Baicalin Regulates Neuronal Fate Decision in Neural Stem

Baicalin,depression,CNS

ORIGINAL

ARTICLE

BaicalinRegulatesNeuronalFateDecisioninNeuralStem/ProgenitorCellsandStimulatesHippocampalNeurogenesisinAdultRats

Peng-WeiZhuang,1,2Guang-ZhiCui,1,2Yan-JunZhang,1,2Mi-XiaZhang,1,2HongGuo,2,3Jin-BaoZhang,1Zhi-QiangLu,1Adejobi-OluwaniyiIsaiah1&Ying-XueLin1

1ChineseMateriaMedicaCollege,TianjinUniversityofTraditionalChineseMedicine,Tianjin,China

2TianjinStateKeyLaboratoryofModernChineseMedicine,TianjinUniversityofTraditionalChineseMedicine,Tianjin,China3InstituteofTraditionalChineseMedicine,TianjinUniversityofTraditionalChineseMedicine,Tianjin,China

Keywords

Balcalin;Cognitivefunctions.;Ischemicstroke;Neuralstem/progenitorcells;Neurogenesis;Neuronalfate.

Correspondence

Y.-J.Zhang,ChineseMateriaMedicaCollege,TianjinUniversityofTraditionalChineseMedicine,No.312AnshanxiRoad,NankaiDistrict,Tianjin300193,China.Tel.:+86-22-5959-6223;Fax:+86-22-5959-6153;E-mail:zyjsunye@http://wendang.chazidian.com

Received3September2012;revision19November2012;accepted20November2012.

SUMMARY

Background:Recentstudiesrevealedthatbaicalin,a avonoidcompoundderivedfromtherootofScutellariabaicalensisGeorgi,couldpromoteneurondifferentiationofNSPCsaftercommencingthedifferentiationprocessinvitro.However,thismaynotbethemostef ca-ciousstrategytodeterminatecellfate.Here,wehaveinvestigatedwhetherbaicalincanin uenceearlyeventsofneurongenerationandstimulateadultneurogenesis.Results:TransientexposureofNSPCstobaicalinduringproliferationcouldactivateMash1toalterthedifferentialfateandincreasetheproportionofcellsexpressingneuronalmarkers.Sevendaysafter,ratswereexposedtotransientcerebralischemia,theyweretreatedfor3weekswithbaicalin,BrdUlabelingstudyshowedthatexposuretobaicalinincreasedthenumberofnewlygeneratedcellsinhippocampus,BrdU/NeuNdoublestaininganalysisindicatedthatbaicalincouldpromotenewneuronproductionaftercerebralischemia.Additionally,Morriswatermazetestshowedthatdelayedpostischemictreatmentwithbaicalinimprovedcognitiveimpairment.Conclusions:Theseresultsidentifytheexistenceofasinglemole-cule,baicalin,whichcanspecifytheneuronalfateofmultipotentNSPCsandstimulateneu-rogenesis,makingitapromisingcandidatefordevelopingclinicallyrelevantstrategiestomanipulateneuronalfateofNSPCsforbrainrepair.

doi:10.1111/cns.12050

The rsttwoauthorscontributedequallytothiswork.

Introduction

Neuralstem/progenitorcells(NSPCs)areexcellentcandidatesfordevelopingtherapeuticstrategiestorepairtheinjuredcentralner-voussystem(CNS)[1].TheneuronsinthehippocampuswouldbeinjuredfollowingwiththeproliferationofNSPCsaftercerebralischemia.However,acurrentlimitationinthisstrategyisthatthemajorityofNSPCstendtodifferentiateintoastrocytes,whicharethoughttobeundesirableastheycaninhibitneuriteoutgrowthandcontributetoregenerativefailurewhenaglialscarisformed[2].TheabilitytopredeterminetheneuronalfateofNSPCswouldbebene cialtoNSPCstherapy.InducingastableandpredictableprogramofneuralcellfateinNSPCsisanimportantgoalforutiliz-

ingthesecellsforthetreatmentofneurologicaldisorder.Recentstudieshavedemonstratedthatlocalsignalingfactorsspecifycellfate[3–5].And Akerblometal.[6]reportedthatmiR-124isaneuronalfatedeterminantinthesubventricularzone.These nd-ingsindicatedthattheabilitytodirectNSPCscellfatemayhavetherapeuticpotential.

