal combination (100mgkg bwt) was given orally to rabbits of this group once dailyfor three weeks

Group IV (Preventive Group) Rabbits of this group werepretreated with plant combination 100mgkg bwt once dailyfor three weeks and then treated with two consecutive doses


Table 1 Conditions used for HPLC analysis

Column Shim-Pack CLC-ODS (C-18) 25 cm times 46mm5 120583m

Mobile phaseGradient A (H2O AAmdash94 6 pH = 227) B(CAN 100) 0ndash15min = 15 B 15ndash30 = 45B 30ndash45 = 100 B

Flow rate 1mLminDetector UV-visible detector 280 nmTemperature RTRange Bipolar 1250mV 10 samples per secDetection Gradient

of salbutamol (60mgkg) orally Blood samples were taken toevaluate any effect of herbal combination

Group V (Curative Groups) Rabbits were treated with sal-butamol (60mgkg) for two days to induce cardiotoxic-ity Then these cardiointoxicated rabbits were treated with200mgkg bwt of plant combination once daily for fivedays and blood samples were collected daily to check theposttreatment effect of herbal mixture

Group VI (Standard Curative Group (Synthetic Drug)) Rab-bits were treated orally with salbutamol (60mgkg) for twodays to induce cardiotoxicity Then these cardiointoxicatedrabbits were treated with a standard drug (Norvasc andCapoten) once daily for five days and blood samples werecollected daily

3 Biochemical Assessment

31 Estimation of Cardiac Biomarkers Blood samples weretaken from the jugular vein of rabbits and serum was sepa-rated for analysis of different cardiac biomarkers like lactatedehydrogenase (LDH) creatine kinase-MB fraction (CK-MB) aspartate transaminase (AST) and alanine transam-inase (ALT) Among lipids total cholesterol triglyceridelow density lipoprotein (LDL) and high density lipopro-tein (HDL) were also estimated All these analyses wereperformed with commercially available kits using chemistryanalyzer (Semar S 1000-elite)

32 Estimation of Antioxidant Enzymes in Heart TissuesAfter experimental period animals were slaughtered andheart tissues were separated and washed with isotonic salineThe tissues were homogenized in 10 ice cold phosphatebuffer (pH = 7) Then this mixture was centrifuged andsupernatant was collected for analysis of antioxidant enzymeslike SOD CAT and GPx by following the method of Hameedet al [19]

4 Toxicological Studies

41 Gross Pathology of Experimental Animal Gross pathol-ogy of experimental animals was performed under thesupervision of a veterinary doctor Changes in weight and

structure of heart kidneys liver stomach and lungs werenoted

42 Histopathological Analysis Histopathological analysiswas performed on the apical portion of the heart lungskidney and liver Fresh tissues of these organs were excisedand fixed in 10 formalin for 24 hours Sections were cut into5 120583m thickness and stained with hematoxylin and eosin Thesections were mounted and observed under light microscopewith magnification of 200x for histological changes

43 Statistical Analysis The results were expressed as meanplusmn standard error of mean for three rabbits in each groupThestatistical analysis was performed using Minitab 160 Analy-sis was made using one-way analysis of variance (ANOVA)followed by Tukeyrsquos comparison test 119875 value of lt005 wasconsidered statistically significant

5 Results

51 HPLC Profile of Polyphenolic Contents The amount ofpolyphenols identified in different medicinal plants has beenshown in Figure 1

Highest amount of caffeic acid was present in gemmoArjun (4352mg100 g of plant extract) followed by Crataegus(2326mg100 g) Black Pepper (1851mg100 g) and Cactus(1361mg100 g)

Highest amount of Chlorogenic Acid was found inCactus grandiflorus (Cactus) that was 11429mg100 g of plantextract while the concentration of Chlorogenic Acid was9118mg100 g in Black Pepper 5816mg100 g in gemmoArjun and 2409mg100 g in Crataegus Maximum amountof Ferulic acid was present in Crataegus (9328mg100 g)followed by Cactus and Black Pepper in which the amount ofFerulic acid was 9067mg100 g and 6935mg100 g of plantextract respectively P-Coumaric acid acid was only presentin Crataegus (1568mg100 g) and was absent in all otherplants

52 Effect of Herbal Combination on Cardiac Markers(Enzyme) and Lipids Cardioprotective potential of herbalcombination was assessed through curative and preventivemodes of treatment

53 Preventive Cardioprotective Potential In preventivemode of treatment herbal combination was fed orally forthree weeks to experimental animals After that salbutamolwas given (60mgkg bwt) for two consecutive days toinduce oxidative stress which could untimely lead to cellnecrosis ventricular arrhythmia and myocardial infarctionthat was confirmed by positive troponin test Troponins arestructural proteins of cardiac muscles which are secretedinto blood with myocardial injury and are good markers formyocardial cell necrosis and myocardial infarction

Salbutamol significantly (119901 lt 005) increased the level ofcardiac biomarker enzymes (CK-MB AST ALT and LDH)in salbutamol induced control group as compared to animalsof normal control Increased level of these enzymes was due


Table 2 Preventive cardioprotective effect of herbal combination on cardiac enzymes in different experimental groups

Groups CK-MB (IUL) LDH (IUL) AST (IUL) ALT (IUL)Normal control 355 plusmn 032 5458 plusmn 224 3726 plusmn 037 456 plusmn 041Salbutamol control group 804 plusmn 047lowast 8595 plusmn 357lowast 1135 plusmn 083lowast 1407 plusmn 063lowast

Base line group 228 plusmn 027 5397 plusmn 401 368 plusmn 054 495 plusmn 084

Herbal mixture + (salbutamol) 382 plusmn 048 5515 plusmn 207 397 plusmn 055 624 plusmn 105

Results are expressed as Mean plusmn Standard Error of Mean (SEM) for 119899 = 3lowastSignificantly different from normal controlSignificantly different from salbutamol control

Table 3 Preventive cardioprotective effect of herbal combination on lipid profile in different experimental groups

Groups Cholesterol (mgdL) Triglyceride (mgdL) LDL (mgdL) HDL (mgdL)Normal control group 42 plusmn 045 1185 plusmn 143 26 plusmn 034 456 plusmn 047Salbutamol control group 862 plusmn 039lowast 3424 plusmn 164lowast 576 plusmn 063lowast 324 plusmn 036lowast


Herbal mixture + salbutamol 555 plusmn 083 2038 plusmn 054 295 plusmn 047 437 plusmn 031


0

2

4

6

8

10

12

14

Caffeic acid Chlorogenicacid

Ferulic acid P-Coumaricacid

Plant phenolicsTA (g)C

Cr

Con

c in

mg100

g of

pla

nt ex

trac

t

PN

Figure 1 HPLC analysis of polyphenolic contents of four medicinalplants

to the oxidative stress and myocardial cell necrosis causedby salbutamol Prior administration of herbal mixture atthe dose of 100mgkg significantly (119901 lt 005) maintainedthe salbutamol induced elevated level of cardiac enzymesA significant (119901 lt 005) increase was observed in thelevels of lipid profile (LDL cholesterol and triglycerides)in salbutamol induced control group as compared to nor-mal control indicating hyperlipidemia while level of HDLwas decreased in salbutamol induced control group Herbalcombination prevented the increase of lipids in preventivegroup showing the lipid lowering effect of herbal supernatantHerbal mixture also restored level of HDL whereas rabbits ofbase line group showed nonsignificant changes in the level ofcardiac biomarkers (Tables 2 and 3)

54 Curative Cardioprotective Potential In curative mode oftreatment oxidative cardiotoxicity (myocardial cell necrosis)

was induced in rabbits by giving orally two consecutive dosesof salbutamol which significantly (119901 lt 005) increasedthe level of cardiac biomarkers (CK-MB LDH AST andALT) and lipids of experimental animals This increasedlevel was then subsequently decreased gradually by treatingthe animals with herbal mixture After five days treatmentanimals were almost completely recovered indicating thecardioprotective potential of herbal combinationThe cardio-protective potential of herbal combination was comparablewith synthetic standard drug Five days treatment of cardiointoxicated rabbits with herbal combination also maintainedsalbutamol induced elevated level of lipids Herbal combina-tion restored the lipid level better than synthetic cardiopro-tective drug (Tables 4 and 5)

55 Effect of Herbal Mixture on Myocardial AntioxidantsEnzymes Results of antioxidant enzymes demonstrated thatthe level of all the three enzymes superoxide dismutase(SOD) catalase and glutathione peroxidase was decreasedsignificantly (119901 lt 005) in salbutamol induced control groupas compared to the animals of normal control group indi-cating high oxidative stress Treatment of rabbits with herbalmixture restored the level of antioxidant enzymes Polyphe-nolics rich herbal combination exhibited better potential incurative mode of treatment (Table 6)


Toxicological study was performed through gross pathologyand histopathological examination

61 Gross Pathology Results of gross pathology of variousorgans of different experimental groups of rabbits are givenin Tables 7 and 8 These results demonstrated that the weightof different body organs of salbutamol induced control groupwas increased remarkably (119901 lt 005) as compared to animals


Table 4 Curative cardioprotective effect of herbal combination on cardiac marker (enzymes) in different experimental groups

Enzyme Day Normal control Salbutamol control Salbutamol + herbal mixture Standard drug

CK-MB (IUL)

1 353 plusmn 050 803 plusmn 132lowast 593 plusmn 049 678 plusmn 1062 345 plusmn 035 815 plusmn 142lowast 5767 plusmn 054 612 plusmn 1673 361 plusmn 054 837 plusmn 212lowast 483 plusmn 076 573 plusmn 232

4 327 plusmn 062 852 plusmn 137lowast 3925 plusmn 053 498 plusmn 210


AST (IUL)




ALT (IUL)

1 45 plusmn 143 1424 plusmn 123lowast 139 plusmn 187 1473 plusmn 3102 433 plusmn 162 1429 plusmn 154lowast 136 plusmn 243 1356 plusmn 2733 427 plusmn 145 1437 plusmn 302lowast 933 plusmn 256 1338 plusmn 2744 455 plusmn 156 1418 plusmn 231lowast 8367 plusmn 212 113 plusmn 2435 473 plusmn 176 1442 plusmn 213lowast 6033 plusmn 198 698 plusmn 345

LDH (IUL)

1 5452 plusmn 243 8592 plusmn 435lowast 7476 plusmn 471 8105 plusmn 7232 5495 plusmn 287 8596 plusmn 384lowast 6097 plusmn 254 7715 plusmn 6343 5428 plusmn 261 8573 plusmn 471lowast 588 plusmn 378 634 plusmn 9334 5472 plusmn 354 8551 plusmn 342lowast 567 plusmn 932 5885 plusmn 783



Table 5 Curative cardioprotective effect of herbal combination on lipids in different experimental groups


Cholesterol (mgdL)




Triglyceride (mgdL)



LDL (mgdL)



HDL (mgdL)




Table 6 Level of antioxidant enzymes (Unitsg of wt) in different experimental groups of rabbit

Antioxidantenzyme Control Salbutamol control Herbal mixture + salbutamol

(preventive)Salbutamol + herbal mixture

(curative) Standard drug

Superoxidedismutase(SOD)

9542 plusmn 054 4973 plusmn 064lowast 6645 plusmn 069 9968 plusmn 086 44 54 plusmn 047

Catalase 40307 plusmn 087 6100 plusmn 058lowast 6200 plusmn 047 40000 plusmn 174 93743 plusmn 146

Peroxidase 8103 plusmn 132 730 plusmn 104lowast 1800 plusmn 176 600 plusmn 126 12057 plusmn 173


Table 7 Weight of different body organs of different experimentalgroups

Groups Heart Liver Lungs KidneyRight Left

Normal control 25 206 47 5 51Salbutamol control 51lowast 342lowast 11lowast 72lowast 81lowast

Preventive group 25 202 51 48 49

Curative group 33 338 75 52 44

Standard drug 28 411 91 5 53Results are expressed as Mean plusmn Standard Error of Mean (SEM) for 119899 = 3lowastSignificantly different from normal controlSignificantly different from salbutamol control

of normal control The weight of body organs was normal inrabbits treated with herbal combination

62 Histopathological Examination of Cardiac Tissues Thehistopathological architecture of heart from different exper-imental groups showed series of variations (Figure 2) Inthe normal control group myocardial fibers were arrangedregularly with clear striation No apparent degeneration ornecrosis was observed (Figure 2(a)) Histological section ofsalbutamol treated heart showed severe necrotic and degener-ative changes and hyperchromatic and pyknotic nuclei as wellas fibroblastic hyperplasia and thick connective tissue pro-liferation (Figure 2(b)) Heart tissues were normal in rabbitstreated with herbal combination Mild necrotic changes incardiomyocytes were observed in curative mode of treatment(Figure 2(c)) An insignificant necrosis was examined in theheart of preventive group (Figure 2(d)) Rabbits of base linegroup also showed normal results

7 Discussion

The present study revealed both imperative curative andpreventive ways of cardioprotective potential It explainedthe cardioprotective potential of herbal mixture of fourplants in widely used catechol amine-induced model ofmyocardial cell necrosis in rabbits In the present researcha significant (119901 lt 005) increase was observed in thelevel of cardiac enzymes (CK-MB LDH AST and ALT)in salbutamol (catechol amine) induced control group ascompared to animals of normal control group Salbutamol

which has structural similarities with Isoproterenol (ISO) isa synthetic catecholamine and120573- adrenergic receptor agonistAt high dose it has the ability to destruct myocardial cells andproduce cardiotoxicity in experimental animals as a result ofdisturbance in physiological balance between production offree radicals and antioxidant defense system [20] Increasesin the level of these enzymes were due to their leakagefrom the damaged heart tissues into the blood stream duringmyocardial necrosis because of myofibril degeneration andmyocyte necrosis [21 22] It also caused cardiac dysfunctionand increased lipid peroxidation alongwith an increase in thelevel of myocardial lipids and altered activities of the cardiacmarkers and antioxidant enzymes [23 24]

Treatment of different groups of rabbits with herbalmixture significantly reduced the salbutamol-induced secre-tion of all cardiac diagnostic marker enzymes (CK-MBLDH AST and ALT) This decreased level or reduction inthe secretion of enzymes could be of enzymes could bedue to repairing and maintenance of the myocardial cellsmembrane Curative and preventive treatment of rabbitswith polyphenolic enriched herbal combination significantlydecreased the elevated cardiac enzyme Polyphenols arepotent antioxidant neutralizing lipid free radicals and pre-vent decomposition of hydroperoxides into free radicals [2526] Their cardioprotective potential may be due to scaveng-ing of highly oxidized metabolites produced by salbutamoland stabilization of heart membrane by herbal combinationwith a consequent decrease in the leakage of these markers[21] The tendency of these cardiac markers to become nearthe normal levels in prior and posttreated group is a clearmanifestation of the cardioprotective potential of the herbalcombination

Significant (119901 lt 005) elevated levels of total choles-terol triglycerides and low density lipoproteins (LDL) wereobserved in salbutamol induced control group indicat-ing salbutamol induced hyperlipidemia Highly oxidativemetabolites of catecholamines lead lipid peroxidation whichis the major destructive reaction in cellular mechanism ofthe myocardial ischemia Highly oxidative metabolite ofcatecholamines like isoproterenol and salbutamol acceleratesrate of peroxidation inmembrane phospholipids and releasesfree fatty acids into plasma by the action of phospholipaseA2 and it is a main causative aspect of salbutamol-inducedhyperlipidemia [20] The treatment of experimental animalswith herbal mixture decreased salbutamol induced high levelof lipids With both ways of treatment the (preventive and


Table 8 Gross pathology of different groups of experimental rabbits


Normal control Normal Normal Normal Normal NormalSalbutamol control Enlarged hard and necrosis Normal Congested Slight necrosis congested Hemorrhage and congestedPreventive Normal Normal Normal Normal NormalCurative Slightly congested Normal Normal Normal NormalStandard drug Normal Normal Congested Normal Slight necrosis

(a) (b)

(c) (d)

Figure 2 Histopathological architecture of heart of different experimental groups

curative) the levels of lipid profile reduced closer to thenormal level because of the remedial action of herbal combi-nationThe level of HDLwas decreased in salbutamol controlgroup indicating the reduction of good cholesterol but inboth curative and preventive group the HDL level increasedsignificantly (119901 lt 005) which is comparable with the normalcontrol It is hypothesized thatHDL can eradicate cholesterolfrom atheroma within arteries and transfer it back to theliver for excretion or reutilization That is why HDL-boundcholesterol is sometimes called ldquogood cholesterolrdquo A highlevel of HDL-C protects against cardiovascular diseases andlow HDL cholesterol levels increase the risk of heart diseases[27] Same trend of lipid profile was observed in manyprevious findings [16 23 28ndash31] It is also obvious from

the present findings that the prepared herbal combinationgave overall better results as compared to the standard drugsbecause of its powerful antioxidant and nontoxic nature

Level of antioxidant enzymes was significantly (119901 lt005) lower in salbutamol induced control group Antioxidantenzymes are biomarker of oxidative stress Production ofhighly reactive free radical species inhibited the activitiesof antioxidant enzymes [32] Glutathione antioxidant systemplays a fundamental role in cellular defense against reactivefree radicals and other oxidant species It protects themyocar-dial cellular membrane against oxidative damage by regulat-ing the redox status of proteins in the cell surface membrane[4 22] In the present case decreased superoxide dismutase(SOD) activity in salbutamol control group may be due to


excessive formation of superoxide anions or the decreasedremoval of superoxide anion which can be harmful to themyocardium The activities of H

2O2scavenging enzymes

(CAT and peroxidase) also decreased significantly (119901 lt 005)after the induction of salbutamol to the experimental rabbitsThe activities of these enzymes can be explained by the factthat excessive superoxide anion may inactivate SOD thusresulting in activation of H

2O2scavenging enzymes [4 28]

Pretreatment of rabbits with herbal combination restoredthe level of endogenous antioxidant enzymes SOD CATand peroxidase Posttreatment of experimental animals withherbal mixture helped to regain the level of these enzymesnear to normalThis can be correlated to the free radical scav-enging potential of the herbal combination which protectedthe rabbits from reactive oxygen species Several studies havereported the increase of endogenous antioxidants by herbalformulation or plants extracts in cardiovascular diseases [3334]

Grosshistopathological examination of different bodyorgans such as heart liver lungs and kidney proved thesafe cardioprotective potential of herbal combination Resultsof histopathological analysis are in line with many previousstudies [35ndash39] and illustrated the cardioprotective potentialand nontoxic nature of herbal combination

8 Conclusion

The herbal combination prepared by mixing the appropriateratio of four medicinal plants was administered to the rabbitssuffering from salbutamol induced myocardial cell necrosisthrough both preventive and curativemode of treatments Allthese four plants have been already evaluated individuallyby our research group for the cardioprotective potential Inthe present study the green combination of the medicinalplants was made which showed better synergistic cardiopro-tective potential Bioactive compounds present in differentplants exert synergistic biofunctionalities in combination byinteracting with one another rather than acting alone Thisherbal combination can be used as an alternative effectivedrug for the treatment of cardiovascular diseases because ofits enriched polyphenolic contents and synergic cardiopro-tective potential


The authors do not have any conflict of interests with otherpeople or organizations

Acknowledgment

The authors are grateful to Higher Education Commission ofPakistan for all financial support (no PM-IPFPHRDHEC20124009) of this study

References

[1] R K Srivastav H H Siddiqui T Mahmood and FAhsan ldquoEvaluation of cardioprotective effect of silk cocoon

(Abresham) on isoprenaline-induced myocardial infarctionin ratsrdquo Avicenna Journal of Phytomedicine vol 3 no 3 pp216ndash223 2013

[2] A Upaganlawar H Gandhi and R Balaraman ldquoIsoproterenolinduced myocardial infarction protective role of natural prod-uctsrdquo Journal of Pharmacology and Toxicology vol 6 no 1 pp1ndash17 2011

[3] N Jahan K U Rahman and S Ali ldquoCardioprotective andantilipidemic potential of Cyperus rotundus in chemicallyinduced cardiotoxicityrdquo International Journal of Agriculture andBiology vol 14 no 6 pp 989ndash992 2012

[4] S Ojha J Bhatia S Arora M Golechha S Kumari andD S Arya ldquoCardioprotective effects of Commiphora mukulagainst isoprenaline-induced cardiotoxicity a biochemical andhistopathological evaluationrdquo Journal of Environmental Biologyvol 32 no 6 pp 731ndash738 2011

[5] W Kchaou F Abbes H Attia and S Besbes ldquoIn vitro antiox-idant activities of three selected dates from Tunisia (Phoenixdactylifera L)rdquo Journal of Chemistry vol 2014 Article ID367681 8 pages 2014

[6] J Liu K Peter D Shi et al ldquoAnti-inflammatory effects of thechinese herbal formula sini tang in myocardial infarction ratsrdquoEvidence-based Complementary and Alternative Medicine vol2014 Article ID 309378 10 pages 2014

[7] M Quinones M Miguel and A Aleixandre ldquoBeneficial effectsof polyphenols on cardiovascular diseaserdquo PharmacologicalResearch vol 68 no 1 pp 125ndash131 2013

[8] E Souri G Amin H Farsam and M B Tehrani ldquoScreening ofantioxidant activity and phenolic content of 24 medicinal plantextractsrdquo Daru vol 16 no 2 pp 83ndash87 2008

[9] I Mohanty S K Gupta and D S Arya ldquoAntiapoptotic andcardioprotective effects of a herbal combination in rats withexperimental myocardial infarctionrdquo International Journal ofIntegrative Biology vol 1 no 3 pp 178ndash188 2007

[10] T S Zima L Fialova O Mestek et al ldquoOxidative stressmetabolism of ethanol and alcohol-related diseasesrdquo Journal ofBiomedical Science vol 8 no 1 pp 59ndash70 2001

[11] S V kumar G Saritha and M Fareedullah ldquoRole of antioxi-dants and oxidative stress in cardiovascular diseasesrdquo Annals ofBiological Research vol 1 no 3 pp 158ndash173 2010

