1SCIENTIFICREPORTS}?6903?wv?wvy~?zw{~?vw}?v}zv}?w
???????????????????????????????
????????????????????????
?????????????????????????
???????????????????????????
??????????????????????????????
???????????????????
?????Yan
w
á????He
x
á??????Wan
w
á??????Xu
w
,Huilin ???
w
á ??????
y
á??????Bi
x
&
???Jia
y
???????????????????????????????????????????????????????????????????????????
??a????????????????????????????????????????????????????????????????????????????á
?????????????????????????????????????????????????????????????????????????
??????????????????????????????????????á??? ?????á??????????????????????
???????????????????????????????????????????????????z??????????????????
??????????????????????????????????¤??????????????????????á?????????????¤??????
??????????????????????????????????????? ????????????????????????????????á
?????????????????????????????a????????????????á???????????????????????????????????
????????????????????????????????????????????????????????????????????????????
?????????????????????????????y??????y?????????????????????????????
???????yβ
?yβ????????????????????????????????????????|vv??wxvv????á????
?????¤?????????????????????????????????????????????????????????????????????????????
?????????????????????????????????????????????????????????????????????????¤??????
????á?????????????????????????????????? ???????????????????á?????????????????????
???????y
?yβ?????????
Depressionisacommonillnessworldwide,withanestimated350millionpeopleofallagesaf_fectedaccording
toareportofWHOin2014
1
,itisalsotheleadingcauseofdisabilityandisamajorcontributortotheoverall
globalburdenofdisease.Depressionisaheterogeneoussyndromeandmayresultinpoorerwell-being.Atits
worst,depressioncanleadtosuicide
2
.Ingeneral,mostclinicalsymptomsofmajordepressivedisorder,suchas
delusions,anxiety,irritabilityorinsomnia,canbeef_fectivelytreatedbycurrentpsychopharmacologicaltreat-
ments.Nevertheless,cognitivedef_icits(likeadiminishedabilitytothinkorconcentrate,orindecisiveness),which
representcoredef_icitsofdepression
3
,maypersistinpatientsevenwhendepressivesymptomshaveabatedor
disappearedandsignif_icantlyaf_fecttheindividual’ssocialandoccupationalfunction.Indeed,meta-analyseshave
shownthatcognitivedef_icitsarestillpresentinremittedpatients
4
.Forthisreason,cognitiveimpairmentemerges
asapotentialtargetforbothpharmacologicalandpsychosocialtreatments,withthef_inalgoalofimproving
functioning.
BDNFisamemberofthenervegrowthfactorfamilyandisexpressedintheadultmammaliancentraland
peripheralnervoussystem,particularlyinthehippocampusandcortex
5
.T_hevastmajorityoftheliteraturelink-
ingneurotrophinstomooddisordersdealswithBDNF,otherneurotrophinsshowingonlyveryminorrole
6–8
.
Inaddition,clinicalstudieshaveshownthatserumBDNFlevelsandhippocampalvolumereductionsinelderly
w
??????????????????????????????????á?????????????????????????????á????????wvyá???????á
wwvvw|á?????
x
??????????????á?????????????????????????????á????????wvyá???????áwwvvw|á
?????
y
??????? ????????? ?????????á?????????????????????????????á????????wvyá???????á
wwvvw|á?????????? ???????????????????????????????????????????????????????????????????????????
??????????????????? ?
???????????????????;wx|????)
Received:15February2017
Accepted:28June2017
Publishedonline:31July2017
www.nature.com/scientificreports/
2SCIENTIFICREPORTS}?6903?wv?wvy~?zw{~?vw}?v}zv}?w
individualsarecloselycorrelatedwithmemoryloss
9,10
,andBDNFmayrescuecognitiveimpairmentsandlearn-
ingdef_icitsinAlzheimer’sdisease
11,12
.PreviousreportsindicatedthatchangesinBDNFlevelareimplicated
inthepathophysiologyofcognitivedeclineindepressionandneurodegenerativedisorders
13
.BDNFexertsits
pro-survivalef_fectsbybindingitshighaf_f_inityreceptorTrkB,toactivateBDNF-TrkBsignaling
14
.Behavioral
responsestoantidepressantshavebeenabolishedinanimalsinwhichBDNF-TrkBsignalingisinhibited
15
.
Itisalsoinvolvedinpropofol-inducedlearningandmemoryimpairments
16
.T_hus,theseresultssuggestthat
BDNF-TrkBsignalingplaysacriticalroleinthemolecularmechanismsofantidepressantsandcognition
enhancement.BDNF-TrkBdownstreamsignaling,includingERK/CREBandPI3K/AKT/GSK-3βpathways,can
modulateneurotransmitterrelease,cellviability,apoptosisandpostsynapticresponses,andthesefunctionsare
closelyassociatedwithdepression
17,18
andlearningability
19
.
About30%ofpatientssuf_feringfromamajordepressivedisorderdonotrespondsuf_f_icientlytoestablish
pharmacological,psychotherapeutic,orsomatictreatments
20
.Inaddition,dif_ferentstudiesconcludethatonly
30–40%ofpatientsthatareoptimallytreatedwithf_irstlineantidepressantsachieveremissionandmorethanone
thirdofpatientswithdepressionareclassif_iedastreatment-resistantdepression(TRD)
21
.Inordertodealwiththe
mentionedlimitationsshownbycurrentantidepressantdrugs,traditionalChinesemedicine(TCM)isattract-
ingincreasingattentionasamethodformeetingthedemandsforhigherremissionrate,fasteronset,persistent
antidepressantaction,andfeweradverseef_fects
22–24
.Schisandrachinensis(Trucz.)Bail.isafamoustraditional
Chinesemedicinewhichisusuallyusedintheclinicwiththefunctionsofinducingastringency,replenishing
andpromotingtheproductionofbodyf_luidandtonifyingthekidneystorelievementalstrain
25
.Dibenzo[a,c]
cyclooctadienelignansarethemainchemicalcomponentsinSchisandrachinensisextract(SCE),andtheseare
showingvariouspharmacologicalactivities,ofwhichtheirantioxidative,neuroprotective,anti-cancer,vasorelax-
antandcytoprotectivepropertiesareamongthemoststudiedones
26–28
.OurgrouppreviousreportedthatSCE
couldproduceantidepressant-likeef_fectsindif_ferentdepressedmodels
29,30
,andamelioratecognitiondef_icits
31
.
