Antidepressant-likeeffectsandcognitiveenhancementofSchisandrachinensisinchronicunpredictablemildstressmiceanditsrelatedmechanism
五味子对慢性不可预见性轻度应激小鼠的抗抑郁样作用和认知增强作用及其相关机制
TingxuYan1,BosaiHe2,ShutongWan1,MengjieXu1,HuilinYang1,FengXiao3,KaishunBi2&YingJia3
Abstract
TheaimofthisstudywastoevaluatewhetherSchisandrachinensisextract(SCE)administrationinfluenceschronicunpredictablemildstress(CUMS)-induceddepressionandcognitiveimpairment,andexploresunderlyingmechanisms.Sucrosepreferencetest(SPT)andforcedswimmingtest(FST)wereusedforassessingdepressivesymptoms,andY-maze,Morriswatermazewereusedforevaluatingcognitionprocesses.TheresultsshowedthatCUMS(4weeks)waseffectiveinproducingbothdepressionandmemorydeficitsinmice.Additionally,CUMSexposuresignificantlydecreasedbrainderivedneurotrophicfactor(BDNF)levelsinhippocampusasindicatedbyELISA,immunohistochemistryandimmunofluorescenceassays,accompaniedbydown-regulatedtyrosinekinasereceptorB(TrkB)/cAMP-responseelementbindingprotein(CREB)/extracellularsignal-regulatedkinase(ERK)andphosphatidylinositol3kinase(PI3K)/proteinkinaseB(AKT)/glycogensynthasekinase-3β(GSK-3β)signalingpathways.ChronicadministrationofSCE(600or1200?mg/kg,i.g.)significantlypreventedalltheseCUMS-inducedbehavioralandbiochemicalalterations.ItsuggestedthatSCEcouldimprovethedepression-likeemotionalstatusandassociatedcognitivedeficitsinCUMSmice,whichmightbemediatedbyregulationofBDNFlevelsinhippocampus,aswellasup-regulatingofTrkB/CREB/ERKandPI3K/AKT/GSK-3βpathways.
本研究的目的是评估五味子提取物（SCE）的施用是否影响慢性不可预见性轻微刺激（CUMS）诱导的抑郁和认知障碍,并且探索其潜在机制。蔗糖偏好测验（SPT）和强迫游泳实验（FST）被用来评估抑郁症状，Y型迷宫和莫里斯水迷宫被用来评估认知过程。结果显示，CUMS（4星期）对老鼠的抑郁症状和记忆缺失有作用。此外，在酶联免疫吸附测定（ELISA）、免疫组织化学和免疫荧光检测的显示下，CUMS暴露显著减少了海马体中脑源性神经营养因子（BDNF）的水平,同时伴随着酪氨酸激酶受体B(TrkB)/cAMP应答元件结合蛋白（CREB）/细胞外信号调节激酶（ERK）和磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（AKT）/糖原合成酶激酶-3β（GSK-3β）信号通路的一系列下调。慢性控制下的SCE（600or1200?mg/kg,i.g.）明显抑制了这些CUMS诱导的行为和生化变化。综合表明，SCE能够改善CUMS小鼠的抑郁情感状态并且和认知缺陷有联系，可能是受海马体BDNF水平的调节和TrkB/CREB/ERK和PI3K/AKT/GSK-3β通路的上调所影响。
Introduction
Depressionisacommonillnessworldwide,withanestimated350millionpeopleofallagesafectedaccordingtoareportofWHOin20141,itisalsotheleadingcauseofdisabilityandisamajorcontributortotheoverallglobalburdenofdisease.Depressionisaheterogeneoussyndromeandmayresultinpoorerwell-being.Atitsworst,depressioncanleadtosuicide2.Ingeneral,mostclinicalsymptomsofmajordepressivedisorder,suchasdelusions,anxiety,irritabilityorinsomnia,canbeefectivelytreatedbycurrentpsychopharmacologicaltreatments.Nevertheless,cognitivedefcits(likeadiminishedabilitytothinkorconcentrate,orindecisiveness),whichrepresentcoredefcitsofdepression3,maypersistinpatientsevenwhendepressivesymptomshaveabatedordisappearedandsignificantlyaffecttheindividual’ssocialandoccupationalfunction.Indeed,meta-analyseshaveshownthatcognitivedefcitsarestillpresentinremittedpatients4.Forthisreason,cognitiveimpairmentemergesasapotentialtargetforbothpharmacologicalandpsychosocialtreatments,withthefinalgoalofimprovingfunctioning.
抑郁症是一种全球广泛的常见疾病，据WHO报道称至2014年估计有3.5亿各年龄段的人深受其害，而且它也是造成残疾的主要因素和全球疾病负担的一大主力贡献者。抑郁症是一种病情复杂的综合症状，可能会导致较差的体质。更糟糕的是，抑郁症会导致自杀的发生。一般来说，重度抑郁症的大多数临床表现有妄想、焦虑、烦躁或失眠，可以被现在的精神药物治疗有效地解决。然而，认知功能障碍（如思考能力减弱或集中或犹豫不决），作为抑郁症核心障碍的体现，即使当抑郁综合征状减少或者消失，它可能仍然存留在病人身上，明显地影响到个体的社会功能和职业能力。事实上，荟萃分析显示，认知障碍仍然出现在已经被治疗的病人身上。因此，认知损伤恢复成为药理治疗和心理治疗的潜在目标，改善健康是最终的目标。
BDNFisamemberofthenervegrowthfactorfamilyandisexpressedintheadultmammaliancentralandperipheralnervoussystem,particularlyinthehippocampusandcortex5.ThevastmajorityoftheliteraturelinkingneurotrophinstomooddisordersdealswithBDNF,otherneurotrophinsshowingonlyveryminorrole6-8.Inaddition,clinicalstudieshaveshownthatserumBDNFlevelsandhippocampalvolumereductionsinelderlyindividualsarecloselycorrelatedwithmemoryloss9,10,andBDNFmayrescuecognitiveimpairmentsandlearningdeficitsinAlzheimer’sdisease11,12.PreviousreportsindicatedthatchangesinBDNFlevelareimplicatedinthepathophysiologyofcognitivedeclineindepressionandneurodegenerativedisorders13.BDNFexertsitspro-survivaleffectsbybindingitshighaffinityreceptorTrkB,toactivateBDNF-TrkBsignaling14.BehavioralresponsestoantidepressantshavebeenabolishedinanimalsinwhichBDNF-TrkBsignalingisinhibited15.Itisalsoinvolvedinpropofol-inducedlearningandmemoryimpairments16.Tus,theseresultssuggestthatBDNF-TrkBsignalingplaysacriticalroleinthemolecularmechanismsofantidepressantsandcognitionenhancement.BDNF-TrkBdownstreamsignaling,includingERK/CREBandPI3K/AKT/GSK-3βpathways,canmodulateneurotransmitterrelease,cellviability,apoptosisandpostsynapticresponses,andthesefunctionsarecloselyassociatedwithdepression17,18andlearningability19.