[12] F Ahsan H H Siddiqui T Mahmood R K Srivastav andA Nayeem ldquoEvaluation of cardioprotective effect of Coleusforskohlii against isoprenaline induced myocardial infarction inratsrdquo Indian Journal of Pharmaceutical and Biological Researchvol 2 no 1 pp 17ndash25 2014

[13] X L Wang ldquoPotential herb-drug interaction in the preventionof cardiovascular diseases during integrated traditional andwestern medicine treatmentrdquo Chinese Journal of IntegrativeMedicine vol 21 no 1 pp 3ndash9 2015

[14] S K Verma V Jain D Verma and R Khamesra ldquoCratae-gus oxyacanthamdasha cardioprotective herbrdquo Journal of HerbalMedicine and Toxicology vol 1 no 1 pp 65ndash71 2007

[15] R K Verma S E Haque and K K Pillai ldquoCactus grandiflorusa homeopathic preparation has protective effect against doxoru-bicin induced cardiomyopathy in ratsrdquo International Journal ofPhytopharmacology vol 3 no 3 pp 281ndash290 2012

[16] N Jahan K U Rehman S Ali and I A Bhatti ldquoAntioxidantactivity of gemmo therapeutically treated indiginous medicinalplantsrdquoAsian Journal of Chemistry vol 23 no 8 pp 3461ndash34702011


[17] F Khursheed K U Rehman M S Akhtar M Z U H Dogarand B Khalil ldquoComparative antilipidemic effects of nativeand gemmo-treated Withania somnifera (Asghand) extractsrdquoJournal of Applied Pharmaceutical Science vol 1 no 2 pp 47ndash59 2010

[18] M S Pak-Dek A Osman N G Sahib et al ldquoEffects ofextraction techniques on phenolic components and antioxidantactivity of Mengkudu (Morinda citrifolia L) leaf extractsrdquoJournal of Medicinal Plants Research vol 5 no 20 pp 5050ndash5057 2011

[19] A Hameed T M Shah B M Atta M A Haq and HSayed ldquoGamma irradiation effects on seed germination andgrowth protein content peroxidase and protease activity lipidperoxidation in desi and kabuli chickpeardquo Pakistan Journal ofBotany vol 40 no 3 pp 1033ndash1041 2008

[20] V S Panda and S R Naik ldquoEvaluation of cardioprotectiveactivity of Ginkgo biloba and Ocimum sanctum in rodentsrdquoAlternative Medicine Review vol 14 no 2 pp 161ndash171 2009

[21] A G Beaulah M A Sadiq V Sivakumar and J R SanthildquoCardioprotective activity of methanolic extract of Croton spar-cifloruson isoproterenol induced myocardial infarcted wistaralbino ratsrdquo Journal of Medicinal Plants Studies vol 2 no 6 pp1ndash8 2014

[22] K H Sabeena Farvin R Anandan S H S Kumar K S ShinyT V Sankar and T KThankappan ldquoEffect of squalene on tissuedefense system in isoproterenol-induced myocardial infarctionin ratsrdquo Pharmacological Research vol 50 no 3 pp 231ndash2362004

[23] M Murugesan M Ragunath S Nadanasabapathy R Revathiand V Manju ldquoProtective role of fenugreek on isoproterenolinduced myocardial infarction in ratsrdquo International ResearchJournal of Pharmacy vol 3 no 2 pp 211ndash216 2012

[24] S Ittagi V K Merugumolu and R S Siddamsetty ldquoCardiopro-tective effect of hydroalcoholic extract of Tecoma stans flowersagainst isoproterenol induced myocardial infarction in ratsrdquoAsian Pacific Journal of Tropical Disease vol 4 no 1 pp S378ndashS384 2014

[25] H-Y Li Z-B Hao X-L Wang L Huang and J-P Li ldquoAntiox-idant activities of extracts and fractions from Lysimachiafoenum-graecum Hancerdquo Bioresource Technology vol 100 no2 pp 970ndash974 2009

[26] A Rohman S Riyanto N Yuniarti W R Saputra R UtamiandW Mulatsih ldquoAntioxidant activity total phenolic and totalflavaonoid of extracts and fractions of red fruit (Pandanusconoideus Lam)rdquo International FoodResearch Journal vol 17 no1 pp 97ndash106 2010

[27] O I Oyewole I G Adanlawo and R O Arise ldquoSerum andtissue lipid profile in wistar rats administered leaf extract ofFicusexasperatardquo Annals of Biological Research vol 4 pp 288ndash291 2013

[28] F Kousar N Jahan K U Rehman and S Nosheen ldquoCardiopro-tective potential of Coriandrum sativumrdquo Plant Science Journalvol 1 no 1 pp 1ndash6 2012

[29] R Sivakumar R Rajesh S Budhan et al ldquoAntilipideimiceffect of chitosan against experimentally induced myocardialinfarction in ratsrdquo Journal of Cell and Animal Biology vol 1 no4 pp 71ndash77 2007

[30] M A Kareem G S Krushna S A Hussain and K L DevildquoEffect of aqueous extract of nutmeg on hyperglycaemia hyper-lipidaemia and cardiac histology associated with isoproterenol-induced myocardial infarction in ratsrdquo Tropical Journal ofPharmaceutical Research vol 8 no 4 pp 337ndash344 2009

[31] K Adi K Metowogo A Mouzou et al ldquoEvaluation of cardio-protective effects of Parkia biglobosa (JacqBenth) mimosaceaestem barkrdquo Journal of Applied Pharmaceutical Science vol 3 no2 pp 60ndash64 2013

[32] M Eshaghi S Zare N Banihabib V Nejati F Farokhi andP Mikaili ldquoCardioprotective effect of Cornus mas fruit extractagainst carbon tetrachloride induced-cardiotoxicity in albinoratsrdquo Journal of Basic and Applied Scientific Research vol 2 no11 pp 11106ndash11114 2012

[33] I Mohanty D S Arya A Dinda K K Talwar S Joshi and SK Gupta ldquoMechanisms of cardioprotective effect of Withaniasomnifera in experimentally induced myocardial infarctionrdquoBasic and Clinical Pharmacology amp Toxicology vol 94 no 4 pp184ndash189 2004

[34] S N Goyal S Arora A K Sharma et al ldquoPreventiveeffect of crocin of Crocus sativus on hemodynamic bio-chemical histopathological and ultrastuctural alterations inisoproterenol-induced cardiotoxicity in ratsrdquo Phytomedicinevol 17 no 3-4 pp 227ndash232 2010

[35] F Fathiazad A Matlobi A Khorrami et al ldquoPhytochemicalscreening and evaluation of cardioprotective activity of ethano-lic extract of Ocimum basilicum L (basil) against isoproterenolinduced myocardial infarction in ratsrdquo DARU Journal of Phar-maceutical Sciences vol 20 no 1 article 87 2012

[36] I R Mohanty S K Gupta D S Arya N Mohanty andY Deshmukh ldquoMedicinal herbs can play significant role inattenuation of ischemia and reperfusion injuryrdquo Journal ofHomeopathy and Ayurvedic Medicine vol 3 pp 2ndash5 2013

[37] S Sahreen M R Khan and R A Khan ldquoHepatoprotectiveeffects of methanol extract of Carissa opaca leaves on CCl

4

-induced damage in ratrdquo BMC Complementary amp AlternativeMedicine vol 11 article 48 2011

[38] K Yousefi F Fathiazad H Soraya M Rameshrad N Maleki-Dizaji and A Garjani ldquoMarrubium vulgare L methanolicextract inhibits inflammatory response and prevents cardiomy-ocyte fibrosis in isoproterenol-induced acutemyocardial infarc-tion in ratsrdquo BioImpacts vol 4 no 1 pp 21ndash27 2014

[39] S Hina K Rehman Z H Dogar et al ldquoCardioprotective effectof gemmotherapeutically treated Withania somnifera againstchemically induced myocardial injuryrdquo Pakistan Journal ofBotany vol 42 no 3 pp 1487ndash1499 2010

Research ArticleHinokitiol Negatively Regulates Immune Responses throughCell Cycle Arrest in Concanavalin A-Activated Lymphocytes

Chi-Li Chung12 Kam-Wing Leung3 Wan-Jung Lu4 Ting-Lin Yen4 Chia-Fu He4

Joen-Rong Sheu4 Kuan-Hung Lin45 and Li-Ming Lien67

1Division of Pulmonary Medicine Department of Internal Medicine Taipei Medical University Hospital Taipei 110 Taiwan2School of Respiratory Therapy College of Medicine Taipei Medical University Taipei 110 Taiwan3Department of Dentistry Yuanrsquos General Hospital Kaohsiung 802 Taiwan4Department of Pharmacology and Graduate Institute of Medical Sciences College of Medicine Taipei Medical UniversityTaipei 110 Taiwan5Central Laboratory Shin Kong Wu Ho-Su Memorial Hospital Taipei 111 Taiwan6School of Medicine College of Medicine Taipei Medical University Taipei 110 Taiwan7Department of Neurology Shin Kong Wu Ho-Su Memorial Hospital Taipei 111 Taiwan

Correspondence should be addressed to Kuan-Hung Lin d102092002tmuedutw and Li-Ming Lien m002177msskhorgtw

Received 30 September 2014 Revised 12 February 2015 Accepted 16 February 2015

Academic Editor Attila Hunyadi

Copyright copy 2015 Chi-Li Chung et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Autoimmune diseases are a group of chronic inflammatory diseases that arise from inappropriate inflammatory responsesHinokitiol isolated from the wood of Chamaecyparis taiwanensis engages in multiple biological activities Although hinokitiolhas been reported to inhibit inflammation its immunological regulation in lymphocytes remains incompleteThus we determinedthe effects of hinokitiol on concanavalin A- (ConA-) stimulated T lymphocytes from the spleens of mice In the present study theMTT assay revealed that hinokitiol (1ndash5120583M) alone did not affect cell viability of lymphocytes but at the concentration of 5 120583Mit could reduce ConA-stimulated T lymphocyte proliferation Moreover propidium iodide (PI) staining revealed that hinokitiolarrested cell cycle of T lymphocytes at the G0G1 phase Hinokitiol also reduced interferon gamma (IFN-120574) secretion from ConA-activated T lymphocytes as detected by an ELISA assay In addition hinokitiol also downregulated cyclin D3 E2F1 and Cdk4expression and upregulated p21 expression These results revealed that hinokitiol may regulate immune responses In conclusionwe for the first time demonstrated that hinokitiol upregulates p21 expression and attenuates IFN-120574 secretion in ConA-stimulatedT lymphocytes thereby arresting cell cycle at the G0G1 phase In addition our findings also indicated that hinokitiol may providebenefits to treating patients with autoimmune diseases

1 Introduction

Mature lymphocytes must proliferate intensely and repeat-edly to provide a rapid immune response and generateimmunological memory [1] Cell proliferation is amandatoryprocess for immune-system function However unregulatedor excessive immune responsesmay cause immune-mediatedinflammatory diseases (IMIDs) such as rheumatoid arthritisCrohnrsquos disease systemic lupus erythematosus (SLE) andmultiple sclerosis [2 3] These diseases are commonly Tlymphocyte-mediated disorders Although the pathogenic

mechanisms underlying the development of these diseasesare not entirely clear studies have proposed that increasedlymphocyte cycling or defective apoptosis may cause break-down of immune tolerance and autoimmunity as well aslymphoma generation [1ndash3] Thus controlling the cell cycleof lymphocytes may be an effective therapeutic strategy fortreating patients with IMIDs

The cell cycle inhibitor p21 which belongs to the CipKipfamily interferes with cycling by inhibiting all cyclin-dependent kinases (CDKs) involved in the G1S phasethereby controlling cell proliferation and tumorigenesis in



various cell types [4] In addition p21 deficiencywas reportedto enhance T lymphocyte activation and proliferation and toinduce autoimmune manifestations [5] Suppression of p21promotesmalignant T lymphocyte proliferation inmalignantCD30+ T lymphocytes [6] Thus p21 may play a critical rolein autoimmune diseases and tumorigenesis by regulating Tlymphocyte activation and proliferation

Hinokitiol is a naturally occurring compound isolatedfrom the wood of Chamaecyparis taiwanensis [7] Hinokitiolhas been used in hair tonics tooth pastes cosmetics and foodas an antimicrobial agent [8] Moreover hinokitiol engagesin multiple biological activities including anticancer andanti-inflammatory activities [9 10] Studies have reportedthat hinokitiol suppresses tumor growth by inhibiting cellproliferation and inducing apoptosis or autophagy in variouscancer cell lines [9 11ndash13] It was also reported to suppresstumor necrosis factor 120572 production by inhibiting NF-120581Bactivity in lipopolysaccharide-stimulated macrophages [10]In our previous study we demonstrated that hinokitiolexhibits potent antiplatelet activity [14]

Although hinokitiol has been reported to engage in mul-tiple biological activities the regulation of lymphocytes byhinokitiol has not been fully investigated In our preliminarystudy we determined that hinokitiol can arrest the cell cycleof T lymphocytesThus we evaluated the effects of hinokitiolin concanavalin A- (ConA-) activated T lymphocytes isolatedfrom the spleens of mice


21 Materials Hinokitiol was purchased from Sigma (StLouis MO) The anticyclin D3 anti-E2F1 anti-Cdk4 andanti-GAPDH polyclonal antibodies (pAbs) and anti-p21monoclonal antibody (mAb) were purchased from GeneTex(Irvine CA)The PI-annexin V-FITC kit was purchased fromBioLegend (San Diego CA) The Mouse Interferon Gamma(IFN-120574) ELISA Ready-SET-Go kit was purchased fromeBioscience (San Diego CA) The Hybond-P polyvinyli-dene difluoride membrane an enhanced chemiluminescence(ECL)western blotting detection reagent and analysis systemthe horseradish peroxidase- (HRP-) conjugated donkey anti-rabbit immunoglobulin G (IgG) and the sheep anti-mouseIgG were purchased from Amersham (BuckinghamshireUK) Hinokitiol was dissolved in 05 dimethyl sulfoxide(DMSO) and stored at 4∘C until used

22 Mice Theprotocols conformed to the Guide for the Careand Use of Laboratory Animals (NIH publication number85ndash23 1996) Briefly male BALBc mice (6ndash8 weeks oldapproximately 20ndash25 g) were purchased from BioLASCOTaiwanCo Ltd and fed in the animal house of TaipeiMedicalUniversity

23 Lymphocyte Preparation The spleen was asepticallyremoved from each mouse and placed in a sterile petri dishcontaining the RPMI 1640 medium Single-cell suspensionswere prepared by gently disrupting the spleen on a sterilewire meshThe cell suspensions were centrifuged at 300 g for

5min and red blood cells were then lysed using the ACK(ammonium-chloride-potassium) lysis buffer (15mL) andsubsequently 1x phosphate buffered saline (PBS 20mL)Thelymphocyte pellets were collected through centrifugation at300 g for 5min and suspended with RPMI containing 5heat-inactivated fetal bovine serum (Gibco)The cell viabilitywas determined according to trypan blue exclusionThe cellswere prepared at an appropriate density depending on thescale of each experiment

24 Cell Viability Cell proliferation was evaluated using acolorimetric assay Cell viability was measured by conduct-ing a 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazoliumbromide (MTT) assay In brief cells (3 times 105 cellswell) werecultured in 96-well plates and incubated with a vehicle orhinokitiol (1 2 or 5120583M) for 24 or 48 h MTT (5mgmL) wasadded and the cells were incubated for an additional 1 h Thecells were then lysed in 400120583L of DMSO The absorbancewas measured at 570 nm by using a microplate reader Eachexperiment was performed in triplicate and repeated at leastthree times

25 Cytokine Secretion according to ELISA Assay Theamounts of secreted IFN-120574 protein were quantified usingthe Mouse IFN-120574 ELISA Ready-SET-Go kit (eBioscienceSan Diego CA) Recombinant IFN-120574 was used to generate astandard curve which was employed in calculating the IFN-120574concentrations of all samples All procedures were performedaccording to the manufacturerrsquos instructions (eBioscience)

26 Flow Cytometric Analysis Cells were cultured in 24-wellplates After reaching 80 confluence the cells were treatedwith a vehicle or hinokitiol (1 2 or 5120583M) for 48 h The cellswere washed twice with PBS detached and centrifuged Thecells (1 times 106) were then resuspended with 05mL of PBS andthen added to propidium iodide (PI 50 120583gmL) for 15minat room temperature in the dark before flow cytometricanalysis was conducted Finally the cells were filtered on anylon mesh filter The samples were analyzed using a flowcytometer (Becton Dickinson FACScan Syst San Jose CA)Each experiment was repeated at least three times

27 Immunoblotting Cells (1 times 107) were cultured in 6-wellplates After reaching 80 confluence the cells were treatedwith a vehicle or hinokitiol (1 2 or 5120583M) for 24 h After thereactions the cells were collected and lysed with 70 120583L of alysis buffer Samples containing 40 120583g of protein were sepa-rated by conducting sodium dodecyl sulfate polyacrylamidegel electrophoresis The proteins were electrotransferred bya Bio-Rad semidry transfer (Hercules CA) The membraneswere blocked with TBST (10mM Tris-base 100mM NaCland 001 Tween 20) containing 5 BSA for 1 h and thenprobed with various primary antibodies Membranes wereincubatedwith theHRP-linked anti-mouse IgG or anti-rabbitIgG (diluted 1 3000 in TBST) for 1 h Immunoreactive bandswere detected using an ECL system Semiquantitative resultswere obtained by scanning reactive bands and quantifyingthe optical density of each band by using videodensitometry


0

20

40

60

80

100

120

DMSO 1 2 5

Cel

l via

bilit

y (

)

24h48h

(a)

Cel

l via

bilit

y (

)

0

100

200

300

400

ConAHinokitiol 1 2 5

minus

minus minus

+ + + +

lowast

(b)

0

500

1000

1500

2000

2500


minus

minus minus

+ + + +

IFN

-120574(p

gm

L)

lowastlowast

(c)

Figure 1 Effects of hinokitiol on cell viability and interferon gamma (IFN-120574) secretion in ConA-activated T lymphocytes Cells were treatedwith hinokitiol (1ndash5120583M) in the absence or presence of ConA (10120583gmL) for 24 or 48 h (a b) Cell viability was determined using a MTTassay (119899 = 4) (c) The level of IFN-120574 was measured by an ELISA assay (119899 = 3) Data (b c) are presented as the mean plusmn SEM (lowast119875 lt 005 andlowastlowast

119875 lt 001 compared with solvent control (DMSO) 119875 lt 005 and 119875 lt 001 compared with the ConA-treated group)

(Bio-profil Biolight Windows Application V200001 VilberLourmat France)

28 Data Analysis The experimental results are expressedas the mean plusmn SEM and are accompanied by the numberof observations The data were assessed by conducting ananalysis of variance When this analysis indicated significantdifferences among the group means further comparisonswere made using the Newman-Keuls method 119875 lt 005indicated statistical significance

3 Results

31 Hinokitiol Reduces the Viability and Cytokine Secretion ofLymphocytes In the present study an MTT assay was usedto evaluate the cell viability and proliferation of lymphocytesAs shown in Figure 1(a) hinokitiol at the concentrations of 1

2 and 5 120583M did not affect the viability of lymphocytes aftertreatment for 24 and 48 h indicating that hinokitiol (le5 120583M)did not exhibit cytotoxicity to lymphocytes Figure 1(b) showsthatConA treatment (10120583gmL) for 24 h induced lymphocyteproliferation which was reversed by 5120583Mhinokitiol indicat-ing that hinokitiol inhibits ConA-induced cell proliferationof lymphocytes In addition we determined the influenceof hinokitiol on the levels of IFN-120574 secreted from ConA-stimulated T lymphocytes (Figure 1(c))

32 Hinokitiol Arrests the Cell Cycle at the G0G1 Phase PIstaining was used to determine the effect of hinokitiol on thecell cycle in ConA-activated lymphocytes Following ConAstimulation for 48 h quiescent lymphocytes (G0) begancycling The population of the G0G1 phase decreased 229and the population of the S and G2M phases increased231 upon ConA treatment compared with nontreatment


DMSO ConA

G0G1

S-G2M

G0G1

S-G2M

G0G1

S-G2M

S-G2M

G0G1 G0G1

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

S-G2M

ConA + hinokitiol 1

ConA + hinokitiol 2 ConA + hinokitiol 5

(a)

0

10

20

30

40

50

0

20

40

60

80

100

Subp

opul

atio

n (

)

Subp

opul

atio

n (

)

G0G1 S + G2M

lowastlowast

lowastlowast


minus

minus minus

+ + + + ConAHinokitiol 1 2 5

minus

minus minus

+ + + +

(b)

Figure 2 Effects of hinokitiol on the cell cycle in ConA-activated T lymphocytes Cells were treated with hinokitiol (1ndash5120583M) in the absenceor presence of ConA (10120583gmL) for 48 h (a) Cell cycle was determined by PI staining under a flow cytometry (b) The panel shows thepopulation of the G0G1 and S-G2M phases Data (b) are presented as the mean plusmn SEM (119899 = 3 lowastlowast119875 lt 001 compared with solvent control(DMSO) 119875 lt 001 compared with the ConA-treated group)

(resting) these changes were reversed by 5 120583M hinokitiol(Figures 2(a) and 2(b)) Hinokitiol markedly arrested the cellcycle at the G0G1 phase in ConA-stimulated lymphocytes(Figure 2(a)) Compared with ConA treatment 5 120583Mhinoki-tiol treatment increased the population of theG0G1 phase by24 and reduced the population of the S andG2M phases by252 (Figures 2(a) and 2(b))

33 Hinokitiol Downregulates the Expression of the CyclinD3 Cdk4 and E2F1 Proteins and Upregulates the Expres-sion of the p21 Protein The processes of cell cycling arecomplex and involve positive regulators such as cyclin D3Cdk4 and E2F1 and negative regulators such as p21 Theseproteins were determined in this study Our data revealedthat 5 120583M hinokitiol significantly inhibited ConA-induced