However,thepotentialneuroprotectiveef_fectsofSCEagainsttheCUMS-induceddepressionandcognitivedef_i-
cits,aswellasthemechanismsremaintobeclarif_ied.T_hus,inthisstudy,weusedtheCUMSmodeltoinvestigate
theantidepressant-likeactivityandcognitiveimpairmentef_fectsofSCE,moreover,theBDNF/TrkB/ERK/CREB
andPI3K/AKT/GSK-3βsignalingpathwaysweredeterminedtoillustratetheactionmechanismofSCE.
??????
???????????????????????????????????AsshowninFig.uni00A01,Posthocanalysisrevealedthat
a4-weeksCUMSexposuresignif_icantlyreducedthepercentageofsucroseconsumptioninthestressedmicein
comparisonwiththecontrolgroup(p<0.01).However,long-termtreatmentwithSCE(600mg/kgor1200mg/
kg)increasedthesucrosepreferenceascomparedtotheCUMScontrolmice(p<0.01,p<0.01,respectively).
Asoneapproachtoinvestigatetheantidepressantef_fectsofSCE,thesucrosepreferencetest,anindicatorof
anhedonia,wasappliedaf_ter4weeksofCUMSexposure.Suchincreasesinsucroseconsumptionsuggestapotent
antidepressant-likeef_fectofSCEwithintheseCUMS-exposedmice.
???????????????????????????T_hereisnosignif_icanceamongallgroupsinthetotaltraveldis-
tancewhichindicatedthatCUMSanddrugtreatmentdidnotalterthelocomotoractivityofthetestanimal
(Tableuni00A01).
??????????????????????????????? ?Consistentwiththesucroseconsumptiontest,Post
hocanalysisindicatedthat4-weeksofCUMSexposuresignif_icantlyincreasedimmobilitytimescomparedto
thecontrolmice(Fig.uni00A02).T_heseef_fectswerereversedbychronicSCEtreatment(600mg/kgor1200mg/kg),with
immobilitydurationsnowbeingsignif_icantlydecreasedascomparedtotheCUMScontrolgroupwithregardto
Figure1.Ef_fectsofSCEadministrationonthesucrosepreferenceintheSPT.T_hedatarepresentedthevaluesof
mean±S.E.M.from10mice/group.p<0.01vs.CUMSgroup.
##
p<0.01vs.Controlgroup.
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3SCIENTIFICREPORTS}?6903?wv?wvy~?zw{~?vw}?v}zv}?w
immobilitytime(p<0.01,p<0.01,respectively).T_heseresultscomplementthoseofthesucrosepreferencetest,
againshowinganantidepressant-likeef_fectofSCEasbaseduponthesedecreasedimmobilitytimesinthisFST.
????????? ?????????AnalysesofthespontaneousalternationpercentagewithinYmazetask
showedsignif_icantoveralldif_ferencesbetweenallgroups(Fig.uni00A03).BothdosesofSCE(600mg/kgor1200mg/kg),
butespecially600mg/kg,signif_icantlyimprovedmemoryformationinCUMS-inducedmiceascomparedtothe
controlmice.T_hechangesinthespontaneousalternationpercentagesofCUMS-inducedmiceexposedtoSCE
arenotrelatedtothechangesinmotoractivity,asevidencedbythenumberofarmentriesascomparedtothe
controlmice.
????????????????????????Asignif_icantdecreaseoftheescapelatencytoreachthesubmerged
platformwasevidencedalongthe5daysofatrainingperiodinallthegroupsintheMWMtest(Tableuni00A02).T_he
CUMSgrouptookmoretimeonthelasttwotrainingdaystoreachtheplatformcomparedwiththecontrolgroup
Groupslocomotionlength(cm/5min)
Control1236±29
CUMS1017±57
Fluoxetine(10mg/kg)1138±76
SCE(300mg/kg)995±79
SCE(600mg/kg)1205±84
SCE(1200mg/kg)1099±59
Table1.Inf_luenceofadministrationofSCEandCUMSprocedureonthelocomotoractivityinmice.
Figure2.Ef_fectsofSCEadministrationontheimobilitytimeintheFST.T_hedatarepresentedthevaluesof
mean±S.E.M.from10mice/group.p<0.05,p<0.01vs.Controlgroup.
#
p<0.05vs.Controlgroup.
Figure3.Ef_fectsofSCEadministrationonY-mazetest,spontaneousalternations(A),numberofarmentries
(B).T_hedatarepresentedthevaluesofmean±S.E.M.from10mice/group.p<0.05vs.CUMSgroup.
##
p<0.01vs.Controlgroup.
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4SCIENTIFICREPORTS}?6903?wv?wvy~?zw{~?vw}?v}zv}?w
intheplacenavigationtest(p<0.05).However,theSCE(600or1200mg/kg)treatedgroupsignif_icantlyamelio-
ratedtheef_fectsofCUMSonescapelatency(p<0.05,p<0.05,p<0.05forthe3
rd
,4
th
and5
th
day,respectively).
