Research Article Resveratrol Regulates Mitochondrial ...downloads.hindawi.com/journals/omcl/2016/6705621.pdf · Research Article Resveratrol Regulates Mitochondrial Biogenesis and

Research ArticleResveratrol Regulates Mitochondrial Biogenesis andFissionFusion to Attenuate Rotenone-Induced Neurotoxicity

Kaige Peng1 Yuan Tao2 Jun Zhang2 Jian Wang1 Feng Ye1

Guorong Dan1 Yuanpeng Zhao1 Ying Cai1 Jiqing Zhao1 Qiang Wu2

Zhongmin Zou1 Jia Cao1 and Yan Sai1

1 Institute of Toxicology College of Preventive Medicine Third Military Medical University Chongqing 400038 China2College of Preventive Medicine Third Military Medical University Chongqing 400038 China

Correspondence should be addressed to Yan Sai sytmmu126com

Received 15 April 2015 Revised 2 August 2015 Accepted 31 August 2015

Academic Editor David Sebastian

Copyright copy 2016 Kaige Peng 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

It has been confirmed that mitochondrial impairment may underlie both sporadic and familial Parkinsonrsquos disease (PD)Mitochondrial fissionfusion and biogenesis are key processes in regulating mitochondrial homeostasis Therefore we exploredwhether the protective effect of resveratrol in rotenone-induced neurotoxicity was associated with mitochondrial fissionfusionand biogenesis The results showed that resveratrol could not only promote mitochondrial mass and DNA copy number but alsoimprovemitochondrial homeostasis and neuron function in rats and PC12 cells damaged by rotenoneWe also observed effects withalterations in proteins known to regulatemitochondrial fissionfusion and biogenesis in rotenone-induced neurotoxicityThereforeour findings suggest that resveratrol may prevent rotenone-induced neurotoxicity through regulating mitochondrial fissionfusionand biogenesis

1 Introduction

Resveratrol (trans-341015840-5-trihydroxystilbene) is a nature-derived compound found in red grapes peanuts and redwine [1]The protective effects of resveratrol have been exten-sively investigated in both in vivo and in vitromodels A widerange of biological and pharmacological properties have beenshown including antioxidant anti-inflammatory antimuta-genic and anticarcinogenic activities [2ndash5]The neuroprotec-tive potential of resveratrol against dopamine- (DA-) inducedapoptosis in neuronal SH-SY5Y cells the 6-OHDA-treatedrat model of Parkinsonrsquos disease (PD) and the MPTP-treatedmouse model of PD has also been confirmed [1 6 7] How-ever the mechanism of resveratrol protection in cellular andanimal PD models has not been clearly elaborated in detail

PD is the second most common neurodegenerative dis-ease characterized by a progressive loss of DA neurons in thesubstantia nigra (SN) [8] Although the etiology of dopamin-ergic neurodegeneration remains unknown it has beenaccepted that PD-related neurodegeneration is the result of

environmental and genetic interactions [8] Rotenone oneof the most widely used pesticides has been suggested asthe primary environmental risk factor for PD [9 10] Long-term and low-dose exposure to rotenone leads to a systemicdefect in mitochondrial complex I and oxidative stress themain contributors to the etiology of PD [9 11] Thereforemitochondria are believed to play an important role inrotenone-induced dopaminergic neurodegeneration [12]

Mitochondria are dynamic organelles continuouslyundergoing fissionfusion and biogenesis to form a networkthat spans the entire area of neuron to meet the demands ofcellular function [13]These processes not only determine thestructure of the entire mitochondrial population in the neu-rons but also influence nearly every aspect of mitochondrialfunction such as the formation of reactive oxygen species(ROS) ATP production and respiration [13 14] Apart frommaintaining normal mitochondrial functions and integritymitochondrial fusionfission as well as mitochondrial bio-genesis has also been associated with cell death mechanisms[15] The dysfunction of mitochondrial fissionfusion and

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 6705621 12 pageshttpdxdoiorg10115520166705621

2 Oxidative Medicine and Cellular Longevity

biogenesis is associated with many diseases in particularneurodegenerative diseases such as Huntingtonrsquos diseaseAlzheimerrsquos disease and PD [15ndash17] In recent years therehave been reports that resveratrol could increase theactivity of mitochondria thereby conferring resistance tomitochondrial dysfunction in various disease states [18] Todate accumulated evidence suggests clinical potential forresveratrol but the precise molecular mechanism for biolog-ical activity in the mitochondria remains poorly understood

In the present study we explored the protective effectof resveratrol on rotenone-induced dopamine neurotoxicityand its action in mitochondrial fissionfusion and biogen-esis We intend to clarify the mechanism of resveratrol inmitochondrial dynamics in dopamine neurotoxicity inducedby rotenone This research will be helpful in explaining thediverse biological and pharmacological properties of resver-atrol

2 Materials and Methods

21 Materials Healthymale Sprague-Dawley rats (5ndash7 weeksold) weighing 180ndash220 g are obtained from the AnimalCenter of theThirdMilitaryMedical University (ChongqingChina) PC12 cells (adrenal gland pheochromocytoma) wereprovided by Shanghai Cell Bank Institute of Biochemistryand Cell Biology Chinese Academy of Sciences Rotenonedimethyl sulfoxide (DMSO) resveratrol and adenosinetriphosphate (ATP) were purchased from Sigma (St LouisMO USA) Except for antibodies of mitochondrial tran-scription factor A (mtTFA Santa Cruz USA) antibodies ofoptic atrophy 1 (OPA1) mitofusin 2 (MFN2) mitochondrialfission 1 (Fis1) dynamin-1-like protein (Drp1) peroxisomeproliferator-activated receptor gamma and coactivator 1alpha (PGC-1120572) were purchased from Abcam (USA) Allother reagents used were of the highest grade available

22 Animal Care and Treatment The male Sprague-Dawleyrats were housed in climate-controlled rooms at 23 plusmn 2∘Chousing temperature on a reverse 12 12 h lightdark cyclewith lights on at 800 Animals were allowed free accessto food and water during the experimental period Ani-mals were divided into three groups control rotenone-exposed and rotenone plus resveratrol treated Between 1and 5 animals per cage were acclimated for 1 week in ourtemperature-controlled animal roomprior to drug treatmentThe rotenone-exposed group (R group) was treated withsubcutaneous injections of rotenone (first 2mgkg everyday for three days second 1mgkg every day for sevendays and then 05mgkg every day for twenty days) Thecontrol group was injected with an equal volume of dimethylsulfoxide for thirty days The resveratrol protection group(R + RSV group) was treated first with intraperitonealinjection of resveratrol (50mgkg every day for thirty days)and then thirty minutes later animals were treated the sameas in the rotenone exposure group All animal procedureswere conducted per the guidelines of the care and use oflaboratory animals and Chinarsquos laws on animal experimen-tation

23 Neurobehavioral Tests Rotarod Performance Test Therotarod test has been used to assess motor coordination andbalance alterations in rodents This test measured the abilityof the rat to preserve balance by holding itself on the rotatingrod [19]Thirty days after drug treatments we performed thislocomotor activity which measured motor coordination inrats The rats were adapted to the bar When all rats couldremain on the rotating rod for approximately 3min each ratwas placed on a horizontal rod rotating at a speed of 12 rpmfor a maximum of 3min The duration of time during whicheach rat was able to maintain its balance walking on the topof the rod was measured The number of rats that fell wasrecorded

24 Tissue Preparation After completion of the behavioraltests on day 31 the rats were sacrificed by decapitationWholebrains were removed quickly and prepared for assay Thebrain was rinsed with chilled saline and the corpus striatumwas dissected on ice according to coordinates indicated in therat brain atlas A portion of the tissueswas used formeasuringthe levels of ROS and ATP Another portion of the tissues wasused for detecting the gene and protein expression levels ofmitochondrial fissionfusion and biogenesis

25 Cell Culture and Treatment PC12 cells were cultured at37∘Cunder an atmosphere of 5CO

2in DulbeccorsquosModified

Eaglersquos Medium (DMEM Invitrogen USA) containing 10horse serum and 5 fetal bovine serum (Hyclone USA)Cells were divided into three groups The rotenone-exposedgroup (R group) was treated with the indicated concentrationof rotenone dissolved in DMSO for 24 h The control groupwas administered equivalent amounts of DMSO for 24 hTheresveratrol protection group (RSV + R group) was pretreatedwith resveratrol for 24 h and then exposed to rotenone for24 h

26 Transfection of siRNA of PGC-1120572 PC12 cells (1times105well)were seeded into 12-well plates containing 500 120583L of completemedium and were incubated for 24 h Then transfection withsiRNA was conducted using a Lipofectamine 2000 reagent asper userrsquos guide After 6 hours the medium was changed toa normal culture medium After another 48 hours the cellswere harvested and sent for isolation of RNA and analysisof PGC-1120572 expression by real-time PCR The sequences areas follows 5-AAGACGGATTGCCCTCATTTG-3 (siPGC-1120572) and 5-AACGUGACACGUUCG GAGAATT-3 (negativecontrol)

27 CCK-8 Assay to Evaluate PC12 Cell Viability PC12cell viability was assayed with the Cell Counting Kit-8(DOJINDO Japan) according to the manufacturerrsquos instruc-tions All results were normalized to optical density val-ues measured from an identically conditioned well withoutmedium

28 Annexin V-FITC to Evaluate PC12 Cell ApoptosisAccording to the instructions of themanufacturer ofAnnexinV-FITC Apoptosis Detection Kit (BD USA) the cells were

Oxidative Medicine and Cellular Longevity 3

Table 1

Sense primer Antisense primermtTFA 51015840-CGCCTGTCAGCCTTAT-31015840 51015840-GACTCATCCTTAGCCTCC-31015840

PGC-1120572 51015840-GACCGTCCAAAGCATTCA-31015840 51015840-GGTTCTTGTCCACGCCTC-31015840

Fis1 51015840-ATCCGTAGAGGCATCGTG-31015840 51015840-GGGAGGAGGAAGAGCAGA-31015840

Drp1 51015840-TCTCCGAGTCCTTTATTG-31015840 51015840-TCTGACCACTTCTTACCG-31015840

OPA1 51015840-AAGAGGCACTTCAAGGTCG-31015840 51015840-TGTATTCGCCAGAACAGGA-31015840

MFN2 51015840-CGTCAAGAAGGATAAGCGACAC-31015840 51015840-CAACCCGCAGGAAGCAAT-31015840

120573-actin 51015840-TGGTGAAGCAGGCATCTGA-31015840 51015840-TGCTGTTGAAGTCGCAGGAG-31015840

harvested for apoptosis analysis After centrifugation andresuspension in staining buffer cells were incubated inAnnexin V-FITC and propidium iodide (PI) for 20min atroom temperature Fluorescence was detected with a FC500flow cytometer (Beckman USA) and data were analyzedusing CXP analysis software (Beckman USA)