00

05

10

15

20

25

30

35

Cyclin D3

GAPDH

Cycli

n D3

(fold

sba

sal)

lowastlowast


minus

minus minus

+ + + +

(a)

0

2

4

6

8

GAPDH

Cdk4

lowastlowast

Cdk4

(fold

sba

sal)


minus

minus minus

+ + + +

(b)

0

1

2

3

4

5

6

GAPDH

E2F1

E2F1

(fold

sba

sal)

lowastlowastlowast


minus

minus minus

+ + + +

(c)

Figure 3 Effects of hinokitiol on positive regulators of the cell cycle Cells were treated with hinokitiol (1ndash5120583M) in the absence or presenceof ConA (10120583gmL) for 24 h The specific antibodies were used to detect (a) cyclin D3 (b) Cdk4 and (c) E2F1 Data (andashc) are presented asthe mean plusmn SEM (119899 = 3 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared with solvent control (DMSO) 119875 lt 005 119875 lt 001 and

119875 lt 0001

compared with the ConA-treated group)

cyclin D3 and Cdk4 expression (Figures 3(a) and 3(b)) anddownregulated the transcriptional factor E2F1 (Figure 3(c))In addition hinokitiol upregulated the cell cycle inhibitor p21(Figure 4(a))

4 Discussion

In the present study we for the first time demonstratedthat hinokitiol negatively regulates immune responses byarresting the G0G1 phase of the cell cycle in ConA-activated


0

2

4

6

8

10

GAPDH

p21


minus

minus minus

+ + + +

lowastlowastlowast

lowastlowast

lowast

p21

(fold

sba

sal)

(a)

Cyclin D3

CDk4

E2F1

Cell proliferation and activation

Autoimmune

S

M

ConA

p21

Hinokitiol

Lymphocytes

G1

G2

IFN-120574

IFN-120574

(b)

Figure 4 Effects of hinokitiol on negative regulators of the cell cycle (a) Cells were treated with hinokitiol (1ndash5120583M) in the presence of ConA(10120583gmL) for 24 h The specific antibody was used to detect p21 Data are presented as the mean plusmn SEM (119899 = 3 lowast119875 lt 005 lowastlowast119875 lt 001and lowastlowastlowast119875 lt 0001 compared with the ConA (alone)-treated group) (b) Schematic illustration of hinokitiol-mediated inhibition of immuneresponses in ConA-activated T lymphocytes Hinokitiol downregulates cyclin D3 Cdk4 and E2F1 expression and upregulates p21 expressionand subsequently arrests the cell cycle at the G0G1 phase Hinokitiol also attenuates IFN-120574 secretion Finally hinokitiol negatively regulatesimmune responses


T lymphocytes Hinokitiol a tropolone-related compoundfound in heartwood cupressaceous plants exhibits multi-ple biological activities including anti-inflammatory antitu-morigenic and antiplatelet activities [9 10 14] However theregulation of lymphocytes by hinokitiol has not been fullyinvestigated Thus in the present study we examined themechanisms underlying the regulation of T lymphocytes byhinokitiol The lectin ConA from the jack bean (Canavaliaensiformis) has been used widely as a T lymphocytes-specificmitogen and to induce the proliferation of lymphocytes [15]Thus we used thismodel to investigate the effect of hinokitiolon T lymphocytes in response to ConA

Dysregulation of the immune system may lead to var-ious chronic diseases such as autoimmune diseases Mostof the damage inflicted by autoimmune diseases is theresult of inappropriate inflammatory responses [16] Failureof self-tolerance is the fundamental cause of autoimmu-nity The principal mechanisms of peripheral tolerance areanergy (functional unresponsiveness) deletion (apoptoticcell death) and suppression by regulatory T cells [17] A pre-vious study reported that increased cell cycling or defectiveapoptosis of lymphocytes may lead to a break of toleranceand autoimmunity [1]The cell cycle is a complex process thatinvolves positive regulators such as cyclins and CDKs andnegative regulators such as CDK inhibitors CDK inhibitorsare classified into two families INK4 and CipKip Duringthe G1-S transition cyclins (D2 and D3) and CDKs (4 and6) are upregulated By contrast the cell cycle inhibitor p21which belongs to the CipKip family interferes with cyclingby inhibiting all CDKs involved in the G1S phase [1]

In the present study we observed that hinokitiol arrestedthe cell cycle of T lymphocytes by suppressing cyclin D3Cdk4 and E2F1 expression and upregulating p21 expressionA study reported that p21 controls T lymphocyte proliferation[18] and Trivedi et al indicated that NK cells inhibit Tlymphocyte proliferation by upregulating p21 resulting incell cycle arrest at the G0G1 phase [19] The findingsof these studies are consistent with our findings that p21upregulation by hinokitiol leads to G0G1 arrest In additionto negatively regulating the cell cycle p21 was reported beassociated with tolerance and systemic autoimmune diseaseLoss of tolerance was observed in p21minusminus mice of whichthe T lymphocytes became more proliferative in response tostimulationThese mice also exhibited an SLE-like syndromecharacterized by the development of anti-DNA antibodiesand glomerulonephritis [18 20] These observations suggestthat hinokitiol prevents autoimmune responses by upregulat-ing p21

In addition IFN-120574 is crucial for immunity to pathogensIFN-120574 is mainly produced in T lymphocytes NKT cells NKcells and B cells [21] T lymphocytes are the major sourcesof IFN-120574 in adaptive immune responses [21] Studies havereported that increased IFN-120574 production is associated withgreater antibacterial and antiviral effects [22 23] Howeveraberrant IFN-120574 expression has been associated with inflam-matory diseases Jaruga et al demonstrated that IFN-120574 playsa vital role in ConA-activated T cell hepatitis by enablingleucocytes to infiltrate the liver [24] Moreover excess IFN-120574

has been associated with chronic autoimmune diseasesincluding inflammatory bowel disease multiple sclerosisdiabetes mellitus and SLE [25 26] Thus we determined theeffect of hinokitiol on IFN-120574 expression in ConA-stimulatedT lymphocytes and observed that hinokitiol significantlyprevented IFN-120574 expression

In clinical practice therapies for autoimmune dis-eases primarily involve using powerful agents chemi-cals or biologics (corticosteroids thiopurines methotrexatecyclosporine and antitumor necrosis factor agents) [27]Such agents suppress the global immune system but fre-quently cause undesirable side effects Certain studies havereported that immunosuppressive drugs can increase the riskof cancer and infectious complications [28ndash31] Regardingthis part we demonstrated that hinokitiol exerts immuno-suppressive effects Moreover previous studies have provedthat hinokitiol engages in antitumor and antibacterial activi-ties Whether these beneficial effects of hinokitiol reduce theincidence of side effects associated with immune suppressionwarrants investigation

In summary we observed that hinokitiol inhibits theactivation and proliferation of T lymphocytes by arresting thecell cycle at the G0G1 phase upregulating p21 expressionand preventing IFN-120574 production (Figure 4(b)) Becauseit engages in multiple biological activities especially anti-inflammatory and antitumorigenic activities hinokitiol mayreduce the unexpected occurrence of side effects during thetreatment of patients with autoimmune diseases Thus theresults of our study suggest that hinokitiol provides benefitsin treating autoimmune diseases


The authors declare that they have no conflict of interests

Acknowledgments

This work was supported by grants from the NationalScience Council Taiwan (NSC102-2320-B-341-001-MY3NSC100-2320-B-038-021-MY3 MOST103-2811-B-038-023and NSC101-2314-B-038-044-MY3) Yuanrsquos General Hospitaland TaipeiMedical University (103-YGH-TMU-01-1) and theShin Kong Wu Ho-Su Memorial Hospital (SKH-8302-101-DR-12 SKH-8302-102-DR-15 SKH-8302-103-NDR-05 andSKH-8302-104-NDR-08) Dr Chi-Li Chung and Dr Kam-Wing Leung contributed equally to this work

References

[1] D Balomenos and A C Martinez ldquoCell-cycle regulation inimmunity tolerance and autoimmunityrdquo Immunology Todayvol 21 no 11 pp 551ndash555 2000

[2] R Beyaert L Beaugerie G van Assche et al ldquoCancer risk inimmune-mediated inflammatory diseases (IMID)rdquo MolecularCancer vol 12 no 1 article 98 2013

[3] A Kuek B L Hazleman andA J K Ostor ldquoImmune-mediatedinflammatory diseases (IMIDs) and biologic therapy a medicalrevolutionrdquo Postgraduate Medical Journal vol 83 no 978 pp251ndash260 2007


[4] C J Sherr and J M Roberts ldquoCDK inhibitors positive andnegative regulators of G1-phase progressionrdquo Genes and Devel-opment vol 13 no 12 pp 1501ndash1512 1999

[5] M-L Santiago-Raber B R Lawson W Dummer et al ldquoRoleof cyclin kinase inhibitor p21 in systemic autoimmunityrdquo TheJournal of Immunology vol 167 no 7 pp 4067ndash4074 2001

[6] Y Wang X Gu G Zhang et al ldquoSATB1 overexpressionpromotes malignant T-cell proliferation in cutaneous CD30+lymphoproliferative disease by repressing p21rdquo Blood vol 123no 22 pp 3452ndash3461 2014

[7] H Suzuki T Ueda I Juranek et al ldquoHinokitiol a selectiveinhibitor of the platelet-type isozyme of arachidonate 12-lipoxygenaserdquo Biochemical and Biophysical Research Communi-cations vol 275 no 3 pp 885ndash889 2000

[8] Y Saeki Y Ito M Shibata Y Sato K Okuda and I TakazoeldquoAntimicrobial action of natural substances on oral bacteriardquoThe Bulletin of Tokyo Dental College vol 30 no 3 pp 129ndash1351989

[9] L H Li P Wu J Y Lee et al ldquoHinokitiol induces DNA damageand autophagy followed by cell cycle arrest and senescence ingefitinib-resistant lung adenocarcinoma cellsrdquo PLoS ONE vol9 no 8 Article ID e104203 2014

[10] S E Byeon Y C Lee J-C Kim J G Han H Y Lee and J YCho ldquoHinokitiol a natural tropolone derivative inhibits TNF-120572 production in LPS-activated macrophages via suppression ofNF-120581Brdquo Planta Medica vol 74 no 8 pp 828ndash833 2008

[11] W-K Wang S-T Lin W-W Chang et al ldquoHinokitiol inducesautophagy in murine breast and colorectal cancer cellsrdquo Envi-ronmental Toxicology 2014

[12] S Liu and H Yamauchi ldquop27-Associated G1 arrest induced byhinokitiol in human malignant melanoma cells is mediated viadown-regulation of pRb Skp2 ubiquitin ligase and impairmentof Cdk2 functionrdquo Cancer Letters vol 286 no 2 pp 240ndash2492009

[13] Y Ido N Muto A Inada et al ldquoInduction of apoptosis byhinokitiol a potent iron chelator in teratocarcinoma F9 cells ismediated through the activation of caspase-3rdquoCell Proliferationvol 32 no 1 pp 63ndash73 1999

[14] K H Lin J R Kuo W J Lu et al ldquoHinokitiol inhibits plateletactivation ex vivo and thrombus formation in vivordquoBiochemicalPharmacology vol 85 no 10 pp 1478ndash1485 2013

[15] N Sharon ldquoLectin receptors as lymphocyte surface markersrdquoAdvances in Immunology vol 34 pp 213ndash298 1983

[16] I R Cohen ldquoActivation of benign autoimmunity as both tumorand autoimmune disease immunotherapy a comprehensivereviewrdquo Journal of Autoimmunity vol 54 pp 112ndash117 2014

[17] J D Rioux andA K Abbas ldquoPaths to understanding the geneticbasis of autoimmune diseaserdquo Nature vol 435 no 7042 pp584ndash589 2005

[18] D Balomenos J Martın-Caballero M I Garcıa et al ldquoThe cellcycle inhibitor p21 controls T-cell proliferation and sex-linkedlupus developmentrdquo Nature Medicine vol 6 no 2 pp 171ndash1762000

[19] P P Trivedi P C Roberts N A Wolf and R H SwanborgldquoNK cells inhibit T cell proliferation via p21-mediated cell cyclearrestrdquo Journal of Immunology vol 174 no 8 pp 4590ndash45972005

[20] C F Arias A Ballesteros-Tato M I Garcıa et al ldquop21CIP1WAF1 controls proliferation of activatedmemory T cells andaffects homeostasis and memory T cell responsesrdquo Journal ofImmunology vol 178 no 4 pp 2296ndash2306 2007

[21] K Schroder P J Hertzog T Ravasi and D A HumeldquoInterferon-gamma An overview of signals mechanisms andfunctionsrdquo Journal of Leukocyte Biology vol 75 no 2 pp 163ndash189 2004

[22] I B Autenrieth M Beer E Bohn S H E Kaufmann and JHeesemann ldquoImmune responses to Yersinia enterocolitica insusceptible BALBc and resistant C57BL6 mice an essentialrole for gamma interferonrdquo Infection and Immunity vol 62 no6 pp 2590ndash2599 1994

[23] A S Major and C F Cuff ldquoEffects of the route of infection onimmunoglobulin G subclasses and specificity of the reovirus-specific humoral immune responserdquo Journal of Virology vol 70no 9 pp 5068ndash5974 1996

[24] B Jaruga F Hong W-H Kim and B Gao ldquoIFN-120574STAT1 actsas a proinflammatory signal in T cell-mediated hepatitis viainduction of multiple chemokines and adhesion molecules acritical role of IRF-1rdquo The American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 287 no 5 pp G1044ndashG1052 2004

[25] J R Schoenborn and C B Wilson ldquoRegulation of interferon-gamma during innate and adaptive immune responsesrdquoAdvances in Immunology vol 96 pp 41ndash101 2007

[26] D Balomenos R Rumold and A N Theofilopoulos ldquoInter-feron-gamma is required for lupus-like disease and lymphoac-cumulation in MRL-lpr micerdquoThe Journal of Clinical Investiga-tion vol 101 no 2 pp 364ndash371 1998

[27] K Orlicka E Barnes and E L Culver ldquoPrevention of infectioncaused by immunosuppressive drugs in gastroenterologyrdquoTher-apeutic Advances in Chronic Disease vol 4 no 4 pp 167ndash1852013

[28] T Hino-Arinaga T Ide R Kuromatsu et al ldquoRisk factors forhepatocellular carcinoma in Japanese patients with autoim-mune hepatitis type 1rdquo Journal of Gastroenterology vol 47 no5 pp 569ndash576 2012

[29] R Das P Feuerstadt and L J Brandt ldquoGlucocorticoids areassociated with increased risk of short-term mortality in hos-pitalized patients with clostridium difficile-associated diseaserdquoThe American Journal of Gastroenterology vol 105 no 9 pp2040ndash2049 2010

[30] W G Dixon K L Hyrich K D Watson et al ldquoDrug-specific risk of tuberculosis in patientswith rheumatoid arthritistreated with anti-TNF therapy results from the British Societyfor Rheumatology Biologics Register (BSRBR)rdquo Annals of theRheumatic Diseases vol 69 no 3 pp 522ndash528 2010

[31] S D Dojcinov G Venkataraman M Raffeld S Pittaluga andE S Jaffe ldquoEBV positive mucocutaneous ulcermdasha study of 26cases associated with various sources of immunosuppressionrdquoThe American Journal of Surgical Pathology vol 34 no 3 pp405ndash417 2010

Research ArticleEffects of the Pinggan Qianyang Recipe onMicroRNA Gene Expression in the Aortic Tissue ofSpontaneously Hypertensive Rats

Guangwei Zhong1 Xia Fang2 Dongsheng Wang1 Qiong Chen2 and Tao Tang2

1 Institute of Integrated Traditional Chinese and Western Medicine Xiangya Hospital Central South UniversityChangsha 410008 China2Department of Geriatrics Xiangya Hospital Central South University Changsha 410008 China

Correspondence should be addressed to Qiong Chen qiongch163com

Received 9 September 2014 Revised 24 January 2015 Accepted 28 January 2015


Copyright copy 2015 Guangwei Zhong et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The present study aimed to investigate the relationship between miRNAs and in spontaneously hypertensive rats (SHR) vascularremodeling and analyze the impact of the Pinggan Qianyang recipe (PQR) on miRNAs Mammalian miRNA microarrayscontaining 509 miRNA genes were employed to analyze the differentially expressed miRNAs in the three groups MiRNAs wereconsidered to be up- or downregulated when the fluorescent intensity ratio between the two groups was over 4-fold Validationof those miRNAs changed in SHR after PQR treatment was used by real-time quantitative RT-PCR (qRT-PCR) Compared withthe normal group a total of 32 miRNAs were differentially expressed by more than twofold among these 18 were upregulated and14 were downregulated in the model group Compared with the normal group there were a number of 17 miRNAs which weresignificantly expressed by more than twofold in the different expressions of 32 miRNAs among these 10 were downregulated and 7were upregulated in the PQR group qRT-PCR verified that miR-20a miR-145 miR-30 and miR-98 were significantly expressed inthe three groupsThese data show that PQR could exert its antihypertensive effect through deterioration of the vascular remodelingprocess The mechanism might be associated with regulating differentially expressed miRNAs in aorta tissue

1 Introduction

Hypertension a lifelong condition is one of the most com-mon cardiovascular diseases Among patients treated by theauthors the prevalence of hypertension in 15 to 69-year-oldpatients is 234 greater than the current estimate of patientswith hypertension in China [1] Because hypertension is animportant risk factor for coronary heart disease and strokedamage to the vital organs such as the heart brain andkidneys can be avoided or minimized by preventing and con-trolling high blood pressure [2] A Chinese medicine scholarhas successfully explored the pathogenesis of spontaneoushypertension and various therapy approaches including thePinggan Qianyang recipe (PQR) a Chinese medicine recipefor calming the liver and suppressing yang [3] PQR whichoriginated from the use of Tianma Guoteng beverages has

been used to treat essential hypertension with satisfactoryresults [4] Recent research has found that Chinese herbalmedicines that involve PQR have a beneficial effect on reduc-ing blood pressure and recovering circadian rhythm in essen-tial hypertension patients [5 6] However the underlyingmechanism of these therapeutic effects remains unknown

miRNAs are a class of highly conserved noncodingsmall-molecule RNAs consisting of about 22 nucleotideseach They adjust protein levels by promoting mRNA degra-dation or inhibiting mRNA translation miRNAs thus partic-ipate in many important biological processes throughout thebody [7 8] miRNAs are involved in cell proliferation differ-entiationmigration and apoptosis [9 10] Cordes et al foundthat reducing miRNA-143 levels could inhibit adipocytedifferentiation in vitro suggesting that miRNAs may play



a significant role in the renin-angiotensin system (RAAS)mdashan important modulator of systemic blood pressure [11]Some miRNAs including miR-1 miR-145 miR-122 miR-221 and miR-222 have been linked to vascular endothelialdysfunction [12] Others have been linked to the regulationof vascular smooth muscle cells these include miR-145 let-7d miR-24 miR-26a and miR-146 [13] The miRNAs miR-1miR-155 and miR-208 have significant effects on the RAAS[14] Therefore a new strategy for hypertension treatmentmight involve maintenance and restoration of stability bytargeting corresponding miRNA expression in the organ ofinterest

To elucidate the association between miRNA expressionand PQR treatment for essential hypertension we carried outanalysis of miRNA gene expression in aortic tissue from SHRthat had received PQR interventionWe tested the hypothesisthat PQRplays an antihypertensive role by regulatingmiRNAexpression in rat aortic tissueThis research may also providenew insights into potential therapeutic targets to prevent andtreat hypertension


21 Animals and Drugs Forty 16-week-old male sponta-neously hypertensive rats (SHR) and 20 male Wistar (WKY)rats (Vital River Laboratory Animal Technology Co LtdBeijing China) of the same age were housed in a sterileenvironment at a temperature of 21 plusmn 1∘C and a relativehumidity of 50 plusmn 10 in a 12-hour day-night cycle Bothgroups of rats had been fed standard rat chow and wateruntil they were 16 weeks old All animal study protocolswere approved by the Animal Care and Use Committee ofCentral SouthUniversity (201303117) and followed the animalmanagement rules set out by the Ministry of Health Chinaand the US National Institutes of Health Guide for the Careand Use of Laboratory Animals The PQR medication recipewas composed of Rhizoma Gastrodiae Ramulus Uncariaecum Uncis Concha Haliotidia Concha Ostreae and RadixAchyranthis Bidentatae all componentswere purchased fromthe Department of Pharmacy Xiangya Hospital CentralSouth University One gram of extract was equal to 425 g ofcrude material

22 Animal Groupings and Treatments The WKY rats andSHR were arbitrarily separated into three groups the normalgroup (119899 = 20) the model group (119899 = 20) and the PQRgroup (119899 = 20) Rats in the PQR group were administeredPQR at a dose of 50mgsdotkgminus1sdotdminus1 by gastrogavageThe otherswere given an equal volume of distilled water For all groupsthe administration course lasted 4 weeks All animals wereused for the miRNA analysis and verification study FortySHR were randomly divided into two groups and were given50mgkg of PQR by gastrogavage once daily for 4 weeksnormal saline was given as the negative control

23 Blood Pressure Detection Systolic blood pressure (SBP)was measured in all rats as previously described [15] Tail-cuff plethysmography (TCP) with a rat tail blood pressuremonitor was used The SBP of each rat was measured five

timesmdashonce before treatment and 1 2 3 and 4 weeks aftertreatment At every time point the mean of the lowest threevalues within 5mmHg was regarded as the SBP value