Intheprobetrial,aprobetestwasconductedbyremovingtheplatformonthelastdayofMWMtask.T_heef_fects
ofSCEtreatmentontheperformanceofspatialprobetrialinmiceareshowninFig.uni00A04.Miceinthecontrolgroup
focusmoretimeinthetargetquadrant,whilemiceintheCUMSgroupfailedtorememberthepreciselocationof
theplatform.TreatmentwithSCE(600or1200mg/kg)reversedthecognitivedef_icitascomparedtotheCUMS
group.
????????? ????????????????????Comparedwiththecontrolgroup,the
CUMSprocedurecouldsignif_icantlydecreasethelevelsofBDNF,pTrkB/TrkB,pCREB/CREBandpERK/ERKin
hippocampus(p<0.05).Meanwhile,SCE600mg/kgand1200mg/kgcoulddramaticallyupregulatetheBDNF/
TrkB/CREB/ERKsignalinginthehippocampusofdrugtreatedmicecomparedtotheCUMSmice(p<0.05)
(Fig.uni00A05A–D).
????????? ????????????????????????????????????????????????????As
showninFig.uni00A06A–F,intergralopticaldensity(IOD)ofBDNF(brownparticles)inCUMSgroupwasremarkably
lowerthanthatincontrolgroup(p<0.01).T_henumberofpositivehippocampalneuronsandIODinSCE(600
or1200mg/kg)weresignif_icantlyhigherthanthoseinCUMSgroup(p<0.05),indicatingthatSCEup-regulated
theBDNFexpressioninhippocampalneuronswhichwasconsistentwiththedataofELISA.
????????? ??????????????????????Immunof_luorescenceassayshowedthatBDNF
(redf_luorescence,Fig.uni00A07A)indepressionmicewassignif_icantlylowercomparedwithcontrolmice.AndSCE(600
or1200mg/kg)groupcouldapparentlyincreasethelevelofBDNFproteinincomparisonoftheCUMSgroup
wereshowninFig.uni00A07Bbasedontheaveragef_luorescenceintensitiesofBDNF.T_heresultswereinlinewiththe
resultsofELISAandimmunohistochemistryassay.
?????????y
?yβ??????????????????Westernblottingresults(Fig.uni00A08)indicated
thatPI3Kwasreducedindepressionmice(p<0.05)andthistendencywasreversedbySCE(600or1200mg/
kg)signif_icantly(p<0.05).T_hedataofAKTandGSK-3βturnedoutthesame,whichindicatedthatPI3K/AKT/
GSK-3βmightbeinvolvedintheantidepressant-likeef_fectofSCE.
???????????????????????????????????????????????H.E.stainingwasperformedtodetect
theneuronalintegrityandorderliness.Inthehippocampus,theneuronsappearedintactandordered,shrinkage,
GroupsDay1Day2Day3Day4Day5
Control83.91±8.0574.52±9.8763.00±9.3942.57±5.7123.62±6.92
CUMS85.49±7.2081.44±9.7672.94±8.4757.91±9.55
#
37.49±8.25
#
Flu(10)88.94±10.5569.98±6.6362.04±13.7746.24±11.0534.13±7.39
SCE(300)80.59±6.3677.14±10.2158.74±7.7649.07±7.2835.14±8.99
SCE(600)85.64±9.0171.47±7.6856.44±10.4933.53±10.1919.75±6.18
SCE(1200)80.39±10.6575.40±9.6957.74±4.3421.49±9.4521.49±11.03
Table2.Ef_fectofSCEontheescapelatency(s)ofCUMS-inducedmiceinwatermazetest.#p<0.05,compared
withcontrolgroup;p<0.05,p<0.01,comparedwithCUMSgroup.
Figure4.Ef_fectsofSCEadministrationontimeinthetargetquadrantofMorriswatermaze.T_hedata
representedthevaluesofmean±S.E.M.from10mice/group.p<0.05vs.CUMSgroup.
##
p<0.01vs.Control
group.
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5SCIENTIFICREPORTS}?6903?wv?wvy~?zw{~?vw}?v}zv}?w
degenerationandnecrosisofthenucleiwerenotobservedinthecontrolmice(Fig.uni00A09A),butshrinkageofnuclei,
swollenandeccentricallydispersedneuronalbodieswerefoundintheCUMSmice(Fig.uni00A09B).However,adminis-
trationofSCEcouldinhibittheabovementionedhistopathologicaldamagessignif_icantly(Fig.uni00A09C,D,E).
?????????
Numerousneuropsychologicalstudieshaveexaminedtheroleofcognitioninpsychopathology,revealingthat
mostpsychiatricdisordersareassociatedwithsomedegreeofcognitiveimpairment
32
.WeusedCUMSproce-
duretomimicthesymptomofdepressionconf_irmedbySPTandFSTinmiceinourbehavioralstudies,which
isconsistentwithpreviousreports
33
.Further,CUMSalsoimpairedthecognitivefunctionofmiceaccordingto
theperformancesofYmazeandMWMtest,whicharetwoclassicalmethodstoassesstheabilityoflearningand
memory.T_heseresultsarewellcoincidentwithpreviousstudies
34,35
.However,withthetreatmentofSCE,the
anhedonia-likebehaviorinsucrosepreferencetestandimmobilitytimeinforcedswimtestwhichbothinduced
byCUMSweresignif_icantincreased,respectively.Ontheotheraspect,thespontaneousalternationinYmaze,
timeinthetargetquadrantandescapelatencyintrainingofSCEmicewereremarkableimprovedcompared
totheCUMSmice.AndNosignif_icantdif_ferencesofthetotaltraveldistanceinthelocomotoractivitytestand
numberofarmentriesinYmazewhichcouldexcludethefalsepositiveresultscausedbythemobilityofmice
duringallthebehavioraltests.Moreover,wefoundasignif_icantcorrelationbetweenthesucrosepreferencevs.
spontaneousalternation,thesucrosepreferencevs.timeinthetargetquadrant,immobilitytimevs.spontaneous
alternationandimmobilitytimevs.timeinthetargetquadrantwhenlinearregressionwasdetermined(Fig.uni00A010).