NSPCsfatecanbedeterminedbyintrinsicsignalsandaffectedbysmallmolecules[7–10].Themainmechanismsoftheneuronalfatespeci cationwouldberegulatingtheproneuralgenes.GeneticstudieshaveprovidedevidencethatproneuralgenesarenecessarytorestricttheneuronalcellfateofNSPCs[11].Neuroge-ninandMasharebHLHtranscriptionfactors,whichinitiateneu-rogenesis[12–14].These ndingsindicatethatbHLHtranscription

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P.-W.Zhuangetal.factorsarethekeypointsinthefatespeci cationofNSPCs.Smallmoleculeshavebeenshowntobeusefulchemicalmaterialsinpromotingneurogenesisandmanipulatingcellfate[9,15].Avari-etyofprimingagentsusedtodirectneuronalfateinNSPCs,suchaslithiumchloride[16], broblastgrowthfactor-2[17],andinsu-lin-likegrowthfactor1[18]hadbeenidenti ed.

Inthesearchfornewtherapeuticsofbraininjuryorneurode-generativedisease,theherbsbeingusedintraditionalmedicinesforneurogenesisarepromisingcandidates.Baicalin(Figure1A)isoneofthepredominant avonoidderivativesisolatedfromthedryrootsofScutellariabaicalensisGeorgi,whichhasprotectiveeffectsagainstcerebralischemia,brainin ammation,andotherbraininjuries[19–21].OurpreviousstudieshaveshowedthatbaicalintreatmentcouldinduceneurondifferentiationofNSPCsafteritscommencingthedifferentiationprocess[22–24],whichhasbeencon rmedbyalateststudy[25].However,thismaynotbethemostef caciousstrategytodeterminatecellfate.AsemphasizedbyVazeyandConnor[16],alternativeavenuefordirectingfatemaybetopretreatNSPCswhiletheymaintaintheirundifferentiatedphenotype.Althoughavarietyofactiveingredi-entsofherbs,suchasginsenosides[26–28],curcumin[29],ferulicacid[30],salvianolicacids[31,32],couldaffectthebiologicalpropertiesofNSPCs.However,duetothepresenceofblood–brainbarrier,someoftheeffectivedrugsinvitrocannotproduceef cacyinvivo.Interestingly,someresearchershaddemonstratedthatbaicalincouldpassthroughtheblood–brainbarrier[33],makingitmorelikelytopromoteneurogenesisinthecentralnervoussystem.

Inthepresentstudy,wedemonstratedthataftertransientexpo-suretobaicalinduringproliferationcouldincreasethenewneu-ronsproductionandimprovethecognitivefunctionsinaratmodelofstroke.Theneuronalfatedeterminationandneurogene-sisstimulatingeffectsofbaicalinmakesitapromisingcandidate

Figure1EffectofbaicalinonthecellviabilityofNSPCs.DissociatedNSC/NPCswereculturedona96-wellplatewiththeindicatedbaicalin(A)concentration(1,1.875,3.75,7.5,15,30lM)for2days.(B)CellproliferationwasmeasuredusingaMTSassaykit.ValueswereexpressedasmeansÆSDofthreeindividualexperiments.n=6,*P<0.05,**P<0.01comparedwithcontrol.

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fordevelopingclinicallyrelevantstrategiestostimulateNSPCsforbrainrepair.

MaterialsandMethods

Animals

PregnantratsandAdultmaleWistarrats(Certi cateNoSCXX2006-0009;VitalRiverLabAnimalTechnologyCo.,Ltd.,Beijing,China)wereusedinthisstudy.Theratswerehousedintempera-ture-controlledconditionswitha12-hlight/darkcyclefollowingsurgery.Theyhadaccesstofoodandwateradlibitum.Allexperi-mentalprocedureswereevaluatedandapprovedbytheanimalethicscommitteeofTianjinUniversityofTraditionalChineseMedicine(TCM-2009-034-E02),andtheanimalcarewasaccord-ingtotheNIHGuidefortheCareandUseofLaboratoryAnimals.

