Ethanol extracts frommerocallis citrina attenuate the decreases of brain-derived neurotrophic factor

depression

JournalofEthnopharmacology144(2012)328–334

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JournalofEthnopharmacology

journalhomepage:http://wendang.chazidian.com/locate/jep

EthanolextractsfromHemerocalliscitrinaattenuatethedecreasesofbrain-derivedneurotrophicfactor,TrkBlevelsinratinducedbycorticosteroneadministration

Li-TaoYia,n,JingLia,Huo-ChenLib,YingZhoub,Bi-FangSua,Ke-FengYangb,MengJiangb,Yan-TingZhanga

ab

DepartmentofChemicalandPharmaceuticalEngineering,CollegeofChemicalEngineering,HuaqiaoUniversity,Xiamen361021,Fujianprovince,PRChinaDepartmentofBiotechnologyandBioengineering,CollegeofChemicalEngineering,HuaqiaoUniversity,Xiamen361021,Fujianprovince,PRChina

articleinfo

Articlehistory:

Received5April2012Receivedinrevisedform27August2012

Accepted6September2012

Availableonline18September2012Keywords:

Hemerocalliscitrina

Antidepressant-likeeffectCorticosterone

Brain-derivedneurotrophicfactorTrkB

abstract

Ethnopharmacologicalrelevance:Hemerocalliscitrina,atraditionalherbalmedicine,hasbeenusedfortheimprovementofbehavioralandemotionalstatusinEastern-Asiacountries.

Aimofthestudy:OurpreviousstudieshavedemonstratedthattheethanolextractsofH.citrina?owers(HCE)reversedthebehavioralalterationsandmonoamineneurotransmitterdysfunctionsinstressedmice.However,therelationofitsantidepressant-likeactionwithneurotrophicmolecularexpressionsremainsunknown.

Materialsandmethods:Toclarifythis,weexploredtheeffectofHCE(32.5,65,130mg/kg,p.o.)onthebehavior,brain-derivedneurotrophicfactor(BDNF)anditsreceptor(TrkB)indepression-likeratsinducedbyexogenousadministrationofthestresshormonecorticosterone(40mg/kg,s.c.).

Results:Itwasobservedthatrepeatedadministrationofcorticosteroneinducedanelevationontheserumcorticosteronelevels,whichcausedtheabnormalitiesobservedinthesucrosepreferencetestandforcedswimmingtest(FST).AdministrationofHCE(65and130mg/kg)reversedthechangesaboveandup-regulatedtheBDNFandTrkBreceptorproteinexpressionsinthebrainregionoffrontalcortexandhippocampus.

Conclusion:These?ndingscon?rmthatHCEproduceanantidepressant-likeeffectincorticosterone-induceddepression-likemodelofratsandthiseffectisatleastpartlymediatedbyBDNF-TrkBsignalinginthefrontalcortexandhippocampus.

&2012ElsevierIrelandLtd.Allrightsreserved.

1.Introduction

HemerocalliscitrinaBaroni(Liliaceae)whichiswidelygrowninChina,JapanandKoreahasbeenusedforfoodandmedicinalpurposesforthousandsofyears.Accordingtothephytochemicalandpharmacologicalstudies,H.citrinamainlycontains?avo-noids,polyphenolsandessentialoilsthatcontributetotheherb’sbiologicaleffects,suchasantibacterial(Zhanetal.,2005),anti-oxidant(LangandLuo,2007)andnitrite-eliminating(Fuetal.,2009)activity.Inaddition,theeffectsofH.citrinasuchasrelievinggloomandimprovingsleepingwererecordedinthefamouspharmaceuticaltextof‘‘CompendiumofMateriaMedica’’,

Abbreviations:ANOVA,analysisofvariance;BDNF,brain-derivedneurotrophicfactor;DA,dopamine;FST,forcedswimmingtest;GR,glucocorticoidreceptor;HCE,ethanolextractsofHemerocalliscitrina;NA,noradrenaline;OFT,open-?eldtest;5-HT,serotonin;TST,tailsuspensiontestn

Correspondingauthor.Tel./fax:þ865926162302.

