Resveratrol Protects against TNF-α-Induced-白藜芦醇（Resveratrol）英文文献

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OPENACCESS

Citation:PanW,YuH,HuangS,ZhuP(2016)ResveratrolProtectsagainstTNF-α-InducedInjuryinHumanUmbilicalEndothelialCellsthroughPromotingSirtuin-1-InducedRepressionofNF-KBandp38MAPK.PLoSONE11(1):e0147034.doi:10.1371/journal.pone.0147034

Editor:AnilKumar,UniversityofMissouri-KansasCity,UNITEDSTATES

Received:September1,2015

Accepted:December28,2015

Published:January22,2016

Copyright:©2016Panetal.Thisisanopenaccessarticledistributedunderthetermsofthe

Creative

CommonsAttributionLicense

,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.

DataAvailabilityStatement:AllrelevantdataarewithinthepaperanditsSupportingInformationfiles.

Funding:ThisstudywassupportedbygrantsfromtheNaturalScienceFoundationofChina(grantno.81373785),theNaturalScienceFoundationofFujianProvince(no.2011J01134;no.2010J01127),andtheProjectionofExcellentYoungDoctorTrainingPlanintheFujianProvincialHealthSystem(no.2013-ZQN-ZD-2).

CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.

RESEARCHARTICLE

ResveratrolProtectsagainstTNF-α-InducedInjuryinHumanUmbilicalEndothelialCellsthroughPromotingSirtuin-1-InducedRepressionofNF-KBandp38MAPK

WeiPan1☯,HuizhenYu1,2,3☯,ShujieHuang,PengliZhu31,3*

1ProvincialClinicalMedicalCollege,FujianMedicalUniversity,Fuzhou,China,2DepartmentofGeriatrics,FujianProvincialHospitalKeyLaboratoryofGeriatrics,FujianMedicalUniversity,Fuzhou,China,3FujianInstituteofClinicalGeriatrics,Fuzhou,China

☯Theseauthorscontributedequallytothiswork.

*zpl7755@126.com

Abstract

Inflammationandreactiveoxygenspecies(ROS)playimportantrolesinthepathogenesisofatherosclerosis.Resveratrolhasbeenshowntopossessanti-inflammatoryandantioxi-dativestressactivities,buttheunderlyingmechanismsarenotfullyunderstood.Inthepres-entstudy,weinvestigatedthemolecularbasisassociatedwiththeprotectiveeffectsofresveratrolontumornecrosisfactor-alpha(TNF-α)-inducedinjuryinhumanumbilicalendo-thelialcells(HUVECs)usingavarietyofapproachesincludingacellviabilityassay,reversetranscriptionandquantitativepolymerasechainreaction,westernblot,andimmunofluores-cencestaining.WeshowedthatTNF-αinducedCD40expressionandROSproductioninculturedHUVECs,whichwereattenuatedbyresveratroltreatment.Also,resveratrolincreasedtheexpressionofsirtuin1(SIRT1);andrepressionofSIRT1bysmall-interferingRNA(siRNA)andtheSIRT1inhibitorEx527reducedtheinhibitoryeffectsofresveratrolonCD40expressionandROSgeneration.Inaddition,resveratroldownregulatedthelevelsofp65andphospho-p38MAPK,butthisinhibitoryeffectwasattenuatedbythesuppressionofSIRT1activity.Moreover,thep38MAPKinhibitorSD203580andthenuclearfactor(NF)-κBinhibitorpyrrolidinedithiocarbamate(PDTC)achievedsimilarrepressiveeffectsasresvera-trolonTNF-α-inducedROSgenerationandCD40expression.Thus,ourstudyprovidesamechanisticlinkbetweenresveratrolandtheactivationofSIRT1,thelatterofwhichisinvolvedinresveratrol-mediatedrepressionofthep38MAPK/NF-κBpathwayandROSpro-ductioninTNF-α-treatedHUVECs.