IntegratedwiththeresultsofH.E.staining,wefoundthatSCEindeedhastheabilitytoimprovethedepressive
symptomsandtheassociatedcognitivedef_icitsef_fectively.
T_heinterrelationshipbetweendepressionandcognitiveimpairmentiscomplexandstillnotwellunderstood.
Inthebrain,BDNFhasbeenimplicatedindevelopment,neuralregeneration,synaptictransmission,synaptic
plasticityandneurogenesis
36
.AlternationinBDNFlevelshavebeenimplicatedinpsychiatricdisorders,includ-
ingdepressionandsubstanceabuse,aswellasneurodegenerativedisorders,suchasAlzheimer’s,Parkinson’sand
Huntington’sdiseases
37
.WefoundthatinthehippocampusofCUMS-inducedmice,BDNFlevelsweresignif-
icantlydecreased,andtheresultsofY-mazeandMWMtestwereshownthatthecognitionimpairmentalong
withthedepressionsymptoms.Af_tertreatmentwithSCE,theBDNFlevelswereupregulated,andthedepression
symptomsandcognitiondeclinewerealleviatedatthesametimeaccordingtothebehavioralresults.Besidesthe
resultsofimmunohistochemistryanalysisandImmunof_luorescenceassay,theaboveallillustratedthatBDNF
hasplayedavitalrolebothindepressionandthelearningobstacletriggeredbyit,whichinlinewiththeprevious
reports
38
.
Tropomyosin-relatedkinaseB(TrkB),aproteintyrosinekinasereceptorandamemberofthelargerfamilyof
Trkreceptors,whichwasexpressedathighlevelsinthebrain,wasidentif_iedastheprimarysignaltransduction
Figure5.Ef_fectsofSCEadministrationonBDNF(A),pTrkB/TrkB(B),pCREB/CREB(C)andpERK/ERK
(D)levelsinhippocampus.T_hedatarepresentedthevaluesofmean±S.E.M.from10mice/group.p<0.05vs.
CUMSgroup.
#
p<0.05vs.Controlgroup.
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6SCIENTIFICREPORTS}?6903?wv?wvy~?zw{~?vw}?v}zv}?w
receptorforBDNF.MultiplelinesofevidencelinkBDNF-TrkBsignalingtothepathophysiologyofMDD,aswell
asthetherapeuticmechanismsofantidepressants
39,40
.Furthermore,extensiveexperimentalevidencesupports
thatBDNFmitigatesdepressivesymptomsmainlybybindingtoTrkB,leadingtoautophosphorylationofTrkB
tyrosineresidues,andactivationofdownstreamsignalingmolecules,includingtheextracellularsignal-regulated
kinase1/2(ERK1/2)knowntophosphorylatecAMPresponseelementbindingprotein(CREB)
41
.Ithasbeen
reportedthatthefull-lengthTrkBautophosphorylationregulateERKonactivationbyBDNF,whichmayincrease
cAMPandactivateCREB-regulatedgenetranscription,andthismechanismfurtherpromotestranscriptionof
BDNF
42
.Inagreementwithpreviousstudies,weobservedthatCUMS-induceddepressivesymptomsandcon-
sequentBDNF/TrkB/CREB/ERK1/2signalingdown-regulationreversedbySCEtreatment,whichindicated
thatSCEcouldincreaseBDNFexpressionbyaf_fectingTrkB/CREB/ERK1/2pathwaytoexertantidepressant-like
ef_fect.Additionally,interactionofBDNFwithTrkBtriggersreceptordimerization,transphosphorylationofintra-
cellulartyrosineresidues,andsubsequentactivationofthethreemajorsignalingpathwaysinvolvingMAPK/
Figure6.ImmunohistochemistryofBDNFproteininthemicehippocampus,Control(A),CUMS(B),SCE
300mg/kg(C),600mg/kg(D),1200mg/kg(E)andIODofBDNF(F).T_hescalebarshows100μm.T_hedata
representedthevaluesofmean±S.E.M.from10mice/group.p<0.05vs.CUMSgroup.
##
p<0.01vs.Control
group.
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7SCIENTIFICREPORTS}?6903?wv?wvy~?zw{~?vw}?v}zv}?w
CREB/ERK,phosphatidylinositol3kinase(PI3K)/AKT,andphospholipaseC-γ
43
.PI3K/AKTandERKsignaling
pathwaysarethemajorTrkB-mediatedsurvivalpathwaysthatpromoteneuronalsurvivalandprotectagainst
apoptosis.BDNF/TrkBsignalingcanpromotefurtherBDNFproductionthroughCREBviaactivationofPI3K/
AKTorERKsignaling,whichisemergingasapositive-feedbackloop
44
.TrkB-inducedPI3Kactivityleadsto
AKTactivation,andonceactivated,AKTleadstoinactivationofGSK-3βatSer-9.Morerecentf_indingsshowed
thatdisruptionofGSK-3phosphorylationbyAKTdecreasesanxietyandreducespronenesstodepressionin
mice
45
andintracellularsignaltransductionsystemasAKT/GSK-3βpathwayhavebeenfoundtobealteredinthe
brainofdepressivepatients
46
.T_hisprovidescompellingevidencethatthePI3K/AKT/GSK-3βsignalingpathway
isanimportantcontributortodepression.HerewedemonstratedthattheSCEtreatmentwasabletoactivate
PI3K/AKT/GSK-3βsignalingpathway,aswelltopromoteincreasedphosphorylatedlevelsofrelatedproteins,
Figure7.Ef_fectsofSCEontheexpressionoftheimmunoreactivityofBDNFinthemousehippocampus.
