Chronic treatment with baicalin prevents the chronic mild stress-induced

baicalin,depression

ProgressinNeuro-Psychopharmacology&BiologicalPsychiatry40(2013)138–143

ContentslistsavailableatSciVerseScienceDirect

ProgressinNeuro-Psychopharmacology&Biological

Psychiatry

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

Chronictreatmentwithbaicalinpreventsthechronicmildstress-induced

depressive-likebehavior:Involvingtheinhibitionofcyclooxygenase-2inratbrain

Yu-ChengLia, ,Ji-DuoShena,JingLib,RuiWangc,ShuoJiaoc,Li-TaoYib,

abc

CollegeofPharmacy,HenanUniversityofTraditionalChineseMedicine,Zhengzhou450008,Henanprovince,PRChina

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

articleinfoabstract

Baicalin,amajorconstituentof avonoidsisolatedfromScutellariaeRadix,hasbeenpreviouslycon rmedtodecreasethedurationofimmobilityinmiceexposedtotheforcedswimmingtest(FST)andtailsuspensiontest(TST).However,itsantidepressanteffectsandmechanismsarestillseldomstudiedinchronicmildstress(CMS)modelofdepression.Inthepresentstudy,weattemptedtoinvestigatetheeffectsofbaicalinonthedepressive-likebehavior,themRNAexpressionandactivityofcyclooxygenase-2(COX-2),aswellasprosta-glandinE2(PGE2)levelsinthefrontalcortexandhippocampus.Moreover,theserumcorticosteronelevelswerealsoexamined.WefoundthatCMSprocedurenotonlydecreasedthesucrosepreferenceandincreasedserumcorticosteronelevels,butalsoelevatedtheactivityandmRNAexpressionofCOX-2,andincreasedPGE2levelsinratbrainregions.Treatmentwithbaicalin(10,20,40mg/kg)preventedtheseabnormalitiesinducedbyCMS.Theseresultscon rmedthatbaicalinexertedantidepressant-likeeffects,andsuggesteditsmechanismsatleastpartiallyrelatedtodeceaseCOX-2activityandexpression,subsequentlyresultedinreductionofPGE2levelsinbrain.Our ndingsmayprovideanewaspecttounderstandtheantidepressantactionofbaicalin,whichistargetedontheCOX-2systeminbrain.

©2012ElsevierInc.Allrightsreserved.

Articlehistory:

Received23July2012

Receivedinrevisedform12September2012Accepted18September2012

Availableonline26September2012Keywords:

AntidepressantBaicalin

Chronicmildstress(CMS)Cyclooxygenase-2(COX-2)ProstaglandinE2(PGE2)

1.Introduction

Depressionisacommonemotionaldisorderandthemonoaminede ciencyandhyperactivityofthehypothalamic–pituitary–adrenal(HPA)axisarethoughttobeinvolvedinthepathogenesisofdepression(Barden,2004;Delgado,2000).Recently,growingevidencessuggeststhatcytokineandin ammationalsoplayanimportantroleinthepath-ogenesisofdepression(Paceetal.,2007).Cyclooxygenase(COX),arate-limitingenzymeintheformationofprostaglandins(PGs)fromarachidonicacid,extensivelyexpressedincentralnervoussystem(CNS)(Kawasakietal.,1993;Kuberaetal.,2011;Toccoetal.,1997).TherearetwomainisoformsofCOXs,COX-1andCOX-2.COX-1isconstitutivelyexpressedinmanycelltypesandCOX-2isusuallyconsid-eredasaninducibleenzymeandup-regulatedfollowingstimulationofvariousstressors(O'Banion,1999;Seibertetal.,1995).Consistentwiththehypothesisthatneuroin ammatoryprocessescontributetotheneuropathologyofpsychosis,increasingamountsofdatasuggeststhatCOX-2mayplayanimportantroleinpathologicalconditionsinCNS,

Abbreviations:CNS,centralnervoussystem;CMS,chronicmildstress;COX-2,cyclooxy-genase-2;FST,forcedswimmingtest;TST,tailsuspensiontest;HPA,hypothalamic–pituitary–adrenal;PGE2,prostaglandinE2.

Correspondingauthor.Tel./fax:+8637165962746. Correspondingauthor.Tel./fax:+865926162302.

