JuanjuanTang,WendaXue,BaomeiXia,etal.InvolvementofnormalizedNMDAreceptorandmTOR-relatedsignalinginrapidantidepressanteffectsofYuejuandketamineonchronicallystressedmice.SciRep,,doi:10./srep.(IF:5.)
前言中医经典方药越鞠丸（Yueju）出自《丹溪心法》，其为朱丹溪于年前创制的解郁名方。越鞠丸由香附（Cyperusrotundus）、川芎（LigusticumChuanxiong）、栀子（Gardeianjasminoides）、苍术（Atractylodeslancea）和神曲（MassaFermentata）五味中药组配而成。越鞠丸具有理气解郁，宽中除满之功效，常用于胸脘痞闷，腹中胀满，饮食停滞，嗳气吞酸。然而，南京中医药大学转化系统生物学与神经科学研究中心教授陈刚领衔的研究小组，于年首次发现越鞠丸具有独特的快速持久抗抑郁作用，并揭示了其改善抑郁脑区失常的蛋白激酶B（Akt）信号通路和持久提升脑源性神经营养因子（BDNF）的机制。
摘要中医经典方药越鞠丸（Yueju）具有与快速抗抑郁原型药氯胺酮（Ketamine）相似的快速持久的抗抑郁效应。本研究旨在评价越鞠丸与氯胺酮在慢性应激大鼠模型中的快速持久抗抑郁效力以及其效力发挥与前额叶N-甲基-D-天门冬氨酸（N-methyl-D-aspartate,NMDA）受体和哺乳动物的雷帕霉素靶蛋白（mammaliantargetofrapamycin,mTOR）相关活性变化之间的关系。慢性轻度应激（chronicmildstress,CMS）将导致糖水偏好实验（sucrosepreferencetest,SPT）、强迫游泳实验、悬尾实验及新环境进食抑制实验的缺失，然而这些缺失可通过越鞠丸和氯胺酮的急性给药而得到不同程度的改善。越鞠丸和氯胺酮给药2小时后，SPT就得以改善，然而给予越鞠丸后其改善效果持续了6天。对于给药6天后的体重恢复，越鞠丸优于氯胺酮。CMS降低了mTOR效应体4E-BP1和p70S6K、其上游的调节子细胞外信号调节激酶（ERK）和蛋白激酶B（Akt）及下游靶点（包括突触蛋白GluR1）的磷酸化。越鞠丸或氯胺酮给药2天后，上述变化得以逆转，然而仅越鞠丸在给药6天后仍可逆转磷酸化Akt。CMS选择性、持久性的增加了NMDA受体亚单位NR1的表达，然而这一结局在氯胺酮或越鞠丸给药2天后得以逆转，然而仅越鞠丸给药6天后上述结局仍旧得以逆转。上述结果表明，NR1和Akt/mTOR信号通路是抑郁重要的治疗靶点。
ABSTRACTYueju,aTraditionalChineseMedicineformula,exhibitedfast-onsetantidepressantresponsessimilartoketamine.ThisstudyfocusedonassessingtherapidandpersistentantidepressantefficacyofYuejuandketamineinchronicallystressedmiceanditsassociationwithalternationsinprefrontalN-methyl-D-aspartate(NMDA)receptorandmammaliantargetofrapamycin(mTOR)-relatedactivity.Chronicmildstress(CMS)ledtodeficitsinsucrosepreferencetest(SPT),forcedswimtest,tailsuspensiontest,andnoveltysuppressedfeedingtest,whichwereimproveddifferentlybyacuteYuejuorketamineadministration.TheimprovementinSPTstartedassoonas2hourspostYuejuandketaminebutlastedfor6daysonlybyYueju.BodyweightwasregainedbyYuejumorethanketamineatpost-drugadministrationday(PAD)6.CMSdecreasedphosphorylationofthemTOReffectors4E-BP1andp70S6K,theirupstreamregulatorsERKandAkt,anddownstreamtargetsincludingsynapticproteinGluR1.YuejuorketaminereversedthesechangesatPAD2,butonlyYuejureversedphosphor-AktatPAD6.CMSselectivelyandlastinglyincreasedNMDAreceptorsubunitNR1expression,whichwasreversedbyketamineorYuejuatPAD2butonlybyYuejuatPAD6.ThesefindingssuggestthatNR1andAkt/mTORsignalingareimportanttherapeutictargetsfordepression.