(A)Hippocampuswerestainedwithspecif_icantibodiesagainstBDNF(red).NucleiwerestainedwithDAPI
(blue).T_hescalebarshows100μm.(B)T_heaveragef_luorescenceintensitiesofBDNFwerequantif_iedaf_terSCE
treatment.T_hedatarepresentedthevaluesofmean±S.E.M.from10mice/group.p<0.05,p<0.01vs.
CUMSgroup.
##
p<0.01vs.Controlgroup.
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8SCIENTIFICREPORTS}?6903?wv?wvy~?zw{~?vw}?v}zv}?w
toimprovethedepressivesyndromesinCUMS-inducedmice.Takenallabovetogether,thesef_indingsleadusto
suggestthatSCEactingasanantidepressantincreasesBDNFlevelsbypromotingTrkB/CREB/ERKpathwayand
activatingPI3K/AKT/GSK-3βpathwaysimultaneously(Fig.uni00A011).
Basedontheformerresearch,depressioncanbeapsychologicalreactiontocognitivedecline,andthusmay
alsoappearasanearlysymptomindementingindividuals
47
.However,recentdatasuggestthatdepression,andin
particularlylate-lifedepression,canalsobeariskfactorforAlzheimer’sdisease
48
.Inourpresentstudy,wecon-
f_irmedthatSCEcouldtreatdepressionandshowadef_initeimprovementofcognition.T_hefurtherandin-depth
studyontheinteractionsbetweendepressionanddementia,andtheseparate,simultaneousef_fectsofSCEon
thesetwokindsofdiseaseswouldbeperformedinthefuturestudy.
Insummary,ourstudyshowsthattheantidepressant-likeef_fectandcognitionimprovementabilityofSCE
dependsonBDNFlevelsraisebyincreasingTrkB/CREB/ERKpathwayandPI3K/AKT/GSK-3βpathwayinthe
meantime.Ourf_indingssuggestthatSCEmightbeapromisingtherapeuticagentofdepression,andfurther
researchisworthtobeinvested.
??????
???????????????????????T_hefruitsofSCEwerepurchasedfromtheTCMshopofTongrentang
(Shenyang,China)andidentifiedbyProfessorYingJia(DepartmentofPharmacognosy,Shenyang
PharmaceuticalUniversity)accordingtotheguidelinesoftheChinesePharmacopoeia(2015).T_hen,thefruitsof
SCEwereexhaustivelyextractedwith95%ethanolatref_luxfor2h3times.Af_terconcentrationinavacuum,the
Figure8.Ef_fectsofSCEadministrationonpPI3K/PI3K(A),pAKT/AKT(B)andpGSK-3β/GSK-3β(C)levels
inhippocampus.T_hedatarepresentedthevaluesofmean±S.E.M.from10mice/group.p<0.05,p<0.01
vs.CUMSgroup.
#
p<0.05,
##
p<0.01vs.Controlgroup.
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9SCIENTIFICREPORTS}?6903?wv?wvy~?zw{~?vw}?v}zv}?w
residuewassuspendedin0.5%sodiumcarboxymethycellulose(CMC-Na)atacertainconcentrationof300mg/
kg,600mg/kgor1200mg/kg.
? ???????????ThechemicalcompositionofSCEwasanalyzedbyusinga
Waters-UPLC-Q-TOF/MSwithanultraviolet/visibledetector(UV/Vis)coupledtoaniontrapmassspectrom-
eterwithanESIinterface.T_heseparationwasachievedonanHSST3Column(100mm×2.1mm,1.8μm).T_he
chromatogramwasrecordedat216nm.MassanalyseswereperformedusinganESIinterfaceinthepositiveion
mode.DatawereperformedwithMasslynxV4.1sof_tware.AsshowninFig.uni00A012andTableuni00A03,fourteenlignanswere
tentativelyidentif_iedbythefullscanonthepositiveionmodeofMS/MSanalysis.Eightmaincompounds(1,4,
7,8,11,12,13and14)ofthoselignanswereidentif_iedwiththeretentiontimeandUVspectraofthereference
substance.
Figure9.Ef_fectsofSCEadministrationonthehistopathologicalchangesinthehippocampusofdepressedmice.
T_hescalebarshows50μm.Control(A),CUMS(B),SCE300mg/kg(C),600mg/kg(D)and1200mg/kg(E).
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10SCIENTIFICREPORTS}?6903?wv?wvy~?zw{~?vw}?v}zv}?w
???????????????????FluoxetinehydrochlorideasapositivecontroldrugwasobtainedfromMelone
PharmaceuticalCo.(Dalian,China).Allotherchemicalsandreagentswereofanalyticalgrade.
???????AdultmaleKunmingmice(weighing20±2g)werepurchasedfromtheExperimentalAnimal
CenterofShenyangPharmaceuticalUniversity(Shenyang,China).Allofthemweremaintainedunderstandard
laboratoryconditionsofconstanttemperature(23±1°C),relativehumidity(50±10%)anda12hlight/dark
cycle(lightfrom7:00a.m.to7:00p.m.)withfoodandwateravailableadlibitumandwereallowedtohabituate
tothenovelenvironmentfor1weekpriortouseinexperiments.T_heexperimentwascarriedoutincompliance
Figure10.Pearson’scorrelationbetweenthesucrosepreferencevs.spontaneousalternation(A),thesucrose
preferencevs.timeinthetargetquadrant(B),immobilitytimevs.spontaneousalternation(C)andimmobility
timevs.timeinthetargetquadrant(D).