29 Mito Tracker Green to Observe the Mitochondrial MassMito Tracker Green (Invitrogen USA) is a mitochondria-selective membrane potential-independent probe [20] Thusmitochondria in cells stained with MitoTracker Green dyedisplay bright green fluorescence making it a tool for deter-mining mitochondrial mass [20ndash22] After treatment PC12cells were rinsed with phosphate-buffered saline (PBS)Thena working solution of the Mito Tracker Green probe wasadded and incubated for 45min while avoiding light PC12cells were then rinsed three times with PBS A confocal lasermicroscope (Leica TCS SP5) was used to observe and photo-graph themitochondrial fluorescence intensity with the sameexcitation and emission wavelengthsThe fluorescence inten-sity was quantified and analyzed with confocal microscopyimage analysis software Mitochondria were classified intofragmented (lt05 120583m) intermediate (05ndash5 120583m) and tubular(gt5 120583m) mitochondria The cells showing 70 fragmentedor intermediate mitochondria were classified as fragmentedcells Several randomfields (ge200 cells per dish) were selectedfrom each sample and analyzed by ImageJ

210 RNA Isolation and RT-PCR Total RNA was isolatedaccording to the manufacturerrsquos instructions of TRIZOL(Invitrogen USA) The quantity and purity of isolated RNAwere analyzed with a NanoDrop ND-1000 spectrophotome-ter The samples of RNA were stored at minus80∘C until analysisAll first-strand cDNA samples were synthesized from 25120583gtotal RNA per 20 120583L reaction using the ReverTra Ace QpcrRT Kit (ToYoBo Japan) Primers against each of the ratgenes were designed using Primer 50 which are shown inTable 1

Real-time PCR (RT-PCR) was performed and the keyparameters were as follows initial denaturation at 94∘C for5min denaturation at 94∘C for 40 seconds annealing at theappropriate annealing temperature for 30 seconds (mtTFA535∘C PGC-1120572 55∘C Fis1 54∘C Drp1 535∘C OPA1 545∘Cand MFN2 565∘C) extension at 72∘C for 45 seconds Theabove three steps ran for 35 cycles followed by 72∘C for5min 120573-actin was used to normalize the RT-PCR results

The annealing temperature of120573-actinwas 55∘CTheamplifiedfragments were analyzed by electrophoresis using 08 and12 agarose gels with ethidium bromide

211 Western Blot Western blot analysis was performed toinvestigate the protein expression of MFN2 OPA1 Drp1 andFis1 Samples of the rat tissue and PC12 cells were sonicatedin ice-cold lysis buffer and centrifuged at 12000 g for 10minThe supernatants were collected Protein concentrations weremeasured using the Bradford Protein Assay Kit Sampleswere boiled in sample loading buffer Equivalent amountsof proteins (10ndash25mg) were loaded in each well of a 15SDS-PAGE gel and the separated proteins were trans-ferred onto PVDF membranes after electrophoresis Blotswere blocked in 5 nonfat dry milk and then incubatedovernight with primary mouse monoclonal antibody againstOPA1 (1 500) mouse monoclonal antibody against MFN2(1 1000) mousemonoclonal antibody against Drp1 (1 1000)mouse monoclonal antibody against Fis1 (1 1000) or mousemonoclonal antibody against 120573-actin (1 1000) After washesin TBST blots were incubated with HRP-conjugated goatanti-mouse secondary antibody The blots were then devel-oped using electrochemiluminescence (ECL) The intensitiesof individual bands of the proteins of interest were thenanalyzed using the BioImaging System The grayscale valueof MFN2 OPA1 Drp1 and Fis1 was normalized to the valueof the standard protein marker (120573-actin) band to determinethe expression level of the protein The experiments wererepeated at least three times independently

212 Mitochondrial DNA Copy Number by Real-Time PCRTotal DNA from PC12 cells was separately isolated byphenol-chloroform extraction with Promega DNA IsolationKits using standard methods To determine mitochondrialDNA (mtDNA) copy number mtDNA was quantifiedby normalizing the mitochondrial-encoded gene mtCol(mitochondrially encoded cytochrome c oxidase 1) to thenuclear-encoded gene Ndufv1 (NADH dehydrogenase(ubiquinone) flavoprotein 1) using RT-PCR and the ΔΔctmethod The sequences of primer pairs designed to amplifymtCo1 as well as the nuclear DNA-encoded internal controlgene Ndufv1 were as follows 51015840-ACCCCCGCTATAACC-CAATATCAGAC-31015840 (F mtco1) 51015840-TGGGTGTCCGAA-GAATCAAAATAG-31015840 (R mtco1) 51015840-CGCCATGACTGG-AGGTGAGGXAG-31015840 (F Ndufv1) and 51015840-GGCCCCGTA-AACCCGATGTCTTC-31015840 (R Ndufv1) The copy number


of mtDNA was then normalized against that of Ndufv1 tocalculate the relative value of mtDNA copy number

213 Detection of Mitochondrial ATP Levels in PC12 CellsMitochondria were extracted from the PC12 cells on icewith Mitochondrial Extraction Kits (Beyotime China) Theextracted mitochondria were incubated with 1mL of chilled06M perchloric acid at 0∘C for 5min The homogenatewas centrifuged at 20000 g for 10min and the pH of thesupernatant was adjusted to pH 65ndash68 using 25M K

2CO3

solutions Next the mixture was centrifuged at 20000 g for10min after standing at 4∘C for 10min The final supernatantwas collected and removed by filtration through Whatmanmembrane filter paper The samples were stored at minus80∘Cuntil analyzed Samples were analyzed with an ODS C18column kept at room temperature with a flow rate of10mLmin and injected with 20120583L of sample The UVdetection wavelength was 254 nm The external standardmethod was used in the experiments The ATP contentwas calculated with the injection volume and the measuredpeak area The concentration of ATP was expressed asmicrograms per gram of protein The Bradford protein assaykit was used to determine the protein concentration of thesamples

214 Determination of ROS Production in PC12 Cells ROSproduction was measured by the oxidation-sensitive fluores-cent probe 2101584071015840-dichlorofluorescein diacetate (DCFH-DA) asmanufacturerrsquos instructions Briefly at the end of treatmentDCFH-DA (10 120583M) was added and incubated in the darkat 37∘C for 30min Fluorescence was monitored by flowcytometry with excitation at 485 nm and emission at 530 nmThe results were expressed as intensity of DCF-fluorescenceof the control group

215 Statistical Analysis Data are expressed as the mean plusmnSEM and were analyzed using SPSS (V1300) Statisticalcomparisons between all groups were performed using One-Way Analysis of Variance (ANOVA) and post hoc compar-isons between groups were completed using Tukeyrsquos test 119875 lt005 were considered statistically significant

3 Results

31 Effects of Resveratrol on the Mortality and RotarodPerformance of Rats The survival rate of rats in the Rgroup was significantly decreased compared with that of thecontrol group (119875 lt 001) However resveratrol pretreatmentenhanced survival rate which was statistically significantrelative to that of the R group (Figure 1(a)) Additionallyas observed in Figures 1(b) and 1(c) all rats in the controlgroup endured 80 s on the rotating rod without fallingHowever rotenone treatment caused disturbance of motorcoordination in rats The duration of time maintainingbalance walking on the top of the rod was decreased andthe number of rats unable to stand on the rotating rodeven during the first set of trials was increased Howeverresveratrol pretreatment decreased the disturbance of motor

coordination with the rate and duration of motion enhancedsignificantly Therefore resveratrol has a protective effect inthe substantia nigra of rats subjected to rotenone-inducedParkinsonrsquos disease

32 Effects of Resveratrol on the Viability Apoptosis andMorphology of PC12 Cells Whether the viability of PC12 cellsexposed to rotenone was protected by resveratrol pretreat-ment was investigated using CCK-8 Kits The results showedthat cell viability was significantly decreased in the rotenone-exposed group compared with that of the control groupHowever resveratrol pretreatment of PC12 cells exposed torotenone influenced viability in a concentration-dependentmanner In our experiment at resveratrol concentrations of10 120583M and 50 120583M there was no difference in PC12 cellviability between the R group and the R + RSV group Whenthe concentration increased to 100120583M 50 120583M and 60 120583Mresveratrol demonstrated a statistically significant protectiveeffect (119875 lt 001)However the protective effect disappearedat a resveratrol concentration of 1000120583M (Figure 2(a))

The morphology of PC12 cells in the control group wassimilar to primary sympathetic neurons in shape with anintegral cell body and long processes When PC12 cells wereexposed to rotenone the cells were damaged the processesbecame shorter and some of the cells became round andpolygonal in shape However pretreatment of PC12 cellswith resveratrol significantly influenced morphology andthe processes appeared obviously longer compared to cellstreated with rotenone alone (Figures 2(b) 2(c) and 2(d))

To further confirm the apoptosis of rotenone PC12 cellswere incubated with Annexin VPI and were analyzed byflow cytometry As shown in Figure 2(e) the percentageof apoptotic cells was increased in the rotenone-exposedgroup compared with the control group while the numberof apoptotic cells was significantly decreased in the R + RSVgroup compared with that of the R group Resveratrol couldobviously alleviate rotenone-induced PC12 cell apoptosis(Figure 2(e))

33 Effects of Resveratrol on Mitochondrial Mass and Mito-chondrial FissionFusion Mitochondrialmasswas quantifiedusing MitoTracker Green Staining was typically observed inperinuclear regions and processes the nucleus was typicallydevoid of staining (Figure 3(a)) MitoTracker Green demon-strated a statistically significant decrease in fluorescenceintensity following treatment with rotenone However therewas a small increase in the fluorescence intensity of PC12cells pretreated with resveratrol compared to the rotenone-exposed group (119875 lt 005) (Figure 3(b)) Furthermore highdefinition images show that mitochondria of control groupwere filamentous with a tubular or thread-like appearance Incomparison rotenone treatment led to a significant increasein the number of fragmented mitochondria with small ring-shapes However resveratrol reversed the morphologicalchanges ofmitochondria (Figure 3(a))The cells showing 70fragmented or intermediate mitochondria were classified asfragmented cellsThe results showed that rotenone treatmentled to a significant increase in the number of fragmented


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Figure 1 RSV improved survival rate and motor ability of rats decreased by rotenone (a) The survival rate of rats in the R group is reducedsignificantly compared to the control and resveratrol pretreatment significantly improves the survival rate of rats exposed to rotenonecompared to the R group (b)The rate ofmotion is decreased in rats exposed to rotenone however the rate ofmotion is significantly increasedin rats pretreated with resveratrol (c) Rotenone leads to a decrease in motion time and resveratrol pretreatment improved the decrease inmotion time induced by rotenone The results are presented as the mean plusmn SEM (119899 = 20group for survival rate 119899 = 6group for motionability) lowastlowast119875 lt 001 versus control group 119875 lt 005 versus R group 119875 lt 001 versus R group

cells compared with control group Resveratrol significantlyreduced the number of fragmented cells compared with Rgroup (Figure 3(c))