Introduction

Thepathogenesisofatherosclerosisisacomplexpathologicalprocessthatinvolvesanongoinginflammatoryresponseinbloodvessels,whichmediatesallstagesofatherosclerosisincluding

ResveratrolonCD40andROSinHUVECs

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initiationandprogression[

1

].Recently,CD40,theBcellsurfaceantigen,hasbeenreportedtobeupstreamofthecytokinesignalingnetwork,whichplaysanimportantroleininflammation.ActivationofCD40byproinflammatorycytokinessuchasCD40L(CD154)hasbeenshowntoaugmenttheexpressionofmatrixmetalloproteinases,procoagulanttissuefactor,chemokines,andcytokines[

2

],whichregulatevariousinflammatoryresponsesintheprocessofatheroscle-rosis[

3

].Therefore,strategiesdesignedtosuppressCD40/CD40Lexpressionmayattenuateinflammation,whichwilleventuallyofferbenefitsduringtheprogressionofatherosclerosis.

Oxidativestressassociatedwithreactiveoxygenspecies(ROS),includingsuperoxide(O2-),hydrogenperoxide(H2O2),hydroxide(OH-),andhypochlorite(OCl-),isalsocrucialtothepathogenesisofatherosclerosis.NADPHoxidase,enzymaticactivationofcytochromeP450,xanthineoxidase,andinflammatoryactivitiesareconsideredtobethemajorsourcesofROS[

4

].ApreviousstudyhasshownthattheactivationofCD40inhibitsendothelialcellmigrationbyincreasingROSproduction[

5

],andanotherstudyhasprovidedevidencethatROSmediateakeyinitiatingstepinthedevelopmentofatherosclerosis[

6

].ThesefindingspointtoaroleforCD40intheregulationofROSgeneration.

Consideringthemajorrolesofinflammationandoxidativeresponsesinthepathogenesisofatherosclerosis,theinflammatoryandoxidativepathwayshavebeenthetherapeutictargetsforatherosclerosis.Studieshaveshownthatlighttomoderateconsumptionofredwinereducestheriskofdevelopingcardiovasculareventsowingtothepolyphenoliccomponentresveratrol[

7

].Indeed,resveratrolhasmanyinterestingproperties,includingtheabilitytoreversedyslipi-demiaandobesity,toinhibithyperglycemiaandhyperinsulinemia,andtoprotectendothelialfunction.Resveratrolalsohasbeenshowntoactonmultiplemoleculartargetsimportantforcelldifferentiationandactivation.Forinstance,Tsuruokaetal.[

8

]havediscoveredthatresvera-trolenhancesmitochondrialfunctionindependentofperoxisomeproliferator-activatedrecep-tor-gammacoactivator1alpha(PGC-1α),andIdoetal.[

9

]havefoundthatresveratrolpreventsoxidativestress-inducedsenescenceandproliferativedysfunctionbyactivatingthe5ʹ-adenosinemonophosphate-activatedproteinkinase(AMPK)–ForkheadBoxO(FOXO)cas-cade.Inaddition,resveratrolhasbeenshowntoregulatevariationsinionchannelsthroughmodulatingthephosphoinositide3-kinase/Akt/endothelialnitricoxidesynthase(eNOS)sig-nalingpathway,thusdecreasingleftarterialfibrosis[

10

].However,theeffectsofresveratrolonCD40expressionandROSgenerationinendothelialcellsarenotfullyunderstood.

Itisknownthatareasonabledoseofresveratrolactivatessirtuin1(SIRT1),aclassIIInico-tinamideadeninedinucleotide(NAD)-dependenthistonedeacetylase[

11

].SIRT1deacetylatesavarietyofsubstratestoregulatethedownstreamsignalingpathways.Ithasbeendemon-stratedthatSIRT1suppressestheexpressionlevelsofinflammatorygenesinavarietyofcelltypesincludingadipocytes[

12

],endothelialcells,smoothmusclecells,andmacrophagestoprotectthecardiovascularsystemfrominjury[

13

].Forinstance,Harijithetal.[

14

]havefoundthattheactivationofinflammasomesisdampenedbySIRT1.Onthecontrary,SIRT1knock-downincreasesintercellularadhesionmolecule-1expressioninendothelialcells[

