Research Article Chilean Strawberry Consumption …downloads.hindawi.com/journals/ecam/2015/320136.pdfResearch Article Chilean Strawberry Consumption Protects against ... Ripefruitsof

Research ArticleChilean Strawberry Consumption Protects againstLPS-Induced Liver Injury by Anti-Inflammatory andAntioxidant Capability in Sprague-Dawley Rats

Sebastian Molinett1 Francisca Nuntildeez2

Mariacutea Alejandra Moya-Leoacuten1 and Jessica Zuacutentildeiga-Hernaacutendez2

1 Instituto de Ciencias Biologicas Universidad de Talca 2 Norte No 685 Talca 3465548 Maule Chile2Departamento de Farmacologıa Escuela de Medicina Universidad de Talca 2 Norte No 685 Talca 3465548 Maule Chile

Correspondence should be addressed to Jessica Zuniga-Hernandez jezunigautalcacl

Received 30 June 2015 Revised 24 August 2015 Accepted 25 August 2015

Academic Editor Jae Youl Cho

Copyright copy 2015 Sebastian Molinett 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 Chilean strawberry fruit has high content of antioxidants and polyphenols Previous studies evidenced antioxidant propertiesby in vitro methods However the antioxidant effect and its impact as functional food on animal health have not been evaluatedIn this study rats were fed with a Chilean strawberry aqueous extract (4 gkg of animal per day) and then subjected to LPS-induced liver injury (5mgkg) Transaminases and histological studies revealed a reduction in liver injury in rats fedwith strawberryaqueous extract compared with the control group Additionally white strawberry supplementation significantly reduced the serumlevels and gene expression of TNF-120572 IL-6 and IL-1120573 cytokines compared with nonsupplemented rats The level of F2-isoprostanesand GSHGSSG indicated a reduction in liver oxidative stress by the consumption of strawberry aqueous extract Altogether theevidence suggests that dietary supplementation of rats with a Chilean white strawberry aqueous extract favours the normalizationof oxidative and inflammatory responses after a liver injury induced by LPS

1 Introduction

The native white Chilean strawberry (Fragaria chiloensisspp chiloensis) is a native species from Chile and thematernal progenitor of the commercial strawberry (Fragariax ananassa Duch) [1] The Mapuche people cultivated thisplant as a nutritive food consumed as fresh or dried fruit orprepared for medicinal purposes [2] During the last decadein Chile there has been an increasing interest in expandingthe culture and production of F chiloensis to gain a niche inthe local and global market as well as take advantage of aresurgent interest in new crops [3]

Berry fruits are known to have an antioxidant effect thatcould prevent different pathological conditions [4] Straw-berry fruits possess a remarkable nutritional compositiondue to their high content of micronutrients such as folatesminerals and vitamins and are also a rich source of phenolicconstituents [5] Particularly white Chilean strawberries have

been described as a good source of phenolic antioxidants[6] Strawberry extracts have higher antioxidant activitycompared with other common consumable fruits [7] Theantioxidant properties of strawberries are mainly due tophenolic compounds [8] and this capability has been provento be effective against superoxide and hydrogen peroxideradicals [9] In vitro studies have shown that white Chileanstrawberry fruit has high free radical scavenging activity[1 8] Despite the potential of this fruit as antioxidant there isa lack of information about the in vivo effects on the oxidativestatus in animal models Nevertheless previous studies havereported that the consumption of commercial strawberryfruit provides beneficial health effects The supplementationof diet with strawberry fruit as a source of antioxidantsreduced the level of blood lipids and the oxidative damage ofLDL in hyperlipidemic subjects [10] Furthermore a hypoc-holesterolemic effect and a decrease in lipid peroxidationwere reported [11] In another study antiplatelet function

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 320136 11 pageshttpdxdoiorg1011552015320136

2 Evidence-Based Complementary and Alternative Medicine

was evidenced by in vitro and in vivo approaches [12] Onthe other hand anti-inflammatory effect has been evidenced[13] in which strawberries are very effective inhibitingcyclooxygenase (ie COX2) a key enzyme in inflammationpathway and a target for different drugs In addition recentstudies suggest that strawberry polyphenols might have arole in cancer prevention and treatment [5] Consideringthat the available knowledge about beneficial health effectof commercial strawberry fruit and the converse scenarioof white Chilean strawberry fruit has a different phenolicprofile it becomes relevant to gain insight into its potentialproperties as functional food

The lipopolysaccharide (LPS) is a major glycolipid com-ponent of the outer cell wall of Gram-negative bacteria madeup from a polysaccharide O-chain and a biologically activelipid-A moiety embedded within the bacterial membrane[14] In rats LPS triggers the rise of cytokines and reactiveoxygen species (ROS) such as superoxide hydroxyl radicalsand peroxynitrite [15] The endotoxemia induced by LPS ischaracterized by the injury of various organs including liverkidney and brain [14] Most of the toxicities observed in LPS-induced liver injury (and systemic) have been attributed toinflammatorymediators produced by activatedmacrophagesincluding tumour necrosis factor-120572 (TNF-120572) and interleukinsIL-1120573 IL-6 IL-8 and IL-12 [16] These toxic mediators caninduce lipid peroxidation oxidative damage and depletion ofintracellular stored antioxidants [17] creating a prooxidativeand inflammatory status Therefore the present study wasdesigned to evaluate the effects of an aqueous solution ofstrawberry fruit orally administrated on the inflammatoryresponse and oxidative status in a LPS-injured liver Biochem-ical changes and histological parameters were analysed

2 Material and Methods

21 Animals Male Sprague-Dawley rats (200ndash250 g) wereobtained from the animal facility of University of TalcaThe animals were housed on 12 h light-dark cycles at 25∘CAnimals received standard animal rodent chow and waterad libitum Care of animals and the experimental protocolsof this study were approved by the Institutional AnimalUse Committee of the University of Talca and conductedin accordance with the recommendations of the EuropeanUnion regarding animal experimentation (Directive of theEuropean Council 86609EC)

22 F chiloensis Fruit Extract Ripe fruits of Fragaria chil-oensis ssp chiloensis (record number 5537 in plant listhttpwwwtheplantlistorg) were collected in December2013 from a commercial orchard at Manzanar Alto PurenIX Region Chile (38∘2101584030210158401015840S 73∘8101584038410158401015840W) After harvestthe fruit was immediately cold transported to the Institute ofBiological Sciences at University of Talca and after freezing itunder liquid nitrogen it was stored at minus80∘C until use Ripe Fchiloensis fruit was homogenized in distilled water (05 gmL)with the help of an Ultraturrax (5min at 4000 rpm) on an icebath The rats were fed twice a day with F chiloensis extractsat the daily dosages of 2 4 and 8 gkg of animal per day

(for the doses-response curve) All the administrations wereperformed with fresh aqueous extracts (Figure 1(a))

23 Animal Preparation and LPS-Induction of Liver InjuryGroups of rats were orally supplemented for 10 days withthe aqueous F chiloensis extract 2 gkg of animal twice a daythat is 4 gkgday of strawberry (Figure 1(b)) while controlrat groups received isovolumetric amounts of saline solution(09 NaCl) At day 10 the animals were challenged to aLPS sepsis induced by a single intraperitoneal rat injectionof 5mgkg LPS (lipopolysaccharide endotoxin Escherichiacoli serotype 0111B4 Sigma-Aldrich San Luis MO USA)from E coli 8 [18] dissolved in 09 NaCl These generatefour experimental groups (a) sal-sal (saline pretreatmentplus a saline LPS vehicle) (b) sal-LPS (saline pretreatmentplus LPS inoculum) (c) Fch-sal (F chiloensis extract duringpretreatment plus LPS vehicle) and (d) Fch-LPS (Fch pre-treatment plus LPS inoculum) Five to six animals composedeach experimental group

After 3 hours of LPS challenge the animals were anes-thetized with an intraperitoneal injection (1mLkg) of zolaz-epam chlorhydrate (25mgmL) and tiletamine chlorhydrate(25mgmL) (Zoletil 50 Virbac SA Carros France) Bloodsamples were obtained by cardiac puncture for serum analy-sis Liver samples were taken from themedial lobes frozen inliquid nitrogen and stored at minus80∘C for further analysis