The mitochondrial mass was determined by mitochon-drial fission and fusion Therefore the regulating signalsin mitochondrial fission and fusion were also investigatedThe results showed that rotenone exposure induced aninhibition of mitochondrial fission and fusion leading toa decrease in expression of both mRNA and protein levelsof genes responsible for mitochondrial fission and fusionHowever resveratrol pretreatment could improve the abilityof mitochondrial fission and fusion by promoting expressionofDrp1 Fis1 OPA1 andMFN2mRNAand protein associatedwith mitochondrial fission and fusion (Figures 3(d) 3(e)3(f) and 3(g)) Thus resveratrol demonstrated a protectiveeffect in mitochondria via enhanced mitochondrial fissionand fusion ability

34 Effects of Resveratrol on mtDNA Copy Number and theExpression and Transcriptional Activity of PGC-1120572 Mito-chondrial DNA (mtDNA) copy number is a critical compo-nent of overall mitochondrial healthThe results showed that

the relative mtDNA copy number was decreased significantlycompared to that of the control group When PC12 cellswere pretreated with resveratrol the mtDNA copy numberincreased compared to the mean value of the PC12 cellsexposed to rotenone alone (Figure 4(f))

Resveratrol-mediated increases in mitochondrial DNAcopy number may be attributed to the promotion of mito-chondrial fission and fusion However the increase mayalso be attained through the regulation of a number oftranscriptional factors and cofactors To assess whetherresveratrol would enhance expression and activity of PGC-1120572 we analyzed the transcription and protein level of thePGC-1120572 gene and its target gene mtTFA which is directlyinvolved in mitochondrial biogenesis The results revealed asignificant decrease in mRNA transcripts and protein levelsof PGC-1120572 and in its downstream target gene (mtTFA) inrotenone-exposed rats (Figures 4(a) and 4(b)) and PC12 cells(Figures 4(c) and 4(d)) compared to the control groupsThe results further confirmed that PGC-1120572 activity andtranscriptional deregulation of its target gene were altered inrats and PC12 cells exposed to rotenone Moreover resver-atrol pretreatment could reverse the inhibition of PGC-1120572


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Figure 2 Effects of resveratrol on PC12 cell viability apoptosis and morphology (a) The results show that rotenone induces a statisticallysignificant decrease in cell viability Resveratrol significantly relieves the damage to PC12 cells induced by rotenone within a concentrationrange (5ndash100 120583M) When the concentration is under 5120583M the protective effect is not significant Protection is concentration-dependenthowever at a concentration of 100 120583M the protective effect of resveratrol on PC12 cell viability was decreased compared to 60 120583M (b) Thenormal (control) morphology of PC12 cells with long processes (c)The processes of PC12 cells in the R group appear much shorter comparedwith that of the control group (d) The injury to PC12 cell morphology was alleviated with resveratrol pretreatment (e) Results show thatrotenone induces an increase of cell apoptosis with statistical significance Resveratrol pretreatment decreases the number of apoptotic cellsinduced by rotenone with significant difference The results are presented as the mean plusmn SEM lowastlowast119875 lt 001 versus control group 119875 lt 001versus R group


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C R R + RSV

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C group R group R + RSV groupC group R group R + RSV group

(g)

Figure 3 The protective effect of resveratrol against rotenone-induced mitochondrial fission and fusion in SD rats and PC12 cells (a b) Thefluorescence intensity indicating mitochondrial mass was calculated and analyzed The results show that rotenone induced a reduction inmitochondrial mass and an increase in the number of fragmented mitochondria with small ring-shapes compared with that in the controlgroup However the decrease in mitochondrial mass and mitochondrial fragmentation is improved by resveratrol pretreatment of PC12cells prior to rotenone exposure (c) The number of fragmented cells is significantly increased in R group compared with that in controlgroup Resveratrol pretreatment significantly reduced the number of fragmented cells compared with R group (d e) In the in vivo modelthe quantification analysis of Drp1 Fis1 OPA1 and MFN2 protein and mRNA levels involved in mitochondrial fission and fusion reveals asignificant decrease in the R groups compared with the control group and an increase in the RSV-pretreated groups exposed to rotenoneshowing resveratrolrsquos protective effect in rotenone-induced neurotoxicity (f g) Protein and mRNA levels are reduced significantly followingrotenone exposure in vitro and resveratrol significantly increased expression compared to the control group Representative immunoblotsof proteins and electrophoretic bands of genes associated with mitochondrial fission and fusion 120573-actin served as the internal control tonormalize the amount of protein andmRNAThe results are presented as themeanplusmn SEMThevalueswere generated from three independentexperiments lowast119875 lt 005 versus control group lowastlowast119875 lt 001 versus control group 119875 lt 005 versus R group 119875 lt 001 versus R group


mtTFA

mtTFA

075

08

085

09

0951

Prot

ein

expr

essio

n (m

TFA

120573-a

ctin

)

PGC-1120572

002040608

112

lowastlowast

lowastlowast

C R R + RSV120573-actin

PGC-1120572

Prot

ein

expr

essio

n(P

GC-

1120572120573

-act

in)


(a)

mtTFAPGC-1120572

000200400600801

012014016

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

000501

01502

025G

ene e

xpre

ssio

n (r

atio

of g

ene 120573-a

ctin

)

lowastlowast lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(b)

mtTFAPGC-1120572

002040608

112

Prot

ein

expr

essio

n (P

GC-

1120572120573

-act

in)

002040608

112

Prot

ein

expr

essio

n(m

tTFA

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(c)

mtTFAPGC-1120572

00102030405060708

Gen

e exp

ress

ion

(mtT

FA 120573

-act

in)

001020304050607

Gen

e exp

ress

ion

(PG

C-1120572

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(d)

Cel

l via

bilit

y (c

ontro

l 100

)

0

50

100

150

Con

trol g

roup

R gr

oup

siPG

C-1120572

$lowastlowast

lowastlowast

R+

RSV

gro

up

siPG

C-1120572

R+

siPG

C-1120572

RRS

V+

+

(e)

002040608

112

Rela

tive M

tDN

A

copy

num

bers

lowastlowast

lowastlowast


(f)

Figure 4 Resveratrol improvesmitochondrial biogenesis inhibited by rotenone Representative immunoblots of proteins and electrophoreticbands of genes involved in mitochondrial biogenesis 120573-actin is used as an internal control to normalize the amount of proteins and mRNA(a b) In vivo model results show that protein and mRNA expression of PGC-1120572 and mtTFA are reduced in rotenone-exposed rats comparedto the control group Resveratrol pretreatment significantly increased protein and mRNA expression of PGC-1120572 and mtTFA which weresuppressed by rotenone treatment (c d) Western blot and RT-PCR analysis show that rotenone inhibited mitochondrial biogenesis in vitroand that resveratrol could relieve the suppression of the two proteins and genes (e) Results show that rotenone induces a decrease of cellviability with statistical significance Resveratrol restores the damage of PC12 cells induced by rotenone with significant difference Howeverafter all three treatments there is no significant change in viability of PC12 cells with PGC-1120572 knocked down compared with that of wild PC12cells (f) The mitochondrial DNA copy number is significantly lower in the R group compared with that in the control group Resveratrolsignificantly enhances mitochondrial DNA copy number in PC12 cells exposed to rotenone The results are presented as the mean plusmn SEMThe values were generated from three independent experiments lowastlowast119875 lt 001 versus control group 119875 lt 005 versus R group 119875 lt 001versus R group $119875 lt 005 versus R + RSV group


0

20

40

60

80

100

120

140

160

180

200


Cell

RO

S flu

ores

cenc

e (co

ntro

l 100

)

lowastlowast

R + RSV group

(a)


ATP

conc

entr

atio

n (c

ontro

l 100

)

0

50

100

150

lowastlowast

R + RSV group

(b)

Figure 5 Effects of resveratrol pretreatment on ROS andATP concentrations in PC12 cells exposed to rotenone (a) ROS formation is inducedin PC12 cells exposed to rotenonewith high fluorescence intensity compared to the control group Resveratrol pretreatment could significantlysuppress ROS formation induced by rotenone (b) Rotenone exposure leads to a significant reduction in the ATP concentration in PC12 cellsThere was an increase in ATP concentration in PC12 cells with resveratrol pretreatment compared to the control group The results arepresented as the mean plusmn SEM lowastlowast119875 lt 001 versus control group 119875 lt 005 versus R group

and mtTFA protein and mRNA expression caused byrotenone (Figure 4) To investigate the role of PGC-1120572 inresveratrolrsquos protection in PC12 cells PGC-1120572 were furtherknocked down Results showed that the viability of PC12 cellswas decreased significantly in siPGC-1120572 + R + RSV groupcompared with that of the R + RSV group (Figure 4(e)) It isindicated that PGC-1120572 downregulation seriously affected theprotective effect of resveratrol on PC12 cell viability Togetherthe results presented here indicate a resveratrol-mediatedincrease in PGC-1120572 transcriptional activity as shown by theupregulation of its target gene This not only was related toPGC-1120572 expression but also suggested a possible effect at aposttranslational level

35 Effects of Resveratrol on ROS Formation and ATP Produc-tion PGC-1120572 is a transcriptional coactivator known as themaster regulator of mitochondrial functions and oxidativemetabolism Because resveratrol can promote the expressionand activity of PGC-1120572 we evaluated the role of resveratrol onoxidative stress and ATP production in PC12 cells exposed torotenoneThe results showed that the ROS level (Figure 5(a))was increased and that ATP production (Figure 5(b)) wasdecreased significantly when PC12 cells were treated withrotenone compared with the control group However if PC12cells were pretreated with resveratrol the ROS productiondecreased (Figure 5(a)) and ATP production (Figure 5(b))increased significantly compared with those of the R groupTherefore resveratrol could reduce oxidative stress and pro-mote ATP production in PC12 cells exposed to rotenone

4 Discussion

Although the etiology of dopaminergic neuronal degenera-tion of PD remains unknownmitochondrial dysfunction hasbeen the focus of studies into the etiology of familial and

sporadic PD [8] Mitochondria are now known to constitutea population of organelles requiring careful balance andintegration of numerous processes including fission-fusionmachinery the regulation of biogenesis migration through-out the cell shape remodeling and autophagy [15 23ndash25]

Here we used in vitro and in vivo PD models to showrotenone-induced loss of dopaminergic neurons and oxida-tive damage in mitochondria decreases in mitochondrialmass andmitochondrial DNA copy number and increases inimpaired mitochondrial fissionfusion and biogenesis Thuswe concluded that rotenone exposure leads to dopaminergicneurodegeneration which is associated with an imbalance ofmitochondrial homeostasis and organelle damage