Figure11.ProposedSCEantidepressant-likemechanismsfortheinteractionamongBDNF,TrkB,CREB,
ERK,PI3K,AKTandGSK-3β.SCEregulatesTrkB/CREB/ERKpathwaytoincreasetheBDNFtoalleviatethe
depressionandcognitivedecline.Ontheotheraspect,BDNFupregulatesPI3K/AKT/GSK-3βpathwayto
amelioratethedepressivesymptomsandcognitiondisabilityinducedbyCUMS.
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11SCIENTIFICREPORTS}?6903?wv?wvy~?zw{~?vw}?v}zv}?w
withtheNationalInstitutesofHealthandinstitutionalguidelinesforthehumanecareofanimalsandwas
approvedbytheAnimalCareCommitteeofShenyangPharmaceuticalUniversity(ProtocolNo.:SYPU-IACUC-
2016C-0921-205).Everyef_fortwasmadetominimizethenumberofanimalsusedandanypainanddiscomfort
experiencedbythesubjects.
??????????CUMSwasperformedaspreviouslydescribed
49
.CUMSconsistedofexposuretoavari-
etyofunpredictablestressors(randomly),including(1)24hfooddeprivation,(2)24hwaterdeprivation,(3)1h
exposuretoanemptybottle,(4)exposuretoanemptycage(withoutsawdustbedding),(5)groupedhousing,(6)
24hsoiledcage(200mlwaterin100gsawdustbedding),(7)levelshakingfor30min,(8)5mincoldswimming
(at5°C),(9)niptailfor1min.Allstresseswereappliedindividuallyandcontinuously,dayandnight.T_hecontrol
animalswerehousedinaseparateroomandhadnocontactwiththestressedgroups.Topreventhabituation
andtoensuretheunpredictabilityofthestressors,allstresseswererandomlyscheduled,repeatedthroughout
the3-weekexperiment.T_hecontrolgroupmicewerelef_tundisturbedexceptfornecessaryproceduressuchas
routinecagecleaning.
???????????????????????????????????????FortestingofthebehavioralandBDNFregu-
lations,themicewererandomlydividedintosixgroups(10animalsforeachgroup):Control-Vehiclegroup,
CUMS-Vehiclegroup,CUMS-f_luoxetine(10mg/kg,i.g.),CUMS-SCEgroup(300mg/kg,i.g.),CUMS-SCEgroup
(600mg/kg,i.g.)andCUMS-SCEgroup(1200mg/kg,i.g.).FortheControl-Vehiclegroup,theanimalswere
injectedwithsameamountof0.5%CMC-Na.Alltheseagentswereadministeredinavolumeof10ml/kg.Before
CUMS,animalswereallowedtohabituatetoallthebehavioraltestsinordertoestablishanindividualbaseline.
T_hesucrosepreferencetestswereconductedtwicebeforeandaf_tertheCUMSprocedure,respectively.Beforethe
tissueisolation,themicewereallowedtotakethetestofallthebehavioraltests.Behavioraltestswereperformed
duringthelightphaseoflight–darkcycle.T_hewholeexperimentalprocedureisshowninFig.uni00A013.
Figure12.UPLCchromatogramofSCE(1)schisandrin;(2)gomisinD;(3)gomisinJ;(4)schisandrolB;
(5)tigloylgomisinH;(6)angeloylgomisinH;(7)schisantherinA;(8)schisantherinB;(9)Schisanhenol;(10)
gomisinE;(11)deoxyschizandrin;(12)gomisinN;(13)SchisandrinB;(14)SchisandrinC.
PeakT
R
(min)MS(m/z)Majorfragments(m/z)Tentativeidentif_ication
14.00432.2148
433.2227[M+H]
+
,
415.2125[M+H-H
2
O]
+
Schisandrin
24.60530.2152
531.2224[M+H]
+
,
548.2492[M+NH4]
+
GomisinD
34.74388.1886389.1964[M+H]
+
GomisinJ
45.04416.1835399.1804[M+H-H
2
O]
+
SchisandrolB
56.08500.2410
501.2477[M+H]
+
,
483.2390[M+H-H
2
O]
+
TigloylgomisinH
66.81500.2410
501.2484[M+H]
+
,
483.2390[M+H-H
2
O]
+
AngeloylgomisinH
79.01536.2046537.2094[M+H]
+
SchisantherinA
89.04514.2203515.2275[M+H]
+
SchisantherinB
99.37402.2042403.2116[M+H]
+
Schisanhenol
1010.44514.2203515.2275[M+H]
+
GomisinE
1112.51416.2199
417.2284[M+H]
+
,
439.2108[M+Na]
+
Deoxyschizandrin
1214.00400.1886401.1962[M+H]
+
GomisinN
1314.38400.1886401.1962[M+H]
+
SchisandrinB
1414.94384.1573
385.1651[M+H]
+
,
407.1471[M+Na]
+
SchisandrinC
Table3.Tentativeidentif_icationofthecompoundsfromSCE.
www.nature.com/scientificreports/
12SCIENTIFICREPORTS}?6903?wv?wvy~?zw{~?vw}?v}zv}?w
??????????????????Sucrosepreferencetest(SPT).Sucrosepreferencetestwascarriedout1daybefore
CUMSandtheendofCUMS.T_hetestwasperformedasdescribedpreviously
50
.Inbrief,72hbeforethetest,
themousewasindividuallyplacedinacagewithtwobottlesofsucrosesolutiontoadaptasucrosesolution(1%,
w/v)for24h;thenonebottleofsucrosesolutionwasreplacedwithwater(24h);af_tertheadaptation,laboratory
miceweredeprivedofwaterandfood(24h).Sucrosepreferencetestwasconductedat9:30a.m.Eachmouse
washousedinindividualcageandfreetoaccesstwobottlescontaining200mlofsucrosesolution(1%w/v)and
200mlofwater,respectively.Af_ter6h,theconsumedweightsofsucrosesolutionandwaterwererecorded.T_he
sucrosepreferencevaluewasobtainedfromthefollowingformula:sucrosepreference(%)=sucroseintake(ml)/
[sucroseintake(ml)+waterintake(ml)]×100%.