E-mailaddresses:litao.yi@http://wendang.chazidian.com,litaoyi@http://wendang.chazidian.com(L.-T.Yi).0378-8741/$-seefrontmatter&2012ElsevierIrelandLtd.Allrightsreserved.http://wendang.chazidian.com/10.1016/j.jep.2012.09.016

themostcompleteandcomprehensivemedicalbookeverwritteninthehistoryoftraditionalChinesemedicine.Clinically,H.citrinahasbeenwidelyusedforthetreatmentofdepressivedisordersinChina(Chenetal.,2008).

Inpreviousstudies,wefoundthattheethanolextractsofH.citrina(HCE)administrationsigni?cantlyreducedtheimmo-bilitytimeinboththeforcedswimmingtest(FST)andtailsuspensiontest(TST)withoutaccompanyingchangesinlocomo-toractivityintheopen-?eldtest(OFT)inmice(Guetal.,2012).Furthermore,wealsodemonstratedthatHCEenhancedserotonin(5-HT)andnoradrenaline(NA)levelsinthefrontalcortexandhippocampusaswellaselevateddopamine(DA)levelsinthefrontalcortex(Guetal.,2012).However,untilnow,theneuro-trophicmolecularexpressionunderlyingtheantidepressant-likeeffectofHCEremainsunclear.

Neurotrophicfactorsarecriticalregulatorsoftheformationandplasticityofneuronalnetworks(LeeandKim,2010).Brain-derivedneurotrophicfactor(BDNF),oneofimportantneuro-trophicfactors,hasalsobeenimplicatedintheetiologyofmajor

depression

L.-T.Yietal./JournalofEthnopharmacology144(2012)328–334329

depressionandthemechanismofantidepressanttreatment(Numakawaetal.,2010).Clinicalstudydemonstratesthatdepres-sionisassociatedwithreducedbrainBDNFanditsreceptor(TrkB)levelsandthatthereductionscanberestoreduptothenormalvaluebyantidepressanttreatment(Thompsonetal.,2011).AnimalstudieshavealsodemonstratedclearevidencethatBDNFsignalingthroughTrkBisinvolvedinthemechanismsofactionofantidepressantagents(Wangetal.,2010;Kutiyanawallaetal.,2011).BDNFandTrkBlevelscanthereforebeusefulmarkersforantidepressant-likeresponse.

Inthepresentstudy,weusedaratdepressionmodelbyrepeatedcorticosteroneinjectionstoinvestigatetheantidepressant-likeeffectofHCE.Furthermore,thestudyexploredwhethertheHCEcouldreversethelossofBDNFanditsreceptorTrkBmRNAandproteinlevelsintheratbrainregionsoffrontalcortexandhippocampus.

2.Materialsandmethods2.1.Animals

MaleSprague-Dawleyrats(220–250g)werepurchasedfromLaboratoryAnimalCenter,FujianMedicalUniversity,FujianProvince,PRChina.Animalsweresinglyhousedunderanormal12-h/12-hlight/darkschedulewiththelightsonat07:00a.m.andhadfreeaccesstotapwaterandfoodpellets.Ambienttempera-tureandrelativehumidityweremaintainedat22721Candat5575%,andgivenastandardchowandwateradlibitumforthedurationofthestudy.Theanimalswereallowed1weektoacclimatizethemselvestothehousingconditionsbeforethebeginningoftheexperiments.AllprocedureswereperformedinaccordancewiththepublishedguidelinesoftheChinaCouncilonAnimalCare(RegulationsfortheAdministrationofAffairsCon-cerningExperimentalAnimals,approvedbytheStateCouncilonOctober31,1988andpromulgatedbyDecreeNo.2oftheStateScienceandTechnologyCommissiononNovember14,1988).2.2.Chemicalsandreagents