In the present study we also have demonstrated thatresveratrol could significantly ameliorate the motor dys-function and attenuate the damage of substantia nigradopaminergic-induced Parkinsonrsquos disease rats It is knownthat a decrease in athletic ability is positively correlatedwith the damage of dopaminergic neurons in the substantianigraThus it is suggested that resveratrol pretreatment has aprotective effect on dopaminergic neurons in rat substantianigra Furthermore the study also showed that resvera-trol treatment could significantly relieve rotenone-inducedincreases in ROS and improve rotenone-induced decreasesin ATP production in PC12 cells All of these data providea mechanistic basis for resveratrol in the clinical treatmentof Parkinsonrsquos disease Thus the potential application ofresveratrol in the treatment of Parkinsonrsquos disease deservesfurther investigation

Therefore combined with other unpublished results col-lected by our lab we have explored the effects of resveratrolon mitochondrial fissionfusion and biogenesis of dopamineneurons in rats and PC12 cells exposed to rotenone Itis known that PGC-1120572 and mtTFA are two key signalingfactors regulating mitochondrial biogenesis which has been


confirmed inmany other studies [26 27] Our results showedthat rotenone suppressed the expression of PGC-1120572 andmtTFA However resveratrol pretreatment could obviouslyimprove mRNA and protein expression of PGC-1120572 andmtTFA in the substantia nigra and in PC12 cells whichwas inhibited by rotenone In addition we further usedPGC-1120572 siRNA to knock down its expression to explorewhether the effect of resveratrol on cell viability is affectedResults showed that cell viability was affected which provedthat resveratrol protection in cells induced by rotenonemight be associated with PGC-1120572 Therefore a decreasein mitochondrial DNA copy number and mitochondrialmass in rotenone-induced neurotoxicity could be the resultof a reduction of mitochondrial biogenesis Inhibition ofmitochondrial biogenesis may contribute to the developmentof rotenone-induced neurotoxicity Furthermore resveratrolcould prevent rotenone-induced neurotoxicity by promotingmitochondrial biogenesis In addition OPA1 MFN2 Drp1and Fis1 proteins were involved in regulating mitochondrialfission and fusion [28] Our results further showed thatrotenone could result in a decrease in OPA1 MFN2 Drp1and Fis1 expression leading to dysfunction in mitochondrialfissionfusion The dysfunction in mitochondrial fission andfusion promoted a decrease in mitochondrial mass whichmay further promote increasedROS formation anddecreasedATP production in rotenone-induced neurotoxicity In ourexperiments the dysfunction of mitochondrial fission andfusion could be alleviated with resveratrol pretreatmentthrough improving the expression ofOPA1MFN2Drp1 andFis1 which were inhibited by rotenone Thus the observedresveratrol-mediated increase in mitochondrial fusion likelyacted to increasemitochondrialDNAcopynumber andmito-chondrial mass which were inhibited by the mitochondrialcomplex I inhibitor rotenone We conclude that resveratrollikely increased mitochondrial fusion not solely throughincreased mitochondrial biogenesis to increase mitochon-drial DNA copy number andmitochondrialmass tomaintainmitochondrial homeostasisThus the neuroprotective effectsof resveratrol on rotenone-induced neurotoxicity were medi-ated through regulation of mitochondrial fissionfusion andbiogenesis Similar results were obtained in a recent study thatdemonstrated that MPTP increased vulnerability with agingby themechanisms underlyingmitochondrial dynamics [29]Therefore these data suggested that resveratrol alleviation ofrotenone-induced damage of dopaminergic neurons could bemediated through regulating the balance of mitochondrialdynamics

It is worth noting that despite using higher doses than thepresent study resveratrol was reported to have no protectiveeffects on rotenone-induced apoptosis in SH-SY5Y cells in aprevious study [30] First we postulated that the differencein protective effects may be related to the use of differentcell lines Through further analysis we found that SH-SY5Ycells were pretreated with resveratrol for 1 h prior to rotenoneexposure as opposed to 24 h in our study Furthermorea paper published recently also reported that resveratrolhad protective effects in SH-SY5Y cells following 24 h pre-treatment by inducing treatment-dependent autophagy [31]Thus a sufficient pretreatment time is required to enable

expression of critical neuroprotective genes and proteins Inaddition to sufficient time based on our experiments theprotection by resveratrol on the viability of PC12 cells exposedto rotenone is more likely confined to a concentrationrange In support of this hypothesis we demonstrated thatincreased fissionfusion and biogenesis protein expression inresponse to resveratrol conferred neuroprotective effects in aconcentration- and time-dependent manner Therefore ourresults demonstrated that resveratrol attenuated rotenone-induced neurotoxicity through increased mitochondrial bio-genesis and maintained the balance of mitochondrial fissionand fusion which may limit ROS production and promoteATP content and thereby acted as a protective agent inrotenone-induced neuronal toxicity

Mitochondrial fissionfusion and biogenesis are dynamicprocesses which regulate mitochondrial homeostasis [15]Recently mitochondrial fissionfusion and biogenesis dys-function have been linked to PD development [8 15]Mitochondrial fissionfusion and biogenesis are active at abasal level in most cells in the body The processes notonly determine the structure of the entire mitochondrialpopulation but also influence nearly every aspect of mito-chondrial function thereby acting as a protectivemechanismin cells [32ndash34] Herein we provided evidence that resver-atrol induced PGC-1120572 and mtTFA expression to augmentmitochondrial biogenesis and influence OPA1MFN2 andFis1Drp1 expression to regulate the balance ofmitochondrialfissionfusion thus maintaining mitochondrial homeostasisand preventing rotenone-induced neuronal degeneration Ofcourse we could not exclude mitophagy also regulatingthe mitochondrial homeostasis which we should do somework on in the future Furthermore resveratrol attenuatedrotenone-induced intracellular ROS generation which alsocorresponded to increased ATP production This study pro-vides novel insight into the cellularmechanisms of resveratrolin preventing neurodegeneration and elucidates potentialdownstream targets for future therapeutic strategies

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by grants from the NSFC (Nat-ural Science Foundation of China) (81473006 81273106) toYan Sai and the Natural Science Foundation of Chongqing(cstc2012jjA10028) to Yan Sai

References

[1] J Blanchet F Longpre G Bureau et al ldquoResveratrol a red winepolyphenol protects dopaminergic neurons in MPTP-treatedmicerdquo Progress in Neuro-Psychopharmacology amp Biological Psy-chiatry vol 32 no 5 pp 1243ndash1250 2008

[2] R Pangeni J K Sahni J Ali S Sharma and S BabootaldquoResveratrol review on therapeutic potential and recentadvances in drug deliveryrdquo Expert Opinion on Drug Deliveryvol 11 no 8 pp 1285ndash1298 2014


[3] V W Dolinsky A Y M Chan I R Frayne P E Light C DesRosiers and J R B Dyck ldquoResveratrol prevents the prohyper-trophic effects of oxidative stress on LKB1rdquo Circulation vol 119no 12 pp 1643ndash1652 2009

[4] K-F Hsu C-L Wu S-C Huang et al ldquoCathepsin L mediatesresveratrol-induced autophagy and apoptotic cell death incervical cancer cellsrdquoAutophagy vol 5 no 4 pp 451ndash460 2009

[5] M Lagouge C Argmann Z Gerhart-Hines et al ldquoResver-atrol improves mitochondrial function and protects againstmetabolic disease by activating SIRT1 and PGC-1alphardquo Cellvol 127 no 6 pp 1109ndash1122 2006

[6] D D Lofrumento G Nicolardi A Cianciulli et al ldquoNeuro-protective effects of resveratrol in an MPTP mouse model ofParkinsonrsquos-like disease possible role of SOCS-1 in reducingpro-inflammatory responsesrdquo Innate Immunity vol 20 no 3pp 249ndash260 2014

[7] F Jin Q Wu Y-F Lu Q-H Gong and J-S Shi ldquoNeuropro-tective effect of resveratrol on 6-OHDA-induced Parkinsonrsquosdisease in ratsrdquo European Journal of Pharmacology vol 600 no1ndash3 pp 78ndash82 2008

[8] J-H Shin V L Dawson and T M Dawson ldquoSnapShotpathogenesis of Parkinsonrsquos diseaserdquo Cell vol 139 no 2 pp440e1ndash440e2 2009

[9] C Perier J Bove M Vila and S Przedborski ldquoThe rotenonemodel of Parkinsonrsquos diseaserdquo Trends in neurosciences vol 26no 7 pp 345ndash346 2003

[10] C M Tanner F Kame G W Ross et al ldquoRotenone paraquatand Parkinsonrsquos diseaserdquoEnvironmental Health Perspectives vol119 no 6 pp 866ndash872 2011

[11] A Spivey ldquoRotenone and paraquat linked to Parkinsonrsquos dis-ease human exposure study supports years of animal studiesrdquoEnvironmental Health Perspectives vol 119 no 6 article A2592011

[12] A Abeliovich ldquoParkinsonrsquos disease mitochondrial damagecontrolrdquo Nature vol 463 no 7282 pp 744ndash745 2010

[13] J R Friedman and J Nunnari ldquoMitochondrial form andfunctionrdquo Nature vol 505 no 7483 pp 335ndash343 2014

[14] J Nunnari and A Suomalainen ldquoMitochondria in sickness andin healthrdquo Cell vol 148 no 6 pp 1145ndash1159 2012

[15] D C Chan ldquoMitochondria dynamic organelles in diseaseaging and developmentrdquo Cell vol 125 no 7 pp 1241ndash12522006

[16] L Cui H Jeong F Borovecki C N Parkhurst N Taneseand D Krainc ldquoTranscriptional repression of PGC-1alpha bymutant huntingtin leads to mitochondrial dysfunction andneurodegenerationrdquo Cell vol 127 no 1 pp 59ndash69 2006

[17] M Milone ldquoMitochondria diabetes and Alzheimerrsquos diseaserdquoDiabetes vol 61 no 5 pp 991ndash992 2012

[18] R Wang Y Y Liu X Y Liu et al ldquoResveratrol protectsneurons and the myocardium by reducing oxidative stress andameliorating mitochondria damage in a cerebral ischemia ratmodelrdquo Cellular Physiology and Biochemistry vol 34 no 3 pp854ndash864 2014

[19] J S Franzone and M C Reboani ldquoStimulating action ofhematoporphyrin on motor coordination and resistance tofatigue in ratsrdquo Archivio per le Scienze Mediche vol 134 no 4pp 403ndash406 1977

[20] J F Buckman H Hernandez G J Kress T V Votyakova S Paland I J Reynolds ldquoMitoTracker labeling in primary neuronaland astrocytic cultures influence of mitochondrial membranepotential and oxidantsrdquo Journal of Neuroscience Methods vol104 no 2 pp 165ndash176 2001

[21] M Agnello G Morici and A M Rinaldi ldquoA method formeasuring mitochondrial mass and activityrdquo Cytotechnologyvol 56 no 3 pp 145ndash149 2008