24 Histological Analysis Liver samples of each animalwere fixed in phosphate-buffered formalin dehydrated inincreasing ethanol concentrations and embedded in paraffinThereafter sections of tissue were cut at 5120583m on a rotatorymicrotome (Leica Ultracut Solms Germany) mounted onclean glass slides and dried overnight at 37∘C Sections werecleared hydrated and stained with haematoxylin and eosinfor histomorphological assessments Blind histopathologicalevaluation was performed in at least ten randomly chosenmicroscopic fields of all histological slides (three to five slidesper each animal assayed) All tissue sections were exam-ined in a Nikon Eclipse 50i microscope (Tokyo Japan) forcharacterization of histopathological changes Photographswere digitalized using micrometrics SE premium 28 versioncamera and software for microscopy

25 Determination of Transaminases Cytokines F2-Isopros-tanes and Glutathione Assays Transaminases levels AST(aspartate transaminase) and ALT (alanine transaminase)were measured using a specific diagnostic kit (SpinReactGirona Spain) and expressed as international unitsL ELISAkits were used for assessment of serum levels (pgmL) ofTNF-120572 IL-6 IL-1120573 and IL-10 cytokines (Biosource Interna-tional Camarillo CA USA) and F2-isoprostanes (CaymanChemical Ann Arbor MI USA) GSH and GSSG contents(120583molg liver) were measured using a specific glutathioneassay kit (Cayman Chemical)

26 RNA Extraction and Quantitative Real-Time PCR TotalRNA was isolated from liver samples by the guanidiniumthiocyanate-phenol-chloroform extraction method (Trizol

Evidence-Based Complementary and Alternative Medicine 3

Point on curve Fch pretreatment

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Figure 1 Experimental protocol for Fch pretreatment (a) Curve doses-response animals were given 10 days Fch (2 4 and 8 gkgday) (b)Protocol of animal design groups after analysis of curve doses-response Animals were given 10 days Fch (4 gkgday) After the period ofFch administration group of control (light grey bars) and Fch-treated (dark grey bars) animals were subject to LPS-induced liver injury thusconforming the experimental groups Blood and liver samples were obtained at 3 hours of LPS challenger (or LPS-vehicle control)

Life Technologies Carlsbad CA USA) The extracted RNAwas treated with DNAse (Turbo DNA-free kit AmbionLife Technologies) to eliminate DNA contamination TheRNA quantity and purity were estimated at 260280 nmby NanoDrop 1000 Spectrophotometer (Thermo ScientificWaltham MA USA) and the cDNA was synthesized with afirst strand cDNA synthesis kit (Thermo Scientific WalthamMA USA) Quantitative PCR was performed in a StratageneMx3000P (Agilent Technologies Santa Clara CA USA)Each reaction consisted of 20 120583L containing 2120583L of (1 10diluted) cDNA 1 120583L of primer mix 10 120583M 10 120583L of SybrGreen PCR Master Mix 2X (Stratagene Agilent Technolo-gies) and nuclease free water to reach the final reactionvolume The cycling conditions were 1 cycle of denaturationat 95∘C for 5min followed by 40 two-segment cycles ofamplification (95∘C15 sec 60∘C45 sec) and a final meltingcycle (95∘C1min 55∘C30 sec and 95∘C30 sec) From eachexperimental group (5-6 animals) three liver samples wererandomly chosen from which RNA was independently iso-lated and cDNA prepared qPCR runs were performed usingthree technical replicates and the mean was used for furtheranalysis

The primers used for quantitative PCR analysis wereTNF-120572 forward GGTTCCGTCCCTCTCATACA reverseAGACACCGCCTGGAGTTCT IL-6 forward ACTGGT-CTGTTGTGGGTGGT reverse AGTTGCCTTCTTGGG-ACTGA IL-1120573 forward GCTGTGGCAGCTACCTATGCT-ACCTATGTCTTG reverse AGGTCGTCATCATCCCAC-GAG IL-10 forward GCTGTGGCAGCTACCTATGTC-TTG reverse AGGTCGTCATCATCCCACGAG GAPDHforward GGCCTCTCTCTTGCTCTCAGTA reverse TTC-TCAAGCTCATTTCCTGGTA The expression levels werenormalized by ΔΔCT method using GAPDH as referencegene and the relative expression was calibrated and assignedvalue one to sal-sal condition

27 Statistical Analysis All values correspond to means plusmnstandard error of themean (SEM) or standard deviation (SD)

The data were evaluated with GraphPad Prism 6 software (LaJolla CA USA) The statistical significance of differences foreach parameter among the groups was evaluated by Tukeyrsquostest for unpaired data or one-way ANOVA A 119901 value of lessthan 005 was considered significant

3 Results

31 Protective Effect of F chiloensis in Acute LPS-Induced LiverInjury It has been reported that a LPS challenge inducesa liver injury [19 20] A significant increase in serum ASTand ALT transaminases was observed as expected in ourexperimental conditions (Figure 2) The effect of severalFch feeding dosages (05 1 and 2 g per day of Fch extract)was tested for their capability to decrease AST and ALTexacerbation after 3 h of LPS challenge (Figure 2(a)) Astrong reduction in transaminases was found at a dosageof 4 gkgday (2 doses of 2 gkg of animal Fch) or higherdosages A dosage of 4 gkgday of fruit extract was employedin further experiments

The supplementation of F chiloensis extract on diet priorto LPS challenge avoids the exacerbation in serum AST(Figure 2(b)) and ALT (Figure 2(c)) activities and valuescomparable to those in nonchallenged rats were determinedControl rats subjected to LPS single injection exhibited a 78-and 43-fold increase (119901 lt 005) in serum AST and ALTactivities respectively in comparison to sal-sal treatmentand importantly this increment in activity was suppressedby Fch supplementation In agreement with these dataliver histological assessments showed normal liver morphol-ogy in sal-sal and Fch-sal groups (Figures 3(a) and 3(c)resp) Changes in liver architecture (disruption) in extensiveareas hepatocytes necrosis and inflammatory infiltrates wereobserved in LPS challenged animals (Figure 3(b)) On thecontrary the livers of Fch-LPS group displayed normal archi-tecture with minimal or absence of necrosis (Figure 3(d))This indicates thatmorphological changes induced by LPS areprevented by Fch supplementation


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Figure 2 Effect of F chiloensis supplementation on serum transaminase levels after LPS challenge (a) Changes in response to different Fchiloensis fruit dosage (Fch) Values correspond to mean plusmn SEM of at least 4ndash6 animals per experimental group Rats were fed daily with theindicated amount of F chiloensis fruit extract (Fch) for 10 days and then subjected to LPS challenge which consisted in one intraperitonealinjection of LPS (5mgkg) ((b) and (c)) Changes in serumAST and ALT in response to LPS challenge Each bar corresponds to mean plusmn SEMof 5 to 6 different animals per experimental group Significant differences between the groups are indicated by asterisk (119901 lt 005 one-wayANOVA and Tukeyrsquos multiple comparison test)

32 Fragaria chiloensis Reduces the Inflammatory DamageInduced by LPS Cytokines pro- and anti-inflammatory weredetermined by ELISA in serum samples obtained from ratssubjected to LPS challenge In addition gene expressionanalyses of the same cytokines were performed in liversamples by RT-qPCR

The levels of serum TNF-120572 (Figure 4(a)) IL-6 (Fig-ure 4(b)) and IL-1120573 (Figure 4(c)) increased in response toLPS challenge by 23- 18- and 37-fold compared to sal-sal treatment (119901 lt 005) In animals supplemented with Fchiloensis fruit extract the increment of cytokines pro- andanti-inflammatory in response to LPS challenge is preventedTherewere nonsignificant changes in IL-10 in response to LPStreatment (Figure 4(d))