NeurosphereCultureandCellViabilityAssay

NSPCswereisolatedfromthecerebralcortexof13.5-day-embry-onicWistarratsaspreviouslydescribed[34].NSPCswereculturedinneurosphereproliferationmediaconsistingofDMEM/F12sup-plementedwith2%(v/v)B27Supplement(Invitrogen,Carlsbad,CA,USA),20ng/mLEGF(Millipore,Temecula,CA,USA),20ng/mLFGF2(Millipore),and100lg/mLheparin(Sigma,St.Louis,MO,USA).Fortheinvitrocellviabilityassay,CellTiter96®AQu-eousOneSolutionCellProliferationAssaykit(Promega,Char-bonni eres-les-Bains,France)wasusedinthisstudy.NSPCswereplatedat30,000cells/wellina96-wellplateinthepresenceandabsence

ofbaicalin(1,1.875,3.75,7.5,15,30lM).Cellprolifera-tionwasassessedatday2ofcellculture.Accordingtothemanu-facturer’srecommendations,40-lLMTSsolutionwasthenaddedintoeachofthewells,anddatawereobtainedatawavelengthof490nmusingamicroplatereader(FlexStation3;MolecularDevices,Sunnyvale,CA,USA).Thesamevolumeofmediumwithoutcellswasusedasblank.

RT-PCRAnalysis

NSPCswereculturedwithorwithoutbaicalin(7.5,15,30lM)inproliferationmediumfor3days.CellswereharvestedandtotalRNAwasisolatedfromtreatedanduntreatedNSPCswithTRIzol(Roche,Mannheim,Germany).The rststrandofcDNAwassyn-thesizedfrom1lgoftotalRNAusingreversetranscriptase(TIAN-GEN)andrandomprimerasdescribedinthemanufacture’sinstructions.Aftersynthesis,1lLofcDNAwereusedinPCRreac-tionwithgene-speci cprimers.ThesequencesofthePCRprimerpairs(5′to3′)thatwereusedforeachgeneareasfollows:b-tubu-linIII,5′-GGACCTCAACCACCTTGTGT-3′(forward),and5′-AAC-ATGGCCGTAAACTGCTC-3′(reverse);GFAP,5′-ATTCCGCGCCT-CTCCCTGTCTC-3′(forward),5′-GCTTCATCCGCCTCCTGTCTGT-3′(reverse);GAPDH,5′-CCAAGGTCATCCATGACAA-3′(forward),and5′-TGTCATACCAGGAAATGAGC-3′(reverse);RT-vecontrolweresetedforRT-PCRsanalysis(RT-vecontrol=reactionwithH2OinsteadofRNAascontrol).Productswereanalyzedon1.5%agarosegelandvisualizedbyethidiumbromidestaining.Therela-tivequantitativeexpressionsoftissue-speci cmarkerswerecalcu-latedafternormalizationwithGAPDHasahousekeepinggene.

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BaicalinRegulatesNeuronalFateandNeurogenesisReal-timeRT-PCRAnalysis

Real-timeRT-PCRwasperformedinareactionmixturecontain-ingPowerSYBRgreenPCRmastermix(AppliedBiosystems,Carlsbad,CA,USA),gene-speci cprimers(Mash1,5′-CTCG-TCCTCTCCGGAACTGATG-3′(forward),and5′-ATGCTCCCGGA-GGGTGGCAAAA-3′(reverse);Ngn1,5′-ATGCCTGCCCCT-TTGGAGACCT-3′(forward),and5′-TGTAGCCTGGCACAGTC-CTCCT-3′(reverse);Ngn2,5′-CGGGTCAGACGTGGACTACT-3′(forward),and5′-GGCGGGAGAAGGATGGGAAGA-3′(reverse).Quantitativereal-timePCRreactionswererunina7500Fastreal-timePCRsystem(AppliedBiosystems)with7500FastSys-temSequencingdetectionsoftwareversion1.3.1andthethermalpro leof95°Cfor10minfollowedby40cyclesof95°Cfor15secondand60°Cfor1min.Theexpressionlevelofeachgenewasenumeratedinthreeindependenttreatments.TherelativegeneexpressionbetweenbaicalintreatmentandcontrolwascalculatedbytheDDCtmethodwithGAPDHastheinternalcontrol.