24 Histological and Morphological Assay Rats were anes-thetizedwith 10 chloral hydrate (400mgkg intraperitonealinjection) at the end of each week of whole-day drug admin-istration The thoracic aorta below the aortic arch of each ratwas stripped and clipped A portion was fixed in 8 neutralformaldehyde embedded in paraffin sectioned at 5 120583mand stained with the hematoxylin-eosin (HE) and Massonmethods [16] Light microscopy was used to image eachcross-sectional slice of which there were five per rat Eachvascular ring in the perpendicular position and the vesselmedia wall were observed The images were observed undera Leica imaging system (LeicaMicrosystems GmbHWetzlarGermany) The media thickness (MT) and inner diameter(LD)weremeasured and the ratio ofmedia thickness to innerdiameter (MTLD)was calculatedOther parts of the thoracicaorta were removed from the adventitia and were promptlyrefrigerated at minus80∘C for miRNA assay

25 RNA Microarray and Hybridization

RNA Extraction Total RNA was extracted by a one-stepmethod using TRIzol (Invitrogen USA) following the manu-facturer protocol concentrated using isopropanol precipita-tion and quantified using a spectrophotometer and agarosegel electrophoresis The polyethylene glycol (PEG) methodwas used to isolate and purify 50 120583g of total RNA

Fluorescently Labeled miRNA miRCURY LNA array labelingkit (Exiqon Denmark) was used Total RNA (10 120583g) wasadded to 2 120583L of Hy

3fluorescent label solution and 2 120583L of

labeling enzyme mixed by pipetting and then incubated at65∘C for 15min to terminate the labeling process

miRNA Microarray Hybridization A miRCURY LNA arraylabeling kit using Macro Kit (ID 208000V71) and hybridbox II (ID 40080) was purchased from Exiqon Biochipslides and cover slips were purchased from Ambion Inc(USA) miRNA microarray hybridization was performedaccording to the miRCURY LNA array kit instructions10 120583L of total RNA was added to 10 120583L of 2x hybridizationbuffer and incubated for 3ndash5min at 95∘C Then 20120583L of thehybridization solution was placed on a microarray slide andcompletely covered with a Bioarray Lifter Slip coverslip Themicroarray slide was placed into the Hybridization ChamberII in a horizontal orientation and bathed at 60∘C for 16 hFollowing incubation hybridization samples were removedfrom the microarray slides with a wash solution Each of 509miRNAs was detected by three replicate probe spots on eachmicroarray slide for a total of six measurements per miRNAper sample after repeated fluorescence exchange

Image Acquisition andQuantification Eachmicroarray (chip)was rinsed and immediately dried then illuminated by a sin-gle 635 nm beam and scanned by a GenePix 4000B dual laserscanner (Molecular Devices LLC USA) Image files were


saved in TIFF format The data were analyzed by GenePixPro 60 software (Molecular Devices LLC USA) After pre-processing the data were normalized to the same interchipglobal mean Finally the differentially expressed genes wereanalyzed by SAM (Significance Analysis of Microarraysversion 21)We used the following screening conditions falsediscovery rate of lt5 and expression differences of ge2-fold

26 Target Prediction Methods Predicted miRNA targetgenes were determined by four software programs miRanda(httpwwwmicrornaorg)miRBaseTargetDatabase (httpmicrornasangeracuk) and Target Scan (httpwwwtarget-scanorg) [17] Outputs varied among the programs Genespredicted by at least two programs were selected as predictedmiRNA target genes

27 Quantitative RT-PCR Differentially expressed miRNAsselected according to ge2-fold upregulation or downregula-tion by microarray analysis were measured by qRT-PCRusing RNA-tailing and primer extension Briefly 2120583g ofRNA was added to 25U120583L of poly (A) polymerase and1mmolL of ATP and incubated in water for 30min at 37∘CPCR primers were designed according to miRNA sequencesindicated by the aforementioned online software programs(26)U6 small nuclear RNA in the ratswas used as an internalcontrol gene Real-time PCR reactions were amplified ina 96-well PCR fluorescence analyzer (MJ real-time PCRinstrument Bio-Rad Laboratories Inc USA) Samples werepredenatured for 5min at 95∘C denatured for 20 s at 94∘Cannealed for 20 s at 58∘C and extended for 30 s at 72∘C fora total of 40 cycles with each sample analyzed in triplicateThe specific product in each PCR reaction was confirmedby the amplification curve Quantification of relative geneexpression was determined by the standard 2minusΔΔCt methodrelative gene expression = 2minus(ΔCtsampleminusΔCtcontrol)

28 StatisticalAnalysis Allresultsarepresented as themeanplusmnstandard deviation All experiments were repeated threetimes An independent sample 119905-test was applied when onlytwo groups were compared whereas comparisons betweenmore than two groups were made by analysis of variance(ANOVA) followed by a Bonferroni posttest Differenceswere considered significant at the level of 119875 lt 005

3 Results

31 PQR Significantly Decreased SBP At the beginning oftreatment SBP was 126 plusmn 11mmHg in the normal groupand 208 plusmn 14mmHg in the model and PQR groups (119875 lt001) However a decrease in SBP was observed in the PQRgroup after 2 weeks of treatment (119875 lt 005) After 4 weeksof treatment the SBP of the PQR group was approximately45mmHg lower than at the beginning of treatment (Figure 1)

32 Morphology and Histology of Vascular Tissue ChangesMasson and HE staining showed that the aortic tunica mediaof the model group was thicker than that of normal groupand the aortic tunica media of PQR-treated rats was thinner

0 1 2 3 4

0

100

200

300

Normal groupModel groupPQR group

SBP

(mm

Hg)

998771

Week

Figure 1 SBP changes inWKY rats or SHR receiving an ia of PQRor distilled water at various times Data are shown as the mean plusmnSD for twenty rats of each group 119875 values for statistical significancewere as 995333119875 lt 001 compared with the model group 998771119875 lt 005 andX119875 lt 001 compared with the PQR group respectively

Table 1 A260 A280 and A260A280 ratios and miRNA concen-trations

Group A260 A280 Ratio ofA260A280

Concentration(120583guL)

Normal group 057 028 196 0183Model group 106 051 208 0295PQR group 092 047 195 0266

than that of control rats in the model group (Figures 2(a)and 2(b)) As shown in Figures 2(c) and 2(d) both MT andMTLD were higher in the model group than in the normalgroup (MT 1267 plusmn 116 120583m versus 843 plusmn 83 120583m resp 119875 =002 MTLD 192 plusmn 019 versus 123 plusmn 021 resp 119875 = 0009)However both MT and MTLD were significantly lower inthe PQR group than in the model group (MT 1024 plusmn 94 120583mversus 1267 plusmn 116 120583m resp 119875 = 004 MTLD 145 plusmn 022versus 192 plusmn 019 resp 119875 = 003)

33 Quality Assessment of Total RNA We extracted totalRNA from the aortic tissues of all rats The purity of the totalRNA was high as indicated by the A260A280 ratio beinggreater than 190 Quality assessment indicated that the totalRNA met the quality requirement of the miRNA microarrayanalysis (Figure 3 and Table 1)

34 Aberrant Expression of miRNAs in SHR Aortic TissueTo determine which miRNAs are potentially involved in theunderlyingmechanism of PQR treatment for essential hyper-tension we tested miRNA levels in all rats by microarrayanalysis We found that miRNA expression was remarkablyaberrant in the model group compared with that of thenormal group In the model group 32 of the 509 rat aortic


N M PQR

(a)

N M PQR

(b)

PQR group0

50

100

150

P = 002 P = 004

MT

(120583m

)

Model group

m)

Normal group

(c)

Normal group Model group PQR group00

05

10

15

20

25

P = 0009 P = 003

MT

LD

(d)

Figure 2 (a) Masson staining of vascular tissue in each group (400x magnification) (b) HE staining of vascular tissue in each group (400xmagnification) (c) MT (d) MTLD N normal group M model group PQR PQR group MT medial thickness LD luminal diameter

miRNAs analyzed were differentially expressed (119875 lt 001)with 18 miRNAs upregulated and 14miRNAs downregulatedAfter 4 weeks of PQR treatment we found that 17 of the32 aortic miRNAs were differentially expressed seven wereupregulated and 10 were downregulated Significant time

course changes of miRNA expression were observed in theaortic tissue more than 468 miRNAs were dysregulated(down- or upregulated) after PQR treatment (Figure 2(a))All differential expression levels of miRNAs at three timepoints are listed in Figure 4 and Table 2 These data indicate


Table 2 Significantly upregulated and downregulated miRNAs in three groups

miRNA Expression level Modelnormal PQRmodelNormal group Model group PQR group

rno-miRNA-1 363 824 687 227 083rno-miRNA-10ab 85 212 118 249 056rno-miRNA-17-5p 121 933 289 771 031rno-miRNA-20a 327 6216 1215 1901 019rno-miRNA-96 432 7537 2113 1745 029rno-miRNA-126-5p 93 323 356 347 110rno-miRNA-139 197 428 334 217 078rno-miRNA-145 128 786 235 614 030rno-miRNA-153 68 1059 351 1557 033rno-miRNA-186a 355 2136 1788 652 084rno-miRNA-187 264 1366 334 517 024rno-miRNA-196ab 451 2097 612 465 029rno-miRNA-210 253 1988 386 786 019rno-miRNA-218 194 793 548 409 061rno-miRNA-221 225 895 298 398 033rno-miRNA-378 148 1253 387 847 031rno-miRNA-451 345 764 598 221 078rno-miRNA-486 71 235 228 331 097rno-miRNA-556 124 617 235 497 038rno-miRNA-15b 1643 238 289 014 121rno-miRNA-26ab 874 156 479 018 313rno-miRNA-30 795 323 948 041 293rno-miRNA-23ab 235 68 57 029 084rno-miRNA-29b 2562 459 1382 018 301rno-miRNA-98 1351 66 527 005 798rno-miRNA-122 1206 197 786 016 399rno-miRNA-125b 3786 1134 1782 029 157rno-miRNA-142-3p 996 487 469 049 096rno-miRNA-158 1328 298 342 022 115rno-miRNA-21 566 103 1427 018 1385rno-miRNA-330 3225 809 1186 025 147rno-let-7bc 786 174 152 022 087

N M PQR

28 S

18 S

Figure 3 Electrophoresis of total RNA N normal group M modelgroup PQR PQR group

that the development of essential hypertension involves awave of expression of sequential classes of miRNAs Thetemporal regulation of these miRNAs indicates that theymight play an important role in PQR treatment of essentialhypertension

35 Validation of miRNA Microarray Results Using qRT-PCRqRT-PCR is a quantitative and specific method that can beused to distinguish a single nucleotide difference betweenmiRNAs Thus involution was obtained by miChip analysisfor four selected miRNAs that showed either high (miR-145) or low (miR-30) signal intensities or high (miR-20a)or low (miRNA-98) differential expression values amongthe three groups The results of qRT-PCR analysis wereoften more reliable than those of the microarray analysisqRT-PCR showed that miR-145 and miR-20a expression wasdownregulated in the model group compared with theirexpression in the PQR group which was consistent with


(a) (b)

Figure 4 Detection of miRNA by microarray analysis Total RNA extracted from three groups of rat aortic tissue were covalently labeledwith Cy3 (green) and Cy5 (red) and hybridized to the array The microarray slides contained two replicate subarrays (a) Normal group andmodel group (b) model group and PQR group

the microarray results Thus the miRNA expression profilesobtained by qRT-PCR fully support the results of miChipanalysis (Figure 5)

36 Results of miR-20a Target Gene Prediction We also per-formed a predicted target analysis formiRNA-20a which waschosen because it was highly expressed in the model groupand downregulated in the PQR group Potential target geneswere predicted using four software programs (miRandaTargetScan PicTar and DIANA-microT) To reduce falsepositive results genes predicted by at least three of these fourdatabases were selected as differentially expressed miRNAtargets for subsequent analysis Screening resulted in theselection of 38 target genes (Table 3)The target genes ofmiR-20a may be involved in the etiology of vascular remodelingthrough cell proliferation apoptosis migration and differen-tiation

4 Discussion

The observations reported here indicate that the underlyingmechanism of PQR treatment for essential hypertensiondoes not mediate vascular remodeling but strictly regulatesmiRNA expression Our previous studies have shown thatTCM (traditional Chinese medicine) treatment not onlyreduces high blood pressure in hypertension but also reversesboth cardiac and vascular smooth muscle cell hypertrophy[18] In the present study we demonstrated that PQR treat-ment fully prevented the development of hypertension aswell as cardiac hypertrophy and aorta remodeling It hasbeen argued that excessive use of PQR in hypertensionmightinterfere with some anatomical andor functional parametersthat are necessary to prevent blood pressure increase

A range of evidence has demonstrated that miRNAscould be used as clinical biomarkers in essential hypertension[19] The most robust multicenter study that provided suchevidence was conducted in Ghent Belgium and focusedon miRNA analysis of potential prognostic biomarkers in500 neuroblastoma patients [20] Although different tech-nological platforms have been used for miRNA profilingthere is significant overlap between prognostic signaturesdescribed in previous work and several miRNAs that werelater identified by more than three independent studies asbeing downregulated in essential hypertension or associatedwith vascular remodeling (eg miR-221 miR-26a miR-21miR-296-5p and miR-204) [21ndash24]

In the present study a microarray assay was appliedto obtain miRNA expression profiles for thoracic aorta inthree groups of SHR and qRT-PCR was used to verifythe microarray data A total of 32 miRNAs in SHR (18upregulated and 14 downregulated) and 17 miRNAs in thePQR treatment group (7 upregulated and 10 downregulated)were successfully identified Furthermore we also founddifferentially expressed miRNA-20a with 38 potential targetgenes in rats which demonstrated that miRNA expressionmight be significant in PQR treatment for rats with essentialhypertension In our studies the most frequently observedand the most promising miRNAs as potential treatmenttargets are miR-145 [11] and miR-208 [25] We found thatmiR-208 is upregulated in insulin-mediated proliferation ofvascular smooth muscle cells and may promote a switchfrom the G0G1 phase of the cell cycle to the S phase Thedirect target of miR-208 has been shown to be p21 [25]and p21 expression in vascular smooth muscle cells has beenshown to be crucial in limiting vascular proliferation invascular remodeling which is strongly associated with essen-tial hypertension [26] Interestingly some studies [27ndash29]


Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

upqRT-PCRmiChip assay

minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)

Figure 5 Validation of miRNA microarray data by qRT-PCR (a) miR-20a (b) miR-145 (c) miRNA-98 (d) miR-30The relative expressionof four miRNAs was normalized to the expression of the internal control gene (U6)

have shown that miR-143 andmiR-145 play an important rolein switching the phenotypes of smooth muscle cells duringvascular remodeling The function of these miRNAs is likelymediated by the degradation of many transcription factorsincluding KLF4 KLF5 Elk-1 and other transcription factorsinvolved in Jagged-1Notch signaling [30] which have beenlinked to the inhibition of differentiation of smooth musclecells MiR-20a a member of the miR-17ndash92 cluster is a highlyconserved miRNA within a noncoding RNA encoded by thec13 or f25 host gene localized on chromosome 13 [31] Thefunctions of each cluster member in essential hypertensionhave not been clearly established Recently Pin et al foundthat miR-20a can inhibit the expression of MKK3 anddownregulate p38 pathway-mediated and VEGF-induced

endothelial cell migration and angiogenesis [32] miR-20a has also been shown to play an important role invascular remodeling [33] In contrast several function-ally well-characterized miRNAs that had previously beenobserved in other diseases were later identified in SHR forthe first time with a high level of statistical significance indi-cating their potential involvement in essential hypertensionpathogenesis These included miR-20a miR-18b miR-375and miR-215 [34]

In conclusion our study demonstrates that PQR hasbeneficial effects in reducing blood pressure and vascu-lar remodeling in SHR The underlying mechanism mightbe related to the modulation of 18 upregulated and 14downregulated miRNAs in particular miR-20a miR-145


Table 3 Predicted target genes of miRNA-20a

Target gene Accession no Target gene nameZNFX1 NM 021035 Zinc finger NFX1-type containing 1IL25 NM 022789 Interleukin 25MAP3K2 NM 006609 Mitogen-activated protein kinase kinase kinase 2AMPD3 NM 001025390 Adenosine monophosphate deaminase 3GPR137C NM 001099652 G protein-coupled receptor 137CACTBL2 NM 001017992 Actin beta-like 2MFAP3L NM 001009554 Microfibrillar-associated protein 3-likeTRIP11 NM 004239 Thyroid hormone receptor interactor 11DGUOK NM 080918 Deoxyguanosine kinaseMFN2 NM 001127660 Mitofusin 2VPS36 NM 004755 Vacuolar protein sorting 36 homologPLS1 NM 001145319 Plastin 1ARHGAP12 NM 018287 Rho GTPase activating protein 12FZD3 NM 017412 Fizzled family receptor3PDK4 NM 002612 Pyruvate dehydrogenase kinase isozyme 4KIF23 NM 004856 Kinesin family member 23VLDLR NM 003383 Very low density lipoprotein receptorFBXO4B NM 001024680 F-box protein 4BZNF652 NM 014897 Zinc finger protein 652RASD1 NM 016048 RAS dexamethasone-induced 1RS1 NM 000330 Retinoschisin 1TNFRSF21 NM 014452 Tumor necrosis factor receptor superfamily member 21FGL1 NM 004467 Fibrinogen-like 1CCND2 NM 001759 Cyclin D2TMEM133 NM 032021 Transmembrane protein 133LPGAT1 NM 014873 Lysophosphatidylglycerol acyltransferase 1IPO7 NM 006391 Importin 7GUCY1A3 NM 000856 Guanylate cycle 1 souble alpha 3TSPAN9 NM 001168320 Tetraspanin 9KLF12 NM 007249 Kruppel-like factor 12SMOC2 NM 001166412 SPARC related modular calcium binding 2MAP3K3 NM 002401 Mitogen-activated protein kinase kinase kinase 3NRP2 NM 018534 Neuropilin 2SOCS6 NM 004232 Suppressor of cytokine signaling 6SLC16A6 NM 001174166 Solute carrier family 16 member 6 (monocarboxylic acid transporter 7)PRR14L NM 173566 Proline rich 14-likeANO6 NM 001025356 Anoctamin 6ZBTB43 NM 001135776 Zinc finger and BTB domain containing 43

miR-30 andmiR-98We suggest that the target genes of miR-20a may be involved in the etiology of vascular remodel-ing through cell proliferation apoptosis migration anddifferentiation However the underlying mechanisms shouldbe further investigated through basic research and well-controlled clinical trials

5 Conclusion

Taken together our findings indicated that PQR could exertits antihypertensive effect through deterioration of the vascu-lar remodeling process The mechanism might be associated

with regulating differentially expressed miRNAs in aortatissue


The authors claim no conflict of interests involved in thestudy

Acknowledgments

This work was supported by research grants from theNational Natural Science Foundation of China (30506644


and 30407125) andChineseMedicine and Pharmacy PlannedProject ofHunanProvince P R China (2009047 and 201245)The authors thank Dr Joen-Rong Sheu for critical reading ofthis paper

References

[1] D Lloyd-Jones R Adams M Carnethon et al ldquoHeart diseaseand stroke statisticsmdash2009 update A report from the Ameri-can heart association statistics committee and stroke statisticssubcommitteerdquo Circulation vol 119 no 3 pp 480ndash486 2009

[2] FHMesserli BWilliams andE Ritz ldquoEssential hypertensionrdquoThe Lancet vol 370 no 9587 pp 591ndash603 2007

[3] Y-J Lv G-L Liu X-M Ji et al ldquoQindan capsule changesadventitial collagen synthesis in spontaneously hypertensiveratsrdquo Chinese Journal of Integrative Medicine vol 19 no 9 pp689ndash695 2013

[4] G-W Zhong M-J Chen Y-H Luo et al ldquoEffect of Chineseherbal medicine for calming Gan and suppressing hyperactiveyang on arterial elasticity function and circadian rhythm ofblood pressure in patients with essential hypertensionrdquo ChineseJournal of Integrative Medicine vol 17 no 6 pp 414ndash420 2011

[5] G W Zhong Y H Luo L L Xiang et al ldquoClinical efficacystudy on calming liver and restraining Yang formula in treatingpatients with mild or moderate degree of essential hyperten-sionrdquo China Journal of Chinese Materia Medica vol 16 no 9pp 776ndash778 2010

[6] G W Zhong W Li M J Chen et al ldquoEffeets on the vascularremodeling and adiponectin expression in aorta in the spon-taneously hypertensive rats by Chinese herb mixture methodrdquoChinese Journal of Hypertension (China) vol 16 no 9 pp 812ndash816 2008

[7] D P Bartel ldquoMicroRNAs genomics biogenesis mechanismand functionrdquo Cell vol 116 no 2 pp 281ndash297 2004

[8] H-W Hwang and J T Mendell ldquoMicroRNAs in cell prolifera-tion cell death and tumorigenesisrdquo British Journal of Cancervol 94 no 6 pp 776ndash780 2006

[9] T Kunej I Godnic S Horvat M Zorc and G A Calin ldquoCrosstalk between MicroRNA and coding cancer genesrdquo CancerJournal vol 18 no 3 pp 223ndash231 2012

[10] D Catalucci P Gallo and G Condorelli ldquoAdvances in molecu-lar genetics genomics proteomics metabolomics and systemsbiology microRNAs in cardiovascular biology and heart dis-easerdquoCirculation CardiovascularGenetics vol 2 no 4 pp 402ndash408 2009

[11] K R Cordes N T SheehyM PWhite et al ldquoMiR-145 andmiR-143 regulate smooth muscle cell fate and plasticityrdquo Nature vol460 no 7256 pp 705ndash710 2009

[12] S K Gupta C Bang and TThum ldquoCirculating MicroRNAs asbiomarkers and potential paracrinemediators of cardiovasculardiseaserdquo Circulation Cardiovascular Genetics vol 3 no 5 pp484ndash488 2010

[13] S Li J Zhu W Zhang et al ldquoSignature microRNA expressionprofile of essential hypertension and its novel link to humancytomegalovirus infectionrdquo Circulation vol 124 no 2 pp 175ndash184 2011

[14] D Torella C Iaconetti D Catalucci et al ldquoMicroRNA-133controls vascular smoothmuscle cell phenotypic switch in vitroand vascular remodeling in vivordquo Circulation Research vol 109no 8 pp 880ndash893 2011