15

],thuspro-motinginflammation.Assuch,resveratrolisconsideredtobeaprotectivefactorandparticipateinpromotinganti-inflammatoryresponsesthroughSIRT1-mediatedsignalpath-ways.However,themechanismsunderlyingthespecificroleofSIRT1intheCD40/CD40LaxisandROSproductionhavenotbeenreportedindepth.Together,thesefindingspromptedustoinvestigatethesignalingpathwaysinvolvedinmediatingtheresveratrol-directedanti-inflam-matoryreaction,andourfindingswillprovideamolecularbasisforthepotentialapplicationofresveratrolintreatingatherosclerosis.

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MaterialsandMethods

Reagents

Resveratrol(SML0963)wasobtainedfromSigma-Aldrich(St.Louis,MO,USA).Tumornecro-sisfactor-alpha(TNF-α)(300-01A)wasobtainedfromPeproTech(RockyHill,NJ,USA).Endothelialcellmedium,fetalbovineserum,endothelialcellgrowthsupplement,andpenicil-lin/streptomycinsolution(1001)werepurchasedfromSciencellResearchLaboratories(Carls-bad,CA,USA).SB203580(S1076),EX527(S1541),andpyrrolidinedithiocarbamate(PDTC)(S1809)werepurchasedfromSelleckChemicals(Houston,TX,USA).Fluoresceinisothiocya-nate(FITC)-conjugatedaffiniPuregoatanti-rabbitIgG(H+L)(111-095-144)waspurchasedfromJacksonImmunoResearch(WestGrove,PA,USA).Rabbitanti-factorVIII-relatedanti-gen(BA0046)waspurchasedfromBoster(Wuhan,China).Anti-CD40(ab47021),SIRT1(ab32441),p65nuclearfactor(NF)-κB(ab7970),phospho-p38MAPK(ab4822)antibodies,BCAproteinassaykit(ab102536)wereobtainedfromAbcam(Abcam,Cambridge,MA,USA).SmallinterferingRNA(siRNA)specificallytargetingSIRT1andCD40andanegativecontrolsiRNA(Nc-siRNA)weresynthesizedbyGenePharma(Shanghai,China).

Cellcultureandimmunocytochemistry

OurstudywasapprovedbytheEthicsCommitteeoftheFujianProvincialHospital(No.K2014-021-01),andallaspectsofthestudywereincompliancewiththeDeclarationofHelsinki.Eachpatientsignedaconsentformbeforeprovidinghumanumbilicalendothelialcells(HUVECs).PrimaryHUVECcultureswereestablishedusingthecollagenasetreatmentmethoddescribedpreviouslybyBaudin[

16

].Briefly,humanumbilicalcordswerecollectedwithin6hofdeliveryandweremaintainedongelatin-coatedT25flasksinendothelialcellmediumsupplementedwith5%fetalbovineserum,50μg/mLendothelialcellgrowthsupplement,and1%penicillin/strepto-mycinsolutionat37°Cinahumidifiedatmosphereof5%CO2/95%air.AllexperimentswereperformedonHUVECsatpassage3to5.

WhentheHUVECsweregrownto80–90%confluence,thecellswerefixedin4%parafor-maldehydefor10minatroomtemperature,followedbythreewasheswithphosphate-bufferedsaline(PBS).Thefixedcellswerepermeabilizedwith0.5%TritonX-100for10minandblockedwith5%bovineserumalbuminfor20min.Then,thecellswereincubatedwiththerabbitanti-factorVIII-relatedantigen(1:50dilution)at4°Covernight.FactorVIIIwasdetectedusingastreptavidinbiotinperoxidasecomplexkitanda3,3’-diaminobenzidinekit.ThestainedcellswerescoredusinganOlympusfluorescencemicroscope(Leica,Germany).