E-mailaddresses:liyucheng@http://wendang.chazidian.com(Y.-C.Li),litaoyi@http://wendang.chazidian.com(L.-T.

Yi).0278-5846/$–seefrontmatter©2012ElsevierInc.Allrightsreserved.

http://wendang.chazidian.com/10.1016/j.pnpbp.2012.09.007

suchasseizures,ischemia,spreadingdepressionandotherdegenera-tivediseases(KoistinahoandChan,2000;Minghetti,2004;Nakayamaetal.,1998).ElevatedCOX-2expressionandactivitywerealsofoundinbrainofpatientswithAlzheimer'sdisease(Nogawaetal.,2003).Fur-thermore,thehippocampalCOX-2mRNAexpressionwasup-regulatedinaratmodelofdepression(Cassanoetal.,2006).A2–6hofimmobi-lizationstresscausedanenhancementofCOX-2proteinexpressionincortexandhippocampus(Madrigaletal.,2003).Ontheotherhand,theCOX-2inhibitor,suchascelecoxib,wasusefulintreatmentofde-pression(Johanssonetal.,2012).

ScutellariaeRadix,thedryrootofScutellariaebaicalensisGeorgi,iswidelyusedintraditionalChineseformulationsfortreatingin am-mationandallergicdiseases.NumerousclinicaltrialsshowedthatScutellariaeRadixoritsactiveingredientswereusefulintreatmentofpatientswithdiarrhea(Morietal.,2003),hypercholesterolemia(Choetal.,2005)andlungcancer(Gol'dbergetal.,1997).Baicalin,themajorbioactive avonoidcompoundfromScutellariaeRadix,hasalsobeenshowntoimproveCNS-relatedactivitiessuchasprotectingagainstseizureinducedbraininjury(Caoetal.,2010;Liuetal.,2012),promotingtheneuraldifferentiation(Lietal.,2012a).FurtherresearchesindicatedthattheseeffectsofbaicalinmaybemediatedbyactivationofErk1/2(Lietal.,2011a),orinhibitionofNMDAreceptor-mediated5-lipoxygenaseactivation(Geetal.,2007).

Ourpreviousstudiesindicatedthatboththetotal avonoidsofScutellariabaicalensisandbaicalin(5,6-dihydroxy-7-O-glucuronide

baicalin,depression

Y.-C.Lietal./ProgressinNeuro-Psychopharmacology&BiologicalPsychiatry40(2013)138–143139

avonoidglycosides)decreasedtheimmobilitytimeinthemouseforcedswimmingtest(FST)andtailsuspensiontest(TST)(Lietal.,2011b,2012b).Moreover,previousstudybyWangetal.(2010)alsodemonstratedthatthetotal avonoidsfromScutellariaeRadix(containing81.27%baicalin)obviouslyshortenthedurationofimmo-bilityinFSTandTSTmice,suggestingthatbaicalinmightproduceanantidepressant-likeeffectviaregulationofdopaminesystem.Asmentionedabove,COX-2playsanimportantroleinthepathogenesisofdepression,andbaicalinisknowntoinhibitCOX-2inmultiplediseasemodels(Chengetal.,2012;Kanekoetal.,2009;Kimetal.,2010;Tuetal.,2009,2011).However,thetherapeuticeffectofbaicalinonchronicmildstress(CMS)modelofdepression,andwhethertheantidepressant-likeactionofbaicalinisinvolvedintheinhibitionofCOX-2remainsunknown.Therefore,thepresentstudyaimstoinvesti-gatetheantidepressant-likeeffectofbaicalininaratmodelofdepressioninducedbyCMS.Simultaneously,theeffectsofbaicalinonCOX-2andprostaglandinE2(PGE2)levelsintheratbrainregionsoffrontalcortexandhippocampuswasalsoexamined.

2.3.Chronicmildstressprocedure

TheCMSprocedurewasslightlymodi edfromthatpreviouslyde-scribedbyWillneretal.(1987).Brie y,theweeklystressregimeconsistedoffoodandwaterdeprivation,stroboscopicillumination(150 ashes/min),whitenoise,light/darksuccessionevery2h,over-nightillumination,45°cagetilt,soiledcageandpair-housing(Table1).Allstressorswereappliedindividuallyandcontinuously.Thecontrolratswerehousedinaseparateroomandhadnocontactwiththestressedanimals.Theseratsweredeprivedoffoodandwaterfor18hprecedingeachsucrosetest,butotherwisefoodandwaterwerefreelyavailableinthehomecage.TheCMSprocedurewaslastfor5weeks.