BDNF是神经生长因子家族的一员，在成年哺乳动物中枢和周围神经系统中被表达，特别是在海马体和皮层中。大多数文献指出神经营养因子与情绪混乱之间是通过BNDF联系的，其他的神经营养因子则几乎无作用。此外，临床研究显示，在老年个体中血清BDFF水平下降和海马体体积减小是和失忆紧密联系的，并且对于老年痴呆症疾病BNDF也许能挽救认知障碍和学习缺陷的症状。以前的研究表明BDNF水平的改变被牵涉进抑郁症人群的认知能力下降和神经退行性疾病的病理生理学研究中。BDNF通过结合它的高亲和力受体TrkB，启动BDNF-TrkB信号通路来发挥它的促存活作用。当BDNF-TrkB信号通路被抑制时，动物对抗抑郁药物的行为反应会丧失。它也参与异丙酚诱导的学习和记忆损伤。因此，这些结果显示BDNF-TrkB信号在抗抑郁药和认知增强的分子机制研究中具有很关键的作用。BDNF-TrkB下游信号，包括ERK/CREB和PI3K/AKT/GSK-3β通路，可以调节神经递质的释放、细胞活力、细胞凋亡和突触后效应，并且这些作用与抑郁症和学习能力有着紧密的联系。
About30%ofpatientssufferingfromamajordepressivedisorderdonotrespondsufficientlytoestablishpharmacological,psychotherapeutic,orsomatictreatments20.Inaddition,differentstudiesconcludethatonly30–40%ofpatientsthatareoptimallytreatedwithfirstlineantidepressantsachieveremissionandmorethanonethirdofpatientswithdepressionareclassifiedastreatment-resistantdepression(TRD)21.Inordertodealwiththementionedlimitationsshownbycurrentantidepressantdrugs,traditionalChinesemedicine(TCM)isattractingincreasingattentionasamethodformeetingthedemandsforhigherremissionrate,fasteronset,persistentantidepressantaction,andfeweradverseeffects22-24.Schisandrachinensis(Trucz.)Bail.isafamoustraditionalChinesemedicinewhichisusuallyusedintheclinicwiththefunctionsofinducingastringency,replenishingandpromotingtheproductionofbodyfluidandtonifyingthekidneystorelievementalstrain25.Dibenzo[a,c]cyclooctadienelignansarethemainchemicalcomponentsinSchisandrachinensisextract(SCE),andtheseareshowingvariouspharmacologicalactivities,ofwhichtheirantioxidative,neuroprotective,anti-cancer,vasorelaxantandcytoprotectivepropertiesareamongthemoststudiedones26-28.OurgrouppreviousreportedthatSCEcouldproduceantidepressant-likeeffectsindifferentdepressedmodels29,30,andamelioratecognitiondeficits31.However,thepotentialneuroprotectiveeffectsofSCEagainsttheCUMS-induceddepressionandcognitivedeficits,aswellasthemechanismsremaintobeclarified.Thus,inthisstudy,weusedtheCUMSmodeltoinvestigatetheantidepressant-likeactivityandcognitiveimpairmenteffectsofSCE,moreover,theBDNF/TrkB/ERK/CREBandPI3K/AKT/GSK-3βsignalingpathwaysweredeterminedtoillustratetheactionmechanismofSCE.
大约30%的病人患有重度抑郁症，用建立药理学治疗、心理学治疗和体细胞治疗的方式尚不足以治愈该疾病。此外，不同的研究总结得出，只有30-40％的用一线抗抑郁药治疗的患者达到缓解效果，超过三分之一的抑郁症患者被列为耐药性抑郁症（TRD）。为了应对目前抗抑郁药所表现出来的限制，中医药学（TCM）作为一种满足高缓解率、更快见效、持续抗抑郁和更少副作用的需求的一种治疗方式，正在引起越来越多的关注。五味子（防风属），是一味著名的中药，经常被用于诊所中，具有诱导止血、补充和促进体液生产以及滋补肾脏来缓解精神紧张的作用。二苯并[a,c]环辛二烯木脂素是五味子提取物（SCE）的主要化学成分，它们具有各种药理学作用，其抗氧化，神经保护，抗癌，血管舒张和细胞保护性能是被研究最多的。我们团队曾报道过SCE可以在不同的抑郁模型中产生抗抑郁效果和改善认知障碍。然而，SCE对CUMS诱导的抑郁症和认知缺陷的潜在神经保护作用以及机制仍有待澄清。因此，在这篇论文，我们采用CUMS模型来研究SCE的抗抑郁作用和认知损伤效果，并确定BDNF/TrkB/ERK/CREB和PI3K/AKT/GSK-3β的信号通路来建立SCE的作用机制。
Results
EffectsofSCEonthesucrosepreferenceintheSPT.AsshowninFig.1,Posthocanalysisrevealedthata4-weeksCUMSexposuresignificantlyreducedthepercentageofsucroseconsumptioninthestressedmiceincomparisonwiththecontrolgroup(p<0.01).However,long-termtreatmentwithSCE(600mg/kgor1200mg/kg)increasedthesucrosepreferenceascomparedtotheCUMScontrolmice(p<0.01,p<0.01,respectively).AsoneapproachtoinvestigatetheantidepressanteffectsofSCE,thesucrosepreferencetest,anindicatorofanhedonia,wasappliedafter4weeksofCUMSexposure.Suchincreasesinsucroseconsumptionsuggestapotentantidepressant-likeeffectofSCEwithintheseCUMS-exposedmice.
应用SPT实验得到的SCE对蔗糖偏好的影响结果。如图一，事后分析显示，与对照组相比，4周的CUMS暴露明显减少了应激小鼠对蔗糖消耗的百分比（p<0.01）。然而，与CUMS对照组小鼠相比，使用SCE(600mg/kgor1200mg/kg)长期治疗组增加了蔗糖偏好(p<0.01,p<0.01,分别)。作为一种调查SCE抗抑郁效果的方法，蔗糖偏好实验，即缓解效果的指示器，被应用在CUMS暴露4周后的小组。蔗糖消耗的这种增加表明在这些CUMS暴露的小鼠中SCE具有强效的抗抑郁作用。
Figure1.EffectsofSCEadministrationonthesucrosepreferenceintheSPT.Thedatarepresentedthevaluesofmean±S.E.M.from10mice/group.p?
图一：SPT实验中SCE施用对蔗糖偏好的影响。数据表示10只小鼠/组的平均值±S.E.M。p<0.01与CUMS组比较。##p<0.01与对照组比较。
EffectsofSCEonlocomotoractivity.ThereisnosignificanceamongallgroupsinthetotaltraveldistancewhichindicatedthatCUMSanddrugtreatmentdidnotalterthelocomotoractivityofthetestanimal(Table?1).
SCE对运动行为的影响。所有组中的差异相对总运动长度而言没有意义，表明CUMS和药物治疗不会改变测试动物的运动行为（表一）。
Groups locomotionlength(cm/5min)运动长度 Control 1236±29 CUMS 1017±57 Fluoxetine(10mg/kg)氟西汀 1138±76 SCE(300mg/kg) 995±79 SCE(600mg/kg) 1205±84 SCE(1200mg/kg) 1099±59 Table1.InfluenceofadministrationofSCEandCUMSprocedureonthelocomotoractivityinmice.
表一：SCE和CUMS控制对小鼠运动活动的影响
EffectsofSCEontheimmobilitytimeintheFST.Consistentwiththesucroseconsumptiontest,Posthocanalysisindicatedthat4-weeksofCUMSexposuresignificantlyincreasedimmobilitytimescomparedtothecontrolmice(Fig.?2).TheseeffectswerereversedbychronicSCEtreatment(600mg/kgor1200mg/kg),withimmobilitydurationsnowbeingsignificantlydecreasedascomparedtotheCUMScontrolgroupwithregardtoimmobilitytime(p<0.01,p<0.01,respectively).Theseresultscomplementthoseofthesucrosepreferencetest,againshowinganantidepressant-likeeffectofSCEasbaseduponthesedecreasedimmobilitytimesinthisFST.