On the other hand the levels of TNF-120572 IL-6 and IL-1120573 mRNA were also increased in LPS treated animals andnonsupplemented (48- 3189- and 144-fold resp 119901 lt 005)compared to nontreated (sal-sal) (Figures 5(a) 5(b) and5(c)) Furthermore the increment in TNF-120572 IL-6 and IL-1120573 transcripts in response to LPS was prevented in animalssupplemented with F chiloensis aqueous fruit extract valueswere significantly decreased by 339- 149- and 18-foldrespectively in the Fch-LPS group compared to sal-LPS

group No changes in mRNA levels of IL-10 were observedamong the different experimental groups (Figure 5(d))

33 Liver Oxidative Stress Related Parameters Induced byLPS Are Diminished by F chiloensis Supplementation Thelevels of plasma 8-isoprostanes increased in response to LPSchallenge (87-fold) compared to sal-sal treatment (Figure 6)In animals supplemented with F chiloensis aqueous extractthe increment in 8-isoprostanes in response to LPS challengeis avoided

The content of hepatic reduced glutathione (GSH) dimin-ished (23) in response to LPS challenge compared tocontrol (sal-sal) treatment (Figure 7(a)) The GSH contentin animals supplemented with F chiloensis aqueous extractwas similar to controls without LPS inoculum howevera significant enhancement in GSH is observed after LPSchallenge (Figure 7(a) inset)

On the other hand liver glutathione disulphide (GSSG)levels increased (236) in response to LPS challenge com-pared to sal-sal treatment (Figure 7(b)) However in responseto LPS the animals pretreated with F chiloensis achieved a netreduction in GSSG levels compared to nonsupplemented ratssubjected to LPS challenge (Figure 7(b) inset)


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Figure 3 Effects of F chiloensis supplementation in liver morphology after LPS challenge Representative liver sections stained withhaematoxylin-eosin from different experimental conditions sal-sal (saline pretreatment plus a saline LPS vehicle) sal-LPS (salinepretreatment plus LPS inoculum) Fch (Fch pretreatment plus LPS vehicle) and Fch-LPS (Fch pretreatment plus LPS inoculum) A totalof 5-6 different animals per experimental group were evaluated original magnification times100 scale bar is 100120583m

4 Discussion

The focus of this study was to investigate the acute effects ofLPS-induced hepatic damage on the inflammatory responseand oxidative stress status and the possible protection offeredby the oral administration of an aqueous extract of thenative Chilean white strawberry Typically a portion ofthe released endotoxin (LPS) is absorbed into the portalcirculation and delivered to the liver where it is quicklycleared by intrahepatic Kupffer cells [21] thus the liver playsa central role in the regulation of entry and metabolism ofLPS inoculumThe model of LPS has been previously shownto result in a reproducible acute inflammatory response withincrements in cytokines and oxidative stress mitochondrialdysfunction and early biochemical changes associated withorgan dysfunction similar to the changes observed in human[22] Mostly the hepatic and systemic sepsis (toxicities)induced by LPS have been attributed to an overproductionof reactive oxygen species (ROS) and the release of chemicalmediators such as peroxide nitric oxide and proinflamma-tory cytokines (massive release of TNF-120572 IL-1120573 and IL-6)which are all formed as a result of the binding of LPS toToll-like receptor 4 (TLR-4) on the surface of Kupffer cells[16 23] As a response hepatocytes strive to clear LPS whichis followed by inflammation hepatocellular apoptosis andeven necrosis [24]

Oxidative stress is a well-known mechanism of LPS-induced hepatic injury and produces a redox imbalancewhich may result in depletion of endogenous antioxidantsuch as the antioxidant enzymes alteration of GSH redoxstatus [14] and generation of 8-isoprostanes free radical-catalysed products from lipid peroxidation (arachidonic acidderivatives) [25] The antioxidant defence system and itsequilibrium become necessary especially during infection oragainst an oxidative insult There are reports in which plantextracts infusions or isolated compounds have been usedto reverse or prevent the hepatotoxicity and the oxidativestress generated by LPS with beneficial effects associatedwith antioxidant and anti-inflammatory properties Rooibosaqueous [14] Eucalyptus globulus leaf extract [26] polyphe-nol extract of Hibiscus sabdariffa [27 28] and fermentedBarley extract [29] In this study we reported that a singleinjection of LPS resulted in hepatic injury as indicated bythe elevation in the levels of serum ALT and AST describedas circulating markers of hepatocyte injury The hepaticmarker enzymes are cytoplasmic in physiological conditionbut are usually leaked into circulation when liver damageoccurs due to an alteration in membrane integrity [14 30]Results from the current study showed that supplementationof diet with the aqueous extract of native Chilean whitestrawberry for 10 days prior to the LPS challenge at a doseof 4 gkgday diminished the induced damage in the liver


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Figure 4 Effects of F chiloensis on serum cytokine levels in response to LPS challenge TNF-120572 (a) IL-6 (b) IL-1120573 (c) and IL-10 (d) levels Eachbar corresponds to mean plusmn SEM of 5-6 different animals per experimental group Significant differences between the groups are indicated byasterisk 119901 lt 005 one-way ANOVA and Tukeyrsquos multiple comparison test

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Figure 5 Effect of Fch supplementation on cytokine gene expression in liver tissue TNF-120572 (a) IL-6 (b) IL-1120573 (c) and IL-10 (d) mRNAs weredetermined by RT-qPCR and normalized to GAPDH Each bar corresponds to mean plusmn SEM of 5-6 different animals per experimental groupSignificant differences between the groups are indicated by asterisk 119901 lt 005 one-way ANOVA and Tukeyrsquos multiple comparison test


Table 1 Total content of anthocyanins flavonols ellagic acid (mg100 g fresh weight) and scavenging activity in strawberry fruits

Fragaria chiloensis ssp chiloensis F x ananassa cv ChandlerTotal anthocyaninslowast 22 279Total flavonolslowast 178 176Total ellagic acidlowast 15729 3745 Scavenging activity (DPPH)lowastlowast 793 642lowastModified from Simirgiotis et al 2009 [1]lowastlowastModified from Simirgiotis and Schmeda-Hirschmann 2010 [6] and Cheel et al 2007 [8]

100

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Plas

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s (pg

mL)

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Fch + LPS

Figure 6 Effect of Fch supplementation on the plasma levels of8-isoprostanes after hepatic LPS challenge Values correspond tomeans plusmn SEM of 5-6 different animals per experimental groupSignificant differences between the groups are indicated by asterisk119901 lt 005 ANOVA and the Tukey multiple comparison test

Several studies have suggested that the phytochemical con-tent and antioxidantfree radical scavenging effect of fruitsand vegetables contribute to their protective effect againstchronic and degenerative diseases [31] The contribution ofvitamin C to the total antioxidant activity of twelve differentfruits analysed was estimated as being lt15 [7] Strawberryextracts were found to have higher antioxidant activitythan extracts from other studied fruits However vitaminC is not the only one that contributes to the antioxidantactivity of fruits and vegetables The antioxidant propertiesof strawberries have been shown to be mainly due to highcontent of phenolic compounds more than to vitamin C [31ndash33] Wang and Jiao [9] evidenced that strawberry aqueousextract exhibited a high level of antioxidant capacity againstfree radical species including superoxide radicals hydrogenperoxide hydroxyl radicals and singlet oxygen also it isimportant to mention that actually there does not exist anystudy that links the hepatoprotection with the antioxidantcapability of strawberry

The polyphenolic composition specifically flavonoid andphenolic compounds of different strawberries cultivars dif-fers amongst them [32 34 35] In addition the content ofphenolic compounds differs significantly amongst strawberryspecies and subspecies and determines the free radical scav-enging activity of these fruits [8] The main phenolic com-pounds in the Chilean white strawberry are ellagic acid andhexahydroxydiphenolic acid-based hydrolysable tannins andprocyanidins as well as flavonol glycosides from quercetinand kaempferol [6] (Table 1) The protective effect of F

chiloensis extract observed in our study may be due to theability of its phenolic content to stabilize and maintain theintegrity of the hepatocyte as well as repair damage tissueby stimulating hepatocyte regeneration and modulation ofKupffer cell activation

