1,25（OH）2D3对谷氨酸诱导HT-22细胞损伤保护作用研究.pdf 7页

'中国科技论文在线http://www.paper.edu.cnProtectiveEffectof1,25(OH)2D3onGlutamate-Induced#CytotoxicityinHT-22Cell12341**5MaShujie,WangJianrong,HeJinjiang,LiBingyan,ZhangZengli(1.DepartmentofLaborHygieneandEnvironmentalHealth,SchoolofPublicHealthofSoochowUniversity,Suzhou215123;2.TheFifthAffiliatedHospitalofXinjiangMedicalUniversity,Urumqi830011;3.ShanghaiEntry-ExitInspectionandQuarantineBureauHongkouOffice,Shanghai200135;104.DepartmentofNutritionandFoodHygiene,SchoolofPublicHealth,SoochowUniiversity,Suzhou215123)Abstract:1,25(OH)2D3deficiencymaybeassociatedwithaffectivedisorders,Parkinson"sdisease,Alzheimer"sdiseaseandcognitiveabilities.Toexploretheprotectiveeffectsof1,25(OH)2D3againstglutamate-induceddamageinamouse-derivedhippocampalHT-22cellsandunderlyingpotential15mechanism.ThecellsurvivalratewasdeterminedwithCellCountingKit-8.Cellcycle,apoptosisandreactiveoxidativespecies(ROS)releaseweredetectedbyFACstechnology.Resultsshowedthatpretreatment1,25(OH)2D3effectivelyincreaseHT-22cellviabilityuponglutamateexposure.Thismightbeassociatedwiththefactthat1,25(OH)2D3significantlyreducedthepercentageofG0/G1circles(G0/G1%)(p<0.01)，increasedtheproportionofSphase(S%)andG2/Mphase(G2/M%)cells20(p<0.01).Moreover,apoptosisinHT-22cellsshowedthelatecellapoptosisratewasdecreased(p<0.05)inglutamategroupincubatedwith1,25(OH)2D3(i.e.combinationgroup)comparedwiththeglutamateonlygroup.Therewasaninverseassociationbetweentheconcentrationof1,25(OH)2D3andapoptosisrate.Meanwhile,theROSreleasewaselevatedinthecombinationgroup(p<0.05).Ourstudyindicatesthat1,25(OH)2D3mightadverselyaffectglutamate-inducedapoptosisintheHT-2225cells，thismightbeowingtothefactthat1,25(OH)2D3couldincreasecellproliferationanddecreaselateapoptosisrate.1,25(OH)2D3mightbeapromisingtherapeuticagentforthetreatmentofdepression.Keywords:1,25-dihydroxyvitaminD3;Glutamate;HT-22cells;Neurotoxicity300Introduction[1]Depressionisaverycommonchronicdiseaseofthecentralnervoussystem,whichischaracterizedbyhighprevalence,highrecurrentrate,highmorbidity,highsuiciderate,lowrecognition[2;3;4]rateandlowtreatmentrate.Hippocampusandforebrainrelatedbrainatrophy,reducedthe[5;6;7]numberofhippocampalneurons,hippocampalnerveaplasiaandthereleaseofglutamate35increasewerefoundindepressionpatients.VitaminD3,aneedednutrientsforbodygrowthanddevelopment，playsacriticalroleinmanydifferentphysiologicalprocesses，whichclassicalroleis[8]boneandcalciummetabolism.VitaminD3istakenupfromthedietorproducedintheskinfrom[9]7-dehydrocholesterolbyexposuretoultravioletradiation.Subsequently,vitaminD3isconvertedto25-hydroxyvitaminD3intheliverandto1,25-dihydroxyvitaminD3[1,25(OH)2D3],theactiveform[10]40ofvitaminD3inthekidney.WiththedeepdevelopmentofvitaminD3studies，thestudyonnon-classicalrolehasdrawnincreasingattentioninrecentyears，especiallytheregulationofthecentral