与蔗糖消耗测试一致，事后分析表明，与对照小鼠相比，4周的CUMS暴露明显增加了不动时间（图2）。慢性SCE治疗（600mg/kg或1200mg/kg）可逆转这些作用，与CUMS对照组相比，静止期持续时间显着降低（p<0.01，p<0.01）。这些结果补充了蔗糖偏好试验的结果，再次显示了基于这种FST中这些减少的不动时间的SCE的抗抑郁效应。
Figure2.EffectsofSCEadministrationontheimmobilitytimeintheFST.Thedatarepresentedthevaluesofmean±S.E.M.from10mice/group.p<0.05,p<0.01vs.Controlgroup.#p<0.05vs.Controlgroup.
图二：SCE治疗对FST的发生时间的影响。数据表示10只小鼠/组的平均值±S.E.M.p<0.05，p<0.01与对照组比较。#p<0.05与对照组比较。
EffectsofSCEonYmazetest.AnalysesofthespontaneousalternationpercentagewithinYmazetaskshowedsignificantoveralldifferencesbetweenallgroups(Fig.?3).BothdosesofSCE(600mg/kgor1200mg/kg),butespecially600mg/kg,significantlyimprovedmemoryformationinCUMS-inducedmiceascomparedtothecontrolmice.ThechangesinthespontaneousalternationpercentagesofCUMS-inducedmiceexposedtoSCEarenotrelatedtothechangesinmotoractivity,asevidencedbythenumberofarmentriesascomparedtothecontrolmice.
在Y迷宫任务中自发交替百分比的分析显示所有组之间显著的总体差异（图3）。与对照小鼠相比，两种剂量的SCE（600mg/kg或1200mg/kg），特别是600mg/kg，显着改善了CUMS诱导的小鼠的记忆形成。施加SCE的CUMS诱导的小鼠的自发交替百分比的变化与运动活动的变化无关，与对照小鼠相比，进臂的次数可以证明。
Figure3.EffectsofSCEadministrationonY-mazetest,spontaneousalternations(A),numberofarmentries(B).Thedatarepresentedthevaluesofmean±S.E.M.from10mice/group.p<0.05vs.CUMSgroup.##p<0.01vs.Controlgroup.
图三：Y迷宫试验中SCE治疗的效果，自发变化（A），手臂进入数量（B）。数据表示10只小鼠/组的平均值±S.E.M.p<0.05与CUMS组比较。##p<0.01与对照组比较。
EfectsofSCEonMorriswatermaze.Asignifcantdecreaseoftheescapelatencytoreachthesubmergedplatformwasevidencedalongthe5daysofatrainingperiodinallthegroupsintheMWMtest(Table?2).TheCUMSgrouptookmoretimeonthelasttwotrainingdaystoreachtheplatformcomparedwiththecontrolgroupintheplacenavigationtest(p<0.05).However,theSCE(600or1200mg/kg)treatedgroupsignificantlyamelioratedtheeffectsofCUMSonescapelatency(p<0.05,p<0.05,p<0.05forthe3rd,4thand5thday,respectively).Intheprobetrial,aprobetestwasconductedbyremovingtheplatformonthelastdayofMWMtask.TheeffectsofSCEtreatmentontheperformanceofspatialprobetrialinmiceareshowninFig.?4.Miceinthecontrolgroupfocusmoretimeinthetargetquadrant,whilemiceintheCUMSgroupfailedtorememberthepreciselocationoftheplatform.TreatmentwithSCE(600or1200mg/kg)reversedthecognitivedeficitascomparedtotheCUMSgroup.
SCE对Morris水迷宫的影响。在对所有组进行为期5天训练的MWM测试中，证明了到达淹没平台的逃生潜伏期显着降低（表2）。在定位游泳测试中，与对照组相比，CUMS小组在最后两个训练日花费了更多的时间来达到平台（p<0.05）。
然而，SCE（600或1200mg/kg）治疗组显著改善了CUMS对逃生潜伏期的影响（p<0.05，p<0.05，p<0.05，分别为第3，第4和第5天）。在探索实验中，通过在MWM测试的最后一天移动平台来进行探索测试。SCE治疗对小鼠空间探索试验效果的影响如图4所示。对照组小鼠集中在目标区域花费了更多的时间，而CUMS组小鼠则未能记住平台的精确位置。与CUMS组相比，用SCE（600或1200mg/kg）治疗逆转了认知缺陷。
Table2.EffectofSCEontheescapelatency(s)ofCUMS-inducedmiceinwatermazetest.#p<0.05,comparedwithcontrolgroup;p<0.05,p<0.01,comparedwithCUMSgroup.
表2.SCE对水迷宫试验中CUMS诱导小鼠逃生潜伏期的影响。#p<0.05，与对照组比较;p<0.05，p<0.01，与CUMS组相比。
Figure4.EffectsofSCEadministrationontimeinthetargetquadrantofMorriswatermaze.Thedatarepresentedthevaluesofmean±S.E.M.from10mice/group.p<0.05vs.CUMSgroup.##p<0.01vs.Controlgroup.
图4：SCE施用对Morris水迷宫中到达目标象限的时间的影响。数据表示10只小鼠/组的平均值±S.E.M.p<0.05鱼CUMS组对比。##p<0.01与对照组比较。
EffectsofSCEonBDNF/TrkB/CREB/ERKsignalingpathways.Comparedwiththecontrolgroup,theCUMSprocedurecouldsignificantlydecreasethelevelsofBDNF,pTrkB/TrkB,pCREB/CREBandpERK/ERKinhippocampus(p<0.05).Meanwhile,SCE600mg/kgand1200mg/kgcoulddramaticallyupregulatetheBDNF/TrkB/CREB/ERKsignalinginthehippocampusofdrugtreatedmicecomparedtotheCUMSmice(p<0.05)(Fig.?5A–D).
SCE对BDNF/TrkB/CREB/ERK信号通路的影响。与对照组相比，CUMS过程可以显着降低海马体BDNF，pTrkB/TrkB，pCREB/CREB和pERK/ERK的水平（p<0.05）。同时，与CUMS小鼠相比，SCE600mg/kg和1200mg/kg可显著上调药物治疗小鼠的海马体BDNF/TrkB/CREB/ERK信号（图5A-D）。
Figure5.EffectsofSCEadministrationonBDNF(A),pTrkB/TrkB(B),pCREB/CREB(C)andpERK/ERK(D)levelsinhippocampus.Thedatarepresentedthevaluesofmean±S.E.M.from10mice/group.p<0.05vs.CUMSgroup.#p<0.05vs.Controlgroup.
图5：SCE给药对海马BDNF（A），pTrkB/TrkB（B），pCREB/CREB（C）和pERK/ERK（D）水平的影响。数据表示10只小鼠/组的平均值±S.E.M.p<0.05与CUMS组对比。#p<0.05与对照组对比。
EffectsofSCEonBDNFinthemousehippocampusbyimmunohistochemistryanalysis.AsshowninFig.6A–F,intergralopticaldensity(IOD)ofBDNF(brownparticles)inCUMSgroupwasremarkablylowerthanthatincontrolgroup(p<0.01).ThenumberofpositivehippocampalneuronsandIODinSCE(600or1200mg/kg)weresignificantlyhigherthanthoseinCUMSgroup(p<0.05),indicatingthatSCEup-regulatedtheBDNFexpressioninhippocampalneuronswhichwasconsistentwiththedataofELISA.