2.4.Sucrosepreferenceanddrugadministrations

BeforethebeginningofCMSprocedure,allratsweregiven1%sucrosesolutionfor24h.Then,bothsucrosesolutionandfreshwaterweremadeaccessibletotheratsforanother24h.Afterdeprivedofdrinkingfor23h,theratsweregivenboth1%sucrosesolutionandfreshwaterfor1hagain.Afterthissucroseconsumptiontrainingphase,theanimalswererandomlydividedinto6groups(10ratspergroup):Control,CMS,CMS- uoxetine(positivecontrol,7mg/kg),andCMS-baicalinatdoseof10mg/kg,20mg/kgand40mg/kg.Fivegroupsexceptcontrolwereex-posedtotheCMSprocedurefor5weeks.Alldrugsweresuspendedinfreshwater,andadministeredbygavageoncedailyat11:00a.m.for5weeks.ThroughtheperiodofCMSandtreatment,sucrosepreferencetestwasconductedfollowingan18-hourfoodandwaterdeprivationat11:00a.m.everyTuesday.Sucrosepreferencewascalculatedassucrosepreference(%)=sucroseintake(ml)/[sucroseintake(ml)+waterin-take(ml)]×100%.Thetreatmentprotocolofdosesandadministrationrouteusedforbaicalinand uoxetinewereadoptedaccordingtoourpreviousstudies(Lietal.,2009,2012b).

2.Materialsandmethods2.1.Materials

BaicalinwaspurchasedfromNanjingZelangMedicalTechnologyCo.Ltd(Nanjing,P.R.China).Thepurityofbaicalinwas98.1%,checkedbyhigh-performanceliquidchromatography(HPLC).Fluoxetinehydrochlo-ridewasfromChangzhouSiyaoPharmaceuticalsCo.,Ltd.(Changzhou,P.R.China).AllprimersusedinthisstudyweredesignedandsynthesizedbySangonBiotechCo.Ltd.(Shanghai,P.R.China).TrizolreagentwasfromInvitrogen(Carlsbad,USA).ReversetranscriptaseMoloneyMurineLeukemiaVirus(M-MLV)usedforcDNAsynthesiswasfromPromegaCorporation(Madison,USA).AllotherreagentsusedinRT-PCRwerefromSangonBiotechCo.Ltd.(Shanghai,P.R.China).

2.5.Bloodandbraintissuessamplecollection

2.2.Animals

MaleWistarrats,180–220gweight,werepurchasedfromtheLaboratoryAnimalCenterofFujianProvince,China.Animalswereallowedtoadaptoneweekbeforetheexperimentstarted.Allratswereindividuallyhoused(cagesize:30×20×16cm),withfoodandwaterfreelyavailable,andmaintainedona12hdark–lightcycle(withthelightsonat07:00hlocatetime)underregulatedtem-peratureconditions(22±2°C),exceptasdescribedbelow.Allproce-dureswereperformedinaccordancewiththepublishedguidelinesoftheChinaCouncilonAnimalCare(RegulationsfortheAdministrationofAffairsConcerningExperimentalAnimals,approvedbytheStateCouncilonOctober31,1988andpromulgatedbyDecreeNo.2oftheStateScienceandTechnologyCommissiononNovember14,1988).

Afterthelaststress,allratsweredecapitatedbetween11:00a.m.and1:00p.m.toavoid uctuationofhormonelevels.Bloodsampleswerecollectedimmediately.Thewholefrontalcortexandwholehip-pocampusineachhemispherewererapidlyseparatedonice-plateandstoredat 80°Cuntilanalysis.ThelocationsoffrontalcortexandhippocampusweredescribedasinPaxinosandWatson'sbook(1998).

2.6.Serumcorticosteroneleveldetermination

Bloodwerecollectedoniceandseparatedinarefrigeratedcentrifugeat4°C.Serumcorticosteronelevelsweremeasuredusinganenzymeimmunoassaykit(EnzoLifeSciences,PlymouthMeeting,USA).

Table1

ThescheduleofCMSprocedure.