[15] X-P Li Y-H Luo G-W Zhong L-L Xiang and Y-H LildquoPharmacodynamic studies on formula for calming the liverand suppressing yang in treating spontaneous hypertensionratsrdquo China Journal of Traditional Chinese Medicine and Phar-macy vol 26 no 4 pp 710ndash715 2011

[16] E L Schiffrin ldquoRemodeling of resistance arteries in essentialhypertension and effects of antihypertensive treatmentrdquo Amer-ican Journal of Hypertension vol 17 no 12 pp 1192ndash1200 2004

[17] A Krek D Grun M N Poy et al ldquoCombinatorial microRNAtarget predictionsrdquo Nature Genetics vol 37 no 5 pp 495ndash5002005

[18] G-W Zhong W Li Y-H Luo et al ldquoEffects of the calmingliver and suppressing yang method on proliferation and theexpression of heat shock protein 27 in vascular smooth musclecells of spontaneously hypertensive ratsrdquo Chinese Journal ofGerontology vol 29 no 2 pp 385ndash388 2009

[19] Y DrsquoAlessandra P Devanna F Limana et al ldquoCirculatingmicroRNAs are new and sensitive biomarkers of myocardialinfarctionrdquo European Heart Journal vol 31 no 22 pp 2765ndash2773 2010

[20] J Bienertova-Vasku P Mazanek R Hezova et al ldquoExtensionof microRNA expression pattern associated with high-riskneuroblastomardquo Tumor Biology vol 34 no 4 pp 2315ndash23192013

[21] N J Leeper A Raiesdana Y Kojima et al ldquoMicroRNA-26ais a novel regulator of vascular smooth muscle cell functionrdquoJournal of Cellular Physiology vol 226 no 4 pp 1035ndash10432011

[22] H Kang B N Davis-Dusenbery P H Nguyen et al ldquoBonemorphogenetic protein 4 promotes vascular smooth musclecontractility by activatingmicroRNA-21 (miR-21) which down-regulates expression of family of dedicator of cytokinesis(DOCK) proteinsrdquoThe Journal of Biological Chemistry vol 287no 6 pp 3976ndash3986 2012

[23] X Liu Y Cheng J Yang L Xu and C Zhang ldquoCell-specificeffects of miR-221222 in vessels molecular mechanism andtherapeutic applicationrdquo Journal of Molecular and CellularCardiology vol 52 no 1 pp 245ndash255 2012

[24] R-R Cui S-J Li L-J Liu et al ldquoMicroRNA-204 regulatesvascular smooth muscle cell calcification in vitro and in vivordquoCardiovascular Research vol 96 no 2 pp 320ndash329 2012

[25] Y Zhang Y Wang X Wang et al ldquoInsulin promotes vascularsmooth muscle cell proliferation via microRNA-208-mediateddownregulation of p21rdquo Journal of Hypertension vol 29 no 8pp 1560ndash1568 2011

[26] E M Jeon H C Choi K Y Lee K C Chang and Y J KangldquoHemin inhibits hypertensive rat vascular smooth muscle cellproliferation through regulation of cyclin D and p21rdquo Archivesof Pharmacal Research vol 32 no 3 pp 375ndash382 2009

[27] B N Davis-Dusenbery M C Chan K E Reno et al ldquoDown-regulation of Kruppel-like Factor-4 (KLF4) by microRNA-143145 is critical for modulation of vascular smooth musclecell phenotype by transforming growth factor-120573 and bonemorphogenetic protein 4rdquo The Journal of Biological Chemistryvol 286 no 32 pp 28097ndash28110 2011

[28] M Xin E M Small L B Sutherland et al ldquoMicroRNAsmiR-143 and miR-145 modulate cytoskeletal dynamics andresponsiveness of smooth muscle cells to injuryrdquo Genes ampDevelopment vol 23 no 18 pp 2166ndash2178 2009

[29] Y Cheng X Liu J Yang et al ldquoMicroRNA-145 a novelsmoothmuscle cell phenotypic marker andmodulator controls


vascular neointimal lesion formationrdquoCirculation Research vol105 no 2 pp 158ndash166 2009

[30] JM Boucher SM Peterson SUrs C Zhang andL Liaw ldquoThemiR-143145 cluster is a novel transcriptional target of Jagged-1Notch signaling in vascular smooth muscle cellsrdquo Journal ofBiological Chemistry vol 286 no 32 pp 28312ndash28321 2011

[31] C Doebele A Bonauer A Fischer et al ldquoMembers of themicroRNA-17-92 cluster exhibit a cell-intrinsic antiangiogenicfunction in endothelial cellsrdquo Blood vol 115 no 23 pp 4944ndash4950 2010

[32] A-L Pin F Houle M Guillonneau E R Paquet M J Simardand J Huot ldquomiR-20a represses endothelial cell migration bytargeting MKK3 and inhibiting p38 MAP kinase activation inresponse to VEGFrdquo Angiogenesis vol 15 no 4 pp 593ndash6082012

[33] D Frank J Gantenberg I Boomgaarden et al ldquoMicroRNA-20a inhibits stress-induced cardiomyocyte apoptosis involvingits novel target Egln3PHD3rdquo Journal of Molecular and CellularCardiology vol 52 no 3 pp 711ndash717 2012

[34] J Song D Kim C-H Chun and E-J Jin ldquoMicroRNA-375a new regulator of cadherin-7 suppresses the migration ofchondrogenic progenitorsrdquoCellular Signalling vol 25 no 3 pp698ndash706 2013

Research ArticleAntrodia camphorata Potentiates Neuroprotection againstCerebral Ischemia in Rats via Downregulation ofiNOSHO-1Bax and Activated Caspase-3 and Inhibition ofHydroxyl Radical Formation

Po-Sheng Yang12 Po-Yen Lin23 Chao-Chien Chang4 Meng-Che Yu5 Ting-Lin Yen5

Chang-Chou Lan6 Thanasekaran Jayakumar5 and Chih-Hao Yang2

1Department of Surgery Mackay Memorial Hospital and Mackay Medical College Taipei Taiwan2Department of Pharmacology School of Medicine Taipei Medical University Taipei Taiwan3Cardiovascular Division Department of Surgery Yuanrsquos General Hospital Kaohsiung Taiwan4Department of Cardiology Cathay General Hospital Taipei Taiwan5Graduate Institute of Medical Sciences College of Medicine Taipei Medical University Taipei Taiwan6Sheen Chain Biotechnology Co Ltd Taipei Taiwan

Correspondence should be addressed toThanasekaran Jayakumar tjaya 2002yahoocoinand Chih-Hao Yang chyangtmuedutw

Received 28 August 2014 Accepted 20 October 2014


Copyright copy 2015 Po-Sheng Yang et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Antrodia camphorata (A camphorata) is a fungus generally used inChinese folkmedicine for treatment of viral hepatitis and cancerOur previous study foundA camphorata has neuroprotective properties and could reduce stroke injury in cerebral ischemia animalmodels In this study we sought to investigate the molecular mechanisms of neuroprotective effects of A camphorata in middlecerebral artery occlusion (MCAO) rats A selective occlusion of the middle cerebral artery (MCA) with whole blood clots wasused to induce ischemic stroke in rats and they were orally treated with A camphorata (025 and 075 gkgday) alone or combinedwith aspirin (5mgkgday) To provide insight into the functions of A camphorata mediated neuroprotection the expression ofBax inducible nitric oxide synthase (iNOS) haem oxygenase-1 (HO-1) and activated caspase-3 was determined by Western blotassay Treatment of aspirin alone significantly reduced the expressions of HO-1 (119875 lt 0001) iNOS (119875 lt 0001) and Bax (119875 lt001) in ischemic regions The reduction of these expressions was more potentiated when rats treated by aspirin combined with Acamphorata (075 gkgday) Combination treatment also reduced apoptosis asmeasured by a significant reduction in active caspase-3 expression in the ischemic brain compared to MCAO group (119875 lt 001) Moreover treatment of A camphorata significantly (119875 lt005) reduced fenton reaction-induced hydroxyl radical (OH∙) formation at a dose of 40mgmL Taken together A camphoratahas shown neuroprotective effects in embolic rats and the molecular mechanisms may correlate with the downregulation of BaxiNOS HO-1 and activated caspase-3 and the inhibition of OH∙ signals

1 Introduction

Stroke denotes to a rapidworldwide neurological impairmentthat victims may grieve paralysis and speech disorder as wellas loss of cognizance due to either ischemia or hemorrhageIt is considered as one of the leading causes of death and dis-ability worldwide [1] Currently intravascular techniques and

thrombolytic agents have remarkably decreased functionaldeficits Although there are good improvements establishedin treatment there is still little that can be done to preventstroke-related brain damageTherefore active prevention andcontrol of stroke are of great clinical value Aspirin is themostwidely used drug for the prevention of secondary strokeHowever the incidence of cerebral haemorrhage and other



bleeding events are major issues while recurrent stroke iscontrolled by this treatment [2] Thus research has beenfocused on finding alternative drugs that may act on differentpathways that have been used to recover them from thegroup of inflammation necrosis and apoptosis all of whichare associated in ischemic stroke [3] Natural products are aprolific source of bioactive agents of different structure andvarying biological activities In the search for neuroprotectiveagents from natural sources a number of plant extracts andseveral natural products isolated from them have beenreported to provide neuroprotection against ischemic stroke[4]

Antrodia camphorata is being used as the complementaryand alternative medicines and it grows only on the innerheartwood wall of the endangered species Cinnamomumkanehirai Hay (Lauraceae) [5ndash7] A camphorata has longbeen used in Taiwanese folk medicine for abdominal painchemical intoxication diarrhea hypertension itchy skin andhepatoma [8] Studies have demonstrated that A camphor-ata induces significant apoptosis of human promyelocyticleukemia (HL-60) cells [9] and its extracts may be used as anadjuvant antitumor agent for human hepatoma cells whichare resistant to most other antitumor agents Our previousstudy had shown that A camphorata possesses antioxidanteffects against carbon tetrachloride- (CCl

4-) induced hepatic

injury in vivo via mediating free radical scavenging activ-ities [10] A camphorata also has shown to reduce H

2O2-

induced lipid peroxidation and enhance hepatic glutathione-dependent enzymes upon protecting CCl

4-induced damage

on rat liver [11] Despite the fact that our very recent studyhas demonstrated that A camphorata has neuroprotectiveeffect against ischemic stroke in rats through reducinginfarct volume and improves neurobehavioral scores andregulating blood perfusion without increasing hemorrhagictransformation [12] themolecularmechanism of action ofAcamphorata in this effect is remained obscured Thus in thisstudy we investigated the effects and possible mechanisms ofaction of A camphorata on ischemic stroke in rats


21 Plant Material Well Shine Biotechnology DevelopmentCo Pvt Ltd Taipei Taiwan provided the extracts of Acamphorata for this study

22 Animals Male Wistar rats (250ndash300 g) were used todetermine the effects of A camphorata alone or in combi-nation with aspirin against MCAO induced brain damageAnimal care and the general protocols for animal use wereapproved by the Institutional Animal Care and Use Commit-tee (IACUC) of Taipei Medical University All animals wereclinically normal free of apparent infection or inflammationand showed no neurological deficits while they were checkedbefore undergoing the experimental procedures

23MCAO-Induced Ischemia As demonstrated in our previ-ous studies an autologous blood clot was administered in rats

forMCAO-induced ischemia [13ndash15] In brief 06mL of arte-rial blood was withdrawn from a femoral catheter by using 1-mL syringe and the blood was immediately injected into PE-10 tubes The tubes were kept at 4∘C for 22 h and the thread-like clots were removed and placed in a saline-filled dishTheclots were then washed to remove blood cells Washed clotswere transferred to fresh dishes and the washing process wascontinued until the saline remained clear The cleared clotsections were cut into 30mm long fragments and then drawnup with the saline solution into a PE-10 catheter

At the time of surgical procedure animals were anesthe-tized with a mixture of 75 air and 25 O

2gases containing

3 isoflurane The common carotid artery (CCA) was iden-tified and approximately 1 cm of the external carotid artery(ECA)was ligated and cut Consequently the pterygopalatineartery (PA) was clamped with a 10 mm microaneurysmclamp and the CCAwas similarly clamped before the carotidbifurcation The internal carotid artery (ICA) was thenclamped between the carotid bifurcation and the PA Afterthat the PE-50 catheter containing the clot was introducedapproximately 5mm into the previously cut ECA and tied inplace with sutures The ICA clamp was removed and the clotwas flushed into the ICA over a period of approximately 5s The PA clamp was removed and the rat was left in thiscondition for 1 h

24 Experimental Procedure Rats were randomly separatedinto six groups at 1 hr after MCA occlusion (1) a sham-oper-ated group (2) a group orally treated with an isovolumetricsolvent (distilled water) for 60 days followed by throm-boembolic occlusion (3) and (4) groups orally treated withA camphorata (025 and 075 gkgday) alone for 60 days fol-lowed by thromboembolic occlusion respectively (5) and (6)groups treated with A camphorata (025 and 075 gkgday)and aspirin (5mgkgday) followed by thromboembolicocclusion respectively An observer blinded to the identity ofthe groups assessed the neurological deficits after reperfusionby forelimb akinesia test

25 Immunoblotting Assay Expressions of HO-1 iNOS Baxand active caspase-3 in the ischemic brain at 24 h afterthromboembolic occlusion-reperfusion injury were analyzedby immunoblotting as described by our previous study [14]Thromboembolic occlusion-insulted and sham-operated ratswere anesthetized with chloral hydrate (400mgkg ip) andthen the apex of the heart was penetrated with a profusioncannula inserted through the left ventricle into the ascendingaorta Perfusion with ice-cold PBS was performed and anincision was made in the right atrium for venous drainageBrains were freshly removed and sectioned coronally intofour sequential parts from the frontal lobe to the occipitallobe The third of four parts of the right hemisphere was sep-arately collected snap-frozen in liquid nitrogen and stored atminus70∘C The frozen tissues were placed in homogenate bufferand homogenized and then sonicated for 10 s three times at4∘C The sonicated samples were subjected to centrifugation(10000timesg)


The supernatant (50 120583g protein) was subjected to sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to polyvinylid-enedifluoride (PVDF) membranes (045120583m Hybond-PAmersham) After incubation in blocking buffer and beingwashed three times with TBST buffer (10mMTris-base100mMNaCl and 01 Tween 20 pH 75) blots weretreated with an anti-HO-1 polyclonal antibody (pAb 1 1000RampDMinneapolisMN) an anti-iNOSmonoclonal antibody(mAb 1 3000 BD Biosciences San Jose CA) an anti-BaxpAb (1 1000 Cell Signaling Beverly MA) and an anti-active caspase-3 pAb (1 250 Biovision Mountain View CA)or an anti-120572-tubulin mAb (1 2000 Santa Cruz Biotech-nology Santa Cruz CA) in TBST buffer overnight Blotswere subsequently washed with TBST and incubated with asecondary horseradish peroxidase- (HRP-) conjugated goatanti-mouse mAb or donkey anti-rabbit immunoglobulin G(IgG)(Amersham) for 1 h Blots were then washed and theimmunoreactive protein was detected using film exposedto enhanced chemiluminescence (ECL) detection reagents(ECL+ system Amersham) The bar graph depicts the ratiosof semiquantitative results obtained by scanning reactivebands and quantifying the optical density using video den-sitometry (Bio-1D vers 99 image software)

26 Measurement of Hydroxyl Radical (HO∙) Formationby Electron Spin Resonance (ESR) Spectrometry The ESRmethod used a Bruker EMX ESR spectrometer (BillericaMA USA) as described previously [16] In brief a Fentonreaction solution (50 120583M FeSO

4+ 2mM H

2O2) was pre-

treated with a solvent control (01DMSO) orA camphorata(20 and 40mgmL) for 10min The rate of hydroxyl radical-scavenging activity was defined by the following equationinhibition rate = 1 minus [signal height (A camphorata)signalheight (solvent control)]

27 Data Analysis Experimental results are expressed asthe mean plusmn SEM and are accompanied by the number ofobservations The experiments were assessed by the methodof analysis of variance (ANOVA) If this analysis indicatedsignificant differences among the group means then eachgroup was compared using the Newman-Keuls method A 119875value of lt005 was considered statistically significant

3 Results

31 A camphorata Inhibits iNOS and HO-1 Expression inThromboembolic Cerebral Tissues To examine the effect ofA camphorata in the ischemic brain we measured theexpression of iNOS and HO-1 in thromboembolic occlusion-insulted cerebral tissues As shown in Figure 1 iNOS wasmore evidenced in tissues of thromboembolic occlusion-reperfusion injury than the level obtained in the corre-sponding area of the sham-operated group Treatment ofA camphorata and aspirin alone at a respective doses of075 gkg and 5mgkg significantly (119875 lt 0001) diminishediNOS expression compared to the MCAO-untreated ratsMoreover a combined treatment of A camphorata with

Relat

ive l

evels

of i

NO

S (fo

lds

basa

l)

0

1

2

3

4

5

6

MCAO

iNOS

Antrodia camphorate(075gkg)

Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

120572-tubulin

lowastlowastlowast

Figure 1 Effects of the extracts of A camphorata combined withaspirin on the expressions of iNOS in cerebral homogenates 24 hafter thromboembolic stroke in rats Fresh brains from each grouprats were removed and sectioned coronally into four sequential partsof the frontal lobe to the occipital lobe The third of four sequentialparts of the ischemic-injured hemisphere was separately collectedhomogenized and centrifugedThe supernatant (50120583g protein) wasthen subjected to SDS-PAGE and transferred onto membranes foranalysis of iNOS expressions Data are presented as the mean plusmnSEM lowastlowastlowast119875 lt 0001 compared to the sham-operated group and119875 lt 0001 compared to the MCAO group

aspirin apparently potentiated A camphorata mediated sup-pression of iNOS expression

A study has revealed that HO-1 is a key player for drugsupon neuroprotection in transient MCAO model [17] Inthis study Western blot was done to investigate whether Acamphorata affects the level of HO-1 expression The resultsshowed that A camphorata and aspirin alone significantly(119875 lt 0001) reduced the expression of HO-1 protein inbrain tissues of MCAO-induced rats (Figure 2) Howeverthis protein expression was not changed whenA camphoratawas treated with aspirin since HO-1 expression seemed quitesimilar as appeared in their individual treatment

32 A camphorata Reduces Aspirin-Mediated Suppressionof Bax-1 and Active Caspase-3 Expressions in Thromboem-bolic Cerebral Tissues Bax is the proapoptotic member andcaspase-3 is the most abundant cysteine protease in the brainand is acutely cleaved and activated in neurons in the earlystages of reperfusion leading to cell apoptosis In this studythe expression levels of these apoptotic proteins which areconsidered as the most important determining factors for thefate of cell and tissues in response to apoptotic stimulationswere determined We found a significant increase in the


HO-1Re

lativ

e lev

els o

f HO

-1 (f

olds

bas

al)

0

10

20

30

40

50

60

70

MCAO

120572-tubulin

lowastlowastlowast

Antrodia camphorate(075gkg)Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

Figure 2 Effects of the extracts of A camphorata combined withaspirin on the expressions of HO-1 in cerebral homogenates 24 hafter thromboembolic stroke in rats Data are presented as the meanplusmn SEM lowastlowastlowast119875 lt 0001 compared to the sham-operated group and119875 lt 0001 compared to the MCAO group

expressions of Bax (119875 lt 001) and active caspase-3 (119875 lt 001)in the injured hemisphere of the MCAO rats as comparedto the level obtained in the corresponding area of the sham-operated group (Figures 3(a) and 3(b)) Despite the factthat the individual treatment of aspirin suppresses both theexpressions of Bax and activated caspase-3 proteins therate of inhibition was potentiated when the treatment wascombined with A camphorata

33 A camphorata Reduces In Vitro OH∙ Formation Todetermine the efficacy of A camphorata upon inhibiting fen-ton reaction-induced OH∙ formation in vitro a cell-per-meative ROS-sensitive dye DCFDA (nonfluorescent in areduced state but fluorescent upon oxidation by ROS) wasused [16] In this study we found that OH∙ was producedduring the fenton reaction very obviously Interestingly treat-ment with A camphorata (40mgmL) markedly inhibitedthe fenton reaction induced OH∙ (Figure 4) however noeffects were observed whenA camphorata is treated at a con-centration of 20mgmL

4 Discussion

Our recent study has demonstrated thatA camphorata showsneuroprotective effect against ischemic insults in MCAOmodel through a mechanism of blood perfusion regulationwithout increasing hemorrhagic transformation This treat-ment also reduced infarct volume in the focal ischemic brain

injury and improves neurological outcomes In this studywe investigated the possible molecular mechanisms of Acamphorata on the observed neuroprotective effect Theresults were found that an extract of A camphorata pos-sesses neuroprotective effect via antiapoptotic and anti-inflammatory effects and reducesOH radical formation in ratthromboembolic stroke

Recently researchers have been attracted to notice thehypothesis that secondary brain damages from hemoglobinas well as its byproducts such as ferrous iron releasedafter heme degradation [18] Heme or hemin released fromhemoglobin accumulates in intracerebral hemorrhage (ICH)[19] and the increased hemin induces HO-1 the rate-limitingenzyme in the oxidative degradation of free heme [20] Highlevels of heme metabolites such as ferrous iron resulted inneuronal cell death Although HO-1 serves a cytoprotectivefunction [21] reports of protective effects of HO-1 inhibitorsin experimental ICH models support the idea that HO-1 is amediator of neurotoxicity in ICH [22 23] and an attractivetherapeutic target for ICH

In this study we found thatA camphorata exerted neuro-protective effects by reducing theMCAO-induced expressionof HO-1 As reported by Chen et al [24] the induction ofHO-1 has been correlated with an experimental model ofMCAOandHO-1 knockoutmice are reported to be protectedfrom brain injury and functional impairment by ICH [25]Our results showed that reduced expression of HO-1 byA camphorata protects the MCAO-induced ischemic braininjury Several reports proposed that a decrease of HO-1expression by HO-1 inhibitor may provide a protective effectagainst stroke in various animal models [26 27] RecentlyHuang et al reported that treatment of vitamin C offersneuroprotection via reducing HO-1 activity in methamphet-amine-induced neurotoxicity in neuronal cells [28] Com-bined with the current data these reports suggest thatmodulation of HO-1 might have a potential as a new therapyfor stroke