CorticosteronewaspurchasedfromTCIDevelopmentCo.,Ltd.(Tokyo,Japan).FluoxetinehydrochloridewaspurchasedfromChangzhouSiyaoPharmaceuticalsCo.,Ltd.(Changzhou,PRChina).Allprimersusedinthisstudyweredesignedandsynthe-sizedbySangonBiotechCo.Ltd.(Shanghai,PRChina).Theanti-BDNFandanti-TrkBantibodyandtherespectivesecondaryantibodywerepurchasedfromSantaCruzBiotechnologyInc.(SantaCruz,USA).TrizolreagentwaspurchasedfromInvitrogen(Carlsbad,USA).ReversetranscriptaseMoloneyMurineLeukemiaVirus(M-MLV)usedforcDNAsynthesiswasfromPromegaCorporation(Madison,USA).AllotherreagentsusedinRT-PCRandwesternboltwerepurchasedfromSangonBiotechCo.Ltd.(Shanghai,PRChina).2.3.PlantmaterialandHPLCanalysis

FlowersofH.citrinaBaroni(Liliaceae)waspurchasedfromXiamenWal-MartandauthenticatedbyCheng-FuLi,DepartmentofPharmacy,XiamenHospitalofTraditionalChinesemedicine(VoucherspecimennumberHU/CE-04281).

SincerutinisthemaincomponentofH.citrina(Yangetal.,2006),a?ngerprintwasanalyzedinHPLCusingrutinasanindex.AShimadzuProminenceLC-20AHPLCsystemequippedwithanUVdetectorwasused.QualitativeandquantitativeanalysesofrutinwereperformedusinganAgilentC18column(4.6mMÂ250mM,5mm),andthecolumntemperatureswerekeptat251C.Alineargradientelutionpro?lewasusedinourstudy[0–20min:methanolto0.5%aceticacidwatersolution(v/v)ratioof20/80

Fig.1.HPLCchromatogramsofHemerocalliscitrina(A)andthestandardcom-poundofrutin(B).

to30/70;20–30min:30/70;30–70min:30/70to80/20].Flowratesofelutioninbothcaseswere1ml/min.Theinjectionvolumefortheanalytewas10ml.Thedetectionwavelengthwassetat254nmforrutin.TherawmaterialH.citrinawasextractedwithmethanolat251Cfor30min.Thecontentofthemarkersub-stancerutininH.citrinawascalculatedas0.39%(Fig.1AandB).

2.4.Preparationoftheethanolextracts

Driedsample(500g)wascutintosmallpiecesandextractedthreetimesinare?uxcondenserfor4hwith3Lof75%ethanolbysonicationatroomtemperature(25721C).Thesolutionswerecombined,?ltered,concentratedunderreducedpressureandlyophilizedintopowders.The?nalyieldwas7.65%(w/w).

2.5.Drugtreatments

Differentgroupsofrats,7animalspergroup,wereusedfordrugtreatmentandtests.Doseswerecalculatedasmg/kgbodyweight.Alltheexperimentalanimalsincludingcontrolanddrug-treatmentgroupsweresimultaneouslydeprivedfoodbutnotwater1hpriortodrugadministration.Theaimoffoodwith-drawnpriortodrugadministrationwasjusttoensurethebioavailabilityofdrug.Inothertimeperiods,allanimalshadfreeaccesstofood.

ThestandarddoseofHCEinrat(130mg/kg)wasbasedonourpreviousmousestudy(Guetal.,2012)andcalculatedonthebasisofbodysurfacearearatiobetweenratandmouse.Inaddition,consideringthelong-termtreatmentusedinthepresentstudy,thedosesof32.5,65,130mg/kgevery24hwereselected.

TheprocedureanddoseofcorticosteroneadministrationwasperformedasdescribedinMaoetal.(2012).Corticosterone(40mg/kg),whichwasdissolvedinasalinesolutioncontaining0.1%dimethylsulfoxideand0.1%Tween-80,wasadministratedbysubcutaneously(s.c.)inavolumeof5ml/kgoncedailyfor21days.HCE(32.5,65,130mg/kg)andthepositivedrug?uoxetine(15mg/kg)wereadministeredbyoral(p.o.)gavageinavolumeof10ml/kg30minpriortothecorticosteroneinjectionfor21days.