[11;12;13]nervoussystemmetabolismanddevelopmentarebeingpaidmoreattentionthanbefore.AnumberofstudieshaveshownthatvitaminD3hasbeenimplicatedinmanyacuteandchronic[12;14;15;16]neurologicaldisorders,includingdepressionandAlzheimer"sdisease(AD).Morerecent[17]45findingssuggestthat1,25(OH)2D3mayaffectbraindevelopmentandfunction.Inthestudy,micehippocampusneuroncelllinesHT-22waspretreatedwithglutamateandsubsequentlyintervenedwith1,25(OH)2D3.Theaimofthestudywastoexploretheprotectiveeffectof1,25(OH)2D3onFoundations:theNationalNaturalScienceFoundationgrants（No.81372981,81372979）Briefauthorintroduction:MaShujie(1991-),Female,Graduation,nourishmentandchronicdiseaseCorrespondanceauthor:ZhangZengli(1967-),Male,Professor,Nourishmentandchronicdisease.E-mail:zhangzengli@suda.edu.cn-1- 中国科技论文在线http://www.paper.edu.cnglutamate-induceddamageintheHT-22cellsanditspotentialmechanism,providinganewmethodfornervoussystemdiseasepreventionandcontrol.501MaterialsandMethods1.1ReagentsHippocampalHT-22cellwasagiftfromDr.XingshunXu(SooChowUniversity,Suzhou,Jiangsu,China).Dulbecco’smodifiedEagle’sMedium(DMEM)andfetalbovineserum(FBS)wereobtainedfromGibcoCorporation(Gibco,USA).Penicillin-streptomycinsolutionand0.25%trypsin-EDTA55solutionwerepurchasedfromInvitrogenLifeTechnologies(Carlsbad,CA,USA).1,25(OH)2D3,glutamate(Glu)andPropyliodideorganism(PI)weretheproductofSigma-Aldrich(St.Louis,MO,-5USA).1,25(OH)2D3wasdissolvedinethanolandstoredinaconcentratedsolution(10mol/L)at-80˚C.CellCountingKit-8(CCK-8)wasboughtfromJapan"schemicalresearchinstitute(DojindoLaboratories,Kumamoto,Japan).AnnexinV-FITC/PIapoptosiskitwasobtainedfromBiouniqure60Corporation.Fluorescentprobe(H2DCFDA)wastheproductofInvitrogenCorporation(Carlsbad,CA,USA).1.2CellcultureHippocampalHT-22cellsweremaintainedinDulbecco’sModifiedEagleMedium(Gibco,USA)containing10%fetalbovineserum,1%antibiotics(100U/mLpenicillinand5mg/mLstreptomycin)65andgrownontissueculturedishesor96-wellplates.Thecellswereculturedatapproximately80%confluencyandstarvedinserum-freeDMEM(SF-DMEM)overnight.Removeculturemedium,HT-22cellsweretreatedwithdifferentfactors,respectivelycontrolgroupweretreatedwithanhydrousethanol,glutamategroup(5mmol/L),1,25(OH)2D3grouptreatedwithdifferentconcentrations1,25(OH)2D3(0.5,5,50nM)andglutamate+1,25(OH)2D3groupincludeglutamate5mmol/Land1,25(OH)2D370(0.5,5,50nM).1.3CellViabilityAssayHT-22cellsduringlogarithmicphasewereplantedin96-wellplates(1×103/dish)andmaintainedinaCO2incubatorfor12hat37˚C.Afterthecellsadherentonplate,mediawereremoved,then200µlofDulbecco’sModifiedEagleMediumsupplementedwithvariousconcentrationsof1,25(OH)2D375wasaddedtoeachwell.Followingtheindicatedtreatments,foreachgroup,sixindependentexperimentswereperformed.Cellswerepretreatedfor72hwith1,25(OH)2D3beforestimulationwithglutamate,thenmediawereremovedaftercellswereincubatedwithglutamatefor12h.Furthermore,200µlofDMEMcontaining10%fetalbovineserumwasaddedtoeachwell,andthecellsweremaintainedingrowthmediumfor12h.Andthenrespectivelyadd10µlCCK-8solution,followingcells80wereincubatedfor1h.Theabsorbancewasreadinamicroplatereader(BioTecInstruments,Inc.,Winooski,VT,USA)at450nm.Thepercentageofsurvivingcellswascalculatedasthepercentagedifferenceofthetreatedcellsversusthevehiclecontrols,accordingtothefollowingformula:viabilityrate(%)=Absoftreatedcells/Absofvehiclecontrolcellsx100.Andtheexperimentwasrepeatedatleastthreetimes.851.4CellCycleAnalysisThepercentagesofcellsinG1/G0,S-phase,andG2/MphasesweredeterminedforHT-22cell5linesusingflowcytometry.Cellsatexponentialphasewereseededintoa60-mmPetridish(1×10/dish)andwereincubatedinCO2incubatorat37°Cfor24h.Afterthecellsadherentonplate,mediawereremoved,treatmentgroupincubatedwith1,25(OH)2D3(0.5,5,50nM),glutamate(5mmol/L),90andglutamate(5mmol/L)+1,25(OH)2D3(0.5,5,50nM).Foreachgroup,threeindependentexperimentswereperformed.Cellswerecollectedafterfixinginglutamatefor12h.Thefixedcells-2- 中国科技论文在线http://www.paper.edu.cnwerestainedinpropidiumiodide(PI)buffer.Thesampleswereincubatedfor15minatroomtemperaturepriortocellcycleanalysisusingaFC500flowcytometer(BeckmanCoulter,Fullerton,CA,USA).951.5ApoptosisAssay5HT-22cellswereseededintoa60-mmPetridish(1×10/dish)atadensityof80%confluenceandwereincubatedinCO2incubatorat37°Cfor24h.Followingtreatment,thecellswereharvestedandwashedwithPBS,thensuspendedinPBSwithPIandAnnexinV.Thecellsuspensionswereincubatedinthedarkfor15minat37˚CandthenanalyzedonaFC500flowcytometer.1001.6MeasurementofintracellularreactiveoxygenspeciesTheintracellularaccumulationofROSintheHT-22cellswasdeterminedusingH2DCF-DA.5Cellsatlogarithmicphasewereputintoa60-mmPetridish(1×10/dish)for24h.Afterthecellsadherentonplate,treatmentgroupincubatedwithdifferentfactors,anhydrousethanol,1,25(OH)2D3(0.5,5,50nM),glutamate(5mmol/L),andglutamate(5mmol/L)+1,25(OH)2D3(0.5,5,50nM).105Foreachgroup,therewerethreeindependentexperiments.12hafterglutamatetreatmentandcellswerecollected.ThecellswerewashedtwiceinPBS,andsubsequentlythecellswerestainedwith10µmol/LH2DCFDAfor30min,at4℃inthedark.Fluorescencedatawerecollectedwithanexcitationwavelengthof488nmandanemissionwavelengthof525nm.MeanfluorescenceintensitywasusedasmeasureofROSlevelasdeterminedbyflowcytometer.1101.7AnalysisofthedataAllvaluesreportedwereexpressedasmeans±SEM.AllstatisticalanalyseswereperformedusingtheSPSS18.0software.Statisticaldifferencesamonggroupsweretestedbyone-wayANOVA.ThevalueP<0.05wasconsideredasstatisticallysignificantdifference.115Tab.1Theeffectsof1,25(OH)2D3and/orglutamateoncellcycledistributionGroupG0/G1(%)S/(%)G2/M(%)Control36.10±1.2856.43±2.387.47±1.960.5nM1,25D337.82±5.4755.08±7.787.10±2.33*****5nM1,25D339.56±2.4450.40±3.3710.05±3.44*****50nM1,25D338.34±1.6449.23±3.6512.43±2.16****Control+Glu52.69±4.6240.48±5.176.84±2.18**##**##0.5nM1,25D3+Glu46.89±2.7844.12±2.198.99±1.53**##**##**##5nM1,25D3+Glu45.14±0.5944.80±4.5410.06±3.95**##**##**##50nM1,25D3+Glu41.20±1.4047.70±2.5311.09±1.14Note:Cellswereplatedin60-mmdishesandtreateddifferentconcentrationsof1,25(OH)2D3and/or5mMglutamate(Glu).CellcyclewasdeterminedbyFC500flowcytometerafter12htreatment.