通过免疫组织化学分析，SCE对小鼠海马BDNF的影响。如图6A-F所示，CUMS组BDNF（棕色颗粒）的整体光密度（IOD）明显低于对照组（p<0.01）。SCE（600或1200mg/kg）阳性海马神经元数量和IOD值显著高于CUMS组（P<0.05），说明SCE上调海马神经元BDNF表达，与ELISA的数据一致。
Figure6.ImmunohistochemistryofBDNFproteininthemicehippocampus,Control(A),CUMS(B),SCE300mg/kg(C),600mg/kg(D),1200mg/kg(E)andIODofBDNF(F).Thescalebarshows100μm.Thedatarepresentedthevaluesofmean±S.E.M.from10mice/group.p<0.05vs.CUMSgroup.##p<0.01vs.Controlgroup.
图6：对照组（A），CUMS（B），SCE300mg/kg（C），600mg/kg（D），1200mg/kg（E）和BDNF的IOD值（F）的BDNF蛋白的免疫组织化学图。比例尺显示100μm。数据表示10只小鼠/组的平均值±S.E.M.p<0.05与CUMS对比。##p<0.01与对照组比较。
EffectsofSCEonBDNFinthemousehippocampus.ImmunofluorescenceassayshowedthatBDNF(redfluorescence,Fig.?7A)indepressionmicewassignificantlylowercomparedwithcontrolmice.AndSCE(600or1200mg/kg)groupcouldapparentlyincreasethelevelofBDNFproteinincomparisonoftheCUMSgroupwereshowninFig.?7BbasedontheaveragefluorescenceintensitiesofBDNF.TheresultswereinlinewiththeresultsofELISAandimmunohistochemistryassay.
SCE对小鼠海马BDNF的影响。免疫荧光测定显示抑郁小鼠中的BDNF（红色荧光，图7A）与对照小鼠相比显着降低。与CUMS组相比，在BDNF的平均荧光强度上，SCE（600或1200mg/kg）组可显著增加BDNF蛋白的水平，图7B显示。结果与ELISA和免疫组化检测结果一致。
Figure7.EffectsofSCEontheexpressionoftheimmunoreactivityofBDNFinthemousehippocampus.(A)HippocampuswerestainedwithspecificantibodiesagainstBDNF(red).NucleiwerestainedwithDAPI(blue).Thescalebarshows100μm.(B)TheaveragefluorescenceintensitiesofBDNFwerequantifiedafterSCEtreatment.Thedatarepresentedthevaluesofmean±S.E.M.from10mice/group.p<0.05,p<0.01vs.CUMSgroup.##p<0.01vs.Controlgroup.
图7：SCE对小鼠海马BDNF免疫反应性表达的影响。（A）用针对BDNF的特异性抗体对海马进行染色（红色）。核用DAPI（蓝色）染色。比例尺显示100μm。（B）用SCE定量处理后BDNF的平均荧光强度。数据表示10只小鼠/组的平均值±S.E.M.p<0.05，p<0.01与CUMS组对比。##p<0.01与对照组对比。
EffectsofSCEonPI3K/AKT/GSK-3βsignalingpathways.Westernblottingresults(Fig.?8)indicatedthatPI3Kwasreducedindepressionmice(p<0.05)andthistendencywasreversedbySCE(600or1200mg/kg)significantly(p<0.05).ThedataofAKTandGSK-3βturnedoutthesame,whichindicatedthatPI3K/AKT/GSK-3βmightbeinvolvedintheantidepressant-likeeffectofSCE.
SCE对PI3K/AKT/GSK-3β信号通路的影响。Western印迹结果（图8）表明抑郁症小鼠PI3K降低（p<0.05），并且这个趋势被SCE（600或1200mg/kg）明显逆转（p<0.05）。AKT和GSK-3β的数据结果相同，表明PI3K/AKT/GSK-3β可能参与到SCE的抗抑郁样作用。
Figure8.EffectsofSCEadministrationonpPI3K/PI3K(A),pAKT/AKT(B)andpGSK-3β/GSK-3β(C)levelsinhippocampus.Thedatarepresentedthevaluesofmean±S.E.M.from10mice/group.p<0.05,p<0.01vs.CUMSgroup.#p<0.05,##p<0.01vs.Controlgroup.
图8：SCE给药对pPI3K/PI3K（A），pAKT/AKT（B）和pGSK-3β/GSK-3β（C）水平在海马中的作用。数据表示10只小鼠/组的平均值±S.E.M.p<0.05，p<0.01与CUMS对比。#p<0.05，##p<0.01与对照组对比。
EffectsofSCEonhistopathologicalchangesinhippocampus.H.E.stainingwasperformedtodetecttheneuronalintegrityandorderliness.Inthehippocampus,theneuronsappearedintactandordered,shrinkage,degenerationandnecrosisofthenucleiwerenotobservedinthecontrolmice(Fig.?9A),butshrinkageofnuclei,swollenandeccentricallydispersedneuronalbodieswerefoundintheCUMSmice(Fig.?9B).However,administrationofSCEcouldinhibittheabovementionedhistopathologicaldamagessignificantly(Fig.?9C,D,E).
SCE对海马组织病理学改变的影响。进行HE染色以检测神经元的完整性和有序性。在海马中，神经元看起来完整，并且在对照小鼠中没有观察到细胞核的收缩，变性和坏死（图9A），但在CUMS小鼠中发现细胞核，肿胀和偏心分散的神经元的收缩图9B）。然而，SCE的施用可以显着抑制上述组织病理学损伤（图9C，D，E）。
Figure9.EffectsofSCEadministrationonthehistopathologicalchangesinthehippocampusofdepressedmice.Thescalebarshows50μm.Control(A),CUMS(B),SCE300mg/kg(C),600mg/kg(D)and1200mg/kg(E).
图9.SCE给药对抑郁小鼠海马组织病理学变化的影响。比例尺显示为50μm。对照（A），CUMS（B），SCE300mg/kg（C），600mg/kg（D）和1200mg/kg（E）。
Discussion
Numerousneuropsychologicalstudieshaveexaminedtheroleofcognitioninpsychopathology,revealingthatmostpsychiatricdisordersareassociatedwithsomedegreeofcognitiveimpairment32.WeusedCUMSproceduretomimicthesymptomofdepressionconfirmedbySPTandFSTinmiceinourbehavioralstudies,whichisconsistentwithpreviousreports33.Further,CUMSalsoimpairedthecognitivefunctionofmiceaccordingtotheperformancesofYmazeandMWMtest,whicharetwoclassicalmethodstoassesstheabilityoflearningandmemory.Theseresultsarewellcoincidentwithpreviousstudies34,35.However,withthetreatmentofSCE,theanhedonia-likebehaviorinsucrosepreferencetestandimmobilitytimeinforcedswimtestwhichbothinducedbyCUMSweresignificantincreased,respectively.Ontheotheraspect,thespontaneousalternationinYmaze,timeinthetargetquadrantandescapelatencyintrainingofSCEmicewereremarkableimprovedcomparedtotheCUMSmice.AndNosignificantdifferencesofthetotaltraveldistanceinthelocomotoractivitytestandnumberofarmentriesinYmazewhichcouldexcludethefalsepositiveresultscausedbythemobilityofmiceduringallthebehavioraltests.Moreover,wefoundasignificantcorrelationbetweenthesucrosepreferencevs.spontaneousalternation,thesucrosepreferencevs.timeinthetargetquadrant,immobilitytimevs.spontaneousalternationandimmobilitytimevs.timeinthetargetquadrantwhenlinearregressionwasdetermined(Fig.?10).IntegratedwiththeresultsofH.E.staining,wefoundthatSCEindeedhastheabilitytoimprovethedepressivesymptomsandtheassociatedcognitivedeficitseffectively.