InVitroDifferentiationAssay

Fortheinvitrodifferentiationassay,NSPCswereculturedwithorwithoutbaicalin(7.5,15,30lM;Baicalin>98.5%waspur-chasedfromTianjinZhongxinPharmaceuticalGroupCo.,Ltd.,Tianjin,China)inproliferationmediumfor3days.Then,neuro-sphereswerecollectedandplatedintopoly-l-lysine-coated12-wellplatesindifferentiationculturemediumwithoutgrowthfac-torsorbaicalin.Neurosphereswereallowedtodifferentiatefor3daysbeforebeing xedforimmunocytochemistry.For uores-cenceimmunostaining,cellswere xedwith4%paraformalde-hydefor30min.After xing,cellswereincubatedovernightat4°Cwiththefollowingprimaryantibody:Rabbitanti-MAP-2(1:500;Millipore),Mouseanti-GFAP(1:500;Millipore),followedbyFITC-conjugatedgoatanti-mouseIgG(1:100;Boster,Wuhan,China),andCy3-conjugatedgoatanti-rabbitIgG(1:100;Boster).4′,6-Diamino-2-phenylindole(DAPI;Sigma)wasusedasa uo-rescentnuclearcounterstain.Immunostainedcellswerevisual-izedbyindirect uorescenceunderthe uorescentmicroscope(Leica,Wetzlar,Germany).MAP-2orGFAP-positivecellswerecountedin10randomlyselected eldsfromthreedifferentchambers.

WesternBlottingAssay

ProteinextractswerepreparedandsubjectedtoWesternblotanalysis.Theproteinconcentrationsofthelysatesweredeter-minedwithaBradfordproteinassaykit(Bio-Rad,Hercules,CA,USA)accordingtothemanufacturer’sinstructions.AnequalamountofproteinwasfractionatedbySDS-polyacrylamidegelelectrophoresis(PAGE)andtransferredontopolyvinylidinedi- uoridemembranes.Afterblockingwith5%skimmilkinTBS-T,themembranewasprobedwithgoatanti-actin(1:2000;SantaCruz,CA,USA);mouseanti-mash1(1:1000;Millipore)primaryantibodyinablockingsolutionofnonfatmilk(5%).Secondaryhorseradishperoxidase-conjugatedgoatanti-mouse,goatanti-rabbit(Boster)antibodyinnonfatmilkblockingsolution(5%)wasthenapplied.Theimmunoreactivitywasvisualizedwith

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ECLWesternblottingdetectionreagents(Millipore).Thetotalproteincontentwasnormalizedusingmouseanti-actinantibodies.

InductionoftheTransientGlobalIschemia

AdultmaleWistarratsweighing200–250gweresubjectedtotransientforebrainischemiabyamethodcombiningthosedescribedpreviously[35–37].Inbrief,12hbeforetheinductionofischemia,ratswereanesthetizedwithsodiumpentobarbital,thebilateralcarotidarterieswereexposedtofacilitatetheocclud-ingonthefollowingday,andthenthevertebralarterieswereirre-versiblyoccludedbyelectrocoagulation.Thenextday,ischemiawasinducedbybilaterallyoccludingcarotidarterieswithaneu-rysmclips,andcarotidarterieswereclampedfor6minexactly.Ratslosttheirrightingre exduringischemic,theclipswereremovedtorestorecerebralblood ow.Theratsthatremaintheirrightingre exin1minafteroccludingofthebothcarotidarterieswereconsideredtobethefailureofischemiaandeliminated.Sham-operatedratswereanesthetized,thecarotidarterieswereisolated,buttheywerenotclamped.

DrugAdministration

Thestudywascarriedoutonratsdividedintothreegroups.(1)Shamgroup+vehicle(sham),(2)Ischemicgroup+vehicle(vehicle),and(3)Ischemicgroup+baicalin(Baicalin).Baicalinwasdilutedinsalineattheconcentrationof100mg/mL(Stocksolution)andincubatedunderagitationfor1hat37°C.Thesolu-tionwasthen lteredwith0.22-mm lter.Baicalinwereadminis-teredintraperitoneally(i.p.)atadoseof50mg/kgbodyweight,oncedailyfor3weeks.Allcontrolsreceivedanamountofvehicleequivalenttodrugtreatmentconditions.