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330L.-T.Yietal./JournalofEthnopharmacology144(2012)328–334

2.6.Sucrosepreferencetest

Thesucrosepreferencetestwasemployedhereintoevaluateanhedonia,oneofthecoresymptomsofmajordepressioninhumans.ThetestwasperformedasdescribedbyMaoetal.(2012).Brie?y,thesucrosepreferencetestwascarriedout24hafterthelastdrugtreatment.Beforethetest,theratsweretrainedtoadapttosucrosesolution(1%,w/v)byplacingtwobottlesofsucrosesolutionineachcagefor24h;thenoneofthebottleswasreplacedwithwaterfor24h.Aftertheadaptationprocedure,theratsweredeprivedofwaterandfoodfor24h.Thesucrosepreferencetestwasconductedat10:00a.m.Theratswerehousedinindividualcagesandgivenfreeaccesstothetwobottlescontaining100mlofsucrosesolution(1%,w/v)and100mlofwater,respectively.After60min,thevolume(ml)ofboththeconsumedsucrosesolutionandwaterwererecorded,andsucrosepreferencewascalculatedassucrosepreference(%)¼sucroseconsumption(ml)/[sucroseconsumption(ml)þwaterconsump-tion(ml)]Â100%.

2.7.Forcedswimmingtest(FST)

TheFSTusedwasthesameasdescribedindetailelsewhere(Porsoltetal.,1978),withsomemodi?cation.Brie?y,thetestwasdonebyplacingaratinaglasscylinder(46cminheight,20cmindiameter)?lledwith30cmhighwater(25721C).Waterwasreplacedbetweeneverytrial.Twoswimmingsessionswereconducted:aninitial15minpretest,followedbya5mintest24hlater.Aratwasconsideredimmobilewheneveritremained?oatingpassivelyinthewaterandonlymakingslightmovementstokeepitsheadabovethewaterline.Thetestsessionswererecordedbyavideocameraandscoredbyanobserverblindtotreatment.

2.8.Bloodsamplingandtissueextraction

AnimalswerekilledbydecapitationonedayafterFST.Toavoid?uctuationsonhormonelevelsduetocircadianrhythm,animalswerebledat12:00–13:00p.m.onthedayofsacri?ce.Thebrainregionsoffrontalcortexandhippocampuswereisolatedimmediately,andthenstoredatÀ801CforlateranalysisofBDNFandTrkBmRNAandproteinlevels.2.9.Serumcorticosteroneassay

Bloodwascollectedoniceandseparatedinarefrigeratedcentrifugeat41C(4000Âgfor10min).Serumwasstoredat–201Cuntilassayswereperformed.Serumcorticosteronelevelsweremeasuredusingacommercialkit(EnzoLifeSciences,PlymouthMeeting,USA)basedonenzymeimmunoassay(Crowther,1995).2.10.RT-PCR

TheprocedureofRT-PCRwasperformedasdescribedindetailelsewhere(Prediger,2001;Panetal.,2010),withsomemodi?ca-tions.TotalRNAwasisolatedfromfrontalcortexorhippocampususingTrizolreagentfollowingthemanufacturer’sinstructions.TheconcentrationandpurityofRNAweremeasuredbytheopticaldensityat260and280nmusingspectrophotometer.Reversetranscriptionwasperformedwith1mgRNAusingM-MLVreversetranscriptaseforcDNAsynthesis.Ampli?cationofcDNAbyPCRwasperformedin25mlreactionscontaining8mlcDNA,1mlforwardand1mlreverseprimers(10mm),2.5mlPCRÂ10buffercontainingMgCl2,0.5mldNTPmixture(10mM),0.5mlTaqpolymerase(2.5U)and11.5mlsterileddH2O.Primers

usedforBDNFwas:50-TGTGACAGTATTAGCGAGTGGGT-30and50-CGATTGGGTAGTTCGGCATT-30,thatusedforTrkBwas50-CTTATGCTTGCTGGTCTTGG-30and50-GGGTATTCTTGCTGCTC-TCA-30.Ingeneral,PCRwasperformedwithapreheatingcycleat941Cfor5min,denaturation,annealingandelongationwerecarriedoutat941Cfor30s,at501Cfor30s(BDNF)or591Cfor60s(TrkB),andat681Cfor15s(BDNF)or721Cfor35s(TrkB),respectively.Thereactionswererepeatedfor40(BDNF)or38(TrkB)cycles.ThePCRproductswereresolvedbya1.5%agarosegelelectrophoresisandquanti?edbytheBio-RadChemiDocXRSGelDocumentationsystemandBio-RadQuantityOnesoftware(Hercules,USA).TheresultswerenormalizedtothemRNAexpressionlevelofGAPDHineachsample.2.11.Westernblot