许多神经心理学研究已经研究了认知在精神病理学中的作用，揭示了大多数精神失调与一定程度的认知障碍有关。我们使用CUMS程序来模拟我们在行为研究中已被SPT和FST小鼠证实的抑郁症状，这和之前的研究是一致的。此外，根据Y迷宫和MWM这两种评估学习和记忆能力的经典方法的测试结果显示，CUMS也会损害小鼠的认知功能。这些结果与以前的研究是符合的34,35。然而，在SCE的治疗下，在由CUMS诱导的蔗糖偏好测试和固定时间的强迫游泳测试中，快感缺失行为分别显著增加。另一方面，与CUMS小鼠相比，SCE小鼠的Y迷宫自发交替、到达目标象限的时间和训练的逃生潜伏期得到显著改善。并且在运动行为测试中的总奔跑路程和在Y迷宫测试中的入口分支数目没有明显的差异，可以排除小鼠在所有行为测试中的不定性造成的假阳性结果。此外，当在确定线性回归时，我们发现在蔗糖偏好和自主选择、蔗糖偏好和目标象限时间、固定时间和自主选择以及固定时间和目标象限时间之间存在着明显的相干性（图10）。整合HE染色的结果，我们发现SCE确实具有有效改善抑郁症状和相关认知缺陷的能力。
Figure10.Pearson’scorrelationbetweenthesucrosepreferencevs.spontaneousalternation(A),thesucrosepreferencevs.timeinthetargetquadrant(B),immobilitytimevs.spontaneousalternation(C)andimmobilitytimevs.timeinthetargetquadrant(D).
图10：在蔗糖偏好与自发交替之间的皮尔森相关性（A），蔗糖偏好与目标象限中时间的比较（B），不动时间与自发交替的比较（C）和不动时间与目标象限时间的比较（D）。
Theinterrelationshipbetweendepressionandcognitiveimpairmentiscomplexandstillnotwellunderstood.Inthebrain,BDNFhasbeenimplicatedindevelopment,neuralregeneration,synaptictransmission,synapticplasticityandneurogenesis36.AlternationinBDNFlevelshavebeenimplicatedinpsychiatricdisorders,includingdepressionandsubstanceabuse,aswellasneurodegenerativedisorders,suchasAlzheimer’s,Parkinson’sandHuntington’sdiseases37.WefoundthatinthehippocampusofCUMS-inducedmice,BDNFlevelsweresignificantlydecreased,andtheresultsofY-mazeandMWMtestwereshownthatthecognitionimpairmentalongwiththedepressionsymptoms.AftertreatmentwithSCE,theBDNFlevelswereupregulated,andthedepressionsymptomsandcognitiondeclinewerealleviatedatthesametimeaccordingtothebehavioralresults.BesidestheresultsofimmunohistochemistryanalysisandImmunofluorescenceassay,theaboveallillustratedthatBDNFhasplayedavitalrolebothindepressionandthelearningobstacletriggeredbyit,whichinlinewiththepreviousreports38.
抑郁症和认知功能障碍之间的相互关系是复杂的，且尚未弄清楚。在大脑中，BDNF涉及神经发育、神经再生、突触传递、突触可塑性和神经发生。BDNF水平的变化涉及到精神障碍，包括抑郁症和药物滥用，以及神经退行性疾病如阿尔兹海默症、帕金森症和亨廷顿病。我们发现在CUMS诱导的小鼠的海马体中，BDNF水平显著减少，并且Y迷宫和MWM测试的结果显示认知功能障碍伴随着抑郁症状。经SCE治疗后，BDNF水平得到上调，同时在行为测试结果中发现抑郁症状和认知功能障碍得到缓解。除了免疫组织化学分析和免疫荧光检测的结果，以上所有结果都说明BDNF在抑郁和学习障碍中扮演了至关重要的角色，这也符合以前的报导。
Tropomyosin-relatedkinaseB(TrkB),aproteintyrosinekinasereceptorandamemberofthelargerfamilyofTrkreceptors,whichwasexpressedathighlevelsinthebrain,wasidentifiedastheprimarysignaltransductionreceptorforBDNF.MultiplelinesofevidencelinkBDNF-TrkBsignalingtothepathophysiologyofMDD,aswellasthetherapeuticmechanismsofantidepressants39,40.Furthermore,extensiveexperimentalevidencesupportsthatBDNFmitigatesdepressivesymptomsmainlybybindingtoTrkB,leadingtoautophosphorylationofTrkBtyrosineresidues,andactivationofdownstreamsignalingmolecules,includingtheextracellularsignal-regulatedkinase1/2(ERK1/2)knowntophosphorylatecAMPresponseelementbindingprotein(CREB)41.Ithasbeenreportedthatthefull-lengthTrkBautophosphorylationregulateERKonactivationbyBDNF,whichmayincreasecAMPandactivateCREB-regulatedgenetranscription,andthismechanismfurtherpromotestranscriptionofBDNF42.Inagreementwithpreviousstudies,weobservedthatCUMS-induceddepressivesymptomsandconsequentBDNF/TrkB/CREB/ERK1/2signalingdown-regulationreversedbySCEtreatment,whichindicatedthatSCEcouldincreaseBDNFexpressionbyaffectingTrkB/CREB/ERK1/2pathwaytoexertantidepressant-likeeffect.Additionally,interactionofBDNFwithTrkBtriggersreceptordimerization,transphosphorylationofintracellulartyrosineresidues,andsubsequentactivationofthethreemajorsignalingpathwaysinvolvingMAPK/CREB/ERK,phosphatidylinositol3kinase(PI3K)/AKT,andphospholipaseC-γ43.PI3K/AKTandERKsignalingpathwaysarethemajorTrkB-mediatedsurvivalpathwaysthatpromoteneuronalsurvivalandprotectagainstapoptosis.BDNF/TrkBsignalingcanpromotefurtherBDNFproductionthroughCREBviaactivationofPI3K/AKTorERKsignaling,whichisemergingasapositive-feedbackloop44.TrkB-inducedPI3KactivityleadstoAKTactivation,andonceactivated,AKTleadstoinactivationofGSK-3βatSer-9.MorerecentfindingsshowedthatdisruptionofGSK-3phosphorylationbyAKTdecreasesanxietyandreducespronenesstodepressioninmice45andintracellularsignaltransductionsystemasAKT/GSK-3βpathwayhavebeenfoundtobealteredinthebrainofdepressivepatients46.ThisprovidescompellingevidencethatthePI3K/AKT/GSK-3βsignalingpathwayisanimportantcontributortodepression.HerewedemonstratedthattheSCEtreatmentwasabletoactivatePI3K/AKT/GSK-3βsignalingpathway,aswelltopromoteincreasedphosphorylatedlevelsofrelatedproteins,toimprovethedepressivesyndromesinCUMS-inducedmice.Takenallabovetogether,thesefindingsleadustosuggestthatSCEactingasanantidepressantincreasesBDNFlevelsbypromotingTrkB/CREB/ERKpathwayandactivatingPI3K/AKT/GSK-3βpathwaysimultaneously(Fig.?11).