Kupffer cells have well-defined roles in the coordinationof hepatic inflammation in a number of diseases [36 37]During endotoxemia LPS is detected by Toll-like receptor 4(TLR4) on sentinel cell (endothelium Kupffer cells etc) thatinitiates the subsequent inflammatory response includingneutrophil recruitment to the liver [37] LPS binding toKupffer cells initiates a cascade of events that upregulatesexpression of the inflammatory cytokines including TNF-120572 stimulating the production of ROS and reactive nitrogenintermediates by activatedmacrophages causing liver damagedue to the oxidative stress Additionally LPS induces migra-tion of activated polymorphonuclear leukocytes (PMNs)extending and keeping the liver injury [37] In the presentstudy LPS challenge significantly increased levels of TNF-120572IL-1120573 and IL-6 and the oral administration of F chiloensisprior to LPS challenge was able to reduce the incrementin serum cytokines and the expression level in the liverof related genes at a comparable level compared to that innonchallenged animals Interestingly these results are con-sistent with the data reported for green tea polyphenols [38]quercetin [39] and resveratrol [40]These studies have shownan anti-inflammatory effect associated with the control ofsignal transduction with the reduction in the expression ofproinflammatory proteins (ie COX-2) inducible tumournecrosis factor-120572 (TNF-120572) and nitric oxide synthase

On the other hand we did not find any difference inthe levels of IL-10 cytokines although IL-10 is produced bymacrophages as a negative-feedback mechanism to dampenuncontrolled production of inflammatory cytokines andexcessive inflammation during infection IL-10 is a potentanti-inflammatory cytokine with a broad effect on bothinnate and adaptive immune system [41] IL-10 could down-regulate TNF-120572 aswell as other cytokines by suppressing theirgene expression in an autocrine-like feedback loop [42] Asour observations showed that the levels of IL-10 were similarbetween control and treated groups these could indicate thatthe inhibition of the other cytokines observed by F chiloensissupplementation may be independent of the activation ofthe feedback mediated by IL-10 Thus we speculate thatthe protective effect detected is due to the antioxidantcapability of F chiloensis extract and another molecularmechanism associated with LPS signalling pathwayThis idea


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Figure 7 Effects on serum glutathione levels in response to LPS challenge (a) Reduced (GSH) and (b) oxidized glutathione (GSSG) levelsValues represent the means plusmn SEM of 5-6 different animals per experimental group Significant differences between the groups are indicatedby asterisk (119901 lt 005 ANOVA and Tukeyrsquos multiple comparison test) Inset net effects of the LPS-induced liver injury in the contentof glutathione with or without pretreatment with Fch (B)-(A) correspond to the difference between serum levels in LPS and the controlcondition and (D)-(C) correspond to the difference between Fch-sal and Fch-LPS group of rats

is reinforced by the observed GSHGSSG normalization andanti-inflammatory effect

The impairment of the antioxidants defence system is acritical step in LPS-induced injury Evidence has shown thatLPS insult is characterized by change in tissue and circulatingantioxidant enzymersquos level and antioxidant molecules likeGSH [14] We found a modulation of the antioxidant systemin the LPS-challenged rats consuming F chiloensis with thenormalization of GSHGSSG ratio and a depletion of 8-isoprostane levels Reduced glutathione (GSH) is the majornonprotein thiol in plant and animal cells GSH is recognizedas a highly effective antioxidant compound as its scavengingand antioxidant properties allow the neutralization of ROSspecies It is essential for the regulation of a variety of cellularfunctions [14] and a consistent and sufficient GSH level canprevent LPS-induced damage [43]The ability to improve theGSHGSSG ratio shown by F chiloensis can be ascribed tothe capability of the different fruit antioxidant componentsto quench free radicals to upregulate the synthesis of GSH

and to restore the antioxidant status results comparablewith the total in vitro scavenging activity determined pre-viously (Table 1) The glutathione regulation observed isconsistent with the low levels of 8-isoprostanes observed inLPS challenged rats supplemented with F chiloensis extractIsoprostanes comprise a group of free radical-catalysed per-oxidation products of arachidonic acid in a COX (cyclooxy-genase) independent mechanism They are seen as oxidativestress markers and potentially mediate some of the adverseeffects of oxidant injury [25 44] ROS have been reported todrive inflammatory cytokines responses by amechanism thatcomprises nuclear factor kappa B transcription factor (NF120581B)that regulates several cytokines including IL-1120573 and IL-6 [45]Some antioxidants such as MitoQ MitoE and melatonin canreduce IL-1120573 and IL-6 levels and decrease NF120581B activationeffect observed in a rat model with acute LPS sepsis [22]A recent study suggested that total flavonoids extractedfrom flowers of Abelmoschus manihot (L) Medic (TFA)protectedmice against carbon tetrachloride- (CCl

4-) induced


liver injury through antioxidant stress and anti-inflammatoryeffects which decreased the MDA level and elevated thecontent of GSH in the liver as compared to those in theCCl4group Meanwhile the inflammatory mediators (eg

TNF-alpha IL-1120573 and NO) were inhibited by TFA treatmentboth at the serum and mRNA levels [46] On the other sidequercetin alleviates inflammation after short-term treatmentin high-fat-fed (HFD) mice Increased nuclear import ofNF120581B and elevated expressions of proinflammatory markerswere further manifestations in the HFD group All thesechanges were reversed in the quercetin-treated groups withsignificant improvement of antioxidant activity compared tothe HFD group [47] These data are consistent with the factthat NF120581B could be the transcription factor that regulatesthe pathways of hepatoprotection observed in F chiloensissupplementation The Pi3KAKTNF120581B signalling pathwayis involved in LPS-induced injury moreover the NF120581Bsignalling pathway is known to play a critical role in sepsis-associated organ failure [18 48] It has been reported thatthe flavonoid apigenin inhibits LPS-induced inflammationthrough inhibition of NF120581B activation by hypophosphoryla-tion of ser536 in the p65 subunit in in vivomodel [48] Thusfurther investigation may be warranted to design the relationbetweenNF120581B and the liver protection observed in this study

Alternatively the potential blocking of lipopolysaccha-ride binding protein (LBP)mechanismmight be related to theanti-inflammatory observed results where LBP is an acutephase protein which plays an important role in lipopolysac-charide (LPS) signalling and innate immunity and theirlevel in acute liver injury and liver failure may significantlyaffect the physiological derangements that are observed inacute liver failure liver transplantation common bile ductligation alcoholic liver injury and hemorrhagic shock [4950] However there have been limited studies of the role ofLBP in animal and humanmodels of severe acute liver injury[49] It is generally accepted that low levels of LBP augmentthe cellrsquos response to LPS whereas high levels of LBP havebeen shown to inhibit cell responses to LPS Cells with LBPknockout became refractory to proinflammatory cytokinesand other inflammatory stimuli (LPS and palmitate) Thiseffect was mediated through decreased NF120581B signalling andreversed by exogenous LBP [51] Also the administration ofLBPK95A (LBP inhibitory peptide) attenuates liver injuryafter acetaminophen-induced hepatotoxicity administrationas evidenced by reductions in serum transaminase levelsand decreased centrilobular necrosis with poor Kupffer cellfunction less liver injury was noted in the absence of LBP[50]

5 Conclusions

Our study demonstrates for the first time the hepato-protective activity of a native Chilean strawberry aque-ous extract which maintained hepatocellular membranestructural integrity attenuated hepatic oxidative stress andinhibited inflammatory response in LPS-induced liver injuryThese effects were achieved by normalization of liverparameters redox status (GSHGSSG ratio and decrease

of isoprostanes) and downregulation of cytokines (TNF-a IL-1120573 and IL-6) The results of this study support thedietary supplementation with Chilean strawberry as a novelnoninvasive strategy to protect the liver and other organsagainst LPS injury Further experiments are being conductedby our research group

Conflict of Interests

None of the authors had a conflict of interests

Authorsrsquo Contribution

Jessica Zuniga-Hernandez and Sebastian Molinett designedthis study for which Marıa Alejandra Moya-Leon and Jes-sica Zuniga-Hernandez secured funding Jessica Zuniga-Hernandez Francisca Nunez and Sebastian Molinett per-formed experiments and Jessica Zuniga-Hernandez andSebastian Molinett performed analysis of data JessicaZuniga-Hernandez and Sebastian Molinett wrote the paperwhich was critically revised by Jessica Zuniga-HernandezSebastian Molinett and Marıa Alejandra Moya-Leon Allauthors read and approved the final paper