[22] L F Marques-Santos J G P Oliveira R C Maia and V MRumjanek ldquoMitotracker green is a P-glycoprotein substraterdquoBioscience Reports vol 23 no 4 pp 199ndash212 2003

[23] S Hoppins and J Nunnari ldquoMitochondrial dynamics andapoptosismdashthe ER connectionrdquo Science vol 337 no 6098 pp1052ndash1054 2012

[24] M O Dietrich Z-W Liu and T L Horvath ldquoMitochondrialdynamics controlled by mitofusins regulate Agrp neuronalactivity and diet-induced obesityrdquo Cell vol 155 no 1 pp 188ndash199 2013

[25] Y Tamura K Itoh and H Sesaki ldquoSnapShot mitochondrialdynamicsrdquo Cell vol 145 no 7 pp 1158ndash1158e1 2011

[26] Z Wu P Puigserver U Andersson et al ldquoMechanisms con-trolling mitochondrial biogenesis and respiration through thethermogenic coactivator PGC-1rdquo Cell vol 98 no 1 pp 115ndash1241999

[27] L Z Agudelo T Femenia FOrhan et al ldquoSkeletalmuscle PGC-11205721 modulates kynurenine metabolism and mediates resilienceto stress-induced depressionrdquo Cell vol 159 no 1 pp 33ndash452014

[28] R J Youle and A M van der Bliek ldquoMitochondrial fissionfusion and stressrdquo Science vol 337 no 6098 pp 1062ndash10652012

[29] N Jiang H Bo C Song et al ldquoIncreased vulnerability withaging to MPTP the mechanisms underlying mitochondrialdynamicsrdquo Neurological Research vol 36 no 8 pp 722ndash7322014

[30] G Filomeni I Graziani D De Zio et al ldquoNeuroprotectionof kaempferol by autophagy in models of rotenone-mediatedacute toxicity possible implications for Parkinsonrsquos diseaserdquoNeurobiology of Aging vol 33 no 4 pp 767ndash785 2012

[31] T-K Lin S-D Chen Y-C Chuang et al ldquoResveratrol partiallyprevents rotenone-induced neurotoxicity in dopaminergic SH-SY5Y cells through induction of heme oxygenase-1 dependentautophagyrdquo International Journal of Molecular Sciences vol 15no 1 pp 1625ndash1646 2014

[32] D M Arduıno A R Esteves and S M Cardoso ldquoMitochon-drial fusionfission transport and autophagy in Parkinsonrsquosdisease when mitochondria get nastyrdquo Parkinsonrsquos Disease vol2011 Article ID 767230 13 pages 2011

[33] B Westermann ldquoMitochondrial fusion and fission in cell lifeand deathrdquo Nature Reviews Molecular Cell Biology vol 11 no12 pp 872ndash884 2010

[34] BWestermann ldquoBioenergetic role of mitochondrial fusion andfissionrdquo Biochimica et Biophysica Acta Bioenergetics vol 1817no 10 pp 1833ndash1838 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


biogenesis is associated with many diseases in particularneurodegenerative diseases such as Huntingtonrsquos diseaseAlzheimerrsquos disease and PD [15ndash17] In recent years therehave been reports that resveratrol could increase theactivity of mitochondria thereby conferring resistance tomitochondrial dysfunction in various disease states [18] Todate accumulated evidence suggests clinical potential forresveratrol but the precise molecular mechanism for biolog-ical activity in the mitochondria remains poorly understood

In the present study we explored the protective effectof resveratrol on rotenone-induced dopamine neurotoxicityand its action in mitochondrial fissionfusion and biogen-esis We intend to clarify the mechanism of resveratrol inmitochondrial dynamics in dopamine neurotoxicity inducedby rotenone This research will be helpful in explaining thediverse biological and pharmacological properties of resver-atrol

2 Materials and Methods

21 Materials Healthymale Sprague-Dawley rats (5ndash7 weeksold) weighing 180ndash220 g are obtained from the AnimalCenter of theThirdMilitaryMedical University (ChongqingChina) PC12 cells (adrenal gland pheochromocytoma) wereprovided by Shanghai Cell Bank Institute of Biochemistryand Cell Biology Chinese Academy of Sciences Rotenonedimethyl sulfoxide (DMSO) resveratrol and adenosinetriphosphate (ATP) were purchased from Sigma (St LouisMO USA) Except for antibodies of mitochondrial tran-scription factor A (mtTFA Santa Cruz USA) antibodies ofoptic atrophy 1 (OPA1) mitofusin 2 (MFN2) mitochondrialfission 1 (Fis1) dynamin-1-like protein (Drp1) peroxisomeproliferator-activated receptor gamma and coactivator 1alpha (PGC-1120572) were purchased from Abcam (USA) Allother reagents used were of the highest grade available

22 Animal Care and Treatment The male Sprague-Dawleyrats were housed in climate-controlled rooms at 23 plusmn 2∘Chousing temperature on a reverse 12 12 h lightdark cyclewith lights on at 800 Animals were allowed free accessto food and water during the experimental period Ani-mals were divided into three groups control rotenone-exposed and rotenone plus resveratrol treated Between 1and 5 animals per cage were acclimated for 1 week in ourtemperature-controlled animal roomprior to drug treatmentThe rotenone-exposed group (R group) was treated withsubcutaneous injections of rotenone (first 2mgkg everyday for three days second 1mgkg every day for sevendays and then 05mgkg every day for twenty days) Thecontrol group was injected with an equal volume of dimethylsulfoxide for thirty days The resveratrol protection group(R + RSV group) was treated first with intraperitonealinjection of resveratrol (50mgkg every day for thirty days)and then thirty minutes later animals were treated the sameas in the rotenone exposure group All animal procedureswere conducted per the guidelines of the care and use oflaboratory animals and Chinarsquos laws on animal experimen-tation

23 Neurobehavioral Tests Rotarod Performance Test Therotarod test has been used to assess motor coordination andbalance alterations in rodents This test measured the abilityof the rat to preserve balance by holding itself on the rotatingrod [19]Thirty days after drug treatments we performed thislocomotor activity which measured motor coordination inrats The rats were adapted to the bar When all rats couldremain on the rotating rod for approximately 3min each ratwas placed on a horizontal rod rotating at a speed of 12 rpmfor a maximum of 3min The duration of time during whicheach rat was able to maintain its balance walking on the topof the rod was measured The number of rats that fell wasrecorded

24 Tissue Preparation After completion of the behavioraltests on day 31 the rats were sacrificed by decapitationWholebrains were removed quickly and prepared for assay Thebrain was rinsed with chilled saline and the corpus striatumwas dissected on ice according to coordinates indicated in therat brain atlas A portion of the tissueswas used formeasuringthe levels of ROS and ATP Another portion of the tissues wasused for detecting the gene and protein expression levels ofmitochondrial fissionfusion and biogenesis

25 Cell Culture and Treatment PC12 cells were cultured at37∘Cunder an atmosphere of 5CO

2in DulbeccorsquosModified

Eaglersquos Medium (DMEM Invitrogen USA) containing 10horse serum and 5 fetal bovine serum (Hyclone USA)Cells were divided into three groups The rotenone-exposedgroup (R group) was treated with the indicated concentrationof rotenone dissolved in DMSO for 24 h The control groupwas administered equivalent amounts of DMSO for 24 hTheresveratrol protection group (RSV + R group) was pretreatedwith resveratrol for 24 h and then exposed to rotenone for24 h

26 Transfection of siRNA of PGC-1120572 PC12 cells (1times105well)were seeded into 12-well plates containing 500 120583L of completemedium and were incubated for 24 h Then transfection withsiRNA was conducted using a Lipofectamine 2000 reagent asper userrsquos guide After 6 hours the medium was changed toa normal culture medium After another 48 hours the cellswere harvested and sent for isolation of RNA and analysisof PGC-1120572 expression by real-time PCR The sequences areas follows 5-AAGACGGATTGCCCTCATTTG-3 (siPGC-1120572) and 5-AACGUGACACGUUCG GAGAATT-3 (negativecontrol)

27 CCK-8 Assay to Evaluate PC12 Cell Viability PC12cell viability was assayed with the Cell Counting Kit-8(DOJINDO Japan) according to the manufacturerrsquos instruc-tions All results were normalized to optical density val-ues measured from an identically conditioned well withoutmedium

28 Annexin V-FITC to Evaluate PC12 Cell ApoptosisAccording to the instructions of themanufacturer ofAnnexinV-FITC Apoptosis Detection Kit (BD USA) the cells were


Table 1


















2CO3




3 Results








0

20

40

60

80

100

120


Surv

ival

rate

()

lowastlowast

R + RSV group

(a)

0

20

40

60

80

100

120

Rate

of m

otio

n (

)


lowastlowast

(b)

0

20

40

60

80

100

120

Mot

ion

time (

seco

nds)


lowastlowast

(c)








0

Con

trol g

roup

R gr

oup

02040608

112141618

2

Opt

ical

den

sity

(OD

)


Solv

ent g

roup

R+

RSV

(1)

R+

RSV

(5)

R+

RSV

(10)

R+

RSV

(50)

R+

RSV

(60)

R+

RSV

(100

)RS

V(1

00)

(a)

(b) (c) (d)

103

104

104

105

105

106

106

107

103

104

105

106

107

107 104 105 106 107

Com

p-FL

2-A

FL

2-A

Com

p-FL

2-A

FL

2-A


Con

trol g

roup

R gr

oup

R+

RSV

grou

p

103

104

105

106

107

104 105 106 107

Com

p-FL

2-A

FL

2-A

0

5

10

15

R + RSV group

lowastlowast

Apop

totic

cells

()

(e)



Mito

chon

dria

l mas

sM

orph

olog

yIn

set


(a)

020406080

100120

Con

trol g

roup

The m

itoch

ondr

ial

mas

s

R gr

oup

R+

RSV

grou

p

lowast

(b)

Frag

men

ted

cells

(cel

ls w

ithou

t el

onga

ted

mito

chon

dria

) (

)

020406080

100

Con

trol g

roup

R gr

oup

R+

RSV

grou

plowastlowast

lowast

(c)

Drp1

002040608

112

C group

Fis1

0010203040506

Prot

ein

expr

essio

n

Prot

ein

expr

essio

n

(Fis1

120573-a

ctin

)

OPA1

002040608

112 MFN2

002040608

112

MFN2

Drp1

Fis1

OPA1

C R

(Drp

1120573

-act

in)

lowastlowast





R + RSV

120573-actin

lowastlowast

lowastlowast

lowastlowast

(OPA

1120573

-act

in)

Prot

ein

expr

essio

n

(MFN

2120573

-act

in)

Prot

ein

expr

essio

n

(d)

Figure 3 Continued


MFN2

000501

01502

02503

035OPA1

000501

01502

025

Fis1

000501

01502

02503

03504

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)G

ene e

xpre

ssio

n (r

atio

of g

ene120573-a

ctin

)