原肌球蛋白相关激酶B（TrkB），一种酪氨酸蛋白激酶受体，作为Trk受体大家族的一员，在大脑中被高水平地表达，现已被确认为BDNF的早期信号转导受体。多种证据将BDNF-TrkB信号通路和MDD的病理生理学、抗抑郁药的治疗机制联系起来。而且，广泛实验证据支持BDNF主要通过结合TrkB使TrkB的酪氨酸残基磷酸化和激活下游信号分子，包括已知的磷酸化cAMP反应原件结合蛋白（CREB）的胞外信号调节激酶1/2(ERK1/2)来缓解抑郁症状。据报道，全长的TrkB磷酸化通过激活BDNF来调节ERK，可能会增加cAMP含量和激活CREB调节基因转录，并且这个过程进一步促进BDNF的转录。与以前的研究一致，我们观察到CUMS诱导的抑郁症状和用SCE治疗后随之发生的BDNF/TrkB/CREB/ERK1/2信号下调得到逆转，这表明SCE可以通过影响TrkB/CREB/ERK1/2通路来增加BDNF表达量以发挥抗抑郁作用。需要补充的是，BDNF和TrkB的相互作用触发了受体二聚体化、细胞内氨基酸残基的转磷酸化和随后的三个主要信号通路的激活，包括MAPK/CREB/ERK、磷脂酰肌醇3激酶(PI3K)/AKT和磷脂酶C-γ。PI3K/AKT和ERK信号通路是主要的TrkB介导的促进神经元存活并防止细胞凋亡的自救途径。BDNF/TrkB信号通路可以通过CREB经过PI3K/AKT或ERK信号的激活能够进一步促进BDNF的生产，如此形成了一个正反馈环路。TrkB诱导的PI3K激活导致AKT活化，并且一旦激活，AKT将使Ser-9处的GSK-3β处失活。更多最近的研究结果表明小鼠中AKT对GSK-3磷酸化的终止减少了焦虑和降低了抑郁的倾向性，并且像AKT/GSK-3β类的细胞内信号转导系统已被发现在抑郁症患者的脑中发生了改变。这也为PI3K/AKT/GSK-3β信号通路是抑郁症重要的效力者提供了令人信服的证据。这里我们展示了SCE治疗有可能激活PI3K/AKT/GSK-3β信号通路，以及促进相关蛋白磷酸化水平的提高，以改善CUMS诱导小鼠的抑郁综合征。综上所述，这些研究表明，SCE作为抗抑郁药通过促进TrkB/CREB/ERK通路同时激活PI3K/AKT/GSK-3β通路来增加BDNF水平。
Figure11.ProposedSCEantidepressant-likemechanismsfortheinteractionamongBDNF,TrkB,CREB,ERK,PI3K,AKTandGSK-3β.SCEregulatesTrkB/CREB/ERKpathwaytoincreasetheBDNFtoalleviatethedepressionandcognitivedecline.Ontheotheraspect,BDNFupregulatesPI3K/AKT/GSK-3βpathwaytoamelioratethedepressivesymptomsandcognitiondisabilityinducedbyCUMS.
图11：BDNF，TrkB，CREB，ERK，PI3K，AKT和GSK-3β之间相互作用的模拟的SCE抗抑郁药物机制。SCE调节TrkB/CREB/ERK途径增加BDNF以减轻抑郁症和认知功能下降。另一方面，BDNF上调PI3K/AKT/GSK-3β途径，改善由CUMS引起的抑郁症状和认知障碍。
Basedontheformerresearch,depressioncanbeapsychologicalreactiontocognitivedecline,andthusmayalsoappearasanearlysymptomindementingindividuals47.However,recentdatasuggestthatdepression,andinparticularlylate-lifedepression,canalsobeariskfactorforAlzheimer’sdisease48.Inourpresentstudy,weconfirmedthatSCEcouldtreatdepressionandshowadefiniteimprovementofcognition.Thefurtherandin-depthstudyontheinteractionsbetweendepressionanddementia,andtheseparate,simultaneouseffectsofSCEonthesetwokindsofdiseaseswouldbeperformedinthefuturestudy.
基于之前的研究，抑郁可能是一种认知衰退的心理反应，因此也可能表现为痴呆症患者的早期症状。然而，最新的数据显示抑郁，特别是晚期抑郁症，有可能成为阿尔兹海默症的危险因素。在我们目前研究中，我们证实了SCE可以治疗抑郁症并且明显改善认知功能，今后研究将进一步深入研究抑郁与痴呆之间的相互作用，以及SCE对这两种疾病的单独和同时影响效果。
Insummary,ourstudyshowsthattheantidepressant-likeeffectandcognitionimprovementabilityofSCEdependsonBDNFlevelsraisebyincreasingTrkB/CREB/ERKpathwayandPI3K/AKT/GSK-3βpathwayinthemeantime.OurfindingssuggestthatSCEmightbeapromisingtherapeuticagentofdepression,andfurtherresearchisworthtobeinvested.
总之，我们的研究表明，SCE的抗抑郁效果和认知改善能力依赖于通过同时促进TrkB/CREB/ERK通路和PI3K/AKT/GSK-3β通路致使的BDNF水平的提高。我们的发现表明SCE也许会成为一种有前途的抑郁症治疗剂，并且对其进一步的研究是值得投入的。
Methods
PreparationsofextractofSCE.SCE提取物的制备ThefruitsofSCEwerepurchasedfromtheTCMshopofTongrentang(Shenyang,China)andidentifiedbyProfessorYingJia(DepartmentofPharmacognosy,ShenyangPharmaceuticalUniversity)accordingtotheguidelinesoftheChinesePharmacopoeia(2015).Then,thefruitsofSCEwereexhaustivelyextractedwith95%ethanolatrefluxfor2h3times.Afterconcentrationinavacuum,theresiduewassuspendedin0.5%sodiumcarboxymethycellulose(CMC-Na)atacertainconcentrationof300mg/kg,600mg/kgor1200mg/kg.
UPLC–Q-TOF/MSanalysisofSCE.SCE的uplc-qtof/质谱分析ThechemicalcompositionofSCEwasanalyzedbyusingaWaters-UPLC-Q-TOF/MSwithanultraviolet/visibledetector(UV/Vis)coupledtoaniontrapmassspectrometerwithanESIinterface.TheseparationwasachievedonanHSST3Column(100mm×2.1mm,1.8μm).Thechromatogramwasrecordedat216nm.MassanalyseswereperformedusinganESIinterfaceinthepositiveionmode.DatawereperformedwithMasslynxV4.1software.AsshowninFig.?12andTable?3,fourteenlignansweretentativelyidentifiedbythefullscanonthepositiveionmodeofMS/MSanalysis.Eightmaincompounds(1,4,7,8,11,12,13and14)ofthoselignanswereidentifiedwiththeretentiontimeandUVspectraofthereferencesubstance.
ChemicalsandReagents.药品和试剂FluoxetinehydrochlorideasapositivecontroldrugwasobtainedfromMelonePharmaceuticalCo.(Dalian,China).Allotherchemicalsandreagentswereofanalyticalgrade.