Acknowledgments

The authors wish to thank the School of Medicine Universityof Talca Chile This study was supported by the InternalProject I002974 (Direccion de Investigacion Universidad deTalca) and by the Anillo ACT 1110 project (Conicyt Chile)

References

[1] M J Simirgiotis C Theoduloz P D S Caligari and GSchmeda-Hirschmann ldquoComparison of phenolic compositionand antioxidant properties of two native Chilean and onedomestic strawberry genotypesrdquo Food Chemistry vol 113 no2 pp 377ndash385 2009

[2] C E Finn J B Retamales G A Lobos and J F HancockldquoThe chilean strawberry (Fragaria chiloensis) over 1000 yearsof domesticationrdquoHortScience vol 48 no 4 pp 418ndash421 2013

[3] J B Retamales P D S Caligari B Carrasco and G SaudldquoCurrent status of the Chilean native strawberry and theresearch needs to convert the species into a commercial croprdquoHortScience vol 40 no 6 pp 1633ndash1634 2005

[4] A Zepeda L G Aguayo J Fuentealba et al ldquoBlueberry extractsprotect testis from hypobaric hypoxia induced oxidative stressin ratsrdquo Oxidative Medicine and Cellular Longevity vol 2012Article ID 975870 7 pages 2012

[5] F Giampieri J M Alvarez-Suarez andM Battino ldquoStrawberryand human health effects beyond antioxidant activityrdquo Journalof Agricultural and Food Chemistry vol 62 no 18 pp 3867ndash3876 2014

[6] M J Simirgiotis andG Schmeda-Hirschmann ldquoDeterminationof phenolic composition and antioxidant activity in fruitsrhizomes and leaves of the white strawberry (Fragaria chiloensisspp chiloensis form chiloensis) using HPLC-DAD-ESI-MS andfree radical quenching techniquesrdquo Journal of Food Compositionand Analysis vol 23 no 6 pp 545ndash553 2010


[7] HWang G H Cao and R L Prior ldquoTotal antioxidant capacityof fruitsrdquo Journal of Agricultural and FoodChemistry vol 44 no3 pp 701ndash705 1996

[8] J Cheel C Theoduloz J A Rodrıguez P D S Caligariand G Schmeda-Hirschmann ldquoFree radical scavenging activityand phenolic content in achenes and thalamus from Fragariachiloensis ssp chiloensis F vesca and F x ananassa cv ChandlerrdquoFood Chemistry vol 102 no 1 pp 36ndash44 2007

[9] S Y Wang and H Jiao ldquoScavenging capacity of berry crops onsuperoxide radicals hydrogen peroxide hydroxyl radicals andsinglet oxygenrdquo Journal of Agricultural and Food Chemistry vol48 no 11 pp 5677ndash5684 2000

[10] D J A Jenkins T H Nguyen CW C Kendall et al ldquoThe effectof strawberries in a cholesterol-lowering dietary portfoliordquoMetabolism Clinical and Experimental vol 57 no 12 pp 1636ndash1644 2008

[11] A BasuMWilkinson K Penugonda B Simmons NM Bettsand T J Lyons ldquoFreeze-dried strawberry powder improveslipid profile and lipid peroxidation in women with metabolicsyndrome baseline and post intervention effectsrdquo NutritionJournal vol 8 article 43 2009

[12] A Naemura T Mitani Y Ijiri et al ldquoAnti-thrombotic effect ofstrawberriesrdquo Blood Coagulation and Fibrinolysis vol 16 no 7pp 501ndash509 2005

[13] N P Seeram R A Momin M G Nair and L D BourquinldquoCyclooxygenase inhibitory and antioxidant cyanidin glyco-sides in cherries and berriesrdquo Phytomedicine vol 8 no 5 pp362ndash369 2001

[14] O R Ajuwon O Oguntibeju and J L Marnewick ldquoAmelio-ration of lipopolysaccharide-induced liver injury by aqueousrooibos (Aspalathus linearis) extract via inhibition of pro-inflammatory cytokines and oxidative stressrdquo BMC Comple-mentaryampAlternativeMedicine vol 14 no 1 pp 392ndash403 2014

[15] O Bayraktar N Tekin O Aydın F Akyuz A Musmul and DBurukoglu ldquoEffects of S-allyl cysteine on lung and liver tissuein a rat model of lipopolysaccharide-induced sepsisrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 388 no 3 pp327ndash335 2014

[16] S Sun H Zhang B Xue et al ldquoProtective effect of glutathioneagainst lipopolysaccharide-induced inflammation and mortal-ity in ratsrdquo Inflammation Research vol 55 no 11 pp 504ndash5102006

[17] H Sebai M Ben-Attia M Sani E Aouani and N Ghanem-Boughanmi ldquoProtective effect of resveratrol on acuteendotoxemia-induced nephrotoxicity in rat through nitricoxide independent mechanismrdquo Free Radical Research vol 42no 11-12 pp 913ndash920 2008

[18] X-J Li G-X Zhang N Sun Y Sun L-Z Yang and Y-J DuldquoProtective effects of erythropoietin on endotoxin-related organinjury in ratsrdquo Journal of Huazhong University of Science andTechnology [Medical Sciences] vol 33 no 5 pp 680ndash686 2013

[19] M D Wheeler ldquoEndotoxin and kupffer cell activation inalcoholic liver diseaserdquo Alcohol Research amp Health vol 27 no4 pp 300ndash306 2003

[20] X-J Li G-X Zhang N Sun Y Sun L-Z Yang and Y-J DuldquoProtective effects of erythropoietin on endotoxin-related organinjury in ratsrdquo Journal of Huazhong University of Science andTechnology vol 33 no 5 pp 680ndash686 2013

[21] H-Y Zhang D-W Han Z-F Zhao et al ldquoMultiple pathogenicfactor-induced complications of cirrhosis in rats a new modelof hepatopulmonary syndrome with intestinal endotoxemiardquo

World Journal of Gastroenterology vol 13 no 25 pp 3500ndash35072007

[22] D A Lowes N R Webster M P Murphy and H F GalleyldquoAntioxidants that protect mitocondria reduce interleukin-6and antioxidative stress improve mitocondrial function andreduce biochemicalmarkers of organ dysfunction in a ratmodelof acute sepsisrdquo British Journal of Anaesthesia vol 110 no 3 pp472ndash480 2013

[23] Y Ohsaki H Shirakawa K Hiwatashi Y Furukawa T Mizu-tani andM Komai ldquoVitamin K suppresses lipopolysaccharide-induced inflammation in the ratrdquo Bioscience Biotechnology andBiochemistry vol 70 no 4 pp 926ndash932 2006

[24] Y Wang L-N Gao Y-L Cui and H-L Jiang ldquoProtectiveeffect of danhong injection on acute hepatic failure inducedby lipopolysaccharide and D-galactosamine in micerdquo Evidence-Based Complementary and Alternative Medicine vol 2014Article ID 153902 8 pages 2014

[25] T Klein K Neuhaus F Reutter and RM Nusing ldquoGenerationof 8-epi-prostaglandin F2120572 in isolated rat kidney glomeruli bya radical-independent mechanismrdquo British Journal of Pharma-cology vol 133 no 5 pp 643ndash650 2001

[26] K Sugimoto S Sakamoto K Nakagawa et al ldquoSuppressionof inducible nitric oxide synthase expression and ameliorationof lipopolysaccharide-induced liver injury by polyphenoliccompounds inEucalyptus globulus leaf extractrdquo FoodChemistryvol 125 no 2 pp 442ndash446 2011

[27] S Bharrhan K Chopra and P Rishi ldquoVitamin E supplementa-tionmodulates endotoxin-induced liver damage in a ratmodelrdquoAmerican Journal of Biomedical Sciences vol 2 pp 51ndash62 2010