Celltreatment

ToinduceaninflammatoryresponseintheHUVECs,thecellsweretreatedwithdifferentconcen-trationsofTNF-α(0,1,10,or20μg/L)for24h.ToexploretheeffectofresveratrolonTNF-α-inducedcellinjury,thecellswerepretreatedwithdifferentconcentrationsofresveratrol(0,5,10,and20μM)for2hinthepresenceorabsenceofTNF-α(10μg/L)for24h.Toexplorethepoten-tialsignalingpathwaysinvolvedintheresveratrol-mediatedcellprotection,thecellsweretreatedwithEx527(10μM),SB203580(10μM),orPDTC(20μM),respectively,intheabsenceorpres-enceofresveratrolandTNF-α.

Cellviabilityassay

Cellviabilitywasassessedusingacellcountingkit-8(CCK-8)assay,accordingtothemanufac-turer’srecommendations.Briefly,cells(1×104cells/well)wereplatedonto96-wellplatesandtreatedwithdrug-containingmediafor24h,CCK-8(10μL)wasaddedtothemediafora2-h

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incubation,andtheabsorbancewasmeasuredat450nmwithanenzyme-linkedimmunosor-bentassayplatereader(Thermo,Boston,MA,USA).

RNAinterference

HUVECswereseededon6-wellplatesandthentransientlytransfectedwithsynthesizedsmall-interferingRNA(siRNA)targetinghumanSIRT1andCD40oranegativecontrolsiRNA(Nc-siRNA).ThesiRNAandLipofectamine2000transfectionagentwereseparatelydilutedwithserum-freemedium,accordingtothemanufacturer’sinstructions.Then,thecellsweretrans-fectedwithsiRNA-Lipofectaminecomplexes,andthesilencingeffectofthesiRNAwasevalu-atedbyreversetranscriptionandquantitativepolymerasechainreaction(RT-qPCR)aswellaswesternblotat48haftertransfection.

FlowcytometryanalysisofROS

Aftereachtreatment,HUVECs(1×106/mL)wereincubatedfor20minat37°Cwithorwith-outthecellpermeablefluorescentandchemiluminescentprobe2’-7’-dichlorodihydroofluores-ceindiacetate(DCFH-DA).Afterincubation,thecellswerewashedtwicewithsterilePBS,theROSlevelsweredetectedbyfluorescence-activatedcellsorting(FACSCalibur,BecktonDickin-son,Oxford,UK),andtheresultswereanalyzedbyCellQuestsoftware(BDBiosciences,SanJose,CA,USA).

FlowcytometryanalysisofCD40expression

Resveratrolanddifferentsignalingpathwayinhibitorswereincubatedwithcellswithorwith-outTNF-αinthemediafor24h.Thereafter,thecellswereharvestedandincubatedwithananti-CD40antibodyfor1hat37°C,followedbyincubationwithhumanFITC-conjugatedgoatanti-rabbitIgGfor30minat4°C.Anisotype-matchedantibodywasusedasacontrol.TheCD40levelsweredetectedbyfluorescence-activatedcellsorting(FACSCalibur,BecktonDick-inson,Oxford,UK),andtheresultswereanalyzedbyCellQuestsoftware(BDBiosciences,SanJose,CA,USA).Themeanfluorescenceintensitywascalculatedasdescribedpreviously.

Westernblotanalysis

Totalcelllysateswereextractedwithice-coldRIPAbufferandmeasuredusingaBCAproteinassaykit.Cellextractswerefractionatedbyelectrophoresison10%sodiumdodecylsulfate–polyacrylamidegelsandtransferredtopolyvinylidenefluoridemembranesfor2hat100V.Themembranesweresubsequentlyblockedwith5%nonfatdrymilkfor1hatroomtempera-tureandovernightat4°CwithprimaryantibodiesagainsthumanSIRT1(1:200dilution),CD40(1:800dilution),NF-κBp65(diluted1:500),p38MAPK(diluted1:800),orβ-actin(1:400dilution),respectively.Themembraneswerethenincubatedwithhorseradishperoxi-dase-conjugatedanti-rabbitsecondaryantibody(1:5000dilution)for1hatroomtemperature.Proteinbandswerevisualizedbytheenhancedchemiluminescence(Gibco,InvitrogenLifeSci-ence,UK).QuantificationofbandintensitywascarriedoutusingImageJsoftware(NationalInstitutesofHealth).