Animals.AdultmaleKunmingmice(weighing20±2g)werepurchasedfromtheExperimentalAnimalCenterofShenyangPharmaceuticalUniversity(Shenyang,China).Allofthemweremaintainedunderstandardlaboratoryconditionsofconstanttemperature(23±1°C),relativehumidity(50±10%)anda12hlight/darkcycle(lightfrom7:00a.m.to7:00p.m.)withfoodandwateravailableadlibitumandwereallowedtohabituatetothenovelenvironmentfor1weekpriortouseinexperiments.TheexperimentwascarriedoutincompliancewiththeNationalInstitutesofHealthandinstitutionalguidelinesforthehumanecareofanimalsandwasapprovedbytheAnimalCareCommitteeofShenyangPharmaceuticalUniversity(ProtocolNo.:SYPU-IACUC2016C-0921-205).Everyeffortwasmadetominimizethenumberofanimalsusedandanypainanddiscomfortexperiencedbythesubjects.
CUMSprocedure.CUMS过程CUMSwasperformedaspreviouslydescribed49.CUMSconsistedofexposuretoavarietyofunpredictablestressors(randomly),including(1)24hfooddeprivation,(2)24hwaterdeprivation,(3)1hexposuretoanemptybottle,(4)exposuretoanemptycage(withoutsawdustbedding),(5)groupedhousing,(6)24hsoiledcage(200mlwaterin100gsawdustbedding),(7)levelshakingfor30min,(8)5mincoldswimming(at5°C),(9)niptailfor1min.Allstresseswereappliedindividuallyandcontinuously,dayandnight.Thecontrolanimalswerehousedinaseparateroomandhadnocontactwiththestressedgroups.Topreventhabituationandtoensuretheunpredictabilityofthestressors,allstresseswererandomlyscheduled,repeatedthroughoutthe3-weekexperiment.Thecontrolgroupmicewereleftundisturbedexceptfornecessaryproceduressuchasroutinecagecleaning.
如先前描述的那样执行CUMS。CUMS包括暴露于各种不可预测的压力源（随机），包括（1）24小时食物剥夺，（2）24小时缺水，（3）1小时暴露于空瓶，（4）暴露于空笼（不含锯屑寝具），（5）组合住房，（6）24小时污染笼（200毫升水，100克锯屑床垫），（7）水平震动30分钟，（8）5分钟冷游泳（5度C），（9）咬合尾巴1分钟。所有的压力都是单独和连续地应用于白天和黑夜。将对照动物放在单独的房间中，与受压组不接触。为了避免习惯并确保压力源的不可预测性，所有压力都是随机安排的，在整个3周的实验中重复。对照组小鼠不受干扰，除了必要的程序，例如常规的笼子清洁。
Drugadministrationandexperimentalgroups.药物使用和实验组FortestingofthebehavioralandBDNFregulations,themicewererandomlydividedintosixgroups(10animalsforeachgroup):Control-Vehiclegroup,CUMS-Vehiclegroup,CUMS-fluoxetine(10mg/kg,i.g.),CUMS-SCEgroup(300mg/kg,i.g.),CUMS-SCEgroup(600mg/kg,i.g.)andCUMS-SCEgroup(1200mg/kg,i.g.).FortheControl-Vehiclegroup,theanimalswereinjectedwithsameamountof0.5%CMC-Na.Alltheseagentswereadministeredinavolumeof10ml/kg.BeforeCUMS,animalswereallowedtohabituatetoallthebehavioraltestsinordertoestablishanindividualbaseline.ThesucrosepreferencetestswereconductedtwicebeforeandaftertheCUMSprocedure,respectively.Beforethetissueisolation,themicewereallowedtotakethetestofallthebehavioraltests.Behavioraltestswereperformedduringthelightphaseoflight–darkcycle.ThewholeexperimentalprocedureisshowninFig.?13.
对于行为和BDNF规定的测试，将小鼠随机分为6组（每组10只动物）：对照-载体组，CUMS-载体组，CUMS-氟西汀（10mg/kg，ig），CUMS-SCE（300mg/kg，ig），CUMS-SCE组（600mg/kg，ig）和CUMS-SCE组（1200mg/kg，ig）。对照对照组，用相同量的0.5％CMC-Na注射动物。所有这些试剂以10ml/kg的体积施用。在CUMS之前，允许动物习惯所有的行为测试，以建立一个单独的基线。蔗糖优选试验分别在CUMS程序之前和之后进行两次。在组织分离之前，允许小鼠进行所有行为测试。在光暗周期的光照阶段进行行为测试。整个实验过程如图13所示。
Behavioralanalyses.Sucrosepreferencetest(SPT).Sucrosepreferencetestwascarriedout1daybeforeCUMSandtheendofCUMS.Thetestwasperformedasdescribedpreviously50.Inbrief,72hbeforethetest,themousewasindividuallyplacedinacagewithtwobottlesofsucrosesolutiontoadaptasucrosesolution(1%,w/v)for24h;thenonebottleofsucrosesolutionwasreplacedwithwater(24h);aftertheadaptation,laboratorymiceweredeprivedofwaterandfood(24h).Sucrosepreferencetestwasconductedat9:30a.m.Eachmousewashousedinindividualcageandfreetoaccesstwobottlescontaining200mlofsucrosesolution(1%w/v)and200mlofwater,respectively.After6h,theconsumedweightsofsucrosesolutionandwaterwererecorded.Thesucrosepreferencevaluewasobtainedfromthefollowingformula:sucrosepreference(%)=sucroseintake(ml)/[sucroseintake(ml)+waterintake(ml)]×100%
Inneropen-fieldbehaviortest(OFT).内部露天行为测试（OFT）InordertoruleoutthepossibilitythatthealterationintheimmobilitytimeintheFSTwasduetointerferenceofthelocomotoractivity,spontaneouslocomotoractivityofeachmousewasobservedinanopenfiledexperimentalvideoanalysissystem(ZS-ZFT,HuaibeiZhenghuaBio-ApparatusCo.Ltd,China).Theapparatuswasplacedinadarkenedandsoundattenuatedtestingroom.ThetotalpathofSpontaneouslocomotivewasevaluatedovera5minperiod51,52.
Forcedswimtest(FST).TheFSTwasconductedandthetotalimmobilitywasestimatedinaccordancewiththemethodsdescribedpreviously53withslightchanges.Inbrief,eachmousewasforcedtoswiminacylindricalglasscontainer(diameter20cm×height50cm)with30cmofwater(22±1°C)for6min,andthedurationofimmobilityduringthelast4minwasdetected.Themicewereconsideredasimmobilewhentheymaintainedfloatinginthewaterwithoutescapingfromthecontainer,makingonlythemovementnecessarytokeeptheirheadsabovethewater.Theparameterwasrecordedbytrainedobserverswhowereblindtotheexperimentalgroups.
Y-mazetest.Y-mazetestasameasureofimmediatespatialworkingmemorywhichisaformofshort-termmemory54.TheY-mazeisconsistedofthreearmsatequalangles(30cmlength×5cmwidth×12cmhigh).Micewereplacedattheendofonearmandallowedtomovefreelythroughthemazefor6min.Anarmentrywascountedwhenthehindpawsofthemousewerecompletelywithinthearm.Theseriesofarmentrieswererecordedvisuallyandthepercentagealternationwascalculated.Aspontaneousalternationwasdefinedassuccessiveentriesintothethreearms,i,e.,ABC,CAB,orBCAbutnotCBC.Thepercentagealternationwascalculatedastheratioofactualtopossiblealternations(definedasthetotalnumberofarmentriesminustwo)multipliedby100asshownbythefollowingequation:Alternation%=[Numberofalternations/(Totalarmentries?2)]×100.Thenumberofarmentrieswasalsousedasanindicatoroflocomotoractivity.