[28] E-S Kao J-D Hsu C-J Wang S-H Yang S-Y Cheng andH-J Lee ldquoPolyphenols extracted from Hibiscus sabdariffa Linhibited lipopolysaccharide-induced inflammation by improv-ing antioxidative conditions and regulating cyclooxygenase-2expressionrdquo Bioscience Biotechnology and Biochemistry vol 73no 2 pp 385ndash390 2009

[29] P E Giriwono H Shirakawa H Hokazono T Goto and MKomai ldquoFermented barley extract supplementationmaintainedantioxidative defense suppressing lipopolysaccharide-inducedinflammatory liver injury in ratsrdquo Bioscience Biotechnology andBiochemistry vol 75 no 10 pp 1971ndash1976 2011

[30] J Zuniga F Venegas M Villarreal et al ldquoProtection againstin vivo liver ischemia-reperfusion injury by n-3 long-chainpolyunsaturated fatty acids in the ratrdquoFree Radical Research vol44 no 8 pp 854ndash863 2010

[31] I M Heinonen A S Meyer and E N Frankel ldquoAntioxidantactivity of berry phenolics on human low-density lipoproteinand liposome oxidationrdquo Journal of Agricultural and FoodChemistry vol 46 no 10 pp 4107ndash4112 1998

[32] K J Meyers C B Watkins M P Pritts and R H LiuldquoAntioxidant and antiproliferative activities of strawberriesrdquoJournal of Agricultural and Food Chemistry vol 51 no 23 pp6887ndash6892 2003

[33] J A Vinson X Su L Zubik and P Bose ldquoPhenol antioxidantquantity and quality in foods fruitsrdquo Journal of Agricultural andFood Chemistry vol 49 no 11 pp 5315ndash5321 2001

[34] S YWangW Zheng andG J Galletta ldquoCultural system affectsfruit quality and antioxidant capacity in strawberriesrdquo Journal ofAgricultural and Food Chemistry vol 50 no 22 pp 6534ndash65422002

[35] S H Hakkinen and A R Torronen ldquoContent of flavonolsand selected phenolic acids in strawberries and Vaccinium


species influence of cultivar cultivation site and techniquerdquoFood Research International vol 33 no 6 pp 517ndash524 2000

[36] T Traeger M Mikulcak C Eipel et al ldquoKupffer cell depletionreduces hepatic inflammation and apoptosis but decreases sur-vival in abdominal sepsisrdquoEuropean Journal of Gastroenterologyand Hepatology vol 22 no 9 pp 1039ndash1049 2010

[37] B McDonald C N Jenne L Zhuo K Kimata and P KubesldquoKupffer cells and activation of endothelial TLR

4

coordinateneutrophil adhesion within liver sinusoids during endotox-emiardquoAmerican Journal of Physiology Gastrointestinal and LiverPhysiology vol 305 no 11 pp G797ndashG806 2013

[38] L E Rhodes G Darby K A Massey et al ldquoOral green teacatechin metabolites are incorporated into human skin andprotect against UV radiation-induced cutaneous inflammationin association with reduced production of pro-inflammatoryeicosanoid 12-hydroxyeicosatetraenoic acidrdquo British Journal ofNutrition vol 110 no 5 pp 891ndash900 2013

[39] S Pastore D Lulli P Fidanza et al ldquoPlant polyphenols regulatechemokine expression and tissue repair in human keratinocytesthrough interaction with cytoplasmic and nuclear componentsof epidermal growth factor receptor systemrdquo Antioxidants ampRedox Signaling vol 16 no 4 pp 314ndash328 2012

[40] S Pastore D Lulli A I Potapovich et al ldquoDifferential mod-ulation of stress-inflammation responses by plant polyphenolsin cultured normal human keratinocytes and immortalizedHaCaT cellsrdquo Journal of Dermatological Science vol 63 no 2pp 104ndash114 2011

[41] S S Iyer A A Ghaffari and G Cheng ldquoLipopolysaccharide-mediated IL-10 transcriptional regulation requires sequentialinduction of type I IFNs and IL-27 in macrophagesrdquo Journal ofImmunology vol 185 no 11 pp 6599ndash6607 2010

[42] U Jaffer R GWade and T Gourlay ldquoCytokines in the systemicinflammatory response syndrome a reviewrdquoHSR Proceedings inIntensive Care and Cardiovascular anesthesia vol 2 pp 161ndash1752010

[43] Y Zhao P Zhou B Liu et al ldquoProtective effect of suberoy-lanilide hydroxamic acid against lipopolysaccharide-inducedliver damage in rodentsrdquo Journal of Surgical Research vol 194no 2 pp 544ndash550 2015

[44] E Moita A Gil-Izquierdo C Sousa et al ldquoIntegrated analysisof COX-2 and iNOS derived inflammatory mediators in LPS-stimulated RAW macrophages pre-exposed to Echium plan-tagineum L bee pollen extractrdquo PLoS ONE vol 8 no 3 ArticleID e59131 2013

[45] E Naik and V M Dixit ldquoMitochondrial reactive oxygenspecies drive proinflammatory cytokine productionrdquo Journal ofExperimental Medicine vol 208 no 3 pp 417ndash420 2011

[46] G Ai Q Liu W Hua Z Huang and D Wang ldquoHepatopro-tective evaluation of the total flavonoids extracted from flowersofAbelmoschusmanihot (L)Medic In vitro and in vivo studiesrdquoJournal of Ethnopharmacology vol 146 no 3 pp 794ndash802 2013

[47] N Das K Sikder S Bhattacharjee et al ldquoQuercetin alleviatesinflammation after short-term treatment in high-fat-fed micerdquoFood and Function vol 4 no 6 pp 889ndash898 2013

[48] X Zhang G Wang E C Gurley and H Zhou ldquoFlavonoidapigenin inhibits lipopolysaccharide-induced inflammatoryresponse through multiple mechanisms in macrophagesrdquo PLoSONE vol 9 no 9 Article ID e107072 2014

[49] G L Su R J Fontana K Jinjuvadia J Bayliss and S C WangldquoLipopolysaccharide binding protein is down-regulated duringacute liver failurerdquoDigestive Diseases and Sciences vol 57 no 4pp 918ndash924 2012

[50] G L Su L M Hoesel J Bayliss M R Hemmila and S CWang ldquoLipopolysaccharide binding protein inhibitory peptideprotects against acetaminophen-induced hepatotoxicityrdquoAmer-ican Journal of Physiology-Gastrointestinal and Liver Physiologyvol 299 no 6 pp G1319ndashG1325 2010

[51] J M Moreno-Navarrete X Escote F Ortega et al ldquoLipopoly-saccharide binding protein is an adipokine involved in theresilience of the mouse adipocyte to inflammationrdquo Diabetolo-gia 2015

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


was evidenced by in vitro and in vivo approaches [12] Onthe other hand anti-inflammatory effect has been evidenced[13] in which strawberries are very effective inhibitingcyclooxygenase (ie COX2) a key enzyme in inflammationpathway and a target for different drugs In addition recentstudies suggest that strawberry polyphenols might have arole in cancer prevention and treatment [5] Consideringthat the available knowledge about beneficial health effectof commercial strawberry fruit and the converse scenarioof white Chilean strawberry fruit has a different phenolicprofile it becomes relevant to gain insight into its potentialproperties as functional food

The lipopolysaccharide (LPS) is a major glycolipid com-ponent of the outer cell wall of Gram-negative bacteria madeup from a polysaccharide O-chain and a biologically activelipid-A moiety embedded within the bacterial membrane[14] In rats LPS triggers the rise of cytokines and reactiveoxygen species (ROS) such as superoxide hydroxyl radicalsand peroxynitrite [15] The endotoxemia induced by LPS ischaracterized by the injury of various organs including liverkidney and brain [14] Most of the toxicities observed in LPS-induced liver injury (and systemic) have been attributed toinflammatorymediators produced by activatedmacrophagesincluding tumour necrosis factor-120572 (TNF-120572) and interleukinsIL-1120573 IL-6 IL-8 and IL-12 [16] These toxic mediators caninduce lipid peroxidation oxidative damage and depletion ofintracellular stored antioxidants [17] creating a prooxidativeand inflammatory status Therefore the present study wasdesigned to evaluate the effects of an aqueous solution ofstrawberry fruit orally administrated on the inflammatoryresponse and oxidative status in a LPS-injured liver Biochem-ical changes and histological parameters were analysed