*p<0.05and**p<0.01,versuscontrolgroup;#p<0.05and##p<0.01,versusglutamategroup.-3- 中国科技论文在线http://www.paper.edu.cn120Fig.1Theeffectsof1,25(OH)2D3oncellviabilityinglutamate-treatedHT-22cells.Note:Cellswereplatedina96-wellplatesandtreateddifferentconcentrationsof1,25(OH)2D3and/or5mMglutamate(Glu).CellviabilitywasdeterminedbyaCellCountingKit-8assayafter12htreatment.*p<0.05and**p<0.01,versusglutamategroup.125Fig.2Theeffectsof1,25(OH)2D3and/orglutamateonapoptosisinHT-22cells.Note:Cellswereplatedin60-mmdishesandtreateddifferentconcentrationsof1,25(OH)2D3and/or5mMglutamate(Glu).CellapoptosiswasdeterminedbyFC500flowcytometerafter12htreatment.*p<0.05and**p<0.01,versusglutamategroup.130Fig.3Theeffectsof1,25(OH)2D3and/orglutamateonROSproductioninHT-22cells.Note:Cellswereplatedin60-mmdishesandtreateddifferentconcentrationsof1,25(OH)2D3and/or5mMglutamate(Glu).CellapoptosiswasdeterminedbyFC500flowcytometerafter12htreatment.*p<0.05,versuscontrolgroup;#p<0.05,versusglutamategroup.-4- 中国科技论文在线http://www.paper.edu.cn1352Results2.1Theeffectsof1,25(OH)2D3andglutamateonHT-22cellssurvivalrateToquantifytheprotectiveeffectof1,25(OH)2D3,HT-22cellsweretreatedwithvariousconcentrationsof1,25(OH)2D3for72hinthepresenceof5mmol/Lglutamate.CellviabilitywasmeasuredbytheCCK-8assay.Comparedwiththecontrol,1,25(OH)2D3alonedidnotaffectcell140viability.Afterincubationwithglutamatefor12h,1,25(OH)2D3reducedglutamate-inducedcelldeathinadose-dependentmanner(p<0.05)(Fig.1).2.2Theeffectsof1,25(OH)2D3andglutamateonHT-22cellcycleComparedwiththevehiclecontrol,glutamatesignificantlyincreasedthepercentageofcellsinG0/G1phase(p<0.01),accompaniedbyareductionofcellsinSandG2/Mphase.Afterbeingtreated1451,25(OH)2D3,asignificantdecreaseintheproportionofcellsinSphase(P<0.01),togetherwithasignificantincreaseintheproportionofcellsintheG0/G1(P<0.05)andG2/M(P<0.01)phaseswerefound.Followingtreatmentwithvariousconcentrationsof1,25(OH)2D3(0.5,5,50nM)and5mMglutamate,thedistributionofG0/G1phaseinHT-22cellswasslowlyreduced,whilecellsinSandG2/Mphasegraduallyincreased(Tab.1).1502.3Theeffectsof1,25(OH)2D3andglutamateonHT-22cellsApoptosisWhenHT-22cellswereincubatedwith1,25(OH)2D3inthepresenceofglutamatefor72h,itwasfoundthatglutamate-inducedincreaseofcellapoptosiswaspreventedby1,25(OH)2D3(0.5–50nM)inadose-dependentmanner(Fig.2).Lateapoptoticratein1,25(OH)2D3/glutamatecombinedgroupwassignificantlylowerthantheonlyglutamategroup(P<0.01).Itsuggeststhat1,25(OH)2D3can155inhibitglutamate-inducedapoptosis.2.4Theeffectsof1,25