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

Drp1

000200400600801

012




C R R + RSV

MFN2

Drp1

Fis1

OPA1

120573-actin

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(e)

OPA1

Drp1

MFN2

Fis1

Drp1

002040608

1

MFN2

002040608

1OPA1

002040608

112

Fis1

002040608

112

lowastlowast

lowastlowast

lowastlowast

lowastlowast

120573-actin

C R R + RSV

(Drp

1120573

-act

in)

Prot

ein

expr

essio

n(O

PA1120573

-act

in)

Prot

ein

expr

essio

n

(MFN

2120573

-act

in)

Prot

ein

expr

essio

n(F

is1 120573

-act

in)

Prot

ein

expr

essio

n



(f)

Drp1

0010203040506

Gen

e exp

ress

ion

(Drp

1120573

-act

in) Fis1

044046048

05052054056

Gen

e exp

ress

ion

(Fis1

120573-a

ctin

)

MFN2

002040608

1

Gen

e exp

ress

ion

(MFN

2120573-a

ctin

) OPA1

002040608

112

Gen

e exp

ress

ion

(OPA

1120573-a

ctin

)

lowastlowast

lowastlowast

lowastlowast

OPA1

Drp1

MFN2

Fis1

120573-actin

C R R + RSV

lowastlowast



(g)



mtTFA

mtTFA

075

08

085

09

0951

Prot

ein

expr

essio

n (m

TFA

120573-a

ctin

)

PGC-1120572

002040608

112

lowastlowast

lowastlowast


PGC-1120572

Prot

ein

expr

essio

n(P

GC-

1120572120573

-act

in)


(a)

mtTFAPGC-1120572

000200400600801

012014016

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

000501

01502

025G

ene e

xpre

ssio

n (r

atio

of g

ene 120573-a

ctin

)


C R R + RSV

mtTFA

120573-actin

PGC-1120572


(b)

mtTFAPGC-1120572

002040608

112

Prot

ein

expr

essio

n (P

GC-

1120572120573

-act

in)

002040608

112

Prot

ein

expr

essio

n(m

tTFA

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(c)

mtTFAPGC-1120572

00102030405060708

Gen

e exp

ress

ion

(mtT

FA 120573

-act

in)

001020304050607

Gen

e exp

ress

ion

(PG

C-1120572

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(d)

Cel

l via

bilit

y (c

ontro

l 100

)

0

50

100

150

Con

trol g

roup

R gr

oup

siPG

C-1120572

$lowastlowast

lowastlowast

R+

RSV

gro

up

siPG

C-1120572

R+

siPG

C-1120572

RRS

V+

+

(e)

002040608

112

Rela

tive M

tDN

A

copy

num

bers

lowastlowast

lowastlowast


(f)



0

20

40

60

80

100

120

140

160

180

200


Cell

RO

S flu

ores

cenc

e (co

ntro

l 100

)

lowastlowast

R + RSV group

(a)


ATP

conc

entr

atio

n (c

ontro

l 100

)

0

50

100

150

lowastlowast

R + RSV group

(b)




4 Discussion













Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 1


















2CO3




3 Results








0

20

40

60

80

100

120


Surv

ival

rate

()

lowastlowast

R + RSV group

(a)

0

20

40

60

80

100

120

Rate

of m

otio

n (

)


lowastlowast

(b)

0

20

40

60

80

100

120

Mot

ion

time (

seco

nds)


lowastlowast

(c)








0

Con

trol g

roup

R gr

oup

02040608

112141618

2

Opt

ical

den

sity

(OD

)


Solv

ent g

roup

R+

RSV

(1)

R+

RSV

(5)

R+

RSV

(10)

R+

RSV

(50)

R+

RSV

(60)

R+

RSV

(100

)RS

V(1

00)

(a)

(b) (c) (d)

103

104

104

105

105

106

106

107

103

104

105

106

107

107 104 105 106 107

Com

p-FL

2-A

FL

2-A

Com

p-FL

2-A

FL

2-A


Con

trol g

roup

R gr

oup

R+

RSV

grou

p

103

104

105

106

107

104 105 106 107

Com

p-FL

2-A

FL

2-A

0

5

10

15

R + RSV group

lowastlowast

Apop

totic

cells

()

(e)



Mito

chon

dria

l mas

sM

orph

olog

yIn

set


(a)

020406080

100120

Con

trol g

roup

The m

itoch

ondr

ial

mas

s

R gr

oup

R+

RSV

grou

p

lowast

(b)

Frag

men

ted

cells

(cel

ls w

ithou

t el

onga

ted

mito

chon

dria

) (

)

020406080

100

Con

trol g

roup

R gr

oup

R+

RSV

grou

plowastlowast

lowast

(c)

Drp1

002040608

112

C group

Fis1

0010203040506

Prot

ein

expr

essio

n

Prot

ein

expr

essio

n

(Fis1

120573-a

ctin

)

OPA1

002040608

112 MFN2

002040608

112

MFN2

Drp1

Fis1

OPA1

C R

(Drp

1120573

-act

in)

lowastlowast





R + RSV

120573-actin

lowastlowast

lowastlowast

lowastlowast

(OPA

1120573

-act

in)

Prot

ein

expr

essio

n

(MFN

2120573

-act

in)

Prot

ein

expr

essio

n

(d)

Figure 3 Continued


MFN2

000501

01502

02503

035OPA1

000501

01502

025

Fis1

000501

01502

02503

03504

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)G

ene e

xpre

ssio

n (r

atio

of g

ene120573-a

ctin

)

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

Drp1

000200400600801

012




C R R + RSV

MFN2

Drp1

Fis1

OPA1

120573-actin

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(e)

OPA1

Drp1

MFN2

Fis1

Drp1

002040608

1

MFN2

002040608

1OPA1

002040608

112

Fis1

002040608

112

lowastlowast

lowastlowast

lowastlowast

lowastlowast

120573-actin

C R R + RSV

(Drp

1120573

-act

in)

Prot

ein

expr

essio

n(O

PA1120573

-act

in)

Prot

ein

expr

essio

n

(MFN

2120573

-act

in)

Prot

ein

expr

essio

n(F

is1 120573

-act

in)

Prot

ein

expr

essio

n



(f)

Drp1

0010203040506

Gen

e exp

ress

ion

(Drp

1120573

-act

in) Fis1

044046048

05052054056

Gen

e exp

ress

ion

(Fis1

120573-a

ctin

)

MFN2

002040608

1

Gen

e exp

ress

ion

(MFN

2120573-a

ctin

) OPA1

002040608

112

Gen

e exp

ress

ion

(OPA

1120573-a

ctin

)

lowastlowast

lowastlowast

lowastlowast

OPA1

Drp1

MFN2

Fis1

120573-actin

C R R + RSV

lowastlowast



(g)



mtTFA

mtTFA

075

08

085

09

0951

Prot

ein

expr

essio

n (m

TFA

120573-a

ctin

)

PGC-1120572

002040608

112

lowastlowast

lowastlowast


PGC-1120572

Prot

ein

expr

essio

n(P

GC-

1120572120573

-act

in)


(a)

mtTFAPGC-1120572

000200400600801

012014016

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

000501

01502

025G

ene e

xpre

ssio

n (r

atio

of g

ene 120573-a

ctin

)


C R R + RSV

mtTFA

120573-actin

PGC-1120572


(b)

mtTFAPGC-1120572

002040608

112

Prot

ein

expr

essio

n (P

GC-

1120572120573

-act

in)

002040608

112

Prot

ein

expr

essio

n(m

tTFA

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(c)

mtTFAPGC-1120572

00102030405060708

Gen

e exp

ress

ion

(mtT

FA 120573

-act

in)

001020304050607

Gen

e exp

ress

ion

(PG

C-1120572

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(d)

Cel

l via

bilit

y (c

ontro

l 100

)

0

50

100

150

Con

trol g

roup

R gr

oup

siPG

C-1120572

$lowastlowast

lowastlowast

R+

RSV

gro

up

siPG

C-1120572

R+

siPG

C-1120572

RRS

V+

+

(e)

002040608

112

Rela

tive M

tDN

A

copy

num

bers

lowastlowast

lowastlowast


(f)



0

20

40

60

80

100

120

140

160

180

200


Cell

RO

S flu

ores

cenc

e (co

ntro

l 100

)

lowastlowast

R + RSV group

(a)


ATP

conc

entr

atio

n (c

ontro

l 100

)

0

50

100

150

lowastlowast

R + RSV group

(b)




4 Discussion













Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2CO3




3 Results








0

20

40

60

80

100

120


Surv

ival

rate

()

lowastlowast

R + RSV group

(a)

0

20

40

60

80

100

120

Rate

of m

otio

n (

)


lowastlowast

(b)

0

20

40

60

80

100

120

Mot

ion

time (

seco

nds)


lowastlowast

(c)








0

Con

trol g

roup

R gr

oup

02040608

112141618

2

Opt

ical

den

sity

(OD

)


Solv

ent g

roup

R+

RSV

(1)

R+

RSV

(5)

R+

RSV

(10)

R+

RSV

(50)

R+

RSV

(60)

R+

RSV

(100

)RS

V(1

00)

(a)

(b) (c) (d)

103

104

104

105

105

106

106

107

103

104

105

106

107

107 104 105 106 107

Com

p-FL

2-A

FL

2-A

Com

p-FL

2-A

FL

2-A


Con

trol g

roup

R gr

oup

R+

RSV

grou

p

103

104

105

106

107

104 105 106 107

Com

p-FL

2-A

FL

2-A

0

5

10

15

R + RSV group

lowastlowast

Apop

totic

cells

()

(e)



Mito

chon

dria

l mas

sM

orph

olog

yIn

set


(a)

020406080

100120

Con

trol g

roup

The m

itoch

ondr

ial

mas

s

R gr

oup

R+

RSV

grou

p

lowast

(b)

Frag

men

ted

cells

(cel

ls w

ithou

t el

onga

ted

mito

chon

dria

) (

)

020406080

100

Con

trol g

roup

R gr

oup

R+

RSV

grou

plowastlowast

lowast

(c)

Drp1

002040608

112

C group

Fis1

0010203040506

Prot

ein

expr

essio

n

Prot

ein

expr

essio

n

(Fis1

120573-a

ctin

)

OPA1

002040608

112 MFN2

002040608

112

MFN2

Drp1

Fis1

OPA1

C R

(Drp

1120573

-act

in)

lowastlowast





R + RSV

120573-actin

lowastlowast

lowastlowast

lowastlowast

(OPA

1120573

-act

in)

Prot

ein

expr

essio

n

(MFN

2120573

-act

in)

Prot

ein

expr

essio

n

(d)

Figure 3 Continued


MFN2

000501

01502

02503

035OPA1

000501

01502

025

Fis1

000501

01502

02503

03504

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)G

ene e

xpre

ssio

n (r

atio

of g

ene120573-a

ctin

)