2 Material and Methods

21 Animals Male Sprague-Dawley rats (200ndash250 g) wereobtained from the animal facility of University of TalcaThe animals were housed on 12 h light-dark cycles at 25∘CAnimals received standard animal rodent chow and waterad libitum Care of animals and the experimental protocolsof this study were approved by the Institutional AnimalUse Committee of the University of Talca and conductedin accordance with the recommendations of the EuropeanUnion regarding animal experimentation (Directive of theEuropean Council 86609EC)

22 F chiloensis Fruit Extract Ripe fruits of Fragaria chil-oensis ssp chiloensis (record number 5537 in plant listhttpwwwtheplantlistorg) were collected in December2013 from a commercial orchard at Manzanar Alto PurenIX Region Chile (38∘2101584030210158401015840S 73∘8101584038410158401015840W) After harvestthe fruit was immediately cold transported to the Institute ofBiological Sciences at University of Talca and after freezing itunder liquid nitrogen it was stored at minus80∘C until use Ripe Fchiloensis fruit was homogenized in distilled water (05 gmL)with the help of an Ultraturrax (5min at 4000 rpm) on an icebath The rats were fed twice a day with F chiloensis extractsat the daily dosages of 2 4 and 8 gkg of animal per day

(for the doses-response curve) All the administrations wereperformed with fresh aqueous extracts (Figure 1(a))

23 Animal Preparation and LPS-Induction of Liver InjuryGroups of rats were orally supplemented for 10 days withthe aqueous F chiloensis extract 2 gkg of animal twice a daythat is 4 gkgday of strawberry (Figure 1(b)) while controlrat groups received isovolumetric amounts of saline solution(09 NaCl) At day 10 the animals were challenged to aLPS sepsis induced by a single intraperitoneal rat injectionof 5mgkg LPS (lipopolysaccharide endotoxin Escherichiacoli serotype 0111B4 Sigma-Aldrich San Luis MO USA)from E coli 8 [18] dissolved in 09 NaCl These generatefour experimental groups (a) sal-sal (saline pretreatmentplus a saline LPS vehicle) (b) sal-LPS (saline pretreatmentplus LPS inoculum) (c) Fch-sal (F chiloensis extract duringpretreatment plus LPS vehicle) and (d) Fch-LPS (Fch pre-treatment plus LPS inoculum) Five to six animals composedeach experimental group

After 3 hours of LPS challenge the animals were anes-thetized with an intraperitoneal injection (1mLkg) of zolaz-epam chlorhydrate (25mgmL) and tiletamine chlorhydrate(25mgmL) (Zoletil 50 Virbac SA Carros France) Bloodsamples were obtained by cardiac puncture for serum analy-sis Liver samples were taken from themedial lobes frozen inliquid nitrogen and stored at minus80∘C for further analysis

24 Histological Analysis Liver samples of each animalwere fixed in phosphate-buffered formalin dehydrated inincreasing ethanol concentrations and embedded in paraffinThereafter sections of tissue were cut at 5120583m on a rotatorymicrotome (Leica Ultracut Solms Germany) mounted onclean glass slides and dried overnight at 37∘C Sections werecleared hydrated and stained with haematoxylin and eosinfor histomorphological assessments Blind histopathologicalevaluation was performed in at least ten randomly chosenmicroscopic fields of all histological slides (three to five slidesper each animal assayed) All tissue sections were exam-ined in a Nikon Eclipse 50i microscope (Tokyo Japan) forcharacterization of histopathological changes Photographswere digitalized using micrometrics SE premium 28 versioncamera and software for microscopy

25 Determination of Transaminases Cytokines F2-Isopros-tanes and Glutathione Assays Transaminases levels AST(aspartate transaminase) and ALT (alanine transaminase)were measured using a specific diagnostic kit (SpinReactGirona Spain) and expressed as international unitsL ELISAkits were used for assessment of serum levels (pgmL) ofTNF-120572 IL-6 IL-1120573 and IL-10 cytokines (Biosource Interna-tional Camarillo CA USA) and F2-isoprostanes (CaymanChemical Ann Arbor MI USA) GSH and GSSG contents(120583molg liver) were measured using a specific glutathioneassay kit (Cayman Chemical)

26 RNA Extraction and Quantitative Real-Time PCR TotalRNA was isolated from liver samples by the guanidiniumthiocyanate-phenol-chloroform extraction method (Trizol




2 2 LPS

3 4 LPS

4 8 LPS

10 (days)

3(h)

0 0

(a)


LPS

LPS



Fch-LPS Fch

10 300(days) (h)

(b)






3 Results




800

600

400

200

0Seru

m tr

ansa

min

ase a

ctiv

ity (U

L)

lowast

05 10 15 2000


ALTAST

(a)

800

600

400

200

0

lowast

Seru

m A

ST ac

tivity

(UL

)


200

150

100

50

0

lowast


Seru

m A

LT ac

tivity

(UL

)

(c)










Sal-sal

100120583m

(a)

Sal-LPS

100120583m

(b)

Fch-sal

100120583m

(c)

Fch-LPS

100120583m

(d)


4 Discussion




60

40

20

0

lowast


Seru

m T

NF-120572

leve

ls (p

gm

L)

(a)

lowast120

60

80

100

40

20

0


Seru

m IL

-6 le

vels

(pg

mL)

(b)

lowast100

60

80

40

20

0


Seru

m IL

-1120573

leve

ls (p

gm

L)

(c)

3

4

5

2

1


Seru

m IL

-10

leve

ls (p

gm

L)

(d)


lowast

4

40

60

2

0


TNF-120572

relat

ive e

xpre

ssio

n

(a)

lowast

4

400

300

500

2


IL-6

relat

ive e

xpre

ssio

n

(b)

4

12

6

8

10

2

0


IL-1120573

relat

ive e

xpre

ssio

n

lowast


10

20

15

05

00

IL-1

0 re

lativ

e exp

ress

ion

(d)





100

200

150

50

0


Plas

ma F

2-iso

pros

tane

s (pg

mL)

lowast

Fch + LPS








6

4

2

0Live

r GSH

cont

ent (120583

mol

g li

ver)


lowast15

Inset

00

05

10

Live

r GSH

cont

ent (120583

mol

g li

ver)

(B-A)


(a)

(b)


(D-C)minus15

minus10

minus05

p lt 005

3

2

1

0Live

r GSS

G co

nten

t (120583

mol

g li

ver)


lowast Inset

00

05

10

Live

r GSS

G co

nten

t diff

eren

ce(120583

mol

g li

ver)

(B-A)



(D-C)minus10

minus05

p lt 005





4-) induced





5 Conclusions







Acknowledgments


References










































4





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2 2 LPS

3 4 LPS

4 8 LPS

10 (days)

3(h)

0 0

(a)


LPS

LPS



Fch-LPS Fch

10 300(days) (h)

(b)






3 Results




800

600

400

200

0Seru

m tr

ansa

min

ase a

ctiv

ity (U

L)

lowast

05 10 15 2000


ALTAST

(a)

800

600

400

200

0

lowast

Seru

m A

ST ac

tivity

(UL

)


200

150

100

50

0

lowast


Seru

m A

LT ac

tivity

(UL

)

(c)










Sal-sal

100120583m

(a)

Sal-LPS

100120583m

(b)

Fch-sal

100120583m

(c)

Fch-LPS

100120583m

(d)


4 Discussion




60

40

20

0

lowast


Seru

m T

NF-120572

leve

ls (p

gm

L)

(a)

lowast120

60

80

100

40

20

0


Seru

m IL

-6 le

vels

(pg

mL)

(b)

lowast100

60

80

40

20

0


Seru

m IL

-1120573

leve

ls (p

gm

L)

(c)

3

4

5

2

1


Seru

m IL

-10

leve

ls (p

gm

L)