?????????????????????????? ?Inordertoruleoutthepossibilitythatthealterationinthe
immobilitytimeintheFSTwasduetointerferenceofthelocomotoractivity,spontaneouslocomotoractivity
ofeachmousewasobservedinanopenf_iledexperimentalvideoanalysissystem(ZS-ZFT,HuaibeiZhenghua
Bio-ApparatusCo.Ltd,China).T_heapparatuswasplacedinadarkenedandsoundattenuatedtestingroom.T_he
totalpathofSpontaneouslocomotivewasevaluatedovera5minperiod
51,52
.
????????????? ?T_heFSTwasconductedandthetotalimmobilitywasestimatedinaccordancewith
themethodsdescribedpreviously
53
withslightchanges.Inbrief,eachmousewasforcedtoswiminacylindrical
glasscontainer(diameter20cm×height50cm)with30cmofwater(22±1°C)for6min,andthedurationof
immobilityduringthelast4minwasdetected.T_hemicewereconsideredasimmobilewhentheymaintained
f_loatinginthewaterwithoutescapingfromthecontainer,makingonlythemovementnecessarytokeeptheir
headsabovethewater.T_heparameterwasrecordedbytrainedobserverswhowereblindtotheexperimental
groups.
??????????Y-mazetestasameasureofimmediatespatialworkingmemorywhichisaformofshort-term
memory
54
.T_heY-mazeisconsistedofthreearmsatequalangles(30cmlength×5cmwidth×12cmhigh).
Micewereplacedattheendofonearmandallowedtomovefreelythroughthemazefor6min.Anarmentry
wascountedwhenthehindpawsofthemousewerecompletelywithinthearm.T_heseriesofarmentrieswere
recordedvisuallyandthepercentagealternationwascalculated.Aspontaneousalternationwasdef_inedassucces-
siveentriesintothethreearms,i,e.,ABC,CAB,orBCAbutnotCBC.T_hepercentagealternationwascalculated
astheratioofactualtopossiblealternations(def_inedasthetotalnumberofarmentriesminustwo)multipliedby
100asshownbythefollowingequation:Alternation%=[Numberofalternations/(Totalarmentries?2)]×100.
T_henumberofarmentrieswasalsousedasanindicatoroflocomotoractivity.
???????????????????Aspatiallearningandmemorytestwasperformedbythemethodof
Morriswithminordif_ference
55
.T_heMorriswatermazeconsistedofalargecirculartank(120cmindiameter,
40cminheight)whichwasdividedgeographicallyintofourquadrants(I,II,IIIandIV).Ablackplatform(9cm
indiameter,30cminheight)wassubmerged1cmbelowthewatersurfaceandf_ixedatthemidpointoftheIV
quadrant.T_hewaterwascoloredwithnon-toxicblackinkandwasmaintainedat23±2°C.T_hetankwasplaced
inadimlylit,soundprooftestroomwithvariousvisualcuesfornavigation.T_heMorriswatermazetestwascom-
posedoftrainingtrialsandprobetest.Beforetest,eachanimalwasscreenedinMWMaccordingtothespeedand
swimmingstate,andawhiteplasticplatformwasusedtoevaluatethevisibleabilityofmice.Inthetrainingtrials,
themicewereplacedinthewaterfacingthepoolwallfromtwodif_ferentstartingpointseverydayandallowed
toswimfreelytoseekthehiddenplatformforamaximum90s.Ifamousefailedtof_indtheplatformwithin90s,
itwasguidedbyanexperimentertotheplatformtostudy30s.T_hetrainingtrialslastedf_iveconsecutivedays.
T_heaverageescapelatencyofeachmouseperdaywasrecorded.T_heprobetestwasperformedwithoutplatform
af_terthe5-daytrainingf_inished.T_hepercentageoftimespentofswimminginthetargetquadrantandthetimes
crossedwheretheplatformwasoriginallylocatedweremeasuredforeachmouse
56
.
???????????????????Tissuesamplecollection.Af_terthecompletionofthebehavioraltest,themicewere
euthanized.Wholebrainswererapidlyremovedfromthemiceandstoredaccordingtothespecif_icexperimental
procedure.Brainregionsofthehippocampusweredissectedonacoldplateandimmediatelyfrozeninliquid
nitrogen.T_hetissuesampleswerestoredat?80°Cuntilassay.
??????????BDNF,pTrkB,TrkB,Pcreb,CREB,pERKandERKconcentrationsofhippocampus
determinationwereperformedusingtheELISAmethod,accordingtothemouseELISAkitsmanufacturer’s
instructions(ShanghaiEnzyme-linkedBiotechnologyCo.,Ltd,China),respectively.Opticaldensitywasobtained
Figure13.Schematicrepresentationoftime-lineforSCEtreatmentinCUMSinducedexpetimentalanimals.
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13SCIENTIFICREPORTS}?6903?wv?wvy~?zw{~?vw}?v}zv}?w
at450nmusingamicroplatereader(Varioskanf_lash,T_hermoscientif_ic,USA)within15minofstopsolution
addition.