RT-qPCRassay

TotalRNAwasisolatedfromHUVECsusingTrizolreagent,accordingtothemanufacturer’sprotocol.TheintegrityoftheRNAsampleswascheckedbyagarosegelelectrophoresis.ThetotalRNAwasreverse-transcribedintocDNAusingareversetranscriptionkit,followedbyquantitativePCRtodetecttheexpressionlevelsofSIRT1,CD40andNF-κB.β-Actinwasused

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asaninternalcontrol.Thethermalcyclingconditionswereasfollows:30sat95°Cfortheacti-vationofTaqDNApolymerase,followedby40cyclesofamplificationat95°Cfor15s,1minat60°C,and1minat72°C.Thereactionswererunintriplicate.Thespecificoligosusedinthereal-timePCRwereasfollows:SIRT1(forward):5ʹ-GATTAGTAGGCGCTTGATGGT-3ʹ,SIRT1(reverse):5ʹ-TCTTCTAAACTTGGACTCGGCAT-3ʹ,CD40(forward):5ʹ-ACACTGCCACCAGCACAAATAC-3ʹ,CD40(reverse):5ʹ-GATAAAGACCAGCACCAAGAGGAT-3ʹ,NF-κB(forward):5’AGTTTGACGGTGAGCTGGTA3’,NF-κB(reverse):5’GCCTCGGCCTGCCGCAAGCCT3’,β-actin(forward):5ʹ-TGGCACCCAGCACAATGAA-3ʹ,β-actin(reverse):5ʹ-CTAAGTCATAGTCCGCCTAGAAGCA-3ʹ.Geneexpressionvalueswerecalculatedbythe2-ΔΔCTrelativeexpressionmethod.

NF-κBreportergeneassay

HUVECs(3×105cells/well)weretransfectedwithsiRNAtargetingSIRT1andthepGL4.32[luc2P/NF-κB-RE/Hygro]vectorusingLipofectamine.At24hpost-transfection,thecellswerepretreatedwithvehicle,resveratrol,orPDTCasindicatedbeforeTNF-αstimulation.Thelucif-eraseactivitywasdeterminedbytheluciferaseassaykit.

Immunofluorescencestaining

Immunofluorescencestainingwascarriedoutwhenthecellsreached60–80%confluence.Aftereachtreatmentmentionedabove,thecellswerefixedin4%formaldehyde-PBSfor15minatroomtemperatureandthenimmersedin0.3%TritonX-100-PBSfor30min.Thereafter,thefixedandpermeabilizedHUVECswereincubatedwiththeprimaryanti-SIRT1antibody(1:150dilution)at4°Covernight,followedbyanotherincubationwiththepolyclonalFITC-conjugatedgoatanti-rabbitIgG(1:50dilution)for1hatroomtemperatureasdescribedprevi-ously[

17

].CellularnucleiwerestainedwithDAPI(1:1000dilution).Thirtycellsfromsixdif-ferentregionspergroupwererandomlychosenandanalyzedwithaconfocalscanninglasermicroscopeequippedwithanargonlaser(LeicaLasertechnik,Heidelberg,Germany).Thetri-plestainingwasanalyzedusingaZeisslaserscanningmicroscope(LSM510METAwithanAxiovert200microscope).ImagesweretakenusingaPlan-Apochromat63×/1.4oilimmersionobjective.Fluorescenceintensitywasdeterminedbycomputer-basedimageanalysissystemMetaView™softwarebymeasuringtheaveragepixelintensitywithinafixedcircledareaplacedovertherelevantareaofthecell[

18

].

Statisticalanalysis

Datawerecollectedfromthreeindependentexperiments,eachcarriedoutintriplicateandexpressedasthemean±standarderrorofthemean(SEM).Statisticalanalysiswasperformedbymeansofone-wayanalysisofvariancefollowedbytheleastsignificantdifferencetestusingSPSS16.0software(USA).Apvalueof<0.05wasregardedasstatisticallysignificant,and<0.01wasregardedashighlysignificant.