BrdULabelingandAnalysisofInVivoNeurogenesis

BrdU(50mg/kg;Sigma)wasinjectedi.p.totheratsonceevery2hoveraperiodof8hatthe20thdayafterbaicalintreatmenttolabeltheproliferatingcellsafterischemia.Thenextday(21th),ratswereanesthetizedwithetherandperfusedwithPBS,followedbyacold4%paraformaldehydesolution.Brainswerecollectedandpost xedovernightina4%paraformaldehydesolutionat4°C.Coronalsections(20lmthickness)wereobtainedthroughoutthehippocampus.ForanalysisconcerningBrdUorBrdU/NeuNdoublestainimmunohistochemistry,sectionswereincubatedin50%formamide/29salinesodiumcitratefor2hat65°C,fol-lowedbyarinsewithPBS.Sectionswerethenincubatedin2NHClfor30minat37°Ctodenaturedouble-strandedDNA,andrinsedin0.1Mboratebuffer(pH8.5).Afterblockingfor2hwith1%BSAinPBS,sectionswereincubatedovernightat4°Cwithmouseanti-BrdUmonoclonalantibody(1:100;senta),orrabbitanti-NeuNpolyclonalantibody(1:100;Millipore).FollowedbyrinsinginPBS,sectionswereincubatedfor2hatRTwithFITC-conjugatedgoatanti-mouseIgG(1:100;Boster)andCy3-conju-gatedgoatanti-rabbitIgG(1:100;Boster).BrdUorBrdU/NeuNdouble-positivecellswerevisualizedunderthe uorescentmicro-scope(Lica,Germany).

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MorrisWaterMazeTask

ThecognitivefunctionswereexaminedusingtheMorriswatermaze.Ablackcircularpool1.5mindiameterwas lledwithwater(22Æ1°C),andahiddenplatform10cmindiameterwasplacedataconstantpositioninthecenterofoneofthefourquadrantswithinthetank.Ratsineachgroups(n=10)wereallowedtoswimfreelyfor2mintobecomehabituatedtotheapparatus.Fromthenextday,inthehiddenplatformtrials,acquisitiontrialswerecarriedouttwotimesperdayfor5days.Ineachtrial,ratswereplacedintothewaterata xedstartingposition,andthetimetakentoescapeontothehiddenplatformwasmeasured.Ratsweregiven90secondto ndthehiddenplatformduringeachacquisitiontrial.Ifitfailedtolocatetheplatformwithin90second,itwasguidedthere,andtheratwasallowedtostayontheplatformfor20second.Thelatencyto ndingtheescapeplatformwasanalyzedasanindexofspatiallearning.Performancewastested24hafterthe naltrainingdayinaprobetrialduringwhichtheplatformwasremoved,theratwasplacedinthestartanditsbehaviorwasmonitoredfor90second.

ofvariance,andthenposthocleastsigni cantdifference(LSD)testwasemployedforcomparisonsbetweencontrolandothergroups.Toanalyzewatermazeplace-navigationperformance,theaverageescapelatencywascalculatedandevaluatedbyrepeated-mea-suresANCOVA.Differenceswereconsideredstatisticallysigni -cantforP<0.05.

Results

BaicalinUpregulatedNeuronalGenesExpression

ToinvestigatetheeffectofbaicalinontheviabilityofNSPCs,NSPCswereexposedtobaicalinfor2days.TheeffectofbaicalintreatmentsonthegrowthratesoftheNSPCswascomparedusingacellviabilitymeasurement.AsdemonstratedinFigure1B,baic-alinhadapromotingactivityforNSPCsproliferationandexhib-itedadose-dependenteffect.Basedonthecellviabilitystudy,thedosesof7.5,15,30lMwerechosedinthefollowingstudy.Mash1andNgn1arethoughttopositivelyregulateneuronaldevelopment[38,39].Theexpressionofproneuraltranscriptionfactors(Mash1,Ngn1andNgn2)intheproliferatingNSPCsinthepresenceandabsenceofbaicalinwasmeasured.ChangesingeneexpressionintheNSPCstreatedanduntreatedwithbaicalinwereevaluatebyreal-timeRT-PCRtechnique.Inourstudy,theresultsshowedthatMash1mRNAincreasedafterbaicalintreatment(Control0.50Æ0.10%vs.15lMbaicalin1.22Æ0.38%,30l

M

DataAnalysis

DatawereexpressedasmeanÆSD.Statisticalcomparisonbetweendifferenttreatmentswascarriedoutbyone-wayanalysis