Monday

FoodandwaterdeprivationStroboscopicilluminationWhitenoise

Light/darksuccessionevery2hOvernightillumination45°cagetiltSoiledcagePair-housing

16:00→

Tuesday10:00

12:00–20:0020:00→

Wednesday

Thursday08:00–20:00

20:00→

08:00

08:00-20:0020:00→

08:00

08:00–20:00

Friday

Saturday

Sunday

→16:00

08:0020:00→

08:00–20:00

08:00

08:00–20:0020:00→

20:00→

08:00

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140Y.-C.Lietal./ProgressinNeuro-Psychopharmacology&BiologicalPsychiatry40(2013)138–143

2.7.Real-timequantitativePCRanalysis

TotalRNAwasextractedfromfrontalcortexandhippocampususingTrizolreagentfollowingthemanufacturer'sinstructions.Reversetran-scriptionwasperformedusingM-MLVreversetranscriptaseforcDNAsynthesis.Real-timePCRreactionswereperformedusingaSYBRPremixExTaqKitinABI-7500system.TheCOX-2(forward5′-TGCCCAGCACTTCACTC-3′;reverse5′-GGAACAGTCGCTCGTCA-3′,386bp)andtheinternalcontrolGAPDH(forward5′-ACCACAGTCCATGCCATCAC-3′;reverse5′-TCCACCACCCTGTTGCTGTA-3′,452bp)primerswereused.The uorescencesignalwasdetectedattheendofeachcycle.Meltingcurveanalysiswasusedtocon rmthespeci cityoftheproducts.Theresultswereanalyzedbythe2 ΔΔCTmethod(LivakandSchmittgen,2001).

2.8.MeasurementofCOX-2activityandPGE2levels

TheCOX-2activityandPGE2contentsinfrontalcortexandhippocam-pusweremeasuredbyusingcommerciallyenzymeimmunoassaykit(COX-2:Cayman,AnnArbor,USA;PGE2:R&D,Minneapolis,USA).Inparticular,SC-560wasusedinCOX-2activityassaytoinhibitCOX-1.

2.9.Statisticalanalyses

Alldatawereexpressedasmean±S.E.M.DataforsucrosepreferenceandbodyweightwereanalyzedusingarepeatedANOVA,withtreatmentasbetweenfactorsandweekaswithinfactors,andtreatmenteffectwasperformedusingaposthocDunnett'stest.Fortheestimationofbiochem-icalresultsintheCMS,aone-wayANOVAfollowingDunnett'sposthoctestwasperformed.AvalueofPb0.05wasconsideredstatisticallysignif-icantforanalysis.

3.Results

3.1.Effectsofbaicalinonsucrosepreferenceandbodyweight

InordertomonitorthevalidityoftheCMSprocedure,weperformedsucrosepreferencetestweekly.ThesucroseintakeintheCMSprocedurewasshowninFig.1A.AtthebeginningoftheCMSprocedure,sucrosepreferencewassimilarineachgroup.ArepeatedANOVAwithtreatmentasbetweenfactorandweekaswithinfactor,re-vealedasigni canteffectoftreatment[F(5,54)=3.166,Pb0.05],andsig-ni canteffectofweek[F(5,270)=3.852,Pb0.05].Moreover,asigni cantweek×treatmentinteractionwasalsoobserved[F(25,270)=2.118,Pb0.05].Post-hoctestshowedasigni canteffectoftreatmentbetweenthevehicle-CMSand20,40mg/kgbaicalin(Pb0.05,Pb0.01,respectively)or7mg/kg uoxetine(Pb0.01),butnot10mg/kgbaicalin.

Inaddition,one-wayANOVArevealedasigni cantdecreasedsucrosepreferenceintheCMSgroup,whichstartedfromweek2(Pb0.001)andsustainedthefollowingweeks(week3:Pb0.01;week4:Pb0.01;week5:Pb0.001).After5weekstreatment,baicalin(10,20and40mg/kg)and uoxetine(7mg/kg)preventedthisdecreaseinducedbyCMS(baicalin:Pb0.05,Pb0.01,Pb0.001; uoxetine:Pb0.001).

Thebodyweightoftheratsineachgroupdoesnothaveasigni cantdifferenceatthebeginning(Fig.1B).Overthe5-weekexperiment,are-peatedANOVAindicatedno-signi canceoftreatment[F(5,54)=0.036,P>0.05]orweek×treatmentinteraction[F(25,270)=1.028,P>0.05]onbodyweight,butasigni cantweekeffect[F(5,270)=4.374,Pb0.01].Post-hoctestshowedthattherewasnosigni cantdifferenceonthebodyweight.