Results

ResveratrolimprovestheviabilityofHUVECsimpairedbyTNF-α

HUVECsweregrownasaconfluentmonolayerwithcobblestonemorphology(

Fig1A

),andthecytoplasmwasstainedbrownishredafterincubationwithananti-VIIIfactorantigen(

Fig

1B

).Asshownin

Fig2

and

S1Table

,10μg/L,butnot1μg/L,TNF-αinducedasignificantdif-ferenceincellviability,comparedwiththecontrolgroup(*p<0.05vs.control).Therefore,weused10μg/LTNF-αastheproperdosetoinducetheinflammatoryresponse.Sinceahigh

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Fig1.

PrimaryHUVECs(×200).(A)ThemorphologyofprimaryHUVECswasobservedunderaninverted,phase-contrastmicroscope.Thecellswerehomogenous,closelyapposed,large,flat,andpolygonalwithacobblestone-shapedappearance.(B)ThecytoplasmoftheHUVECswasstainedbrown-redafterincubationwitharabbitanti-factorVIIIantigenbytheimmunocytochemicalstainingmethod.ThepurityoftheculturedHUVECswasmorethan95%.

doi:10.1371/journal.pone.0147034.g001

concentrationofresveratrol(>30μM)hasbeenshownpreviouslytoimpaircellviability[

19

],wetreatedHUVECswithresveratrolatconcentrationslessthan30μM(0,5,10,and20μM)toinvestigatetheeffectofresveratrolontheviabilityofHUVECs.While10μMresveratroldidnothaveanysignificanteffectontheviabilityofHUVECs,itgreatlyimprovedthesurvivalofHUVECsimpairedbyTNF-α(#p<0.05vs.TNF-αalone).Similarresultswereobservedwith

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Fig2.

ResveratrolincreasescellviabilityinTNF-α-treatedHUVECs.CellviabilitywasassessedviatheCCK-8method.HUVECswereexposedtodifferentconcentrationsofresveratrol(0,5,10,and20μM)beforetreatmentwithTNF-α(10μg/L).Dataareexpressedasthemean±SEMfromsixindependentexperiments,eachcarriedoutintriplicate.*p<0.05vs.control;#p<0.05vs.TNF-αalone.

doi:10.1371/journal.pone.0147034.g002

20μMresveratrol.TheseobservationssuggestthatresveratrolimprovestheviabilityofHUVECsimpairedbyTNF-αtreatment.

ResveratrolattenuatestheincreasedexpressionofCD40triggeredbyTNF-αstimulationinHUVECs

IncreasedexpressionoftheCD40receptoractivatedbyCD40Lhasbeenshowntoaltertheexpressionlevelsofspecificadhesionmolecules,thuspromotingtheinflammatoryresponse[

20

].Inaddition,increasedexpressionofCD40alsohasbeenshowntobeinvolvedintheTNF-α-inducedinflammatoryresponse[

21

].TodeterminewhetherresveratrolaltersTNF-α-inducedCD40expressioninHUVECs,cellswerepreincubatedwithdifferentconcentrationsofresveratrolpriortoTNF-αstimulation,followedbywesternblotanalysistodeterminetheproteinlevelsofCD40(

Fig3A

).WhileTNF-αsignificantlyinducedtheexpressionofCD40asexpected(**p<0.01vs.control),thisinductionwasdownregulatedbyresveratrolinadose-dependentfashion(##p<0.01vs.TNF-αalone),although10μMresveratroldidnothaveanysignificanteffectonCD40expressionatbaseline.Thus,weconcludethatresveratroltreatmentattenuatestheincreasedexpressionofCD40triggeredbyTNF-αstimulation.