Morriswatermazetest(MWM).AspatiallearningandmemorytestwasperformedbythemethodofMorriswithminordifference55.TheMorriswatermazeconsistedofalargecirculartank(120cmindiameter,40cminheight)whichwasdividedgeographicallyintofourquadrants(I,II,IIIandIV).Ablackplatform(9cmindiameter,30cminheight)wassubmerged1cmbelowthewatersurfaceandfixedatthemidpointoftheIVquadrant.Thewaterwascoloredwithnon-toxicblackinkandwasmaintainedat23±2°C.Thetankwasplacedinadimlylit,soundprooftestroomwithvariousvisualcuesfornavigation.TheMorriswatermazetestwascomposedoftrainingtrialsandprobetest.Beforetest,eachanimalwasscreenedinMWMaccordingtothespeedandswimmingstate,andawhiteplasticplatformwasusedtoevaluatethevisibleabilityofmice.Inthetrainingtrials,themicewereplacedinthewaterfacingthepoolwallfromtwodifferentstartingpointseverydayandallowedtoswimfreelytoseekthehiddenplatformforamaximum90s.Ifamousefailedtofindtheplatformwithin90s,itwasguidedbyanexperimentertotheplatformtostudy30s.Thetrainingtrialslastedfiveconsecutivedays.Theaverageescapelatencyofeachmouseperdaywasrecorded.Theprobetestwasperformedwithoutplatformafterthe5-daytrainingfinished.Thepercentageoftimespentofswimminginthetargetquadrantandthetimescrossedwheretheplatformwasoriginallylocatedweremeasuredforeachmouse56.
Biochemicalanalysis.Tissuesamplecollection.Afterthecompletionofthebehavioraltest,themicewereeuthanized.Wholebrainswererapidlyremovedfromthemiceandstoredaccordingtothespecificexperimentalprocedure.Brainregionsofthehippocampusweredissectedonacoldplateandimmediatelyfrozeninliquidnitrogen.Thetissuesampleswerestoredat?80°Cuntilassay.
ELISAkitsassay.酶联免疫吸附试验。BDNF,pTrkB,TrkB,Pcreb,CREB,pERKandERKconcentrationsofhippocampusdeterminationwereperformedusingtheELISAmethod,accordingtothemouseELISAkitsmanufacturer’sinstructions(ShanghaiEnzyme-linkedBiotechnologyCo.,Ltd,China),respectively.Opticaldensitywasobtainedat450nmusingamicroplatereader(Varioskanflash,Thermoscientific,USA)within15minofstopsolutionaddition.
Westernblot.HippocampaltissueswerehomogenizedinRIPA(150mMsodiumchloride,50mMTris(pH8.0),0.5%sodiumdeoxycholate,0.1%SDS,1%TritonX-100)andPMSF(DalianMelonepharma,China)andkeptonicefor30min.Thetissuehomogenatewascentrifugedat10000gfor20minat4°C.ThesupernatantwasobtainedandusedasthetotalhippocampalandcerebralcortexproteinextractmeasuredbyBCAassaykit(DalianMelonepharma,China)todetermineproteinconcentrationandstoredat?80°Cuntiluse.Samplesweredilutedwithanequalvolumeofloadingbuffer(BeyotimeBiotechCo.,China),andboiledat95°Cfor5min.Approximately50μgofproteinwasloadedineachwellandseparatedin10%or12%SDS-PAGEgels.Theproteinsweretransferredontonitrocellulosemembranes.Themembranesweresaturatedandblockedwith5%fat-freepowderedmilkat37°Cfor1.5handincubatedovernightat4°Cinoneofthefollowingprimaryantibodies,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)Blotswerewashedthreetimesfor30mininTBSTatroomtemperatureandthenincubatedfor1.5hinoneofthefollowingHRP-conjugatedantibodies,whichweredilutedin5%fat-freepowderedmilkinTBS:Anti-rabbitIgG(1:2000,CST,USA)fordetectionoftargetproteins,GADPHandβ-actin.Afterthreetimeswashesfor30mininTBST,immunolabeledproteinbandsweredetectedusingtheECLWesternblotdetectionkit(DalianMelonepharma,China).Graphsofblotswereobtainedinthelinearrangeofdetectionandwerequantifiedforthelevelofspecificinductionbyscanninglaserdensitometry.
Immunohistochemistryassay.Miceweresacrificedbyoverdoseofsodiumpentobarbitoneforimmunohistochemicalanalysis,andthenintracardiallyperfusedwithPBS,followedbychilled4%PFAinPBS.Thebrainwasslicedinto15mmsectionsonacryostatblockedinPBScontaining1%goatserumand0.1%Triton×100,andincubatedat4°Covernightwithanti-BDNF(mouseIgG,1:500,abcam,USA).Afterwashing,aHRPconjugatedgoatanti-rabbitIgGcomplex(1:1000,absin,China)wasappliedfor1h.Colordevelopmentwasperformedwithadiaminobenzidineperoxidasesubstratekit(TL-125-QHD,ThermoFisherScientific,USA).Sectionswereconunterstainedwithhematoxylin.
Immunofluorescenceassay.TissuebiopsiesweredeparaffinizedandpermeabilizedwithPBS/0.1%Triton×100.Antigenretrievalwasperformedbyboilingtheslidesin0.01Mtrisodiumcitratebuffer,pH6,for10min.Sectionswerethenpreincubatedwith10%normalgoatserumcontaining0.2%Triton×100overnightat4°Ctoblocknonspecificbinding.Slideswerethenincubatedovernightat4°Cwithanti-BDNF(mouseIgG,1:100,abcam,USA)in2%serum,sectionswerewashedthreetimesinPBStoremoveunboundprimaryantibody,thenincubatedwithsecondaryantibodyfor1hat37°C.Afterthat,sectionswerewashedthreemoretimesinPBST(PBS,with1%Tween20)andcoveredwithDAPI/Fluorescencequenchingagent(Beyotime,China).SlideswereviewedusingaLeicafluorescencemicroscopecoupledwithacomputerassistedvideocamera(AxioscopeA1,Germany).
Histologyassay.组织学检测Forhistopathology,hematoxylin-eosinandCongoredwereusedasdescribedpreviously57.Afterthebehavioraltestsanimalsweredecapitatedandtheirbrainswereremovedquickly,postfixedin37%formaldehydesolutionfor48h,andthenallthebrainwascutinthecoronalplaneandstainedwithhematoxylinandeosin.Hippocampusneuronswereexaminedinlightmicroscopy.
Statisticalanalysis.统计分析。Resultsareexpressedasmean±SEM.Thesignificancesbetweendifferentgroupswereassessedusingone-wayANOVA,followedbyTukeyHSDpost-hoctestwhensignificantmaineffectswereindicated.Two-wayANOVAwasusedtoanalyzedatafromtheMorriswatermazetrainingtrials.Inallcalculations,p<0.05wasconsideredtobestatisticallysignificant.StatisticalanalysiswasperformedwithSPSSsoftware19.0.
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