Inaddition,one-wayANOVArevealedthatthecontrolgroupgainedmoreweightthanCMSgroup(Pb0.01).Therewerenosigni -cantdifferencesamongtherestofthegroups.

Fig.1.Effectsofbaicalinonsucrosepreference(A)andbodyweight(B)inCMSrats.Datawereexpressedasmean±SEM(n=10).Forstatisticalsigni cance,++Pb0.01,+++

Pb0.001vs.control; Pb0.05, Pb0.01, Pb0.001vs.

CMS-vehicle.

3.2.Effectsofbaicalinonserumcorticosteronelevels

AsshowninFig.2,theserumcorticosteronelevelsinCMSgroupwassigni canthigherthanthatofcontrolgroup(Pb0.01).Chronicad-ministrationwith uoxetine(7mg/kg)andbaicalin(20,40

mg/kg)

Fig.2.EffectsofbaicalinonserumcorticosteronelevelsinCMSrats.Datawereexpressed asmean±SEM(n=10).Forstatisticalsigni cance,++Pb0.01vs.control;Pb0.05, Pb0.01vs.CMS-vehicle.

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Y.-C.Lietal./ProgressinNeuro-Psychopharmacology&BiologicalPsychiatry40(2013)138–143141

signi cantlyinhibitedserumcorticosteronelevels(Pb0.01,Pb0.05,Pb0.01,respectively).

3.3.EffectsofbaicalinonCOX-2mRNAexpressioninthefrontalcortexandhippocampus

ToinvestigatewhetherCOX-2isinvolvedinCMSinduceddepression,wecheckedCOX-2mRNAexpressioninfrontalcortexandhippocampususingreal-timequantitativePCR.AsseeninFig.3A(Frontalcortex)andFig.3B(hippocampus),COX-2mRNAexpressionwassigni cantlyin-creasedabout4foldinCMSratscomparedwiththecontrolgroup(Pb0.01),whichwereinhibitedby20,40mg/kgbaicalin(Frontalcortex:Pb0.01,Pb0.01;Hippocampus:Pb0.05,Pb0.01,respectively)and7mg/kg uoxetine(Frontalcortex:Pb0.01;Hippocampus:Pb0.01,re-spectively)treatment.

3.4.EffectsofbaicalinonCOX-2activityinthefrontalcortexandhippocampus

WefurtherdetectedCOX-2activityinthefrontalcortex(Fig.4A)andhippocampus(Fig.4B).Asexpected,COX-2activitywassigni cantelevatedafterCMSprocedure(Frontalcortex:Pb0.05;Hippocampus:Pb0.01,respectively).Chronicbaicalintreatment(10,20,40mg/kg)re-storedCOX-2activitybothinfrontalcortex(Pb0.01,Pb0.01,Pb0.01,respectively)andhippocampus(Pb0.05,Pb0.01,Pb0.01,respectively).

Fig.3.EffectsofbaicalinonthemRNAexpressionofCOX-2infrontalcortex(A)andhippocampus(B)inCMSrats.Datawereexpressedasmean±SEM(n=6).Forstatisticalsigni cance,++Pb0.01vs.control; Pb0.05, Pb0.01vs.

CMS-vehicle.

Fig.4.EffectsofbaicalinonCOX-2activityinfrontalcortex(A)andhippocampus(B)inCMSrats.Datawereexpressedasmean±SEM(n=7).Forstatisticalsigni cance,+

Pb0.05,+++Pb0.001vs.control; Pb0.05, Pb0.01vs.CMS-vehicle.