Theprocedureofwesternblotwasperformedasdescribedindetailelsewhere(BlancherandJones,2001;Chenetal.,2010),withsomemodi?cations.Brainsampleswerehomogenizedinalysisbuffercontaining50mMTris–HCl(pH7.4),1mMEDTA,150mMNaCl,1%TritonX-100,1%sodiumdeoxycholate,0.1%SDS,1mMtrichostatinA,phosphataseinhibitorcocktail.Thehomogenateswerecentrifugedat14000Âgfor20minat41C,andthesupernatantswerecollectedandstoredatÀ201Cuntilfurtheruse.TheproteinconcentrationwasdeterminedbyaBCAassay.TotalproteinswereseparatedbySDS-PAGEandtransferredtoaPVDFmembrane.Followingblockingin3%BSA/TBSTatroomtemperaturefor1h,themembraneswereincubatedwiththeappropriateprimaryantibodiesat41Covernight(anti-BDNF:1:500,anti-TrkB:1:1000).AfterbeingwashedwithTBSTforthreetimes,themembraneswereincubatedwithanHRP-labeledsecondaryantibody(1:4000).TheblotswerewashedagainforthreetimesbyTBSTbufferandtheimmunoreactivebandsweredetectedbyusingtheenhancedchemiluminescencemethod.WesternblotbandswerescannedandsubsequentlyanalyzeddensitometricallywithBio-RadQuantityOnesoftware(Hercules,USA).TheresultswerenormalizedtotheproteinexpressionlevelofGAPDHineachsample.2.12.Statisticalanalyses

Alldatawereexpressedasmean7S.E.M.Tocompareexperi-mentalandcontrolgroups,weusedone-wayANOVA,followedbypost-hocDunnett’stest.AvalueofPo0.05wasconsideredstatisticallysigni?cantforanalysis.

3.Results

3.1.EffectsofHCEonthesucrosepreference

TheeffectsofHCEonthesucrosepreferenceinratsexposedtorepeatedcorticosteroneadministrationweregiveninFig.2.Corticosteroneadministrationinducedasigni?cantdecreaseinthesucrosepreference(Po0.05).65and130mg/kgHCEreversedthereductiontothebaselinevalue(Po0.05,Po0.01,respec-tively).Fluoxetine,thepositivedrug,alsoincreasedthesucrosepreferenceintheratexposedtocorticosterone(Po0.05).3.2.EffectsofHCEontheimmobilitytime

Repeatedcorticosteronetreatment(s.c.)causedasigni?cantelevationofimmobilitytimeintheratFSTcomparedwiththecontrols(Po0.05).Long-termtreatmentwithHCE(65and130mg/kg)and?uoxetine(15mg/kg)for21dayssigni?cantly

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L.-T.Yietal./JournalofEthnopharmacology144(2012)328–334331

Fig.2.EffectsofHCEonthesucrosepreferenceintheratsexposedtorepeatedcorticosteroneadministration.#Po0.05vscontrolwithvehiclegroup.*Po0.05and**Po0.01vscorticosteronewithvehiclegroup.

Fig.3.EffectsofHCEontheimmobilitytimeintheratFST.#Po0.05vscontrolwithvehiclegroup.**Po0.01vscorticosteronewithvehicle

group.

reducedtheimmobilitytime(Po0.01,Po0.01,Po0.01,respec-tively)tobaselinevalue(Fig.3).

3.3.EffectsofHCEontheserumcorticosteronelevels

Exposuretocorticosteroneresultedinasigni?cantincreaseintheserumcorticosteronelevelscomparedwiththecontrols(Po0.01).Long-termHCE(65and130mg/kg)or?uoxetine(15mg/kg)treatmentsnormalizedtheincreasedlevelsofcorti-costerone(Po0.05,Po0.01,Po0.01,respectively)intheratexposedtorepeatedcorticosteroneadministration(Fig.4).3.4.EffectsofHCEontheBDNFandTrkBreceptorexpressionToinvestigatetheeffectsofHCEonthemRNAexpressionofBDNFandTrkB,whicharetwomoleculescrucialforantidepressant-likebehaviorandneurogenesis,weconductedRT-PCRandwesternblotanalysesafterthecorticosteroneadmin-istrationfor21days.AscanbeseeninFig.5and6exogenouscorticosteronereducedtheBDNFbutnotTrkBmRNAlevelsinthefrontalcortexandhippocampus(Po0.01,Po0.05,respectively).HCE(65and130mg/kg)treatmentsreversedthechangesinthefrontalcortex(Po0.01,Po0.05,respectively)andhippocampus(Po0.05,Po0.001,respectively).

