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10点击：2940EuropeanJournalofPharmac；Contentslistsavailableat；EuropeanJournalofPharmac；journalhomepage:www.else；MolecularandCellularPhar；Morronisideprotectshuman；WenWanga,FanglingSuna,Yi；abDep
EuropeanJournalofPharmacology613(ContentslistsavailableatScienceDirectEuropeanJournalofPharmacologyjournalhomepage:www.elsevier.com/locate/ejpharMolecularandCellularPharmacologyMorronisideprotectshumanneuroblastomaSH-SY5Ycellsagainsthydrogenperoxide-inducedcytotoxicityWenWanga,FanglingSuna,YiAnb,HouxiAia,LiZhanga,WentingHuangb,LinLia,?abDepartmentofPharmacology,XuanwuHospitalofCapitalMedicalUKeyLaboratoryforNeurodegenerativeDiseasesofMinistryofEducation,Beijing100053,ChinaSchoolofLifeScienceandTechnology,BeijingInstituteofTechnology,Beijing100081,ChinaarticleinfoabstractOxidativestress-inducedcelldamagehasbeenimplicatedinavarietyofneurodegenerativedisorders.Morroniside,aniridoridglycosideisolatedfromCornusof?cinalisSieb.EtZucc.,hasshownpotentantioxidantproperties.ThepresentstudyinvestigatedtheprotectiveactionsofmorronisideagainstthecytotoxicityproducedbyexposuretoH2O2(300C500μM)inSH-SY5Ycells.IntracellularaccumulationofCa2+,anddecreasesinmitochondrialmembranepotential(MMP)causedbyaddedH2O2werereducedbymorroniside.IncubationofcellswithH2O2causedamarkeddecreaseinsuperoxidedismutase(SOD)thisdecreasewassigni?cantlyinhibitedbymorroniside.Inaddition,thepercentageofcellsundergoingH2O2-inducedapoptosiswasdecreased,dosedependently,inthepresenceofmorroniside.Theseresultssuggestthatmorronisidehasprotectiveeffectsagainstoxidativestress-inducedneurotoxicprocesses.?2009ElsevierB.V.Allrightsreserved.Articlehistory:Received26August2008Receivedinrevisedform31March2009Accepted8April2009Availableonline18April2009Keywords:MorronisideCytotoxicityNeurodegenerativediseaseOxidativestressCornusof?cinalis1.IntroductionInChinesetraditionalmedicine,Cornusof?cinalisishighlyprizedforitstherapeuticabilities:reducingbloodglucose,immunologicalregulation,antishock,antiarrhythmia,antibiosis,etc.ThetotaliridoidglycosidesofC.of?cinaliscontainingloganin,morronisideandotherminorconstituentspossessanumberofpharmacologicalactivitiessuchasmitigatingthevascularcomplicationsofdiabetesandrepressingimmunity,rheumatoidarthritisandcerebralischemiaCreperfusioninjurythroughmultiplemechanismsofaction(Lietal.,).ItwasreportedthatthetotaliridoidglycosidesarethemainactiveconstituentsofC.of?cinalis.MorronisideisoneofthemostabundantiridoidglycosidesanditschemicalstructureisshowninFig.1.Wehadpreviouslyreportedthatincubationofcellswithmorronisideledtoasigni?cantdose-dependentelevationofcellularglutathione(GSH)accompaniedbyamarkedprotectionagainstH2O2-mediatedtoxicity,andmorronisideat1C100μMinhibitedtheformationofreactiveoxygenspeciesandtheactivationofcaspases-3and-9,andupregulatedBcl-2,whereasnosigni?cantchangesoccurredinBaxlevelsinSH-SY5Ycells(Wangetal.,2008).Inthecurrentstudy,weexploredtheabilityofmorronisidetoprotectagainstH2O2-inducedcytotoxicityinhumanSH-SY5Ycells.HumanneuroblastomaSH-SY5Ycellsthatdifferentiateintoneuron-likecellsaftertreatmentwithretinoicacidwereusedasanexperimentalparadigmbecausetheyaresensitivetoseveralinducersofapoptosisandcytotoxicity,suchasdopamine,glutamateandH2O2(Gaoetal.,2008;Miglioaetal.,2004;Ruffelsetal.,2004;Ubertietal.,2002;Zhangetal.,2007;Zhaoetal.,2008).ThemechanismsunderlyingtheprotectiveeffectsofmorronisideinSH-SY5YcellswereduetoinhibitionoftheaccumulationofintracellularCa2+,aswellastoanormalizationofsuperoxidedismutase(SOD)levels,restorationofthemitochondrialmembranepotential(MMP),andinhibitionofapoptosis.2.Materialsandmethods2.1.MaterialsMEMmedium,fetalbovineserum,andphosphate-bufferedsaline(PBS)wereobtainedfromGibcoLifeTechnologies(Heidelberg,Germany).PropidiumIodide(PI),Rhodamine123(Rh123),bovineserumalbumin(BSA)andlactatedehydrogenase(LDH)activitykitswerepurchasedfromSigma(St.Louis,IL.,USA).Fura2-AMwasfromBiotium.SODwaspur-chasedfromNanjingJianchengBioengineeringInstitute(China).VitaminEwaspurchasedfromXiamenXinsaCo.Ltd(China).Allotherchemicalswereofanalyticalgrade.2.1.1.MorronisideextractionThesarcocarpofC.of?cinaliswaspurchasedfromTong-Ren-TangCompany,Beijing,China,andauthenticatedbyProfessorWenWang.Theair-driedmaterialwaspowderedandextractedwithwater.Theresultingsolutionwasconcentratedtogenerateanaqueousresidue.Theresiduewasextractedwith80%ethanol,andtheresultingsolutionwasagainextractedwithethanol.Afterdissolvingtheresidueinwater,theaqueoussolutionwasconcentratedandchromatographedonan?Correspondingauthor.Tel.:+;fax:+.E-mailaddress:(L.Li)./$Cseefrontmatter?2009ElsevierB.V.Allrightsreserved.doi:10.1016/j.ejphar.20W.Wangetal./EuropeanJournalofPharmacology613(Fig.1.Structureofmorroniside.SP70resincolumnusing20%EtOHastheeluant.The20%EtOHeluatewasconcentratedandthenchromatographedonasilicagelcolumnusingCHCl3CMeOHeluantmixtures(15:1C8:1)toyieldacrudeiridoidglycosidefraction.Thisfractionwasre-chromatographedonasilicagelcolumnusingCHCl3CMeOHeluantmixtures(10:1C6:1)togenerateawhitecrystallineproduct.Highperformanceliquidchromatography(HPLC)analysisconsistedofaYMCCo.(Dinslaken,Germany)C18column(250mm×4.6mm,10μm)atacolumntemperatureof35°C.GlycosideswereelutedwithanacetonitrileCwatermixture(15:85)ata?owrateof1.0ml/minandadetectionwavelengthof240nm.Their?nalpuritywasdeterminedtobe98.5%.2.2.CellcultureandtreatmentHumanneuroblastomaSH-SY5YcellswereculturedinMEMmediumsupplementedwith10%(v/v)fetalbovineserum(FBS),100units/mlpenicillin/streptomycinand15mMHEPESinawater-jacketedincubatorat37°Cinahumidi?edatmosphereof5%CO2and95%air.Cellswerefedevery3daysandsubculturedoncetheyreached80C90%con?uence.SH-SY5Ycellswerepre-treatedfor24hwithdifferentconcentrationsofmorroniside.ThemediawerethenremovedandcellswerewashedoncewithsupplementedMEM.Subsequently,cellswerecontinuouslyculturedinMEMmediacontaining300C500μMH2O2for2C18h.H2O2wasdissolvedintheMEMmedium,whilemorronisidewasdissolvedinPBS,andvitaminEwasfreshlypreparedasastocksolutionindimethylsulfoxide(DMSO,?nalmediumconcentra-tionof0.05%).The?nalconcentrationofDMSO(0.05%,v/v)didnotaffectcellviability.2.3.CytotoxicityassayLactatedehydrogenase(LDH)releasewasmeasuredasaninvitromarkerforcellulartoxicity.Aftercellswereexposedto500μMH2O2inthepresenceofmorronisideorvitaminEfor24h,themediumwascollected.TheamountofLDHreleasedbycellswasdeterminedusinganassaykitaccordingtothemanufacturer'sprotocol,andtheabsorbanceofthesampleswasreadat490nm.LDHleakagewasexpressedasthepercentage(%)ofthetotalLDHactivity(LDHinthemedium+LDHinthecell),accordingtothefollowingequation:%LDHreleased=(LDHactivityinthemedium/totalLDHactivity)×100.2.4.MeasurementofactivityofSODTodeterminetheactivityofSOD,cellswereincubatedin6-wellplateswiththeindicatedconcentrationsofmorronisideorvitaminEfor24h,andthenincubatedinthepresenceof500μMH2O2for18h.Thecellsuspensionwascentrifuged(4000g,5min,4°C),andthecellpelletswerethenultrasonicatedfor10min(every1minwith1minintervals)at4°Cin1mlPBS.SODactivityreactionsystem,containing0.2mlofenzymeextract,2mlofSODIsolution,and1mlofSODIIsolution,wasilluminatedat4000lxfor15min.SODIsolutionisthe0.05Mphosphatebuffer(pH7.8)containing3mg/mlmethionine,0.1mg/mlNBT(nitrobluetetrazolium),and0.05mg/mlEDTA;SODIIsolutionisthe0.05Mphosphatebuffer(pH7.8)containing2.26lg/mlribo?avin.Absorbancewasreadat550nmandtheactivityofSODwascalculatedusingtheformula:[(controlvalue?blankvalue)?(samplevalue?blankvalue)]/(controlvalue?blankvalue)×2×(totalvolume/samplevolume)/proteinconcentration.2.5.MeasurementofintracellularCa2+([Ca2+]i)[Ca2+]iwasmonitoredusingthe?uorescentCa2+-sensitivedye,Fura2-acetoxymethyester(Fura2-AM)(Liuetal.,2001).Acon?uentmonolayerofSH-SY5Ycellswaspre-treatedwith1C100μMmorroni-sideor10μMvitaminEfor24hbeforeincubationinthepresenceof300μMH2O2for18h.Brie?y,cellsweretrypsinized,pelleted,resuspendedinmediumandincubatedwith5μMFura-2/AMinHEPES-buffercontaining(inmM):NaCl110,KCl2.6,MgSO41.0,CaCl21.0,HEPES25andglucose11(pH7.4)for40minat37°Candthenwashedtwicetoremoveanyextracellulardye.Thecellnumberwasadjustedto1millioncells/ml.Alterationsin?uorescenceintensityweremonitoredbya?uorescencespectrophotometer(LS-45,PerkinElmer,USA).Excitationwavelengthwas340/380emissionwavelengthwas510nm.The?uorescenceratio(F340/F380)wascalculatedasanindicatorof[Ca2+]i(HaoandLiu,.Measurementofmitochondrialmembranepotential(MMP)Mitochondrialmembranepotentialwasmonitoredusingthe?uorescentrhodaminedye,Rh123.ThisisacellpermeablecationicdyethatpreferentiallypartitionsintomitochondriabasedonthehighlynegativeMMP.DepolarizationoftheMMPresultsinthelossofRh123fromthemitochondriaandanincreaseinintracellular?uo-rescence(Satohetal.,1997).Rh123wasaddedtocellculturestoachievea?nalconcentrationof10μMfor30minat37°CafterthecellsweretreatedwithmorronisideplusH2O2asdescribedabove.ThecellswerecollectedbypipettingandwashedtwicewithPBSandthenanalyzedby?owcytometry.Thelaserwasadjustedtoemitat480nm,anda530nmlong-pass?lterwasused.2.7.FlowcytometricdetectionofapoptoticcellsCellswerecollectedbycentrifugation(310×g)24hafterH2O2exposureandtheywerewashedwithPBS.Thepelletswereresuspendedinicecold70%ethanoland?xedat4°Cfor24C48h.Cellswerethencentrifuged,andtheethanolwasremovedbywashingthoroughlywithPBS.Thecellpelletswereresuspendedin1mlDNAstainingreagentcontaining50μg/mlRNase,0.1%tritonX-100,0.1mMEDTA(pH7.4),and50μg/mlpropidiumiodide(PI).Stainingwasstableat4°Cfor30min(Telfordetal.,1991).Red?uorescence(DNA)wasdetectedthrougha563C607nm?lterusingaFACS440?owcytometer(BectonDickinson,FranklinLakes,NJ,USA).Forthe?owcytometrichistograms,apoptoticcellsexhibitedDNA?uorescenceinthesubdiploidregions,whicharewellseparatedfromthenormalG1peak.Tenthousandcellsineachsamplewereanalyzedandthepercentageofapoptoticcellsaccumulatedinthesub-G1peakwascalculated.2.8.StatisticalanalysisAllresultswereexpressedasmean±S.E.M.ofatleastsixinde-pendentexperiments,andanalyzedusingone-wayANOVA.Whereasigni?cantdifferencewasdetected,theLSDpost-testwasapplied.Pb0.05wasdeemedtoindicateasigni?cantdifference.3.Results3.1.Morronisideidenti?cationMorroniside,intheformofawhitecrystal,possessedmassspec-trometry(MS)valuesofm/z425+(M+NH4)and405?(M-H).The1H-NMRspectrum(400MHz,D2O)andthe13C-NMRspectrum(100MHz,D2O)showedasignalpattern(Wangetal.,2008)thatwasidenti?edasthemorronisidecompound(Fig.1)bycomparingitsspectraldatawiththosepreviouslyreported(Yangetal.,2005).Itspuritywas98.5%asdeterminedbyHPLC(Fig.2).W.Wangetal./EuropeanJournalofPharmacology613(1Fig.2.TheanalysiswasconductedbyRP-HPLC,andconsistedofaYMC-C18column(250mm×4.6mm,10μm)withacolumntemperatureof35°C.TheeluatewasacetonitrileCwater(15:85)witha?owrateof1.0ml/minandadetectionwavelengthat240nm.Itspuritywas98.5%.3.2.MorronisideprotectedSH-SY5YcellsagainstH2O2-inducedcytotoxicityToinvestigatetheprotectiveeffectsofmorroniside,anassaytodetectLDHwasused.LDHisastablecytoplasmicenzymepresentinallcells,anditisrapidlyreleasedintothecellculturesupernatantupondamagetotheplasmamembrane.Thus,anincreaseinthenumberofdeadorplasmamembrane-damagedcellsresultsinanincreaseinLDHactivityintheculturesupernatant.AsshowninFig.3,asigni?cantincreaseinLDHreleasewasobservedafteran18hexposureto500μMH2O2,therebyre?ectinganincreaseincelltoxicity.MorronisideandvitaminEsigni?cantlyattenuatedthisincreaseinLDHrelease.3.3.ProtectiveeffectsbymorronisideoninducibilityofSODAsshowninFig.4,theactivityofSODinSH-SY5Ycellsexposedto500μMH2O2for18hwassigni?cantlylower(?40%)thanvaluesforthenormalcontrolgroup.Morronisidesigni?cantlyenhancedSODactivityinthecellspre-treatedwithH2O2.3.4.MorronisideinhibitedH2O2-induced[Ca2+]iincreasesinSH-SY5YcellsInaKrebs'solutioncontainingcalcium,300μMH2O2exposureinducedanincreaseinthe?uorescenceratio(F340/F380)from100