3.5.EffectsofbaicalinonPGE2levelsinthefrontalcortexandhippocampusTheeffectsofbaicalinand uoxetineonPGE2contentinthefrontalcortexandhippocampuswereshowninFig.5AandB.CMScausedasigni cantincreaseinPGE2contentinfrontalcortex(Pb0.01)andhippocampus(http://wendang.chazidian.comparedwithCMS-vehiclerats,chronictreatmentwith10,20and40mg/kgbaicalinsigni cantlyattenuatedCMS-inducedincreasedPGE2inadose-dependentmannerbothinfrontalcortex(Pb0.05,Pb0.01,Pb0.01,respectively)andhippocampus(Pb0.05,Pb0.01,Pb0.01,respectively).The uoxetineat7mg/kgalsosigni cantlyreducedtheCMS-inducedPGE2contentinfrontalcortexandhippocampus(Pb0.01,Pb0.01,respectively).4.Disccusion

Chronicstressisanimportantprecipitantfactorindepression,andthechangesinvariousbodysystemsthatoccurredindepressionaresimilartothoseobservedinresponsetostress(Leonard,2001b).CMS,themostpromisingandvaluablerodentmodelofdepression,arewidelyusedtoresearchandscreenforantidepressants(Willneretal.,1987).Inaddition,asproposedbyWillner,CMSappearsmoresuitableforstudyingthemechanismsofantidepressantdrugscomparedtoacutestressmodels(Willner,1997).IntheCMSprocedure,animalsareexposedtodifferentkindsofmildstresseveryday,whichmimicschronicstressfullifeeventsandresultsinanhedonia,acoresymptomofhumandepression.Asaresult,thedecreasedsucrosepreferenceindexinducedbyCMSprocedurecanberestoredbytherapeuticallyeffectivedrugsforthetreatmentofdepression(Zhangetal.,2010).Therefore,wein-vestigatedtheantidepressant-likeeffectandmechanismsofchronicbaicalintreatmentinthismodel.Ourresultsfoundthata

reduction

baicalin,depression

142Y.-C.Lietal./ProgressinNeuro-Psychopharmacology&BiologicalPsychiatry40(2013)138–143

Fig.5.EffectsofbaicalinonthePGE2levelsinfrontalcortex(A)andhippocampus(B)inCMSrats.Datawereexpressedasmean±SEM(n=7).Forstatisticalsigni cance,++

Pb0.01vs.control; Pb0.05, Pb0.01vs.CMS-vehicle.

ofsucrosepreferenceinducedbyCMSprocedurewassigni cantlyattenuatedbychronictreatmentwithbaicalin,whichfurthercon rmedtheantidepressant-likeeffectofbaicalin.

Additionally,ourresultsshowedthatCMScausedareductionofbodyweight,whichwasconsistentwithotherreports(Edgaretal.,2002;LiuandZhou,2012;Zhangetal.,2010).However,severalpreviousstudiesalsoreportedthatCMSexertedonlyslightornoeffectonbodyweightofrats(Firstetal.,2011;Gamaroetal.,2008).Thesediscrepanciesmaybeproducedbymanyfactors,suchasdifferentstrainsofanimals,exper-imentalconditions,stressedtimeandsoon(Pothionetal.,2004).

Inrecentyears,thebiologicalresearchofdepressionhadmovedbeyondthemonoamineshypothesis.Agrowingbodyofevidencessupportsthatdepressionisalsorelatedtothedysfunctionofimmunesystems(Leonard,2001a;LicinioandWong,1997).Excessivesecretionofpro-in ammatorycytokinesresultedin‘sicknessbehavior,’thesymptomofwhichwassimilartothoseofdepression,indicatingtheroleofcytokinesinhumandepression(Dantzer,2006).Forexample,increasedpro-in ammatorycytokinesreleaseincludinginterleukin-1β(IL-1β)andtumornecrosisfactorα(TNFα),wereobservedindepressedpatients(Maes,1999).Inaddition,previousstudydemonstratedthatexpressionofCOX-2mRNAandproteinisoftenenhancedinvarioushumancelltypesstimulatedbypro-in ammatorycytokinessuchasIL-1βandTNFα(Kuwanoetal.,2004).Thesereportscon rmedtherolesofCOX-2inregulatingin ammatoryresolution(GilroyandColville-Nash,2000).Asaresult,neuroprotectiveeffectsofCOX-2inhib-itorhadbeenobservedinvariousCNS-relateddisorders(Dhiretal.,2006;Huetal.,2005;Kumarietal.,2007)includingdepression(Neryetal.,2008).Inclinicaltrails,celecoxib(aCOX-2inhibitor)exhibitedtherapeuticeffectsinmajordepression:resultsofadouble-blind,ran-domized,placebocontrolled,add-onpilotstudy(Mülleretal.,2006).