Inaddition,BDNFandTrkBproteinlevelsinthefrontalcortex(Po0.05,Po0.05,respectively)andhippocampus(Po0.05,Po0.05,respectively)weredramaticallydecreasedbyrepeatedcorticosteroneadministration.HCEtreatment(65,130mg/kg)alleviatedthechangesinthetwobrainregions.

Fig.4.EffectsofHCEontheserumcorticosteronelevels(onedayafterFST)intheratsexposedtorepeatedcorticosteroneadministration.##Po0.01vscontrolwithvehiclegroup.*Po0.05and**Po0.01vscorticosteronewithvehicle

group.

4.Discussion

Ingeneral,thestressmodelsofdepressionsuchasTST,FSTandchronicmildstressarewidelyadoptedbyresearcherstoevaluatetheantidepressantagentsandtheirrelatedmechanism(Willner,1997;Baietal.,2001;Cryanetal.,2005).However,oneofthebiggestproblemswithexperimenter-appliedstressmodelsisalackofcontroloverindividualdifferencesinresponsivitytophysicalandpsychologicalstressors(SternerandKalynchuk,2010).Insuchcase,amorevaliddepression-likemodelneedstobeestablished.

Itiswell-knownthatelevatedlevelsofglucocorticoidhavebeenimplicatedindepression(Zunszainetal.,2011).Ingeneral,glucocorticoidservesanadaptivefunctioninprotectingtheorganismagainstmanydefensereactionselicitedbyacutestress.Oncereleased,glucocorticoidsactonbodilytissuestolimitnon-essentialfunctionsandmobilizeenergytodealwiththestressor.Glucocorticoidsalsoreachthebrainwheretheyexertaninhibi-toryin?uencetohaltHPAaxisactivitythroughanegativefeed-backinhibitoryregulation(SternerandKalynchuk,2010).Butiftheprolongedstressexists,chronicallyelevatedglucocorticoidlevelswilloccurandimpairthenegativefeedbackinhibition,?nallyleadtodysregulatedemotionalandphysiologicalhome-ostasiswhichisseenintheetiologyofdepression(HermanandCullinan,1997).Anumberofexperimentalstudieshavealsodemonstratedthedepression-likeeffectsoftheexogenousadmin-istrationofglucocorticoids.Ithasbeenobserved,forinstance,thatcorticosteronetreatment(s.c.)induceddepression-likebehaviorsintheFSTandTST(Gourleyetal.,2008;Lauetal.,2011).Long-termadministrationofcorticosteronealsoresultedindepressedphenotype,reducedhippocampalcellproliferationandgranulecelllayervolume,asobservedinpatientssufferingfromdepression(Murrayetal.,2008).Thus,the?ndingssuggestthatadministra-tionofexogenouscorticosteroneisausefulmethodforstudyingtherelationshipbetweenstress,behaviors,glucocorticoids,neuro-trophicfactorsanddepression(SternerandKalynchuk,2010).

Inourpresentstudy,repeatedcorticosteronetreatmentinducedareductionofsucrosepreferenceandanincreaseofimmobilitytime,whichisinlinewithother?ndings(Gourleyetal.,2008;Maoetal.,2012).Thesucrosepreference,whichre?ectsasymptomanhedonia(inabilitytoexperiencepleasure)existingindepressedpatients,canbeimprovedbyantidepres-sants(Willner,1997).Thepresent?ndingsrevealedthatrepeatedadministrationofHCEreversedtheanhedonic-likebehaviorincorticosterone-inducedrats.Intheotherbehavioraltest,themeasurementindeximmobilitytimeoftheFST,whichis

induced

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332L.-T.Yietal./JournalofEthnopharmacology144(2012)328–334