%incontrolsto163%inH2O2-exposedcells.Whenthecellswerepre-treatedwith1,10,and100μMmorronisideor10μMvitaminE,F340/F380wasFig.3.EffectofmorronisideonH2O2-inducedLDHinSH-SY5Ycells.Cellswerepre-treatedwithdifferentconcentrationsofmorroniside(1C100μM)orvitaminE(10μM)for24handthenincubatedintheabsenceorinthepresenceof500μMH2O2for18h.TherelativeLDHreleaseratewasthenmeasured.Therewasasigni?cantincreaseinLDHactivityofcellsreceiving500μMH2O2comparedtocontrolvalues.Valuesrepresentmeans±S.E.M.of6independentexperiments(###Pb0.001comparedwi???Pb0.001comparedwithonlyH2O2treatmentgroup).Fig.4.EffectofmorronisideontheH2O2-inducedactivationofSODinSH-SY5Ycells.Cellswerepre-treatedwith1C100μMmorronisideor10μMvitaminEfor24h,andthenincubatedinthepresenceof500μMH2O2for18h.ProteinwasextractedandtheactivityofSODwasdetectedbyaSODkit.Valuesrepresentmeans±S.E.M.of6differentexperiments(##Pb0.01comparedwi??Pb0.01and?Pb0.05comparedwithonlyH2O2treatmentgroup).reducedfrom163%to143%,124%,110%,and116%,respectively(Fig.5).TheH2O2-inducedincreaseinintracellularCa2+wasdependentonextracellularCa2+,sinceremovalofextracellularCa2+preventedtheH2O2-inducedincreasein[Ca2+]i.3.5.MorronisidepreventedlossofMMPinSH-SY5YcellsToassesstheeffectofmorronisideonthechangesinMMPinducedbyH2O2inSH-SY5Ycells,?owcytometricanalyseswerecarriedoutusingRh123.Thisdyeisalipophiliccation,anditsuptakebymito-chondriaisproportionaltotheMMP.AfterincubationofSH-SY5Ycellswith300μMH2O2for18h,the?uorescenceofRh123increasedto92?ovecontrolvalues,whichrepresentedalossofMMP.Pretreatmentwithdifferentconcentrationsofmorroniside(1,10,and100μM)and10μMvitaminEprotectedcellsagainsttheH2O2-inducedlossofMMP―inmorronisidetreatedcellsthe?uorescencewaselevatedtoonly20?ovecontrolvalues(Fig.6).3.6.MorronisideprotectedSH-SY5YcellsagainstH2O2-inducedapoptosisTotestwhetherH2O2-inducescelldeathviaapoptosis,thepercentageofapoptoticcellswasmeasuredby?owcytometry.Thesub-G1peakfrom?owcytometrydetectionisconsideredanindicatorofcellapoptosis.OurresultsshowedthattreatmentofcellswithFig.5.Effectofmorronisideon[Ca2+]iincreasesinducedby300μMH2O2inSH-SY5Ycells.Cellswerepre-treatedwith1C100μMmorronisideor10μMvitaminEfor24h,thenincubatedinthepresenceof300μMH2O2for18h.Followingtreatment,cellswereloadedwith5μMFura-2/AMovera30minperiodat37°C.[Ca2+]iwasmeasuredbyFura-2/AM340/380nm?uorescenceratio(F340/F380).Valuesrepresentmeans±S.E.M.of6experiments(###Pb0.001vs.??Pb0.01vs.onlyH2O2treatmentgroup).22W.Wangetal./EuropeanJournalofPharmacology613(Fig.6.EffectofmorronisideonH2O2-inducedlossofmitochondrialmembranepotential.Cellsweretreatedwith300μMH2O2for18hperiod.Varyingconcentrations(1C100μM)ofmorronisideor10μMvitaminEwereaddedtothemedium24hbeforeH2O2addition.Followingtreatment,cellswereloadedwithRh123,and?uorescencewasmonitoredovera30minperiodat37°C.TheMMPwasquanti?edthroughRh123?uorescence.Valuesrepresentmeans±S.E.M.of10experiments(##Pb0.01vs.??Pb0.01and*Pb0.05