Intheanimalstudy,Guoetal.(2009)demonstratedthatchroniccelecoxibtreatmentreverseddepressive-likebehaviorinducedbyCMSviareducingCOX-2expressioninbrain.Therefore,ourstudywasalsoundertakentoinvestigatewhetherthelevelsofCOX-2andPGE2invariousbrainregionswereup-regulatedinCMS-inducedratsandwhetherbaicalincouldpreventtheseabnormalities.ThepresentstudyfoundthattheactivityandmRNAexpressionofCOX-2,aswellasPGE2levels,weresimultaneouslyincreasedinfrontalcortexandhip-pocampusintheCMSrats,whichwereconsistentwiththepreviousstudies(Gárateetal.,2011;Wangetal.,2011).Baicalinsigni cantlydecreasedCOX-2expressionandactivityandPGE2levelsinthesetwocriticalbrainregions.Infact,theCOX-2inhibitoryeffectofScutellariaeRadixorbaicalinhadbeenreportedinpreviousstudies.Forexample,ScutellariaeRadixreducedLPS-stimulatedPGE2production(Kanekoetal.,2009).IncreasedCOX-2mRNAexpressionwasattenuatedbybaicalinintheratlivercausedbyischemia/reperfusion(Kimetal.,2010).Inaddition,baicalinimprovedthedetrimentaleffectsonspatialmemoryassociatedwithglobalcerebralischemiabyreducinghippocam-palapoptosisviatheinhibitionofCOX-2expression(Chengetal.,2012).Moreover,baicalinproducedtheneuroprotectiveeffectinaratmodelofpermanentfocalcerebralischemiathroughtheinhibitionofCOX-2expressioninbrain(Tuetal.,2009,2011).Theseresultsabovesuggestedthatbaicalinelicitedantidepressanteffectspartiallythroughat-tenuatingorreversingCMS-inducedabnormalitiesinCOX-2expression.

Itisgenerallyacceptedthatglucocorticoidsplayanessentialroleinresponsestoenvironmentalstressors,servinginitiallytomobilizebodilyresponsestochallengeandultimatelyservingtorestrainneuro-endocrineaswellasimmuneresponses(in ammation)(Paceetal.,2007).Reducedresponsivenesstoglucocorticoidsandimpairedfunction-ingoftheglucocorticoidreceptor,whichwerecommonlyseeninmajordepression(RaisonandMiller,2003),mayinturncontributetoex-cessivein ammationaswellashyperactivityofthehypothalamic-pituitary-adrenal(HPA)axis(Leonard,2001a).Interestingly,ithadbeencon rmedthatinhibitionofCOX-2pathwaysmayrepresentatherapeutictargetfornormalizationofreducedresponsivenesstoglucocorticoidsandimpairedfunctioningoftheglucocorticoidre-ceptor(Huetal.,2005).Partiallyinparallelwiththeactionofcelecoxib(Huetal.,2005),wespeculatedthatbaicalinproducedapositiveeffectonglucocorticoidreceptorsystem(Decreasedserumcorticosteronelevels)byinhibitionofCOX-2anditsdownstreamproductPGE2levels.5.Conclusion

Inconclusion,thepresentstudyinvestigatedtheantidepressant-likeeffectsandmechanismsofbaicalinintheCMSmodelofWistarrats.TheCMSprocedureelevatedCOX-2activityandexpression,aswellasitscatalysatePGE2levelsinfrontalcortexandhippocampus.ChronicbaicalintreatmentdeceasedCOX-2activityandexpression,subsequentlyresultedinreductionofPGE2levelsinbrain,and nallynormalizedtheHPAaxis(atleastreducingcorticosteronelevels).These ndingsmightprovideabasisforexaminingtheneuroimmuneandneuroendocrineinteractionindepressionandbaicalintreatment.6.Contributors

AuthorYu-ChengLidesignedthestudy,participatedintheresearch(Real-timePCR)andwrotethe rstdraftofthemanuscript.Ji-DuoShencontributedtotheanalysisofresultsandpartlyresearch(Real-timePCR).JingLiandRuiWangparticipatedintheestablishmentofCMSmodel.ShuoJiaotookpartinthedeterminationofELISAkits.Li-TaoYidesignedthestudyandrevisedthedraftofthemanuscript.Acknowledgement

TheprojectwassupportedbyNaturalScienceFoundationofEdu-cationDepartmentofHenanProvinceofChina(No.

12B310004),