1.51.5

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Relative BDNF protein levels(normalized by GAPDH)

Relative BDNF mRNA levels(normalized by GAPDH)

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Corticosterone administrationCorticosterone administration

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Fig.5.EffectsofHCEonthefrontalcortexBDNFandTrkBreceptormRNAandproteinexpressionsintheratsexposedtorepeatedcorticosteroneadministration.#Po0.05and##Po0.01vscontrolwithvehiclegroup.*Po0.05,**Po0.01and***Po0.001vscorticosteronewithvehiclegroup.

byswimmingstress,canbedramaticallydecreasedbyabroadspectrumofclinicallyeffectiveantidepressants(Hasco¨etandBourin,2009).Consistentwithourpreviousstudy(Guetal.,2012),HCEalsoreversedtheelevationofimmobilitytimeinducedbycorticosteronetreatment(s.c.)intheratFST.Takentogether,theresultsobtainedfromthebehavioralstudiesagaincon?rmedthatHCEtreatmentexertedanantidepressant-likeeffectintheratstreatedbycorticosterone(s.c.).

Itwasobservedinourstudythatrepeatedcorticosteronetreatment(s.c.)causedanelevationofserumcorticosteronelevelsinrats,whichwasconsistentwiththepreviousstudies(Dwivedietal.,2006;Johnsonetal.,2006).Thismeansthatexogenouscorticosteroneresultsinanabsoluteincreaseincirculatingserumcorticosteronelevels,anindicatorofstressanddepressioninlaboratoryanimals.Thus,thepresentstudyrevealedthatthebehavioralconsequencesofrepeatedcorticosteroneadministra-tionwereaccompaniedbydysregulationoftheHPAaxis.

Glucocorticoidandglucocorticoidreceptor(GR)hasbeenconsideredtobeespeciallyimportantinrelationtoBDNFregula-tion(Numakawaetal.,2009).StressorsstimulatetheactivityoftheHPAaxis,andthenglucocorticoidsincrease,whichleadtothereducedexpressionofBDNF(LeeandKim,2010).Forexample,previousstudyindicateddrinkingcorticosteronedecreasedbrainBDNFexpression(Gourleyetal.,2008).ItwasalsoobservedthatadrenalectomyresultedinanelevationinthelevelsofBDNF(Chaoetal.,1998).Consistentwiththestudies,ourstudyalsofoundthatadministrationofexogenouscorticosteronereducedtheBDNFmRNAandproteinlevelsinthefrontalcortexandhippocampusofrats.Inaddition,increasesofBDNFinbrainhavebeenshowntoattenuatedepression-relatedbehavior(Shirayamaetal.,2002).Moreover,antidepressantincreasesBDNF-inducedhippocampalneurogenesisviaaGR-dependentmechanism(Anackeretal.,2011).Therefore,whenweexaminedtheeffectsofHCEonthecorticosterone-mediateddecreaseinBDNFexpres-sion,theresultsoflong-termHCEtreatmentreversingthereductionsuggestedthatthebehavioralimprovementinthemodelmaybeconcurrentwithincreasedBDNFlevels.

GrowingevidencesuggestsBDNF-TrkBsignalingservesanimportantroleintheregulationofmanyofthebehavioralandmolecularmechanismofantidepressant(Saarelainenetal.,2003).EitherdecreasedBDNFavailabilityorreducedlevelsofTrkBneurotrophinreceptorcouldreduceBDNFsignaling(Fahnestocketal.,2012).Short-orlong-termtreatmentswithantidepressantsactivateTrkBreceptorinmousebrain(Rantam¨akietal.,2007).Therefore,besidesBDNF,weevaluatedtheeffectsofHCEongeneandproteinexpressionsofTrkBreceptorinthefrontalcortexandhippocampus.TheimmunoblottingresultsshowedthatTrkBreceptorproteinsinfrontalcortexandhippocampusweremark-edlydecreasedbyrepeatedcorticosteroneadministrationandreversedbyHCEtreatment.However,therewerenosigni?cantchangesinfrontalcortexandhippocampalTrkBmRNAlevelsafterlong-termcorticosteroneorHCEtreatment.Theresults