(OH)2D3andglutamateonHT-22cellsOxidativeDamageInthenextstudy,wefurtherinvestigatedtheeffectof1,25(OH)2D3andglutamateonthelevelofROS,biomarkersofoxidativestress,inHT-22cells.AsshowninFig.3,theexposuretoglutamateleadtoanevidentlyincreaseinROSlevel,comparedwithcontrol.However,the1,25(OH)2D3treatment160didnotpreventglutamate-inducedincreaseofROSinHT-22cell.3ConclusionRecentstudieshavesuggestedthatglutamateexcesscanresultinaformofcelldeathcalledglutamate-inducedoxytosis.HT-22cells,animmortalizedmousehippocampalcelllinelackingin[18]ionotropicglutamatereceptors,havebeenwidelyusedasamodelforthemechanisticstudyof[19;20]165glutamate-inducedneurotoxicity.Previousexperimentsofclinicalandanimalindicatethatthe[21;22]glutamatelevelsisrelatedwiththeprocessofdepressionhappened.1,25-DihydroxyvitaminD3[1,25(OH)2D3]isthemostactivemetaboliteofvitaminD3.Inrecentyears,thereisgrowingevidencethatoptimalvitaminDlevelsisrequirementforadultbrain.ManystudiesdemonstratethatvitaminDdeficiencymayhasalsobeenassociatedwithaffectivedisorders,Alzheimerdisease,Parkinsondisease,[23;24;25]170andcognitivedecline.Thepresentstudydemonstratesthat1,25(OH)2D3canpreventglutamate-inducedinjuryinaneuronalcelllineinadose-dependentmanner.Thebeneficialeffectof1,25(OH)2D3consistsindecreasinginapoptosisandmaintaincellcycle.Thissuggeststhat1,25(OH)2D3exertedapotentcytoprotectiveactionagainstglutamateinduceddamageinHT-22cell.175Cellgrowthiscloselyrelatedtothecellcycle.Andcellcycleextensionwillleadtoslowercellproliferation.Inthisstudy,glutamatealonewasdemonstratedtosignificantlyincreasethepercentageofG0/G1-phasecellsanddecreasethedistributionofSandG2/M-phasecells.Thisindicatesthat-5- 中国科技论文在线http://www.paper.edu.cnglutamatecausescellcyclearrestattheG0/G1checkpoint.Withrespecttoglutamatealone,afterbeingtreatedwith1,25(OH)2D3for72hbeforeglutamatewasgiven,whichobviouslydropthenumberof180cellsinG0/G1phase,andincreasetheamountofSandG2/Mphaseinadose-responserelationship.Thisresultshowsthat1,25(OH)2D3canameliorateglutamate-inducedG0/G1phasearrest.Thisresultindicatesthat1,25(OH)2D3playaprotectiveeffectforglutamate-inducedcytotoxicitythroughvariousmechanisms.[26]PreviousstudieshaveshownthatvitaminDmightfightoxidativedamage.However,the185resultsofthecurrentstudyindicatethat1,25(OH)2D3-treatedHT-22cellshaveraisedROSlevels.Afteradministratingofglutamate,intracellularROSlevelsaresignificantlyincreased.Combinedtreatmentwith1,25(OH)2D3andglutamateleadtoasignificantlyincreaseinintracellularROSlevelswhicharehigherthanthoseforglutamateonlygroup.Werepeatedtheexperimentseveraltimesandresultswereconsistent.IncreasingROShavebeenlinkedtomultiplepathologies,including190neurodegenerativediseases.However,ROSarealsosuggestedtoregulateawidevarietyofphysiology[27]assignalingmolecules.Oxidativestressisnotpathologicatanysituation.Sometimesitmightbephysiologicalrole.Thereasonwhich1,25(OH)2D3stimulatedROSlevelinHT-22cellinthestudyisnotunderstoodcurrently.Inconclusion,thepresentstudydemonstratesthat1,25(OH)2D3antagonismglutamate-induced195HT-22cellsdeath.VitaminDmightplayanincreasinglyimportantroleinthepreventionandtreatmentofcentralnervoussystemdiseases.AcknowledgementsThestudywasfundedbythepriorityacademicprogramdevelopmentofJiangsuhighereducationinstitutionsandtheNationalNaturalScienceFoundationgrants(81372981,81372979).200References[1]CaspiA,SugdenK,MoffittTE,etal.Influenceoflifestressondepression:moderationbyapolymorphisminthe5-HTTgene[J].Science,2003,301(5631):386-389.[2]KesslerRC,BerglundP,DemlerO,etal.Theepidemiologyofmajordepressivedisorder:resultsfromtheNationalComorbiditySurveyReplication(NCS-R)[J].Jama,2003,289(23):3095-3105.205[3]BakishD.Newstandardofdepressiontreatment:remissionandfullrecovery[J].TheJournalofclinicalpsychiatry,2001,62(Suppl26):5-9.[4]GuzeSB,RobinsE.Suicideandprimaryaffectivedisorders.TheBritishjournalofpsychiatry[J].thejournalofmentalscience,1970,117(539):437-438.[5]ZhuXH,YanHC,ZhangJ,etal.Intermittenthypoxiapromoteshippocampalneurogenesisandproduces210antidepressant-likeeffectsinadultrats[J].TheJournalofneuroscience:theofficialjournaloftheSocietyforNeuroscience,2010,30(38):12653-12663.[6]MervaalaE,FohrJ,KononenM,etal.QuantitativeMRIofthehippocampusandamygdalainseveredepression[J].PsycholMed,2000,30(1):117-125.[7]CotterD,MackayD,LandauS,etal.Reducedglialcelldensityandneuronalsizeintheanteriorcingulate215cortexinmajordepressivedisorder[J].Archivesofgeneralpsychiatry,2001,58(6):545-553.[8]HolickMF.ResurrectionofvitaminDdeficiencyandrickets[J].TheJournalofclinicalinvestigation.2006,116(8):2062-2072.[9]HolickMF.SunlightandvitaminDforbonehealthandpreventionofautoimmunediseases,cancers,andcardiovasculardisease[J].TheAmericanjournalofclinicalnutrition,2004,80(6Suppl):1678S-1688S.220[10]MichigamiT.[VitaminDmetabolismandaction][J].Clinicalcalcium,2007,17(10):1493-1498.[11]NimitphongH,HolickMF.VitaminD,neurocognitivefunctioningandimmunocompetence[J].Currentopinioninclinicalnutritionandmetaboliccare,2011,14(1):7-14.[12]HoogendijkWJ,LipsP,DikMG,etal.Depressionisassociatedwithdecreased25-hydroxyvitaminDandincreasedparathyroidhormonelevelsinolderadults[J].Archivesofgeneralpsychiatry,2008,65(5):508-512.225[13]MayHT,BairTL,LappeDL,etal.AssociationofvitaminDlevelswithincidentdepressionamongageneralcardiovascularpopulation[J].Americanheartjournal,2010,159(6):1037-1043.[14]HoangMT,DefinaLF,WillisBL,etal.Associationbetweenlowserum25-hydroxyvitaminDanddepressioninalargesampleofhealthyadults:theCooperCenterlongitudinalstudy[J].MayoClinicproceedings,2011,86(11):1050-1055.230[15]BerkM,SandersKM,PascoJA,etal.VitaminDdeficiencymayplayaroleindepression[J].Medicalhypotheses,2007,69(6):1316-1319.-6- 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