A study demonstrated that iNOS knock-out mice show-ing reduced brain damage after ischemia because of anincreased expression of iNOS may also contribute toenhanced neuronal injury [29] and there is an evidence thatiNOS plays a role as a mediator in the reduction of infarctsize via late preconditioning [30] A recent study also suggeststhat iNOS may be involved in the inflammatory reactionthat follows cerebral ischemia and iNOS mRNA and enzy-matic activity are expressed in brain after permanent MCAocclusion [31] Treatment with the selective iNOS inhibitorwas reported to be reduced infarct volume suggesting thatiNOS activity contributes to ischemic brain damage [32]A study reported that bioactive constituents of myceliumof A camphorata antroquinonol B 4-acetyl-antroquinonolB 23-(methylenedioxy)-6-methylbenzene-14-diol and 24-dimethoxy-6-methylbenzene-13-diol along with antrodin Dinhibit iNOS activity in lipopolysaccharide- (LPS-) activatedmurine macrophages [33] In the present study we demon-strated that treatment of A camphorata in MCAO-inducedembolic rats significantly reduced the expression of iNOS isharmful to the postischemic brain and may be of worth inthe treatment of cerebral ischemia


BaxRe

lativ

e lev

els o

f Bax

(fol

dsb

asal

)

00

05

10

15

20

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

(a)

Relat

ive l

evel

s of

activ

ated

casp

ase-

3 (fo

lds

basa

l)

00

05

10

15

20

25

Activated

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

caspase-3

(b)

Figure 3 Effects of the extracts of A camphorata combined with aspirin on the expressions of (a) Bax and (b) caspase-3 in cerebralhomogenates 24 h after thromboembolic stroke in rats Data are presented as the mean plusmn SEM lowastlowast119875 lt 001 compared to the sham-operatedgroup and

119875 lt 001 compared to the MCAO group

Apoptosis is also known as programmed cell death whichis an initiative suicide process after the cells receive a signalor stimulation with some other related geneThe Bcl-2 familyproteins are key regulators of apoptosis which include bothantiapoptotic members such as Bcl-2 and the proapoptoticmembers such as Bax It has been suggested that a slightchange in the dynamic balance of Bcl2Bax proteins mayresult either in inhibition or promotion of cell death [34]Apoptosis has been reported to occur after transient cerebralischemia and is regulated by the pro- and antiapoptotic pro-teins and it contributes to ischemic cell damage after stroke[35] Caspase-3 is an essential protein for brain developmentbut it also serves as a crucial mediator of neuronal apoptosis[36] During ischemia caspase-3 is cleaved and activatedwhereupon it degrades multiple substrates in the cytoplasmand nucleus leading to cell death [37] Caspase-3 deficientadult mice reported to be more resistant to ischemic stressboth in vivo and in vitro [37] Therefore it is of great interestto control the activation of Bax and caspase-3 for the potentialtherapeutic treatment of neurological diseases Several stud-ies have demonstrated that treatment of caspase-3 inhibitorsreduced ischemic-induced brain damage [38] A recent studyhas suggested that inhibition of Bcl2Bax ratiomay be a noveltarget for the treatment of stroke [39] and these authorshave shown that chemokine-like factor 1 (CKLF1) a novelC-C chemokine with antibodies displays neuroprotectiveeffects against cerebral ischemia via regulation of apoptosis-related protein expression in ischemic hemisphere In the

present study it has been shown that A camphorata hasneuroprotective effects in MCAO-induced rats via inhibitingBax and caspase-3 expressions

Oxidative stress involves the formation of reactive oxy-gennitrogen species (ROSRNS) which are causal factors inthe neuropathology of stroke [40] Abundant ROS are gener-ated during an acute ischemic stroke through multiple injurymechanisms such as mitochondrial inhibition Ca

2+ over-

load and reperfusion injury [41] Brain ischemia generatessuper oxide radical (O

2

∙) from which H2O2is formed H

2O2

is the source of hydroxyl radical (OH∙) An in vivo studyhas revealed that a dry matter of fermented filtrate (DMF)from A camphorata in submerged culture shows antioxidantlike effects against H

2O2-induced cytotoxicity in HepG2

and carbon tetrachloride- (CCl4-) induced hepatotoxicity

[11] They showed that DMF may play a role in preventingoxidative damage in living systems by upregulating hep-atic glutathione-dependent enzymes to preserve the normalreduced and oxidized glutathione (GSHGSSH) ratio andscavenging free radicals formed during CCl

4metabolism

A previous study was reported that polysaccharidesextracted from fruiting bodies or cultured mycelia of Acamphorata exhibit an antihepatitis B virus effect [42] Inthat study the authors have specified that extracts from cul-tured mycelia of A camphorata inhibit N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate- (PMA-) induced ROS production in peripheralhuman neutrophils (PMN) or mononuclear cells (MNC)


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

3450 3460 3470 3480 3490 3500

[G]

Control

20mgmL

40mgmL

00

02

04

06

08

10

12

ESR

signa

l int

ensit

y (a

u)

Control 20 40

Antrodia camphorata (mgmL)

lowastlowastlowast

lowastHydroxyl radical

Figure 4 Effects of the extracts of A camphorata on hydroxyl rad-ical formation ESR spectra show the effects of A camphorata at40mgmL and significantly inhibit hydroxyl radical formation inthe fenton reaction Data are presented as the mean plusmn SEM lowastlowastlowast119875 lt0001 compared to the control group

OH∙ can be produced from O2under a variety of stress con-

ditions and are involved in numerous cellular disorders suchas inflammations embryo teratogenesis herbicide effects celldeath and killing of microorganisms in pathogen-defensereactions It is generally assumed that OH∙ is generatedin biological systems from H

2O2by the Fenton reaction

[43 44] Therefore in the present study we used fentonreaction to evaluate the hydroxyl radical-scavenging activityof A camphorata by the ESR experiment We found thatA camphorata significantly inhibits OH∙ formation at ahigher concentration of 40mgmL These results proposedthat neuroprotection by A camphorata may be involved atleast partly in the inhibition of free radical formation

In conclusion our recent study was demonstrated thatA camphorata provides neuroprotection against MCAO-induced ischemic stroke via improved neurological func-tional scores and reduced infarct volume without causing

hemorrhagic incidence when it is used in conjunction withaspirin therapy nevertheless the mechanisms underlyingremained intricate Therefore we performed this study andfound that the neuroprotective effect of A camphorata ispossibly via enhanced inhibition of HO-1 followed by theinhibition of inflammatory responses (ie iNOS) and apop-tosis (Bax and activated caspase-3) in the ischemic brain Inaddition neuroprotection byA camphoratamay be involvedat least partly by the inhibition of free radical formation


The authors declare that they have no conflict of interests todisclose

Authorsrsquo Contribution

Po-Sheng Yang and Po-Yen Lin contributed equally to thiswork

Acknowledgment

This work was supported by Grants from the National Sci-ence Council of Taiwan (NSC97-2320-B-038-016-MY3 andNSC100-2320-B-038-021-MY3) and Yuanrsquos General Hospital-Taipei Medical University (103-YGH-TMU-01-3)

References

[1] A Towfighi and J L Saver ldquoStroke declines from third tofourth leading cause of death in the United States historicalperspective and challenges aheadrdquo Stroke vol 42 no 8 pp2351ndash2355 2011

[2] W Hacke M Kaste E Bluhmki et al ldquoThrombolysis withalteplase 3 to 45 hours after acute ischemic strokerdquo The NewEngland Journal ofMedicine vol 359 no 13 pp 1317ndash1329 2008

[3] P H Chan ldquoReactive oxygen radicals in signaling and damagein the ischemic brainrdquo Journal of Cerebral Blood Flow ampMetab-olism vol 21 no 1 pp 2ndash14 2001

[4] Z A Malik M Singh and P L Sharma ldquoNeuroprotectiveeffect of Momordica charantia in global cerebral ischemia andreperfusion induced neuronal damage in diabeticmicerdquo Journalof Ethnopharmacology vol 133 no 2 pp 729ndash734 2011

[5] T Y Song S L Hsu C T Yeh and G C Yen ldquoMycelia fromAntrodia camphorata in submerged culture induce apoptosis ofhuman hepatoma HepG2 cells possibly through regulation offas pathwayrdquo Journal of Agricultural and Food Chemistry vol53 no 14 pp 5559ndash5564 2005

[6] Y L Hsu Y C Kuo P L Kuo L T Ng Y H Kuo and C C LinldquoApoptotic effects of extract from Antrodia camphorata fruitingbodies in human hepatocellular carcinoma cell linesrdquo CancerLetters vol 221 no 1 pp 77ndash89 2005

[7] P C Cheng C Y Hsu C C Chen and K M Lee ldquoIn vivoimmunomodulatory effects of Antrodia camphorata polysac-charides in a T1T2 doubly transgenic mouse model for inhibit-ing infection of Schistosoma mansonirdquo Toxicology and AppliedPharmacology vol 227 no 2 pp 291ndash298 2008

[8] H Nakano S Ikenaga T Aizu et al ldquoHuman metallothio-nein gene expression is upregulated by 120573-thujaplicin possible


involvement of protein kinase C and reactive oxygen speciesrdquoBiological amp Pharmaceutical Bulletin vol 29 no 1 pp 55ndash592006

[9] Y-C Hseu H-L Yang Y-C Lai J-G Lin G-W Chen andY-H Chang ldquoInduction of apoptosis by Antrodia camphoratain human premyelocytic leukemia HL-60 cellsrdquo Nutrition andCancer vol 48 no 2 pp 189ndash197 2004

[10] G Hsiao M-Y Shen K-H Lin et al ldquoAntioxidative and hep-atoprotective effects of Antrodia camphorata extractrdquo Journal ofAgricultural and Food Chemistry vol 51 no 11 pp 3302ndash33082003

[11] T Y Song and G C Yen ldquoProtective effects of fermented filtratefrom Antrodia camphorata in submerged culture against CCl

4

-induced hepatic toxicity in ratsrdquo Journal of Agricultural andFood Chemistry vol 51 no 6 pp 1571ndash1577 2003

[12] Y M Lee C Y Chang T L Yen et al ldquoExtract of Antrodiacamphorata exerts neuroprotection against embolic stroke inrats without causing the risk of hemorrhagic incidencerdquo TheScientific World Journal vol 2014 Article ID 686109 8 pages2014

[13] G Hsiao K H Lin Y Chang et al ldquoProtective mechanismsof inosine in platelet activation and cerebral ischemic damagerdquoArteriosclerosisThrombosis and Vascular Biology vol 25 no 9pp 1998ndash2004 2005

[14] T Jayakumar W-H Hsu T-L Yen et al ldquoHinokitiol a naturaltropolone derivative offers neuroprotection from thromboem-bolic stroke in vivordquo Evidence-based Complementary and Alter-native Medicine vol 2013 Article ID 840487 8 pages 2013

[15] J J Lee W H Hsu T L Yen et al ldquoTraditional Chinesemedicine Xue-Fu-Zhu-Yu decoction potentiates tissue plas-minogen activator against thromboembolic stroke in ratsrdquoJournal of Ethnopharmacology vol 134 no 3 pp 824ndash830 2011

[16] D-S Chou G Hsiao M-Y Shen Y-J Tsai T-F Chen and J-R Sheu ldquoESR spin trapping of a carbon-centered free radicalfrom agonist-stimulated human plateletsrdquo Free Radical Biologyand Medicine vol 39 no 2 pp 237ndash248 2005

[17] S Saleem H Zhuang S Biswal Y Christen and S DoreldquoGinkgo biloba extract neuroprotective action is dependent onheme oxygenase 1 in ischemic reperfusion brain injuryrdquo Strokevol 39 no 12 pp 3389ndash3396 2008

[18] F-P Huang G Xi R F Keep Y Hua A Nemoianu and JT Hoff ldquoBrain edema after experimental intracerebral hem-orrhage role of hemoglobin degradation productsrdquo Journal ofNeurosurgery vol 96 no 2 pp 287ndash293 2002

[19] A H Koeppen A C Dickson and J Smith ldquoHeme oxygenasein experimental intracerebral hemorrhage the benefit of tin-mesoporphyrinrdquo Journal of Neuropathology amp ExperimentalNeurology vol 63 no 6 pp 587ndash597 2004

[20] N G Abraham and A Kappas ldquoPharmacological and clinicalaspects of heme oxygenaserdquo Pharmacological Reviews vol 60no 1 pp 79ndash127 2008

[21] Z-P Teng J Chen L-Y Chau N Galunic and R F ReganldquoAdenoviral transfer of the heme oxygenase-1 gene protectsstriatal astrocytes from heme-mediated oxidative injuryrdquo Neu-robiology of Disease vol 17 no 2 pp 179ndash187 2004

[22] Y Gong H Tian G Xi R F Keep J T Hoff and Y Hua ldquoSys-temic zinc protoporphyrin administration reduces intracere-bral hemorrhage-induced brain injuryrdquo Acta NeurochirurgicaSupplementum vol 96 pp 232ndash236 2006

[23] K R Wagner Y Hua G M de Courten-Myers et al ldquoTin-mesoporphyrin a potent heme oxygenase inhibitor for treat-ment of intracerebral hemorrhage in vivo and in vitro studiesrdquoCellular andMolecular Biology vol 46 no 3 pp 597ndash608 2000

[24] P S Chen C-C Wang C D Bortner et al ldquoValproic acid andother histone deacetylase inhibitors inducemicroglial apoptosisand attenuate lipopolysaccharide-induced dopaminergic neu-rotoxicityrdquo Neuroscience vol 149 no 1 pp 203ndash212 2007

[25] JWang and S Dore ldquoHeme oxygenase-1 exacerbates early braininjury after intracerebral haemorrhagerdquo Brain vol 130 no 6pp 1643ndash1652 2007

[26] K Kawaguchi F Lambein and K Kusama-Eguchi ldquoVascu-lar insult accompanied by overexpressed heme oxygenase-1as a pathophysiological mechanism in experimental neuro-lathyrism with hind-leg paraparesisrdquo Biochemical and Biophysi-cal Research Communications vol 428 no 1 pp 160ndash166 2012

[27] Y Guo Q Wang K Zhang et al ldquoHO-1 induction in motorcortex and intestinal dysfunction in TDP-43 A315T transgenicmicerdquo Brain Research vol 1460 pp 88ndash95 2012

[28] Y-N Huang J-YWang C-T Lee C-H Lin and C-C Lai ldquoL-Ascorbate attenuates methamphetamine neurotoxicity throughenhancing the induction of endogenous heme oxygenase-1rdquoToxicology and Applied Pharmacology vol 265 no 2 pp 241ndash252 2012

[29] C Iadecola F Zhang R Casey M Nagayama and M Eliz-abeth Ross ldquoDelayed reduction of ischemic brain injury andneurological deficits in mice lacking the inducible nitric oxidesynthase generdquo Journal of Neuroscience vol 17 no 23 pp 9157ndash9164 1997

[30] J Imagawa D M Yellon and G F Baxter ldquoPharmacologicalevidence that inducible nitric oxide synthase is a mediator ofdelayed preconditioningrdquo British Journal of Pharmacology vol126 no 3 pp 701ndash708 1999

[31] C Iadecola X Xu F Zhang E E El-Fakahany and M ERoss ldquoMarked induction of calcium-independent nitric oxidesynthase activity after focal cerebral ischemiardquo Journal of Cere-bral Blood Flow and Metabolism vol 15 no 1 pp 52ndash59 1995

[32] C Iadecola F Zhang and X Xu ldquoInhibition of induciblenitric oxide synthase ameliorates cerebral ischemic damagerdquoAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 268 no 1 pp R286ndashR292 1995

[33] S-S Yang G-J Wang S-Y Wang Y-Y Lin Y-H Kuo and T-H Lee ldquoNew constituents with iNOS inhibitory activity frommycelium of Antrodia camphoratardquo Planta Medica vol 75 no5 pp 512ndash516 2009

[34] M S Ola M Nawaz and H Ahsan ldquoRole of Bcl-2 familyproteins and caspases in the regulation of apoptosisrdquoMolecularand Cellular Biochemistry vol 351 no 1-2 pp 41ndash58 2011

[35] S I Savitz J A Erhardt J V Anthony et al ldquoThe novel 120573-blocker carvedilol provides neuroprotection in transient focalstrokerdquo Journal of Cerebral Blood Flow and Metabolism vol 20no 8 pp 1197ndash1204 2000

[36] A G Porter and R U Janicke ldquoEmerging roles of caspase-3 inapoptosisrdquoCell DeathampDifferentiation vol 6 no 2 pp 99ndash1041999

[37] D A Le Y Wu Z Huang et al ldquoCaspase activation and neu-roprotection in caspase-3-deficient mice after in vivo cerebralischemia and in vitro oxygen glucose deprivationrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 99 no 23 pp 15188ndash15193 2002

[38] M Sun and C Xu ldquoNeuroprotective mechanism of taurinedue to up-regulating calpastatin and down-regulating calpain


and caspase-3 during focal cerebral ischemiardquo Cellular andMolecular Neurobiology vol 28 no 4 pp 593ndash611 2008

[39] L L Kong Z Y Wang J Hu et al ldquoInhibition of chemokine-like factor 1 protects against focal cerebral ischemia throughthe promotion of energy metabolism and anti-apoptotic effectrdquoNeurochemistry International vol 76 pp 91ndash98 2014

[40] J T Coyle and P Puttfarcken ldquoOxidative stress glutamate andneurodegenerative disordersrdquo Science vol 262 no 5134 pp689ndash695 1993

[41] S Cuzzocrea D P Riley A P Caputi and D Salvemini ldquoAnti-oxidant therapy a new pharmacological approach in shockinflammation and ischemiareperfusion injuryrdquo Pharmacolog-ical Reviews vol 53 no 1 pp 135ndash159 2001

[42] Y-C Shen C-J Chou Y-H Wang C-F Chen Y-C Chouand M-K Lu ldquoAnti-inflammatory activity of the extracts frommycelia of Antrodia camphorata cultured with water-solublefractions from five different Cinnamomum speciesrdquo FEMSMicrobiology Letters vol 231 no 1 pp 137ndash143 2004

[43] B Halliwell and JM C Gutteridge ldquoBiologically relevantmetalion-dependent hydroxyl radical generation An updaterdquo FEBSLetters vol 307 no 1 pp 108ndash112 1992

[44] E R Stadtman ldquoOxidation of free amino acids and aminoacid residues in proteins by radiolysis and by metal-catalyzedreactionsrdquo Annual Review of Biochemistry vol 62 pp 797ndash8211993







Editorial Board










Contents






















Mao-Hsiung Yen









1 Introduction






N

N


Core

Penumbra














mNSS

ShammodelTMP

Biomarker

MAP-23 d7 d

14 d

14 d




Rats






2O2











3 Results



18

16

14

12

10

8

6

4

2

0

mN

SS

lowast

lowastlowast

ModelTMPSham

3 d 7 d 14 d








Sham

Model

TMP

3 d 7 d 14 d

(a)

50000

40000

30000

20000

10000

0

IOD


ShamModelTMP

3 d 7 d 14 d

(b)








2200

2000

1800

1600

1400

1200

1000

800

600

400

200

0

Tota

l den

driti

c len

gth

(120583m

)

Sham Model TMP



4 Discussion








Sham Model TMP

Basilar

Apical

(a)

12

10

8

6

4

2

0

lowast

Num

ber o

f spi

nes (10120583

m)

ShamModelTMP

Basilar Apical

lowastlowastlowast

(b)






12

11

10

9

8

7

6

5

4

mN

SS

27000 30000 33000 36000 39000 42000


r = minus0619 P = 0032

(a)

12

11

10

9

8

7

6

5

4

mN

SS

1400 1600 1800 2000 2200


r = minus0640 P = 0025

(b)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0705 P = 0010

(c)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0501 P = 0097

(d)




5 Conclusion





Acknowledgment


References













































































1 Introduction








2 Methodology





























5 Results


















0

2

4

6

8

10

12

14




Cr

Con

c in

mg100

g of

pla

nt ex

trac

t

PN












CK-MB (IUL)




AST (IUL)




ALT (IUL)


LDH (IUL)






Cholesterol (mgdL)




Triglyceride (mgdL)



LDL (mgdL)



HDL (mgdL)





















7 Discussion









(a) (b)

(c) (d)












8 Conclusion




Acknowledgment


References








































4













1 Introduction



















0

20

40

60

80

100

120

DMSO 1 2 5

Cel

l via

bilit

y (

)

24h48h

(a)

Cel

l via

bilit

y (

)

0

100

200

300

400


minus

minus minus

+ + + +

lowast

(b)

0

500

1000

1500

2000

2500


minus

minus minus

+ + + +

IFN

-120574(p

gm

L)

lowastlowast

(c)





3 Results





DMSO ConA

G0G1

S-G2M

G0G1

S-G2M

G0G1

S-G2M

S-G2M

G0G1 G0G1

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

S-G2M

ConA + hinokitiol 1


(a)

0

10

20

30

40

50

0

20

40

60

80

100

Subp

opul

atio

n (

)

Subp

opul

atio

n (

)

G0G1 S + G2M

lowastlowast

lowastlowast


minus

minus minus


minus

minus minus

+ + + +

(b)





00

05

10

15

20

25

30

35

Cyclin D3

GAPDH

Cycli

n D3

(fold

sba

sal)

lowastlowast


minus

minus minus

+ + + +

(a)

0

2

4

6

8

GAPDH

Cdk4

lowastlowast

Cdk4

(fold

sba

sal)


minus

minus minus

+ + + +

(b)