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

Drp1

000200400600801

012




C R R + RSV

MFN2

Drp1

Fis1

OPA1

120573-actin

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(e)

OPA1

Drp1

MFN2

Fis1

Drp1

002040608

1

MFN2

002040608

1OPA1

002040608

112

Fis1

002040608

112

lowastlowast

lowastlowast

lowastlowast

lowastlowast

120573-actin

C R R + RSV

(Drp

1120573

-act

in)

Prot

ein

expr

essio

n(O

PA1120573

-act

in)

Prot

ein

expr

essio

n

(MFN

2120573

-act

in)

Prot

ein

expr

essio

n(F

is1 120573

-act

in)

Prot

ein

expr

essio

n



(f)

Drp1

0010203040506

Gen

e exp

ress

ion

(Drp

1120573

-act

in) Fis1

044046048

05052054056

Gen

e exp

ress

ion

(Fis1

120573-a

ctin

)

MFN2

002040608

1

Gen

e exp

ress

ion

(MFN

2120573-a

ctin

) OPA1

002040608

112

Gen

e exp

ress

ion

(OPA

1120573-a

ctin

)

lowastlowast

lowastlowast

lowastlowast

OPA1

Drp1

MFN2

Fis1

120573-actin

C R R + RSV

lowastlowast



(g)



mtTFA

mtTFA

075

08

085

09

0951

Prot

ein

expr

essio

n (m

TFA

120573-a

ctin

)

PGC-1120572

002040608

112

lowastlowast

lowastlowast


PGC-1120572

Prot

ein

expr

essio

n(P

GC-

1120572120573

-act

in)


(a)

mtTFAPGC-1120572

000200400600801

012014016

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

000501

01502

025G

ene e

xpre

ssio

n (r

atio

of g

ene 120573-a

ctin

)


C R R + RSV

mtTFA

120573-actin

PGC-1120572


(b)

mtTFAPGC-1120572

002040608

112

Prot

ein

expr

essio

n (P

GC-

1120572120573

-act

in)

002040608

112

Prot

ein

expr

essio

n(m

tTFA

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(c)

mtTFAPGC-1120572

00102030405060708

Gen

e exp

ress

ion

(mtT

FA 120573

-act

in)

001020304050607

Gen

e exp

ress

ion

(PG

C-1120572

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(d)

Cel

l via

bilit

y (c

ontro

l 100

)

0

50

100

150

Con

trol g

roup

R gr

oup

siPG

C-1120572

$lowastlowast

lowastlowast

R+

RSV

gro

up

siPG

C-1120572

R+

siPG

C-1120572

RRS

V+

+

(e)

002040608

112

Rela

tive M

tDN

A

copy

num

bers

lowastlowast

lowastlowast


(f)



0

20

40

60

80

100

120

140

160

180

200


Cell

RO

S flu

ores

cenc

e (co

ntro

l 100

)

lowastlowast

R + RSV group

(a)


ATP

conc

entr

atio

n (c

ontro

l 100

)

0

50

100

150

lowastlowast

R + RSV group

(b)




4 Discussion













Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120


Surv

ival

rate

()

lowastlowast

R + RSV group

(a)

0

20

40

60

80

100

120

Rate

of m

otio

n (

)


lowastlowast

(b)

0

20

40

60

80

100

120

Mot

ion

time (

seco

nds)


lowastlowast

(c)








0

Con

trol g

roup

R gr

oup

02040608

112141618

2

Opt

ical

den

sity

(OD

)


Solv

ent g

roup

R+

RSV

(1)

R+

RSV

(5)

R+

RSV

(10)

R+

RSV

(50)

R+

RSV

(60)

R+

RSV

(100

)RS

V(1

00)

(a)

(b) (c) (d)

103

104

104

105

105

106

106

107

103

104

105

106

107

107 104 105 106 107

Com

p-FL

2-A

FL

2-A

Com

p-FL

2-A

FL

2-A


Con

trol g

roup

R gr

oup

R+

RSV

grou

p

103

104

105

106

107

104 105 106 107

Com

p-FL

2-A

FL

2-A

0

5

10

15

R + RSV group

lowastlowast

Apop

totic

cells

()

(e)



Mito

chon

dria

l mas

sM

orph

olog

yIn

set


(a)

020406080

100120

Con

trol g

roup

The m

itoch

ondr

ial

mas

s

R gr

oup

R+

RSV

grou

p

lowast

(b)

Frag

men

ted

cells

(cel

ls w

ithou

t el

onga

ted

mito

chon

dria

) (

)

020406080

100

Con

trol g

roup

R gr

oup

R+

RSV

grou

plowastlowast

lowast

(c)

Drp1

002040608

112

C group

Fis1

0010203040506

Prot

ein

expr

essio

n

Prot

ein

expr

essio

n

(Fis1

120573-a

ctin

)

OPA1

002040608

112 MFN2

002040608

112

MFN2

Drp1

Fis1

OPA1

C R

(Drp

1120573

-act

in)

lowastlowast





R + RSV

120573-actin

lowastlowast

lowastlowast

lowastlowast

(OPA

1120573

-act

in)

Prot

ein

expr

essio

n

(MFN

2120573

-act

in)

Prot

ein

expr

essio

n

(d)

Figure 3 Continued


MFN2

000501

01502

02503

035OPA1

000501

01502

025

Fis1

000501

01502

02503

03504

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)G

ene e

xpre

ssio

n (r

atio

of g

ene120573-a

ctin

)

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

Drp1

000200400600801

012




C R R + RSV

MFN2

Drp1

Fis1

OPA1

120573-actin

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(e)

OPA1

Drp1

MFN2

Fis1

Drp1

002040608

1

MFN2

002040608

1OPA1

002040608

112

Fis1

002040608

112

lowastlowast

lowastlowast

lowastlowast

lowastlowast

120573-actin

C R R + RSV

(Drp

1120573

-act

in)

Prot

ein

expr

essio

n(O

PA1120573

-act

in)

Prot

ein

expr

essio

n

(MFN

2120573

-act

in)

Prot

ein

expr

essio

n(F

is1 120573

-act

in)

Prot

ein

expr

essio

n



(f)

Drp1

0010203040506

Gen

e exp

ress

ion

(Drp

1120573

-act

in) Fis1

044046048

05052054056

Gen

e exp

ress

ion

(Fis1

120573-a

ctin

)

MFN2

002040608

1

Gen

e exp

ress

ion

(MFN

2120573-a

ctin

) OPA1

002040608

112

Gen

e exp

ress

ion

(OPA

1120573-a

ctin

)

lowastlowast

lowastlowast

lowastlowast

OPA1

Drp1

MFN2

Fis1

120573-actin

C R R + RSV

lowastlowast



(g)



mtTFA

mtTFA

075

08

085

09

0951

Prot

ein

expr

essio

n (m

TFA

120573-a

ctin

)

PGC-1120572

002040608

112

lowastlowast

lowastlowast


PGC-1120572

Prot

ein

expr

essio

n(P

GC-

1120572120573

-act

in)


(a)

mtTFAPGC-1120572

000200400600801

012014016

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

000501

01502

025G

ene e

xpre

ssio

n (r

atio

of g

ene 120573-a

ctin

)


C R R + RSV

mtTFA

120573-actin

PGC-1120572


(b)

mtTFAPGC-1120572

002040608

112

Prot

ein

expr

essio

n (P

GC-

1120572120573

-act

in)

002040608

112

Prot

ein

expr

essio

n(m

tTFA

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(c)

mtTFAPGC-1120572

00102030405060708

Gen

e exp

ress

ion

(mtT

FA 120573

-act

in)

001020304050607

Gen

e exp

ress

ion

(PG

C-1120572

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(d)

Cel

l via

bilit

y (c

ontro

l 100

)

0

50

100

150

Con

trol g

roup

R gr

oup

siPG

C-1120572

$lowastlowast

lowastlowast

R+

RSV

gro

up

siPG

C-1120572

R+

siPG

C-1120572

RRS

V+

+

(e)

002040608

112

Rela

tive M

tDN

A

copy

num

bers

lowastlowast

lowastlowast


(f)



0

20

40

60

80

100

120

140

160

180

200


Cell

RO

S flu

ores

cenc

e (co

ntro

l 100

)

lowastlowast

R + RSV group

(a)


ATP

conc

entr

atio

n (c

ontro

l 100

)

0

50

100

150

lowastlowast

R + RSV group

(b)




4 Discussion













Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

Con

trol g

roup

R gr

oup

02040608

112141618

2

Opt

ical

den

sity

(OD

)


Solv

ent g

roup

R+

RSV

(1)

R+

RSV

(5)

R+

RSV

(10)

R+

RSV

(50)

R+

RSV

(60)

R+

RSV

(100

)RS

V(1

00)

(a)

(b) (c) (d)

103

104

104

105

105

106

106

107

103

104

105

106

107

107 104 105 106 107

Com

p-FL

2-A

FL

2-A

Com

p-FL

2-A

FL

2-A


Con

trol g

roup

R gr

oup

R+

RSV

grou

p

103

104

105

106

107

104 105 106 107

Com

p-FL

2-A

FL

2-A

0

5

10

15

R + RSV group

lowastlowast

Apop

totic

cells

()

(e)



Mito

chon

dria

l mas

sM

orph

olog

yIn

set


(a)

020406080

100120

Con

trol g

roup

The m

itoch

ondr

ial

mas

s

R gr

oup

R+

RSV

grou

p

lowast

(b)

Frag

men

ted

cells

(cel

ls w

ithou

t el

onga

ted

mito

chon

dria

) (

)

020406080

100

Con

trol g

roup

R gr

oup

R+

RSV

grou

plowastlowast

lowast

(c)

Drp1

002040608

112

C group

Fis1

0010203040506

Prot

ein

expr

essio

n

Prot

ein

expr

essio

n

(Fis1

120573-a

ctin

)

OPA1

002040608

112 MFN2

002040608

112

MFN2

Drp1

Fis1

OPA1

C R

(Drp

1120573

-act

in)

lowastlowast





R + RSV

120573-actin

lowastlowast

lowastlowast

lowastlowast

(OPA

1120573

-act

in)

Prot

ein

expr

essio

n

(MFN

2120573

-act

in)

Prot

ein

expr

essio

n

(d)

Figure 3 Continued


MFN2

000501

01502

02503

035OPA1

000501

01502

025

Fis1

000501

01502

02503

03504

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)G

ene e

xpre

ssio

n (r

atio

of g

ene120573-a

ctin

)

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

Drp1

000200400600801

012




C R R + RSV

MFN2

Drp1

Fis1

OPA1

120573-actin

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(e)

OPA1

Drp1

MFN2

Fis1

Drp1

002040608

1

MFN2

002040608

1OPA1

002040608

112

Fis1

002040608

112

lowastlowast

lowastlowast

lowastlowast

lowastlowast

120573-actin

C R R + RSV

(Drp

1120573

-act

in)