(d)


lowast

4

40

60

2

0


TNF-120572

relat

ive e

xpre

ssio

n

(a)

lowast

4

400

300

500

2


IL-6

relat

ive e

xpre

ssio

n

(b)

4

12

6

8

10

2

0


IL-1120573

relat

ive e

xpre

ssio

n

lowast


10

20

15

05

00

IL-1

0 re

lativ

e exp

ress

ion

(d)





100

200

150

50

0


Plas

ma F

2-iso

pros

tane

s (pg

mL)

lowast

Fch + LPS








6

4

2

0Live

r GSH

cont

ent (120583

mol

g li

ver)


lowast15

Inset

00

05

10

Live

r GSH

cont

ent (120583

mol

g li

ver)

(B-A)


(a)

(b)


(D-C)minus15

minus10

minus05

p lt 005

3

2

1

0Live

r GSS

G co

nten

t (120583

mol

g li

ver)


lowast Inset

00

05

10

Live

r GSS

G co

nten

t diff

eren

ce(120583

mol

g li

ver)

(B-A)



(D-C)minus10

minus05

p lt 005





4-) induced





5 Conclusions







Acknowledgments


References










































4





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















800

600

400

200

0Seru

m tr

ansa

min

ase a

ctiv

ity (U

L)

lowast

05 10 15 2000


ALTAST

(a)

800

600

400

200

0

lowast

Seru

m A

ST ac

tivity

(UL

)


200

150

100

50

0

lowast


Seru

m A

LT ac

tivity

(UL

)

(c)










Sal-sal

100120583m

(a)

Sal-LPS

100120583m

(b)

Fch-sal

100120583m

(c)

Fch-LPS

100120583m

(d)


4 Discussion




60

40

20

0

lowast


Seru

m T

NF-120572

leve

ls (p

gm

L)

(a)

lowast120

60

80

100

40

20

0


Seru

m IL

-6 le

vels

(pg

mL)

(b)

lowast100

60

80

40

20

0


Seru

m IL

-1120573

leve

ls (p

gm

L)

(c)

3

4

5

2

1


Seru

m IL

-10

leve

ls (p

gm

L)

(d)


lowast

4

40

60

2

0


TNF-120572

relat

ive e

xpre

ssio

n

(a)

lowast

4

400

300

500

2


IL-6

relat

ive e

xpre

ssio

n

(b)

4

12

6

8

10

2

0


IL-1120573

relat

ive e

xpre

ssio

n

lowast


10

20

15

05

00

IL-1

0 re

lativ

e exp

ress

ion

(d)





100

200

150

50

0


Plas

ma F

2-iso

pros

tane

s (pg

mL)

lowast

Fch + LPS








6

4

2

0Live

r GSH

cont

ent (120583

mol

g li

ver)


lowast15

Inset

00

05

10

Live

r GSH

cont

ent (120583

mol

g li

ver)

(B-A)


(a)

(b)


(D-C)minus15

minus10

minus05

p lt 005

3

2

1

0Live

r GSS

G co

nten

t (120583

mol

g li

ver)


lowast Inset

00

05

10

Live

r GSS

G co

nten

t diff

eren

ce(120583

mol

g li

ver)

(B-A)



(D-C)minus10

minus05

p lt 005





4-) induced





5 Conclusions







Acknowledgments


References










































4





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Sal-sal

100120583m

(a)

Sal-LPS

100120583m

(b)

Fch-sal

100120583m

(c)

Fch-LPS

100120583m

(d)


4 Discussion




60

40

20

0

lowast


Seru

m T

NF-120572

leve

ls (p

gm

L)

(a)

lowast120

60

80

100

40

20

0


Seru

m IL

-6 le

vels

(pg

mL)

(b)

lowast100

60

80

40

20

0


Seru

m IL

-1120573

leve

ls (p

gm

L)

(c)

3

4

5

2

1


Seru

m IL

-10

leve

ls (p

gm

L)

(d)


lowast

4

40

60

2

0


TNF-120572

relat

ive e

xpre

ssio

n

(a)

lowast

4

400

300

500

2


IL-6

relat

ive e

xpre

ssio

n

(b)

4

12

6

8

10

2

0


IL-1120573

relat

ive e

xpre

ssio

n

lowast


10

20

15

05

00

IL-1

0 re

lativ

e exp

ress

ion

(d)





100

200

150

50

0


Plas

ma F

2-iso

pros

tane

s (pg

mL)

lowast

Fch + LPS








6

4

2

0Live

r GSH

cont

ent (120583

mol

g li

ver)


lowast15

Inset

00

05

10

Live

r GSH

cont

ent (120583

mol

g li

ver)

(B-A)


(a)

(b)


(D-C)minus15

minus10

minus05

p lt 005

3

2

1

0Live

r GSS

G co

nten

t (120583

mol

g li

ver)


lowast Inset

00

05

10

Live

r GSS

G co

nten

t diff

eren

ce(120583

mol

g li

ver)

(B-A)



(D-C)minus10

minus05

p lt 005





4-) induced





5 Conclusions







Acknowledgments


References










































4





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















60

40

20

0

lowast


Seru

m T

NF-120572

leve

ls (p

gm

L)

(a)

lowast120

60

80

100

40

20

0


Seru

m IL

-6 le

vels

(pg

mL)

(b)

lowast100

60

80

40

20

0


Seru

m IL

-1120573

leve

ls (p

gm

L)

(c)

3

4

5

2

1


Seru

m IL

-10

leve

ls (p

gm

L)

(d)


lowast

4

40

60

2

0


TNF-120572

relat

ive e

xpre

ssio

n

(a)

lowast

4

400

300

500

2


IL-6

relat

ive e

xpre

ssio

n

(b)

4

12

6

8

10

2

0


IL-1120573

relat

ive e

xpre

ssio

n

lowast


10

20

15

05

00

IL-1

0 re

lativ

e exp

ress

ion

(d)





100

200

150

50

0


Plas

ma F

2-iso

pros

tane

s (pg

mL)

lowast

Fch + LPS








6

4

2

0Live

r GSH

cont

ent (120583

mol

g li

ver)


lowast15

Inset

00

05

10

Live

r GSH

cont

ent (120583

mol

g li

ver)

(B-A)


(a)

(b)


(D-C)minus15

minus10

minus05

p lt 005

3

2

1

0Live

r GSS

G co

nten

t (120583

mol

g li

ver)


lowast Inset

00

05

10

Live

r GSS

G co

nten

t diff

eren

ce(120583

mol

g li

ver)

(B-A)



(D-C)minus10

minus05

p lt 005





4-) induced





5 Conclusions







Acknowledgments


References










































4





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















100

200

150

50

0


Plas

ma F

2-iso

pros

tane

s (pg

mL)

lowast

Fch + LPS








6

4

2

0Live

r GSH

cont

ent (120583

mol

g li

ver)


lowast15

Inset

00

05

10

Live

r GSH

cont

ent (120583

mol

g li

ver)

(B-A)


(a)

(b)


(D-C)minus15

minus10

minus05

p lt 005

3

2

1

0Live

r GSS

G co

nten

t (120583

mol

g li

ver)


lowast Inset

00

05

10

Live

r GSS

G co

nten

t diff

eren

ce(120583

mol

g li

ver)

(B-A)



(D-C)minus10

minus05

p lt 005





4-) induced





5 Conclusions







Acknowledgments


References










































4





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















6

4

2

0Live

r GSH

cont

ent (120583

mol

g li

ver)


lowast15

Inset

00

05

10

Live

r GSH

cont

ent (120583

mol

g li

ver)

(B-A)


(a)

(b)


(D-C)minus15

minus10

minus05

p lt 005

3

2

1

0Live

r GSS

G co

nten

t (120583

mol

g li

ver)


lowast Inset

00

05

10

Live

r GSS

G co

nten

t diff

eren

ce(120583

mol

g li

ver)

(B-A)



(D-C)minus10

minus05

p lt 005





4-) induced





5 Conclusions







Acknowledgments


References










































4





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusions







Acknowledgments


References










































4





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































4





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















4





















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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