???????????HippocampaltissueswerehomogenizedinRIPA(150mMsodiumchloride,50mMTris(pH
8.0),0.5%sodiumdeoxycholate,0.1%SDS,1%TritonX-100)andPMSF(DalianMelonepharma,China)and
keptonicefor30min.T_hetissuehomogenatewascentrifugedat10000gfor20minat4°C.T_hesupernatant
wasobtainedandusedasthetotalhippocampalandcerebralcortexproteinextractmeasuredbyBCAassaykit
(DalianMelonepharma,China)todetermineproteinconcentrationandstoredat?80°Cuntiluse.
Samplesweredilutedwithanequalvolumeofloadingbuf_fer(BeyotimeBiotechCo.,China),andboiledat
95°Cfor5min.Approximately50μgofproteinwasloadedineachwellandseparatedin10%or12%SDS-PAGE
gels.T_heproteinsweretransferredontonitrocellulosemembranes.T_hemembranesweresaturatedandblocked
with5%fat-freepowderedmilkat37°Cfor1.5handincubatedovernightat4°Cinoneofthefollowingpri-
maryantibodies,whichweredilutedin5%fat-freepowderedmilkinTBS:PI3Kinasep85(19H8)(1:1000,CST,
USA),Akt(pan)(C67E7)(1:1000,CST,USA),GSK-3β(D5C5Z)(1:1000,CST,USA),GADPHRabbitmAb
(1:1000,CST,USA),β-actinRabbitmAb(1:1000,CST,USA).Phospho-GSK-3β(Ser9)(1:1000,CST,USA),
Phospho-Akt(Ser473)(1:2000,CST,USA).Anti-PI3Kp85(phosphorY607)(1:1000,Abcam,USA)Blotswere
washedthreetimesfor30mininTBSTatroomtemperatureandthenincubatedfor1.5hinoneofthefollowing
HRP-conjugatedantibodies,whichweredilutedin5%fat-freepowderedmilkinTBS:Anti-rabbitIgG(1:2000,
CST,USA)fordetectionoftargetproteins,GADPHandβ-actin.Af_terthreetimeswashesfor30mininTBST,
immunolabeledproteinbandsweredetectedusingtheECLWesternblotdetectionkit(DalianMelonepharma,
China).Graphsofblotswereobtainedinthelinearrangeofdetectionandwerequantif_iedforthelevelofspecif_ic
inductionbyscanninglaserdensitometry.
?????????????????????????Miceweresacrif_icedbyoverdoseofsodiumpentobarbitoneforimmuno-
histochemicalanalysis,andthenintracardiallyperfusedwithPBS,followedbychilled4%PFAinPBS.T_hebrain
wasslicedinto15mmsectionsonacryostatblockedinPBScontaining1%goatserumand0.1%Triton×100,
andincubatedat4°Covernightwithanti-BDNF(mouseIgG,1:500,abcam,USA).Af_terwashing,aHRPconju-
gatedgoatanti-rabbitIgGcomplex(1:1000,absin,China)wasappliedfor1h.Colordevelopmentwasperformed
withadiaminobenzidineperoxidasesubstratekit(TL-125-QHD,T_hermoFisherScientif_ic,USA).Sectionswere
conunterstainedwithhematoxylin.
???????????????????????TissuebiopsiesweredeparaffinizedandpermeabilizedwithPBS/0.1%
Triton×100.Antigenretrievalwasperformedbyboilingtheslidesin0.01Mtrisodiumcitratebuf_fer,pH6,for
10min.Sectionswerethenpreincubatedwith10%normalgoatserumcontaining0.2%Triton×100overnight
at4°Ctoblocknonspecif_icbinding.Slideswerethenincubatedovernightat4°Cwithanti-BDNF(mouseIgG,
1:100,abcam,USA)in2%serum,sectionswerewashedthreetimesinPBStoremoveunboundprimaryantibody,
thenincubatedwithsecondaryantibodyfor1hat37°C.Af_terthat,sectionswerewashedthreemoretimesin
PBST(PBS,with1%Tween20)andcoveredwithDAPI/Fluorescencequenchingagent(Beyotime,China).Slides
wereviewedusingaLeicaf_luorescencemicroscopecoupledwithacomputerassistedvideocamera(Axioscope
A1,Germany).
??????????????Forhistopathology,hematoxylin-eosinandCongoredwereusedasdescribedpreviously
57
.
Af_terthebehavioraltestsanimalsweredecapitatedandtheirbrainswereremovedquickly,postf_ixedin37%for-
maldehydesolutionfor48h,andthenallthebrainwascutinthecoronalplaneandstainedwithhematoxylinand
eosin.Hippocampusneuronswereexaminedinlightmicroscopy.
???????????????????Resultsareexpressedasmean±SEM.T_hesignif_icancesbetweendif_ferentgroupswere
assessedusingone-wayANOVA,followedbyTukeyHSDpost-hoctestwhensignif_icantmainef_fectswereindi-
cated.Two-wayANOVAwasusedtoanalyzedatafromtheMorriswatermazetrainingtrials.Inallcalculations,
p<0.05wasconsideredtobestatisticallysignif_icant.StatisticalanalysiswasperformedwithSPSSsof_tware19.0.
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ThisresearchwassupportedbyNationalNaturalScienceFoundationofChina(No.81573580)andKey
LaboratoryofPolysaccharideBioactivityEvaluationofLiaoningProvince.
?????????????????
TingxuYanwrotethemainmanuscripttext,BosaiHeperformedthedatastatistic,MengjieXu,ShutongWan
andHuilingYangperformedtheanimalexperimentsandbiochemicaldetermination,andFengXiaoprepared
f_igures.KaishunBiandYingJiareviewedthemanuscript.
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