Prot

ein

expr

essio

n(O

PA1120573

-act

in)

Prot

ein

expr

essio

n

(MFN

2120573

-act

in)

Prot

ein

expr

essio

n(F

is1 120573

-act

in)

Prot

ein

expr

essio

n



(f)

Drp1

0010203040506

Gen

e exp

ress

ion

(Drp

1120573

-act

in) Fis1

044046048

05052054056

Gen

e exp

ress

ion

(Fis1

120573-a

ctin

)

MFN2

002040608

1

Gen

e exp

ress

ion

(MFN

2120573-a

ctin

) OPA1

002040608

112

Gen

e exp

ress

ion

(OPA

1120573-a

ctin

)

lowastlowast

lowastlowast

lowastlowast

OPA1

Drp1

MFN2

Fis1

120573-actin

C R R + RSV

lowastlowast



(g)



mtTFA

mtTFA

075

08

085

09

0951

Prot

ein

expr

essio

n (m

TFA

120573-a

ctin

)

PGC-1120572

002040608

112

lowastlowast

lowastlowast


PGC-1120572

Prot

ein

expr

essio

n(P

GC-

1120572120573

-act

in)


(a)

mtTFAPGC-1120572

000200400600801

012014016

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

000501

01502

025G

ene e

xpre

ssio

n (r

atio

of g

ene 120573-a

ctin

)


C R R + RSV

mtTFA

120573-actin

PGC-1120572


(b)

mtTFAPGC-1120572

002040608

112

Prot

ein

expr

essio

n (P

GC-

1120572120573

-act

in)

002040608

112

Prot

ein

expr

essio

n(m

tTFA

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(c)

mtTFAPGC-1120572

00102030405060708

Gen

e exp

ress

ion

(mtT

FA 120573

-act

in)

001020304050607

Gen

e exp

ress

ion

(PG

C-1120572

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(d)

Cel

l via

bilit

y (c

ontro

l 100

)

0

50

100

150

Con

trol g

roup

R gr

oup

siPG

C-1120572

$lowastlowast

lowastlowast

R+

RSV

gro

up

siPG

C-1120572

R+

siPG

C-1120572

RRS

V+

+

(e)

002040608

112

Rela

tive M

tDN

A

copy

num

bers

lowastlowast

lowastlowast


(f)



0

20

40

60

80

100

120

140

160

180

200


Cell

RO

S flu

ores

cenc

e (co

ntro

l 100

)

lowastlowast

R + RSV group

(a)


ATP

conc

entr

atio

n (c

ontro

l 100

)

0

50

100

150

lowastlowast

R + RSV group

(b)




4 Discussion













Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Mito

chon

dria

l mas

sM

orph

olog

yIn

set


(a)

020406080

100120

Con

trol g

roup

The m

itoch

ondr

ial

mas

s

R gr

oup

R+

RSV

grou

p

lowast

(b)

Frag

men

ted

cells

(cel

ls w

ithou

t el

onga

ted

mito

chon

dria

) (

)

020406080

100

Con

trol g

roup

R gr

oup

R+

RSV

grou

plowastlowast

lowast

(c)

Drp1

002040608

112

C group

Fis1

0010203040506

Prot

ein

expr

essio

n

Prot

ein

expr

essio

n

(Fis1

120573-a

ctin

)

OPA1

002040608

112 MFN2

002040608

112

MFN2

Drp1

Fis1

OPA1

C R

(Drp

1120573

-act

in)

lowastlowast





R + RSV

120573-actin

lowastlowast

lowastlowast

lowastlowast

(OPA

1120573

-act

in)

Prot

ein

expr

essio

n

(MFN

2120573

-act

in)

Prot

ein

expr

essio

n

(d)

Figure 3 Continued


MFN2

000501

01502

02503

035OPA1

000501

01502

025

Fis1

000501

01502

02503

03504

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)G

ene e

xpre

ssio

n (r

atio

of g

ene120573-a

ctin

)

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

Drp1

000200400600801

012




C R R + RSV

MFN2

Drp1

Fis1

OPA1

120573-actin

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(e)

OPA1

Drp1

MFN2

Fis1

Drp1

002040608

1

MFN2

002040608

1OPA1

002040608

112

Fis1

002040608

112

lowastlowast

lowastlowast

lowastlowast

lowastlowast

120573-actin

C R R + RSV

(Drp

1120573

-act

in)

Prot

ein

expr

essio

n(O

PA1120573

-act

in)

Prot

ein

expr

essio

n

(MFN

2120573

-act

in)

Prot

ein

expr

essio

n(F

is1 120573

-act

in)

Prot

ein

expr

essio

n



(f)

Drp1

0010203040506

Gen

e exp

ress

ion

(Drp

1120573

-act

in) Fis1

044046048

05052054056

Gen

e exp

ress

ion

(Fis1

120573-a

ctin

)

MFN2

002040608

1

Gen

e exp

ress

ion

(MFN

2120573-a

ctin

) OPA1

002040608

112

Gen

e exp

ress

ion

(OPA

1120573-a

ctin

)

lowastlowast

lowastlowast

lowastlowast

OPA1

Drp1

MFN2

Fis1

120573-actin

C R R + RSV

lowastlowast



(g)



mtTFA

mtTFA

075

08

085

09

0951

Prot

ein

expr

essio

n (m

TFA

120573-a

ctin

)

PGC-1120572

002040608

112

lowastlowast

lowastlowast


PGC-1120572

Prot

ein

expr

essio

n(P

GC-

1120572120573

-act

in)


(a)

mtTFAPGC-1120572

000200400600801

012014016

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

000501

01502

025G

ene e

xpre

ssio

n (r

atio

of g

ene 120573-a

ctin

)


C R R + RSV

mtTFA

120573-actin

PGC-1120572


(b)

mtTFAPGC-1120572

002040608

112

Prot

ein

expr

essio

n (P

GC-

1120572120573

-act

in)

002040608

112

Prot

ein

expr

essio

n(m

tTFA

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(c)

mtTFAPGC-1120572

00102030405060708

Gen

e exp

ress

ion

(mtT

FA 120573

-act

in)

001020304050607

Gen

e exp

ress

ion

(PG

C-1120572

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(d)

Cel

l via

bilit

y (c

ontro

l 100

)

0

50

100

150

Con

trol g

roup

R gr

oup

siPG

C-1120572

$lowastlowast

lowastlowast

R+

RSV

gro

up

siPG

C-1120572

R+

siPG

C-1120572

RRS

V+

+

(e)

002040608

112

Rela

tive M

tDN

A

copy

num

bers

lowastlowast

lowastlowast


(f)



0

20

40

60

80

100

120

140

160

180

200


Cell

RO

S flu

ores

cenc

e (co

ntro

l 100

)

lowastlowast

R + RSV group

(a)


ATP

conc

entr

atio

n (c

ontro

l 100

)

0

50

100

150

lowastlowast

R + RSV group

(b)




4 Discussion













Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MFN2

000501

01502

02503

035OPA1

000501

01502

025

Fis1

000501

01502

02503

03504

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)G

ene e

xpre

ssio

n (r

atio

of g

ene120573-a

ctin

)

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

Drp1

000200400600801

012




C R R + RSV

MFN2

Drp1

Fis1

OPA1

120573-actin

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(e)

OPA1

Drp1

MFN2

Fis1

Drp1

002040608

1

MFN2

002040608

1OPA1

002040608

112

Fis1

002040608

112

lowastlowast

lowastlowast

lowastlowast

lowastlowast

120573-actin

C R R + RSV

(Drp

1120573

-act

in)

Prot

ein

expr

essio

n(O

PA1120573

-act

in)

Prot

ein

expr

essio

n

(MFN

2120573

-act

in)

Prot

ein

expr

essio

n(F

is1 120573

-act

in)

Prot

ein

expr

essio

n



(f)

Drp1

0010203040506

Gen

e exp

ress

ion

(Drp

1120573

-act

in) Fis1

044046048

05052054056

Gen

e exp

ress

ion

(Fis1

120573-a

ctin

)

MFN2

002040608

1

Gen

e exp

ress

ion

(MFN

2120573-a

ctin

) OPA1

002040608

112

Gen

e exp

ress

ion

(OPA

1120573-a

ctin

)

lowastlowast

lowastlowast

lowastlowast

OPA1

Drp1

MFN2

Fis1

120573-actin

C R R + RSV

lowastlowast



(g)



mtTFA

mtTFA

075

08

085

09

0951

Prot

ein

expr

essio

n (m

TFA

120573-a

ctin

)

PGC-1120572

002040608

112

lowastlowast

lowastlowast


PGC-1120572

Prot

ein

expr

essio

n(P

GC-

1120572120573

-act

in)


(a)

mtTFAPGC-1120572

000200400600801

012014016

Gen

e exp

ress

ion

(rat

io o

f gen

e120573-a

ctin

)

000501

01502

025G

ene e

xpre

ssio

n (r

atio

of g

ene 120573-a

ctin

)


C R R + RSV

mtTFA

120573-actin

PGC-1120572


(b)

mtTFAPGC-1120572

002040608

112

Prot

ein

expr

essio

n (P

GC-

1120572120573

-act

in)

002040608

112

Prot

ein

expr

essio

n(m

tTFA

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(c)

mtTFAPGC-1120572

00102030405060708

Gen

e exp

ress

ion

(mtT

FA 120573

-act

in)

001020304050607

Gen

e exp

ress

ion

(PG

C-1120572

120573-a

ctin

)

lowastlowast

lowastlowast

C R R + RSV

mtTFA

120573-actin

PGC-1120572


(d)

Cel

l via

bilit

y (c

ontro

l 100

)

0

50

100

150

Con

trol g

roup

R gr

oup

siPG

C-1120572

$lowastlowast

lowastlowast

R+

RSV

gro

up

siPG

C-1120572

R+

siPG

C-1120572

RRS

V+

+

(e)

002040608

112

Rela

tive M

tDN

A

copy

num

bers

lowastlowast

lowastlowast


(f)



0

20

40

60

80

100

120

140

160

180

200


Cell

RO

S flu

ores

cenc

e (co

ntro

l 100

)

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0

50

100

150

lowastlowast

R + RSV group

(b)




4 Discussion













Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















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20

40

60

80

100

120

140

160

180

200


Cell

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S flu

ores

cenc

e (co

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lowastlowast

R + RSV group

(a)


ATP

conc

entr

atio

n (c

ontro

l 100

)

0

50

100

150

lowastlowast

R + RSV group

(b)




4 Discussion













Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120

140

160

180

200


Cell

RO

S flu

ores

cenc

e (co

ntro

l 100

)

lowastlowast

R + RSV group

(a)


ATP

conc

entr

atio

n (c

ontro

l 100

)

0

50

100

150

lowastlowast

R + RSV group

(b)




4 Discussion













Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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