ResveratrolsuppressesTNF-α-triggeredCD40expressionviaactivatingSIRT1inHUVECs

ApreviousstudyhassuggestedthatresveratrolactivatesSIRT1[

22

].Therefore,weanalyzedtheexpressionofSIRT1inHUVECsincubatedwithdifferentconcentrationsofresveratrol(0,5,10,and20μM)intheabsenceorpresenceofTNF-α.Asshownin

Fig4

and

S3Table

,west-ernblotanalysisshowedthattheproteinlevelsofSIRT1weresignificantlydecreaseduponstimulationwith10μg/LTNF-α(*p<0.05vs.control).However,thisdecreasewassignifi-cantlyattenuatedby10and20μMresveratroltreatment(#p<0.05vs.TNF-αalone).Intrigu-ingly,20μMresveratroltreatmentincreasedtheexpressionofSIRT1atbaseline($p<0.05vs.control).Thus,weconcludethatresveratrolimprovestheexpressionofSIRT1,whichwassup-pressedbyTNF-αstimulation.

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Fig3.

ResveratrolattenuatestheTNF-α-inducedCD40expressioninHUVECs.(A)ResveratrolwasaddedatdifferentconcentrationsasindicatedtoTNF-α-stimulatedHUVECs,andCD40expressionwasdeterminedbywesternblot.Therepresentativedatafromthreeindependentassaysareshown.(B)DensitometricanalysisoftheblotsshowninA.Thedataareexpressedasthemean±SEMfromthreeindependentexperiments,eachcarriedoutintriplicate.**p<0.01vs.control;##p<0.05vs.TNF-αalone.

doi:10.1371/journal.pone.0147034.g003

Asshownin

Fig3

and

S2Table

,CD40expressioninHUVECswasdecreasedwithincreas-ingconcentrationsofresveratrolrangingfrom5μMto20μM.Next,weusedaloss-of-functionapproach,i.e.,RNAinterferenceortheSIRT1inhibitorEx527toknockdownSIRT1geneexpressiontoexplorewhetherSIRT1isinvolvedintheresveratrol-mediatedexpressionofCD40.TheHUVECswithtransfectedsiRNAspecificallytargetingSIRT1ortreatmentwithEx527exhibitedsignificantlydecreasedSIRT1expression,andtheknockdownefficiencywasapproximately73.38±0.8363%(

S4Table

,

S5Table

,Figs

5

and

6

).TheintracellularCD40expressioninhibitedbyresveratrolwassignificantlyincreasedinresponsetoSIRT1knock-downortheSIRT1inhibitor(++p<0.01vs.TNF-α+resveratrol).

TofurtherexaminethesubcellularlocationandexpressionofSIRT1inHUVECs,cellswereexposedtoTNF-α(10μg/L)for24hintheabsenceorpresenceof10or20μMresveratrolortransfectedwithSIRT1siRNA,followedbyimmunostainingtodetectSIRT1expression.Asshownin

Fig7

,fluorescentpunctaweresignificantlyincreasedbyresveratrolat10and20μM,buttheseincreasesweresignificantlyattenuatedbySIRT1siRNA.Asshownin

Fig7

,

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Fig4.

ResveratrolincreasestheexpressionofSIRT1inTNF-α-treatedHUVECs.HUVECswereeitheruntreated(control),treatedwithTNF-α(10μg/L)alone,resveratrol(10μM)alone,orcotreatedwithTNF-α(10μg/L)anddifferentconcentrationsofresveratrolasindicatedfor24h.(A)WesternblotanalysiswascarriedoutontheproteinlysatespurifiedfromHUVECsoftheabove-mentionedgroups.β-Actinwasusedasaloadingcontrol.(B)DensitometricanalysisoftheblotsshowninA.Thedataareexpressedasthe

mean±SEMfromthreeindependentexperiments,eachcarriedoutintriplicate.*p<0.05vs.control;

#p<0.05,##p<0.01vs.TNF-αalone;$p<0.05vs.control.

doi:10.1371/journal.pone.0147034.g004

immunofluorescencelabelingwithaspecificantibodyagainstSIRT1demonstratedthatSIRT1ismainlylocalizedinthenucleusbutalsoispresentinthecytoplasmofHUVECs.

ResveratrolsuppressesTNF-α-triggeredROSgenerationviaactivatingSIRT1inHUVECs

Toinvestigatetheanti-oxidanteffectofresveratrol,we