0

1

2

3

4

5

6

GAPDH

E2F1

E2F1

(fold

sba

sal)

lowastlowastlowast


minus

minus minus

+ + + +

(c)


119875 lt 0001



4 Discussion



0

2

4

6

8

10

GAPDH

p21


minus

minus minus

+ + + +

lowastlowastlowast

lowastlowast

lowast

p21

(fold

sba

sal)

(a)

Cyclin D3

CDk4

E2F1


Autoimmune

S

M

ConA

p21

Hinokitiol

Lymphocytes

G1

G2

IFN-120574

IFN-120574

(b)












Acknowledgments


References









































1 Introduction


























3 Results



0 1 2 3 4

0

100

200

300


SBP

(mm

Hg)

998771

Week










N M PQR

(a)

N M PQR

(b)

PQR group0

50

100

150

P = 002 P = 004

MT

(120583m

)

Model group

m)

Normal group

(c)


05

10

15

20

25

P = 0009 P = 003

MT

LD

(d)








N M PQR

28 S

18 S





(a) (b)




4 Discussion





Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References














































1 Introduction







4-) induced hepatic


2O2-


4-induced damage








2gases containing







4+ 2mM H

2O2) was pre-



3 Results


Relat

ive l

evels

of i

NO

S (fo

lds

basa

l)

0

1

2

3

4

5

6

MCAO

iNOS


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

120572-tubulin

lowastlowastlowast






HO-1Re

lativ

e lev

els o

f HO

-1 (f

olds

bas

al)

0

10

20

30

40

50

60

70

MCAO

120572-tubulin

lowastlowastlowast


minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++




4 Discussion







BaxRe

lativ

e lev

els o

f Bax

(fol

dsb

asal

)

00

05

10

15

20

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

(a)

Relat

ive l

evel

s of

activ

ated

casp

ase-

3 (fo

lds

basa

l)

00

05

10

15

20

25

Activated

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

caspase-3

(b)






2+ over-


2


2O2





4metabolism



lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

3450 3460 3470 3480 3490 3500

[G]

Control

20mgmL

40mgmL

00

02

04

06

08

10

12

ESR

signa

l int

ensit

y (a

u)

Control 20 40


lowastlowastlowast













Acknowledgment


References














4










































Editorial Board










Contents






















Mao-Hsiung Yen









1 Introduction






N

N


Core

Penumbra














mNSS

ShammodelTMP

Biomarker

MAP-23 d7 d

14 d

14 d




Rats






2O2











3 Results



18

16

14

12

10

8

6

4

2

0

mN

SS

lowast

lowastlowast

ModelTMPSham

3 d 7 d 14 d








Sham

Model

TMP

3 d 7 d 14 d

(a)

50000

40000

30000

20000

10000

0

IOD


ShamModelTMP

3 d 7 d 14 d

(b)








2200

2000

1800

1600

1400

1200

1000

800

600

400

200

0

Tota

l den

driti

c len

gth

(120583m

)

Sham Model TMP



4 Discussion








Sham Model TMP

Basilar

Apical

(a)

12

10

8

6

4

2

0

lowast

Num

ber o

f spi

nes (10120583

m)

ShamModelTMP

Basilar Apical

lowastlowastlowast

(b)






12

11

10

9

8

7

6

5

4

mN

SS

27000 30000 33000 36000 39000 42000


r = minus0619 P = 0032

(a)

12

11

10

9

8

7

6

5

4

mN

SS

1400 1600 1800 2000 2200


r = minus0640 P = 0025

(b)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0705 P = 0010

(c)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0501 P = 0097

(d)




5 Conclusion





Acknowledgment


References













































































1 Introduction








2 Methodology





























5 Results


















0

2

4

6

8

10

12

14




Cr

Con

c in

mg100

g of

pla

nt ex

trac

t

PN












CK-MB (IUL)




AST (IUL)




ALT (IUL)


LDH (IUL)






Cholesterol (mgdL)




Triglyceride (mgdL)



LDL (mgdL)



HDL (mgdL)





















7 Discussion









(a) (b)

(c) (d)












8 Conclusion




Acknowledgment


References








































4













1 Introduction



















0

20

40

60

80

100

120

DMSO 1 2 5

Cel

l via

bilit

y (

)

24h48h

(a)

Cel

l via

bilit

y (

)

0

100

200

300

400


minus

minus minus

+ + + +

lowast

(b)

0

500

1000

1500

2000

2500


minus

minus minus

+ + + +

IFN

-120574(p

gm

L)

lowastlowast

(c)





3 Results





DMSO ConA

G0G1

S-G2M

G0G1

S-G2M

G0G1

S-G2M

S-G2M

G0G1 G0G1

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

S-G2M

ConA + hinokitiol 1


(a)

0

10

20

30

40

50

0

20

40

60

80

100

Subp

opul

atio

n (

)

Subp

opul

atio

n (

)

G0G1 S + G2M

lowastlowast

lowastlowast


minus

minus minus


minus

minus minus

+ + + +

(b)





00

05

10

15

20

25

30

35

Cyclin D3

GAPDH

Cycli

n D3

(fold

sba

sal)

lowastlowast


minus

minus minus

+ + + +

(a)

0

2

4

6

8

GAPDH

Cdk4

lowastlowast

Cdk4

(fold

sba

sal)


minus

minus minus

+ + + +

(b)

0

1

2

3

4

5

6

GAPDH

E2F1

E2F1

(fold

sba

sal)

lowastlowastlowast


minus

minus minus

+ + + +

(c)


119875 lt 0001



4 Discussion



0

2

4

6

8

10

GAPDH

p21


minus

minus minus

+ + + +

lowastlowastlowast

lowastlowast

lowast

p21

(fold

sba

sal)

(a)

Cyclin D3

CDk4

E2F1


Autoimmune

S

M

ConA

p21

Hinokitiol

Lymphocytes

G1

G2

IFN-120574

IFN-120574

(b)












Acknowledgments


References









































1 Introduction


























3 Results



0 1 2 3 4

0

100

200

300


SBP

(mm

Hg)

998771

Week










N M PQR

(a)

N M PQR

(b)

PQR group0

50

100

150

P = 002 P = 004

MT

(120583m

)

Model group

m)

Normal group

(c)


05

10

15

20

25

P = 0009 P = 003

MT

LD

(d)








N M PQR

28 S

18 S





(a) (b)




4 Discussion





Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References














































1 Introduction







4-) induced hepatic


2O2-


4-induced damage








2gases containing







4+ 2mM H

2O2) was pre-



3 Results


Relat

ive l

evels

of i

NO

S (fo

lds

basa

l)

0

1

2

3

4

5

6

MCAO

iNOS


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

120572-tubulin

lowastlowastlowast






HO-1Re

lativ

e lev

els o

f HO

-1 (f

olds

bas

al)

0

10

20

30

40

50

60

70

MCAO

120572-tubulin

lowastlowastlowast


minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++




4 Discussion







BaxRe

lativ

e lev

els o

f Bax

(fol

dsb

asal

)

00

05

10

15

20

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

(a)

Relat

ive l

evel

s of

activ

ated

casp

ase-

3 (fo

lds

basa

l)

00

05

10

15

20

25

Activated

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

caspase-3

(b)






2+ over-


2


2O2





4metabolism



lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

3450 3460 3470 3480 3490 3500

[G]

Control

20mgmL

40mgmL

00

02

04

06

08

10

12

ESR

signa

l int

ensit

y (a

u)

Control 20 40


lowastlowastlowast













Acknowledgment


References














4







































Editorial Board










Contents






















Mao-Hsiung Yen









1 Introduction






N

N


Core

Penumbra














mNSS

ShammodelTMP

Biomarker

MAP-23 d7 d

14 d

14 d




Rats






2O2











3 Results



18

16

14

12

10

8

6

4

2

0

mN

SS

lowast

lowastlowast

ModelTMPSham

3 d 7 d 14 d








Sham

Model

TMP

3 d 7 d 14 d

(a)

50000

40000

30000

20000

10000

0

IOD


ShamModelTMP

3 d 7 d 14 d

(b)








2200

2000

1800

1600

1400

1200

1000

800

600

400

200

0

Tota

l den

driti

c len

gth

(120583m

)

Sham Model TMP



4 Discussion








Sham Model TMP

Basilar

Apical

(a)

12

10

8

6

4

2

0

lowast

Num

ber o

f spi

nes (10120583

m)

ShamModelTMP

Basilar Apical

lowastlowastlowast

(b)






12

11

10

9

8

7

6

5

4

mN

SS

27000 30000 33000 36000 39000 42000


r = minus0619 P = 0032

(a)

12

11

10

9

8

7

6

5

4

mN

SS

1400 1600 1800 2000 2200


r = minus0640 P = 0025

(b)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0705 P = 0010

(c)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0501 P = 0097

(d)




5 Conclusion





Acknowledgment


References













































































1 Introduction








2 Methodology





























5 Results


















0

2

4

6

8

10

12

14




Cr

Con

c in

mg100

g of

pla

nt ex

trac

t

PN












CK-MB (IUL)




AST (IUL)




ALT (IUL)


LDH (IUL)






Cholesterol (mgdL)




Triglyceride (mgdL)



LDL (mgdL)



HDL (mgdL)





















7 Discussion









(a) (b)

(c) (d)












8 Conclusion




Acknowledgment


References








































4













1 Introduction



















0

20

40

60

80

100

120

DMSO 1 2 5

Cel

l via

bilit

y (

)

24h48h

(a)

Cel

l via

bilit

y (

)

0

100

200

300

400


minus

minus minus

+ + + +

lowast

(b)

0

500

1000

1500

2000

2500


minus

minus minus

+ + + +

IFN

-120574(p

gm

L)

lowastlowast

(c)





3 Results





DMSO ConA

G0G1

S-G2M

G0G1

S-G2M

G0G1

S-G2M

S-G2M

G0G1 G0G1

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

S-G2M

ConA + hinokitiol 1


(a)

0

10

20

30

40

50

0

20

40

60

80

100

Subp

opul

atio

n (

)

Subp

opul

atio

n (

)

G0G1 S + G2M

lowastlowast

lowastlowast


minus

minus minus


minus

minus minus

+ + + +

(b)





00

05

10

15

20

25

30

35

Cyclin D3

GAPDH

Cycli

n D3

(fold

sba

sal)

lowastlowast


minus

minus minus

+ + + +

(a)

0

2

4

6

8

GAPDH

Cdk4

lowastlowast

Cdk4

(fold

sba

sal)


minus

minus minus

+ + + +

(b)

0

1

2

3

4

5

6

GAPDH

E2F1

E2F1

(fold

sba

sal)

lowastlowastlowast


minus

minus minus

+ + + +

(c)


119875 lt 0001



4 Discussion



0

2

4

6

8

10

GAPDH

p21


minus

minus minus

+ + + +

lowastlowastlowast

lowastlowast

lowast

p21

(fold

sba

sal)

(a)

Cyclin D3

CDk4

E2F1


Autoimmune

S

M

ConA

p21

Hinokitiol

Lymphocytes

G1

G2

IFN-120574

IFN-120574

(b)












Acknowledgments


References









































1 Introduction


























3 Results



0 1 2 3 4

0

100

200

300


SBP

(mm

Hg)

998771

Week










N M PQR

(a)

N M PQR

(b)

PQR group0

50

100

150

P = 002 P = 004

MT

(120583m

)

Model group

m)

Normal group

(c)


05

10

15

20

25

P = 0009 P = 003

MT

LD

(d)








N M PQR

28 S

18 S





(a) (b)




4 Discussion





Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References














































1 Introduction







4-) induced hepatic


2O2-


4-induced damage








2gases containing







4+ 2mM H

2O2) was pre-



3 Results


Relat

ive l

evels

of i

NO

S (fo

lds

basa

l)

0

1

2

3

4

5

6

MCAO

iNOS


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

120572-tubulin

lowastlowastlowast






HO-1Re

lativ

e lev

els o

f HO

-1 (f

olds

bas

al)

0

10

20

30

40

50

60

70

MCAO

120572-tubulin

lowastlowastlowast


minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++




4 Discussion







BaxRe

lativ

e lev

els o

f Bax

(fol

dsb

asal

)

00

05

10

15

20

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

(a)

Relat

ive l

evel

s of

activ

ated

casp

ase-

3 (fo

lds

basa

l)

00

05

10

15

20

25

Activated

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

caspase-3

(b)






2+ over-


2


2O2





4metabolism



lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

3450 3460 3470 3480 3490 3500

[G]

Control

20mgmL

40mgmL

00

02

04

06

08

10

12

ESR

signa

l int

ensit

y (a

u)

Control 20 40


lowastlowastlowast













Acknowledgment


References














4





































Editorial Board










Contents






















Mao-Hsiung Yen









1 Introduction






N

N


Core

Penumbra














mNSS

ShammodelTMP

Biomarker

MAP-23 d7 d

14 d

14 d




Rats






2O2











3 Results



18

16

14

12

10

8

6

4

2

0

mN

SS

lowast

lowastlowast

ModelTMPSham

3 d 7 d 14 d








Sham

Model

TMP

3 d 7 d 14 d

(a)

50000

40000

30000

20000

10000

0

IOD


ShamModelTMP

3 d 7 d 14 d

(b)








2200

2000

1800

1600

1400

1200

1000

800

600

400

200

0

Tota

l den

driti

c len

gth

(120583m

)

Sham Model TMP



4 Discussion








Sham Model TMP

Basilar

Apical

(a)

12

10

8

6

4

2

0

lowast

Num

ber o

f spi

nes (10120583

m)

ShamModelTMP

Basilar Apical

lowastlowastlowast

(b)






12

11

10

9

8

7

6

5

4

mN

SS

27000 30000 33000 36000 39000 42000


r = minus0619 P = 0032

(a)

12

11

10

9

8

7

6

5

4

mN

SS

1400 1600 1800 2000 2200


r = minus0640 P = 0025

(b)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0705 P = 0010

(c)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0501 P = 0097

(d)




5 Conclusion





Acknowledgment


References













































































1 Introduction








2 Methodology





























5 Results


















0

2

4

6

8

10

12

14




Cr

Con

c in

mg100

g of

pla

nt ex

trac

t

PN












CK-MB (IUL)




AST (IUL)




ALT (IUL)


LDH (IUL)






Cholesterol (mgdL)




Triglyceride (mgdL)



LDL (mgdL)



HDL (mgdL)





















7 Discussion









(a) (b)

(c) (d)












8 Conclusion




Acknowledgment


References








































4













1 Introduction



















0

20

40

60

80

100

120

DMSO 1 2 5

Cel

l via

bilit

y (

)

24h48h

(a)

Cel

l via

bilit

y (

)

0

100

200

300

400


minus

minus minus

+ + + +

lowast

(b)

0

500

1000

1500

2000

2500


minus

minus minus

+ + + +

IFN

-120574(p

gm

L)

lowastlowast

(c)





3 Results





DMSO ConA

G0G1

S-G2M

G0G1

S-G2M

G0G1

S-G2M

S-G2M

G0G1 G0G1

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

S-G2M

ConA + hinokitiol 1


(a)

0

10

20

30

40

50

0

20

40

60

80

100

Subp

opul

atio

n (

)

Subp

opul

atio

n (

)

G0G1 S + G2M

lowastlowast

lowastlowast


minus

minus minus


minus

minus minus

+ + + +

(b)





00

05

10

15

20

25

30

35

Cyclin D3

GAPDH

Cycli

n D3

(fold

sba

sal)

lowastlowast


minus

minus minus

+ + + +

(a)

0

2

4

6

8

GAPDH

Cdk4

lowastlowast

Cdk4

(fold

sba

sal)


minus

minus minus

+ + + +

(b)

0

1

2

3

4

5

6

GAPDH

E2F1

E2F1

(fold

sba

sal)

lowastlowastlowast


minus

minus minus

+ + + +

(c)


119875 lt 0001



4 Discussion



0

2

4

6

8

10

GAPDH

p21


minus

minus minus

+ + + +

lowastlowastlowast

lowastlowast

lowast

p21

(fold

sba

sal)

(a)

Cyclin D3

CDk4

E2F1


Autoimmune

S

M

ConA

p21

Hinokitiol

Lymphocytes

G1

G2

IFN-120574

IFN-120574

(b)












Acknowledgments


References









































1 Introduction


























3 Results



0 1 2 3 4

0

100

200

300


SBP

(mm

Hg)

998771

Week










N M PQR

(a)

N M PQR

(b)

PQR group0

50

100

150

P = 002 P = 004

MT

(120583m

)

Model group

m)

Normal group

(c)


05

10

15

20

25

P = 0009 P = 003

MT

LD

(d)








N M PQR

28 S

18 S





(a) (b)




4 Discussion





Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References














































1 Introduction







4-) induced hepatic


2O2-


4-induced damage








2gases containing







4+ 2mM H

2O2) was pre-



3 Results


Relat

ive l

evels

of i

NO

S (fo

lds

basa

l)

0

1

2

3

4

5

6

MCAO

iNOS


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

120572-tubulin

lowastlowastlowast






HO-1Re

lativ

e lev

els o

f HO

-1 (f

olds

bas

al)

0

10

20

30

40

50

60

70

MCAO

120572-tubulin

lowastlowastlowast


minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++




4 Discussion







BaxRe

lativ

e lev

els o

f Bax

(fol

dsb

asal

)

00

05

10

15

20

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

(a)

Relat

ive l

evel

s of

activ

ated

casp

ase-

3 (fo

lds

basa

l)

00

05

10

15

20

25

Activated

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

caspase-3

(b)






2+ over-


2


2O2





4metabolism



lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

3450 3460 3470 3480 3490 3500

[G]

Control

20mgmL

40mgmL

00

02

04

06

08

10

12

ESR

signa

l int

ensit

y (a

u)

Control 20 40


lowastlowastlowast













Acknowledgment


References














4











































Contents






















Mao-Hsiung Yen









1 Introduction






N

N


Core

Penumbra














mNSS

ShammodelTMP

Biomarker

MAP-23 d7 d

14 d

14 d




Rats






2O2











3 Results



18

16

14

12

10

8

6

4

2

0

mN

SS

lowast

lowastlowast

ModelTMPSham

3 d 7 d 14 d








Sham

Model

TMP

3 d 7 d 14 d

(a)

50000

40000

30000

20000

10000

0

IOD


ShamModelTMP

3 d 7 d 14 d

(b)








2200

2000

1800

1600

1400

1200

1000

800

600

400

200

0

Tota

l den

driti

c len

gth

(120583m

)

Sham Model TMP



4 Discussion








Sham Model TMP

Basilar

Apical

(a)

12

10

8

6

4

2

0

lowast

Num

ber o

f spi

nes (10120583

m)

ShamModelTMP

Basilar Apical

lowastlowastlowast

(b)






12

11

10

9

8

7

6

5

4

mN

SS

27000 30000 33000 36000 39000 42000


r = minus0619 P = 0032

(a)

12

11

10

9

8

7

6

5

4

mN

SS

1400 1600 1800 2000 2200


r = minus0640 P = 0025

(b)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0705 P = 0010

(c)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0501 P = 0097

(d)




5 Conclusion





Acknowledgment


References













































































1 Introduction








2 Methodology





























5 Results


















0

2

4

6

8

10

12

14




Cr

Con

c in

mg100

g of

pla

nt ex

trac

t

PN












CK-MB (IUL)




AST (IUL)




ALT (IUL)


LDH (IUL)






Cholesterol (mgdL)




Triglyceride (mgdL)



LDL (mgdL)



HDL (mgdL)





















7 Discussion









(a) (b)

(c) (d)












8 Conclusion




Acknowledgment


References








































4













1 Introduction



















0

20

40

60

80

100

120

DMSO 1 2 5

Cel

l via

bilit

y (

)

24h48h

(a)

Cel

l via

bilit

y (

)

0

100

200

300

400


minus

minus minus

+ + + +

lowast

(b)

0

500

1000

1500

2000

2500


minus

minus minus

+ + + +

IFN

-120574(p

gm

L)

lowastlowast

(c)





3 Results





DMSO ConA

G0G1

S-G2M

G0G1

S-G2M

G0G1

S-G2M

S-G2M

G0G1 G0G1

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

S-G2M

ConA + hinokitiol 1


(a)

0

10

20

30

40

50

0

20

40

60

80

100

Subp

opul

atio

n (

)

Subp

opul

atio

n (

)

G0G1 S + G2M

lowastlowast

lowastlowast


minus

minus minus


minus

minus minus

+ + + +

(b)





00

05

10

15

20

25

30

35

Cyclin D3

GAPDH

Cycli

n D3

(fold

sba

sal)

lowastlowast


minus

minus minus

+ + + +

(a)

0

2

4

6

8

GAPDH

Cdk4

lowastlowast

Cdk4

(fold

sba

sal)


minus

minus minus

+ + + +

(b)

0

1

2

3

4

5

6

GAPDH

E2F1

E2F1

(fold

sba

sal)

lowastlowastlowast


minus

minus minus

+ + + +

(c)


119875 lt 0001



4 Discussion



0

2

4

6

8

10

GAPDH

p21


minus

minus minus

+ + + +

lowastlowastlowast

lowastlowast

lowast

p21

(fold

sba

sal)

(a)

Cyclin D3

CDk4

E2F1


Autoimmune

S

M

ConA

p21

Hinokitiol

Lymphocytes

G1

G2

IFN-120574

IFN-120574

(b)












Acknowledgments


References









































1 Introduction


























3 Results



0 1 2 3 4

0

100

200

300


SBP

(mm

Hg)

998771

Week










N M PQR

(a)

N M PQR

(b)

PQR group0

50

100

150

P = 002 P = 004

MT

(120583m

)

Model group

m)

Normal group

(c)


05

10

15

20

25

P = 0009 P = 003

MT

LD

(d)








N M PQR

28 S

18 S





(a) (b)




4 Discussion





Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References














































1 Introduction







4-) induced hepatic


2O2-


4-induced damage








2gases containing







4+ 2mM H

2O2) was pre-



3 Results


Relat

ive l

evels

of i

NO

S (fo

lds

basa

l)