vs.onlyH2O2treatmentgroup).500μMH2O2for24hsigni?cantlyinducedexpressionofthesub-G1peak,indicatinganaccumulationofapoptoticcellsabove60%.Morroniside(1,10,and100μM)and10μMvitaminEsigni?cantlyattenuatedH2O2-inducedapoptoticcellaccumulationinthesub-G1peak(Fig.7).4.DiscussionOxidativestresshadbeenwidelyimplicatedintheneuronalcelldeaththatisassociatedwithavarietyofchronicneurodegenerativedisorderssuchasParkinson'sdisease(Zhangetal.,2000),Alzheimer'sdisease(Behl,1999)andHuntington'sdisease(Alexietal.,2000).Therearenumerousinducersofoxidativestressthatareabletocausecytotoxicityandapoptoticdeath.Forinstance,hydrogenperoxide(H2O2)caninducecytotoxicityandapoptosisinmanydifferentcelltypes,andthiseffectcanbeblockedbytheadditionofantioxidants(JangandSurh,2001;Gaoetal.,2008;Gupta2004;Zhangetal).Inresponsetosuchoxidativedamage,acomplexantioxidantdefencemechanismhasdeveloped,andnaturalantioxidantsserveanimportantroleinthisprocess.Therefore,itisofgreatinteresttotheFig.7.InhibitoryeffectofmorronisideonH2O2-inducedapoptosisinSH-SY5Ycells.Cellswerepre-treatedwith1C100μMmorronisideor10μMvitaminEfor24h,thenincubatedinthepresenceof500μMH2O2for24h.Cellswere?xedwithcold70%ethanolat4°C,andthenresuspendedinPIstain,andthered?uorescencewasdetectedby?owcytometry.Thepercentageofapoptoticcellaccumulationinthesub-G1peakwascalculated.Valuesrepresentmeans±S.E.M.of6experiments(###Pb0.001vs.???Pb0.001,and??Pb0.01vs.onlyH2O2treatmentgroup).healthcarecommunitytosearchforpotentialantioxidantsexistinginnaturalproductsandherbalpreparationsfortherapeuticapplications(Wangetal.,2000;WengandWang,2000).WedemonstratedthatexposureofcellstoH2O2causesarapidincreaseinreactiveoxygenspeciesproductionasdeterminedbyadramaticincreaseintheintensityofDCF-labelledcells(Wangetal.,2008).TheresultspresentedinthisstudyclearlyshowedthatpretreatmentofSH-SY5Ycellswithmorronisidefor24hpriortoexposuretoH2O2for18hresultedinsigni?cantlyincreasedlevelsofSOD.Inadditiontoproducinganincreaseinreactiveoxygenspeciesandconsequentlipidperoxidation,H2O2exposurecausedanelevationofintracellularCa2+levels.ThiscalciumsignallingrequiredextracellularCa2+andledtothedepolarizationoftheMMPinSH-SY5Ycells.TheoccurrenceoflargeincreasesinintracellularCa2+representsadetrimentalconsequenceofoxidativestressmediatedbythegenerationofreactiveoxygenspeciesinthesecells.SustainedelevatedCa2+levelsincellsmayimpairmitochondrialfunctionandmayactivatephospho-lipases,proteases,andendonucleases,leadingtoirreversiblemembrane,organelle,andchromatindamage,andeventuallytocelldeath.Weusedthecationic?uorescentdyeRh123,whichissequesteredinmitochon-driaduetoitshighlynegativeMMPandisreleaseduponmitochondrialdepolarization(Satohetal.,1997).UsingRh123localizationasameasureofmitochondrialintegrity,wefoundthat300μMH2O2ledtomitochondrialmembranedepolarization.MorronisidereducedtheriseofMMP,whichmaybeduetoinhibitionoftheintracellularaccumula-tionofreactiveoxygenspeciesandCa2+.Inthepresentwork,exposureofSH-SY5YcellstoH2O2resultedinsigni?cantincreasesinintracellularreactiveoxygenspeciesandCa2+,andtriggeredDNAdamageasassessedbyaDNAsensitivedyewitha?owcytometer.Thistreatmentcausedasigni?cantsub-G1peakinthecytometryhistograms,andproducedcytotoxicityasevaluatedbyanLDHassay,indicatingthepresenceofanapoptoticcomponentinH2O2-inducedcellinjury.Inaddition,morronisidesigni?cantlyattenuatedtheapoptosistriggeredbyH2O2inSH-SY5Ycells.OnepossibleunderlyingmechanismunderlyingtheeffectivenessofmorronisideinprotectingagainstH2O2neurotoxicitymayinvolveitsstructure(Fig.8).Ifthereisanelectrondonatinggroup,especiallyahydroxylgrouplocatedontheo-orp-positionsofphenoliccompounds,theirantioxidantactivitiesareincreasedgreatly.Also,thisisoneofthemainreasonswhymorronisidehassuchstrongantioxidantactivitiesbecausethesegroupsallowthephenolstomoreeasilydonatehydrogenatomstoactivefreeradicalstointerruptthechainreactionofauto-oxidation(Wangetal.,2008).Insummary,H2O2causesapoptoticandnecroticcelldeathinSH-SY5Ycellsthroughoxidativestress.MorronisideprotectsSH-SY5YcellsfromH2O2-inducedtoxicitybyinterruptingthecelldeathcascadeatthefollowingthreedistinctsteps:(1)blockingreactiveoxygenspeciesproduction,(2)inhibitingCa2+in?ux,(3)andpreventingMMPde-creases.Theelucidationofthemechanismsunderlyingthecytotoxicitycausedbyoxidativestressandtheprotectionbymorronisidemayprovideadditionalinsightsintothemolecularbasisofthechemopro-tectiveeffectsofthisantioxidant.Wepreviouslyreportedthatmorronisidepreventsperoxide-inducedapoptosisbytheinductionofGSHexpression,inhibitionoftheformationofreactiveoxygenspecies,andtheactivationofcaspases-3and9,andupregulationofBcl-2inSH-SY5Ycells(Wangetal.,2008).NowwereportthatmorronisideprotectshumanneuroblastomaSH-SY5Ycellsagainsthydrogenperoxide-inducedcytotoxicity.ThismightFig.8.Mechanismofmorronisidepossessingantioxidanteffectsfromstructure.W.Wangetal./EuropeanJournalofPharmacology613(3helpinthedesignanddevelopmentofdrugsorregimenstocontrolandpreventoxidativestress-mediatedneurotoxicprocess.AcknowledgmentsThisresearchwassupportedbygrantsfromtheProgramFounda-tion:Chinese“973”programfoundation(No:);NationalNaturalScienceFoundationofChina(No:);BeijingNaturalScienceFoundation,China(No:7062031);Base-clinicalFoundationofCapitalMedicalUniversity,China(No:2006JL05);andtheKeyLaboratoryforNeurodegenerativeDiseasefromtheMinistryofEduca-tionFoundation,China(No:802).ReferencesAlexi,T.,Borlongan,C.V.,Faull,R.L.M.,Williams,C.E.,Clark,R.G.,Gluckman,P.D.,Hughes,P.E.,2000.Neuroprotectivestrategiesforbasalgangliadegeneration:Parkinson'sandHuntington'sdiseases.Prog.Neurobiol.60,409C470.Behl,C.,1999.Alzheimer'sdiseaseandoxidativestress:implicationsfornoveltherapeuticapproaches.Prog.Neurobiol.57,301C323.Gao,M.,Zhang,W.C.,Liu,Q.S.,Hu,J.J.,Liu,G.T.,Du,G.H.,2008.Pinocembrinpreventsglutamate-inducedapopto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