Figure2EffectofbaicalinonthebHLHtranscriptionfactorsexpressionandtheneuralfateofNSPCs.ThemRNAorproteinlevelsofMash1treatedwithdesignatedconcentrations(7.5,15,30lM)ofbaicalinanalyzedbyreal-timeRT-PCRorWesternblotting.(A)ThemRNAlevelsquanti edwerecalculatedbytheDDCtmethod,normalizedwithGAPDHinternalcontrol.Mash1expressionweresigni cantlyupregulatedinbaicalin-treatedNSPCscomparedwithcontrol(n=6);(B)Semiquanti edscoresofMash1expressionweresigni cantlyupregulatedinbaicalin-treatedNSPCscomparedwithcontrol,normalizedwithActininternalcontrol(n=6);(C)ThemRNAlevelsofb-tubulinIII,GFAPtreatedwithdesignatedconcentrations(7.5,15,30lM)ofbaicalinfor3dayswereanalyzedbyRT-PCR.ThemRNAlevelsweresemiquanti edbydensitometricmeasurements,normalizedwithGAPDHinternalcontrol(n=6),andexpressedasmeansÆSDofthreeindividualexperiments.ValueswereexpressedasmeansÆSDofthreeindividualexperiments.*P<0.05,**P<0.01comparedwithcontrol.[Correctionmadeafteronlinepublicationon24Jan2013:‘percentoftotalgene’wasremovedfromthey-axislabelinpartAand‘percentoftotalprotein’wasremovedfromthey-axislabelinpartB.]

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baicalin1.54Æ0.09,n=6,P<0.05,P<0.01,respectively;Fig-ure2A).Toprovidefurtherevidencethatbaicalinupregulatedmash1expression,Mash1proteinwasdetectedbyWesternblot-tingassaywithanti-mash1antibody.AsshowninFigure2B,baic-alinsigni cantlyincreasedmash1expressioncomparedwithuntreatedcontrol(Control0.34Æ0.08vs.7.5lMbaicalin0.83Æ0.11,15lMbaicalin0.82Æ0.13,30lMbaicalin0.87Æ0.10,n=6,P<0.01).TheseresultsindicatedthatbaicalinstimulatesneuronaldifferentiationbyactivatingbHLHtranscrip-tionfactors.

BaicalinSpeci edtheNeuralFateofNSPCs

GiventheinductionofbHLHfactorsbybaicalin,inthefollowingresearchweinvestigatedwhetherbaicalintreatmentduringpro-liferationwouldincreaseneuronalfatedetermination.Cellsweresubjectedtobaicalintreatmentfor3daysunderproliferativecon-

ditionsandtheabilityoftheirneuronalfatecommitmentwascomparedbyRT-PCRtechnique,usingspeci cneuronalmarkers.Theexpressionsofb-tubulinIII(neuronmarker)andGFAP(as-trogliamarker)weremeasuredbyRT-PCRtechnique.ResultsshowedthatnobandsweredetectedinRT-vecontrolsamples,andafter3daysofbaicalintreatment,NSPCssigni cantlydecreasedthemRNAexpressionsofGFAP(Control0.98Æ0.21vs.15lMbaicalin0.57Æ0.12,30lMbaicalin0.48Æ0.09,n=6,P<0.01),andbaicalinincreasedtheexpressionofb-tubu-linIII(Control1.02Æ0.16vs.15lMbaicalin1.89Æ0.37,30lMbaicalin2.80Æ0.64,n=6,P<0.01;Figure2C).

Immunostainingwasalsoemployedtoevaluatetheabilityofneuronalfatedeterminationofbaicalin.NSPCsweresubjectedtobaicalintreatmentunderproliferativeconditionsfor3days,afterthatwewithdrewthebaicalin,andmadetheNSPCsadherenttodifferentiatewithoutbaicalinfor3days.Basedonthedifferentia-tionassay,wealsofoundthat,alongwithelevatedproneural

gene

Figure3BaicalinprimedtowardaneuronalfateofNSPCsduringtheirsubsequentdifferentiation.(A)FluorescencemicroscopicphotosshowingtheneuraldifferentionofNSPCstreatedwithdesignatedconcentrations(7.5,15,30lM)ofbaicalinfor3daysinproliferationmedium.Red,MAP-2;Green,GFAP;blue,DAPI.Scalebar,100lm.(B)Baicalinsigni cantlyincreasedthefractionofMAP-2-positivecells.(C)Therewasnosigni cantdifferenceinthefractionofGFAP-positivecells.ThedataexpressedasmeansÆSDofthreeindividualexperiments.n=8,*P<0.05,**P<0.01comparedwithcontrol.

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