0

1

2

3

4

5

6

MCAO

iNOS


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

120572-tubulin

lowastlowastlowast






HO-1Re

lativ

e lev

els o

f HO

-1 (f

olds

bas

al)

0

10

20

30

40

50

60

70

MCAO

120572-tubulin

lowastlowastlowast


minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++




4 Discussion







BaxRe

lativ

e lev

els o

f Bax

(fol

dsb

asal

)

00

05

10

15

20

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

(a)

Relat

ive l

evel

s of

activ

ated

casp

ase-

3 (fo

lds

basa

l)

00

05

10

15

20

25

Activated

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

caspase-3

(b)






2+ over-


2


2O2





4metabolism



lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

3450 3460 3470 3480 3490 3500

[G]

Control

20mgmL

40mgmL

00

02

04

06

08

10

12

ESR

signa

l int

ensit

y (a

u)

Control 20 40


lowastlowastlowast













Acknowledgment


References














4








































Contents






















Mao-Hsiung Yen









1 Introduction






N

N


Core

Penumbra














mNSS

ShammodelTMP

Biomarker

MAP-23 d7 d

14 d

14 d




Rats






2O2











3 Results



18

16

14

12

10

8

6

4

2

0

mN

SS

lowast

lowastlowast

ModelTMPSham

3 d 7 d 14 d








Sham

Model

TMP

3 d 7 d 14 d

(a)

50000

40000

30000

20000

10000

0

IOD


ShamModelTMP

3 d 7 d 14 d

(b)








2200

2000

1800

1600

1400

1200

1000

800

600

400

200

0

Tota

l den

driti

c len

gth

(120583m

)

Sham Model TMP



4 Discussion








Sham Model TMP

Basilar

Apical

(a)

12

10

8

6

4

2

0

lowast

Num

ber o

f spi

nes (10120583

m)

ShamModelTMP

Basilar Apical

lowastlowastlowast

(b)






12

11

10

9

8

7

6

5

4

mN

SS

27000 30000 33000 36000 39000 42000


r = minus0619 P = 0032

(a)

12

11

10

9

8

7

6

5

4

mN

SS

1400 1600 1800 2000 2200


r = minus0640 P = 0025

(b)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0705 P = 0010

(c)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0501 P = 0097

(d)




5 Conclusion





Acknowledgment


References













































































1 Introduction








2 Methodology





























5 Results


















0

2

4

6

8

10

12

14




Cr

Con

c in

mg100

g of

pla

nt ex

trac

t

PN












CK-MB (IUL)




AST (IUL)




ALT (IUL)


LDH (IUL)






Cholesterol (mgdL)




Triglyceride (mgdL)



LDL (mgdL)



HDL (mgdL)





















7 Discussion









(a) (b)

(c) (d)












8 Conclusion




Acknowledgment


References








































4













1 Introduction



















0

20

40

60

80

100

120

DMSO 1 2 5

Cel

l via

bilit

y (

)

24h48h

(a)

Cel

l via

bilit

y (

)

0

100

200

300

400


minus

minus minus

+ + + +

lowast

(b)

0

500

1000

1500

2000

2500


minus

minus minus

+ + + +

IFN

-120574(p

gm

L)

lowastlowast

(c)





3 Results





DMSO ConA

G0G1

S-G2M

G0G1

S-G2M

G0G1

S-G2M

S-G2M

G0G1 G0G1

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

S-G2M

ConA + hinokitiol 1


(a)

0

10

20

30

40

50

0

20

40

60

80

100

Subp

opul

atio

n (

)

Subp

opul

atio

n (

)

G0G1 S + G2M

lowastlowast

lowastlowast


minus

minus minus


minus

minus minus

+ + + +

(b)





00

05

10

15

20

25

30

35

Cyclin D3

GAPDH

Cycli

n D3

(fold

sba

sal)

lowastlowast


minus

minus minus

+ + + +

(a)

0

2

4

6

8

GAPDH

Cdk4

lowastlowast

Cdk4

(fold

sba

sal)


minus

minus minus

+ + + +

(b)

0

1

2

3

4

5

6

GAPDH

E2F1

E2F1

(fold

sba

sal)

lowastlowastlowast


minus

minus minus

+ + + +

(c)


119875 lt 0001



4 Discussion



0

2

4

6

8

10

GAPDH

p21


minus

minus minus

+ + + +

lowastlowastlowast

lowastlowast

lowast

p21

(fold

sba

sal)

(a)

Cyclin D3

CDk4

E2F1


Autoimmune

S

M

ConA

p21

Hinokitiol

Lymphocytes

G1

G2

IFN-120574

IFN-120574

(b)












Acknowledgments


References









































1 Introduction


























3 Results



0 1 2 3 4

0

100

200

300


SBP

(mm

Hg)

998771

Week










N M PQR

(a)

N M PQR

(b)

PQR group0

50

100

150

P = 002 P = 004

MT

(120583m

)

Model group

m)

Normal group

(c)


05

10

15

20

25

P = 0009 P = 003

MT

LD

(d)








N M PQR

28 S

18 S





(a) (b)




4 Discussion





Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References














































1 Introduction







4-) induced hepatic


2O2-


4-induced damage








2gases containing







4+ 2mM H

2O2) was pre-



3 Results


Relat

ive l

evels

of i

NO

S (fo

lds

basa

l)

0

1

2

3

4

5

6

MCAO

iNOS


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

120572-tubulin

lowastlowastlowast






HO-1Re

lativ

e lev

els o

f HO

-1 (f

olds

bas

al)

0

10

20

30

40

50

60

70

MCAO

120572-tubulin

lowastlowastlowast


minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++




4 Discussion







BaxRe

lativ

e lev

els o

f Bax

(fol

dsb

asal

)

00

05

10

15

20

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

(a)

Relat

ive l

evel

s of

activ

ated

casp

ase-

3 (fo

lds

basa

l)

00

05

10

15

20

25

Activated

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

caspase-3

(b)






2+ over-


2


2O2





4metabolism



lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

3450 3460 3470 3480 3490 3500

[G]

Control

20mgmL

40mgmL

00

02

04

06

08

10

12

ESR

signa

l int

ensit

y (a

u)

Control 20 40


lowastlowastlowast













Acknowledgment


References














4





































Contents






















Mao-Hsiung Yen









1 Introduction






N

N


Core

Penumbra














mNSS

ShammodelTMP

Biomarker

MAP-23 d7 d

14 d

14 d




Rats






2O2











3 Results



18

16

14

12

10

8

6

4

2

0

mN

SS

lowast

lowastlowast

ModelTMPSham

3 d 7 d 14 d








Sham

Model

TMP

3 d 7 d 14 d

(a)

50000

40000

30000

20000

10000

0

IOD


ShamModelTMP

3 d 7 d 14 d

(b)








2200

2000

1800

1600

1400

1200

1000

800

600

400

200

0

Tota

l den

driti

c len

gth

(120583m

)

Sham Model TMP



4 Discussion








Sham Model TMP

Basilar

Apical

(a)

12

10

8

6

4

2

0

lowast

Num

ber o

f spi

nes (10120583

m)

ShamModelTMP

Basilar Apical

lowastlowastlowast

(b)






12

11

10

9

8

7

6

5

4

mN

SS

27000 30000 33000 36000 39000 42000


r = minus0619 P = 0032

(a)

12

11

10

9

8

7

6

5

4

mN

SS

1400 1600 1800 2000 2200


r = minus0640 P = 0025

(b)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0705 P = 0010

(c)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0501 P = 0097

(d)




5 Conclusion





Acknowledgment


References













































































1 Introduction








2 Methodology





























5 Results


















0

2

4

6

8

10

12

14




Cr

Con

c in

mg100

g of

pla

nt ex

trac

t

PN












CK-MB (IUL)




AST (IUL)




ALT (IUL)


LDH (IUL)






Cholesterol (mgdL)




Triglyceride (mgdL)



LDL (mgdL)



HDL (mgdL)





















7 Discussion









(a) (b)

(c) (d)












8 Conclusion




Acknowledgment


References








































4













1 Introduction



















0

20

40

60

80

100

120

DMSO 1 2 5

Cel

l via

bilit

y (

)

24h48h

(a)

Cel

l via

bilit

y (

)

0

100

200

300

400


minus

minus minus

+ + + +

lowast

(b)

0

500

1000

1500

2000

2500


minus

minus minus

+ + + +

IFN

-120574(p

gm

L)

lowastlowast

(c)





3 Results





DMSO ConA

G0G1

S-G2M

G0G1

S-G2M

G0G1

S-G2M

S-G2M

G0G1 G0G1

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

S-G2M

ConA + hinokitiol 1


(a)

0

10

20

30

40

50

0

20

40

60

80

100

Subp

opul

atio

n (

)

Subp

opul

atio

n (

)

G0G1 S + G2M

lowastlowast

lowastlowast


minus

minus minus


minus

minus minus

+ + + +

(b)





00

05

10

15

20

25

30

35

Cyclin D3

GAPDH

Cycli

n D3

(fold

sba

sal)

lowastlowast


minus

minus minus

+ + + +

(a)

0

2

4

6

8

GAPDH

Cdk4

lowastlowast

Cdk4

(fold

sba

sal)


minus

minus minus

+ + + +

(b)

0

1

2

3

4

5

6

GAPDH

E2F1

E2F1

(fold

sba

sal)

lowastlowastlowast


minus

minus minus

+ + + +

(c)


119875 lt 0001



4 Discussion



0

2

4

6

8

10

GAPDH

p21


minus

minus minus

+ + + +

lowastlowastlowast

lowastlowast

lowast

p21

(fold

sba

sal)

(a)

Cyclin D3

CDk4

E2F1


Autoimmune

S

M

ConA

p21

Hinokitiol

Lymphocytes

G1

G2

IFN-120574

IFN-120574

(b)












Acknowledgments


References









































1 Introduction


























3 Results



0 1 2 3 4

0

100

200

300


SBP

(mm

Hg)

998771

Week










N M PQR

(a)

N M PQR

(b)

PQR group0

50

100

150

P = 002 P = 004

MT

(120583m

)

Model group

m)

Normal group

(c)


05

10

15

20

25

P = 0009 P = 003

MT

LD

(d)








N M PQR

28 S

18 S





(a) (b)




4 Discussion





Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References














































1 Introduction







4-) induced hepatic


2O2-


4-induced damage








2gases containing







4+ 2mM H

2O2) was pre-



3 Results


Relat

ive l

evels

of i

NO

S (fo

lds

basa

l)

0

1

2

3

4

5

6

MCAO

iNOS


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

120572-tubulin

lowastlowastlowast






HO-1Re

lativ

e lev

els o

f HO

-1 (f

olds

bas

al)

0

10

20

30

40

50

60

70

MCAO

120572-tubulin

lowastlowastlowast


minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++




4 Discussion







BaxRe

lativ

e lev

els o

f Bax

(fol

dsb

asal

)

00

05

10

15

20

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

(a)

Relat

ive l

evel

s of

activ

ated

casp

ase-

3 (fo

lds

basa

l)

00

05

10

15

20

25

Activated

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

caspase-3

(b)






2+ over-


2


2O2





4metabolism



lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

3450 3460 3470 3480 3490 3500

[G]

Control

20mgmL

40mgmL

00

02

04

06

08

10

12

ESR

signa

l int

ensit

y (a

u)

Control 20 40


lowastlowastlowast













Acknowledgment


References














4




















































Mao-Hsiung Yen









1 Introduction






N

N


Core

Penumbra














mNSS

ShammodelTMP

Biomarker

MAP-23 d7 d

14 d

14 d




Rats






2O2











3 Results



18

16

14

12

10

8

6

4

2

0

mN

SS

lowast

lowastlowast

ModelTMPSham

3 d 7 d 14 d








Sham

Model

TMP

3 d 7 d 14 d

(a)

50000

40000

30000

20000

10000

0

IOD


ShamModelTMP

3 d 7 d 14 d

(b)








2200

2000

1800

1600

1400

1200

1000

800

600

400

200

0

Tota

l den

driti

c len

gth

(120583m

)

Sham Model TMP



4 Discussion








Sham Model TMP

Basilar

Apical

(a)

12

10

8

6

4

2

0

lowast

Num

ber o

f spi

nes (10120583

m)

ShamModelTMP

Basilar Apical

lowastlowastlowast

(b)






12

11

10

9

8

7

6

5

4

mN

SS

27000 30000 33000 36000 39000 42000


r = minus0619 P = 0032

(a)

12

11

10

9

8

7

6

5

4

mN

SS

1400 1600 1800 2000 2200


r = minus0640 P = 0025

(b)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0705 P = 0010

(c)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0501 P = 0097

(d)




5 Conclusion





Acknowledgment


References













































































1 Introduction








2 Methodology





























5 Results


















0

2

4

6

8

10

12

14




Cr

Con

c in

mg100

g of

pla

nt ex

trac

t

PN












CK-MB (IUL)




AST (IUL)




ALT (IUL)


LDH (IUL)






Cholesterol (mgdL)




Triglyceride (mgdL)



LDL (mgdL)



HDL (mgdL)





















7 Discussion









(a) (b)

(c) (d)












8 Conclusion




Acknowledgment


References








































4













1 Introduction



















0

20

40

60

80

100

120

DMSO 1 2 5

Cel

l via

bilit

y (

)

24h48h

(a)

Cel

l via

bilit

y (

)

0

100

200

300

400


minus

minus minus

+ + + +

lowast

(b)

0

500

1000

1500

2000

2500


minus

minus minus

+ + + +

IFN

-120574(p

gm

L)

lowastlowast

(c)





3 Results





DMSO ConA

G0G1

S-G2M

G0G1

S-G2M

G0G1

S-G2M

S-G2M

G0G1 G0G1

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

S-G2M

ConA + hinokitiol 1


(a)

0

10

20

30

40

50

0

20

40

60

80

100

Subp

opul

atio

n (

)

Subp

opul

atio

n (

)

G0G1 S + G2M

lowastlowast

lowastlowast


minus

minus minus


minus

minus minus

+ + + +

(b)





00

05

10

15

20

25

30

35

Cyclin D3

GAPDH

Cycli

n D3

(fold

sba

sal)

lowastlowast


minus

minus minus

+ + + +

(a)

0

2

4

6

8

GAPDH

Cdk4

lowastlowast

Cdk4

(fold

sba

sal)


minus

minus minus

+ + + +

(b)

0

1

2

3

4

5

6

GAPDH

E2F1

E2F1

(fold

sba

sal)

lowastlowastlowast


minus

minus minus

+ + + +

(c)


119875 lt 0001



4 Discussion



0

2

4

6

8

10

GAPDH

p21


minus

minus minus

+ + + +

lowastlowastlowast

lowastlowast

lowast

p21

(fold

sba

sal)

(a)

Cyclin D3

CDk4

E2F1


Autoimmune

S

M

ConA

p21

Hinokitiol

Lymphocytes

G1

G2

IFN-120574

IFN-120574

(b)












Acknowledgments


References









































1 Introduction


























3 Results



0 1 2 3 4

0

100

200

300


SBP

(mm

Hg)

998771

Week










N M PQR

(a)

N M PQR

(b)

PQR group0

50

100

150

P = 002 P = 004

MT

(120583m

)

Model group

m)

Normal group

(c)


05

10

15

20

25

P = 0009 P = 003

MT

LD

(d)








N M PQR

28 S

18 S





(a) (b)




4 Discussion





Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References














































1 Introduction







4-) induced hepatic


2O2-


4-induced damage








2gases containing







4+ 2mM H

2O2) was pre-



3 Results


Relat

ive l

evels

of i

NO

S (fo

lds

basa

l)

0

1

2

3

4

5

6

MCAO

iNOS


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

120572-tubulin

lowastlowastlowast






HO-1Re

lativ

e lev

els o

f HO

-1 (f

olds

bas

al)

0

10

20

30

40

50

60

70

MCAO

120572-tubulin

lowastlowastlowast


minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++




4 Discussion







BaxRe

lativ

e lev

els o

f Bax

(fol

dsb

asal

)

00

05

10

15

20

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

(a)

Relat

ive l

evel

s of

activ

ated

casp

ase-

3 (fo

lds

basa

l)

00

05

10

15

20

25

Activated

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

caspase-3

(b)






2+ over-


2


2O2





4metabolism



lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

3450 3460 3470 3480 3490 3500

[G]

Control

20mgmL

40mgmL

00

02

04

06

08

10

12

ESR

signa

l int

ensit

y (a

u)

Control 20 40


lowastlowastlowast













Acknowledgment


References














4









































Mao-Hsiung Yen









1 Introduction






N

N


Core

Penumbra














mNSS

ShammodelTMP

Biomarker

MAP-23 d7 d

14 d

14 d




Rats






2O2











3 Results



18

16

14

12

10

8

6

4

2

0

mN

SS

lowast

lowastlowast

ModelTMPSham

3 d 7 d 14 d








Sham

Model

TMP

3 d 7 d 14 d

(a)

50000

40000

30000

20000

10000

0

IOD


ShamModelTMP

3 d 7 d 14 d

(b)








2200

2000

1800

1600

1400

1200

1000

800

600

400

200

0

Tota

l den

driti

c len

gth

(120583m

)

Sham Model TMP



4 Discussion








Sham Model TMP

Basilar

Apical

(a)

12

10

8

6

4

2

0

lowast

Num

ber o

f spi

nes (10120583

m)

ShamModelTMP

Basilar Apical

lowastlowastlowast

(b)






12

11

10

9

8

7

6

5

4

mN

SS

27000 30000 33000 36000 39000 42000


r = minus0619 P = 0032

(a)

12

11

10

9

8

7

6

5

4

mN

SS

1400 1600 1800 2000 2200


r = minus0640 P = 0025

(b)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0705 P = 0010

(c)

12

11

10

9

8

7

6

5

4

mN

SS

7 8 9 10 11


r = minus0501 P = 0097

(d)




5 Conclusion





Acknowledgment


References













































































1 Introduction








2 Methodology





























5 Results


















0

2

4

6

8

10

12

14




Cr

Con

c in

mg100

g of

pla

nt ex

trac

t

PN












CK-MB (IUL)




AST (IUL)




ALT (IUL)


LDH (IUL)






Cholesterol (mgdL)




Triglyceride (mgdL)



LDL (mgdL)



HDL (mgdL)





















7 Discussion









(a) (b)

(c) (d)












8 Conclusion




Acknowledgment


References








































4













1 Introduction



















0

20

40

60

80

100

120

DMSO 1 2 5

Cel

l via

bilit

y (

)

24h48h

(a)

Cel

l via

bilit

y (

)

0

100

200

300

400


minus

minus minus

+ + + +

lowast

(b)

0

500

1000

1500

2000

2500


minus

minus minus

+ + + +

IFN

-120574(p

gm

L)

lowastlowast

(c)





3 Results





DMSO ConA

G0G1

S-G2M

G0G1

S-G2M

G0G1

S-G2M

S-G2M

G0G1 G0G1

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

300

240

180

120

60

0

0 200 400 600 800 1000

Cou

nts

FL2-A

S-G2M

ConA + hinokitiol 1


(a)

0

10

20

30

40

50

0

20

40

60

80

100

Subp

opul

atio

n (

)

Subp

opul

atio

n (

)

G0G1 S + G2M

lowastlowast

lowastlowast


minus

minus minus


minus

minus minus

+ + + +

(b)





00

05

10

15

20

25

30

35

Cyclin D3

GAPDH

Cycli

n D3

(fold

sba

sal)

lowastlowast


minus

minus minus

+ + + +

(a)

0

2

4

6

8

GAPDH

Cdk4

lowastlowast

Cdk4

(fold

sba

sal)


minus

minus minus

+ + + +

(b)

0

1

2

3

4

5

6

GAPDH

E2F1

E2F1

(fold

sba

sal)

lowastlowastlowast


minus

minus minus

+ + + +

(c)


119875 lt 0001



4 Discussion



0

2

4

6

8

10

GAPDH

p21


minus

minus minus

+ + + +

lowastlowastlowast

lowastlowast

lowast

p21

(fold

sba

sal)

(a)

Cyclin D3

CDk4

E2F1


Autoimmune

S

M

ConA

p21

Hinokitiol

Lymphocytes

G1

G2

IFN-120574

IFN-120574

(b)












Acknowledgments


References









































1 Introduction


























3 Results



0 1 2 3 4

0

100

200

300


SBP

(mm

Hg)

998771

Week










N M PQR

(a)

N M PQR

(b)

PQR group0

50

100

150

P = 002 P = 004

MT

(120583m

)

Model group

m)

Normal group

(c)


05

10

15

20

25

P = 0009 P = 003

MT

LD

(d)








N M PQR

28 S

18 S





(a) (b)




4 Discussion





Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References














































1 Introduction







4-) induced hepatic


2O2-


4-induced damage








2gases containing







4+ 2mM H

2O2) was pre-



3 Results


Relat

ive l

evels

of i

NO

S (fo

lds

basa

l)

0

1

2

3

4

5

6

MCAO

iNOS


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

120572-tubulin

lowastlowastlowast






HO-1Re

lativ

e lev

els o

f HO

-1 (f

olds

bas

al)

0

10

20

30

40

50

60

70

MCAO

120572-tubulin

lowastlowastlowast


minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++




4 Discussion







BaxRe

lativ

e lev

els o

f Bax

(fol

dsb

asal

)

00

05

10

15

20

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

(a)

Relat

ive l

evel

s of

activ

ated

casp

ase-

3 (fo

lds

basa

l)

00

05

10

15

20

25

Activated

120572-tubulin

MCAO


Aspirin (5mgkg)

minus

minus

minus minus

minus minus

minus +

+

+ + +

+

++

lowastlowast

caspase-3

(b)






2+ over-


2


2O2





4metabolism



lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

3450 3460 3470 3480 3490 3500

[G]

Control

20mgmL

40mgmL

00

02

04

06

08

10

12

ESR

signa

l int

ensit

y (a

u)

Control 20 40


lowastlowastlowast













Acknowledgment


References














4





































Documents

Bioactives and Traditional Herbal Medicine for the