结果图1.氯胺酮与越鞠丸治疗CMS大鼠不同时间点的行为效应。对照大鼠给予赋形剂治疗，暴露于CMS的大鼠接受单剂量赋形剂、越鞠丸或氯胺酮治疗。(A)给药2小时、2天及6天后糖水偏好实验结果。(B,C)给药2小时、2天及6天后体重变化情况。(D)给药1天及5天后悬尾实验的静止时间。(E)给药1天及5天后强迫游泳实验的静止时间。(F)给药2天及6天后新环境进食抑制实验期间的进食的延迟情况。(G)给药2天及6天后新环境进食抑制实验期间总的进食量。
Figure1.BehavioraleffectsatdifferenttimepointsafterasingleketamineandYuejutreatmentinCMSmice.Controlanimals(Con)receivedvehicletreatment,andanimalsexposedtoCMSreceivedasingleadministrationofvehicle(Veh),Yueju(YJ)orketamine(Ket).(A)Sucrosepreferencetestat2hours,2daysand6daysafterdrugadministration,respectively.(B,C)Bodyweightchangesat2daysand6dayspostdrugadministration.(D)TheimmobilitytimeintheTSTat1dayand5dayspostdrugadministration.(E)TheimmobilitytimeintheFST1dayand5daysafterdrugadministration.(F)LatencytofeedduringtheNSFtestat2daysand6dayspostdrugadministration.(G)TotalamountoffoodconsumedduringtheNSFtestat2daysand6dayspostdrugadministration.
图2.CMS大鼠接受氯胺酮和越鞠丸治疗2天后mTOR下游效应体活性的变化情况。(A)4E-BP1的磷酸化水平。(B)以总的p70S6K为基准标化后磷酸化p70S6K。(C)以总的mTOR为基准标化后的磷酸化mTOR。
Figure2.AlternationsofactivationofdownstreameffectorsofmTORsignalinginPFCofCMSmiceat2dayspostketamineandYuejuadministration.(A)Phosphorylationof4E-BP1level.(B)Phosphorylatedp70S6K(pp70S6K)normalizedtototalp70S6K.(C)phosphorylatedmTOR(p-mTOR)normalizedtototalmTOR.
图3.CMS大鼠接受氯胺酮和越鞠丸治疗2天后ERK1/2和Akt的变化情况。(A)以总的Akt为基准标化后的磷酸化Akt的光密度分析。(B)以总的ERK1/2为基准标化后的磷酸化ERK1/2。
Figure3.AlternationsofactivationofERK1/2andAktinthePFCofCMSmiceat2dayspostketamineandYuejuadministration.(A)DensitometricanalysisofphosphorylatedAktnormalizedtototalAkt.(B)phosphorylatedERK1/2normalizedtototalERK1/2.
图4.CMS接受氯胺酮和越鞠丸治疗后突触蛋白的变化情况。越鞠丸或氯胺酮单剂量给药后对PFC中蛋白表达水平的影响通过蛋白印迹法加以测定。(A)NR1.(B)NR2A.(C)NR2B.(D)GluR-1.(E)PSD-95.(F)synapsin-1.
Figure4.AlternationsofsynapticproteinsinthePFCofmicebyCMSaswellasketamineandYuejutreatment.ThetreatmenteffectsonproteinexpressionlevelsinthePFCweredeterminedwithwesternblottingat2dayspostasingleadministrationofYuejuorketamine.(A)NR1.(B)NR2A.(C)NR2B.(D)GluR-1.(E)PSD-95.(F)synapsin-1.
图5.CMS大鼠接受氯胺酮和越鞠丸治疗6天后mTOR信号通路及突触蛋白的变化情况。越鞠丸或氯胺酮单剂量给药后对PFC中蛋白表达水平的影响通过蛋白印迹法加以测定。(A)NR1.(B)GluR-1.(C)4E-BP1磷酸化水平.(D)以总的Akt为基准标化后的磷酸化Akt.
Figure5.AlternationsofsynapticproteinsandmTORsignalinginthePFCofCMSmiceat6dayspostketamineandYuejuadministration.ThetreatmenteffectsonproteinexpressionlevelsinthePFCweredeterminedwithwesternblotting.(A)NR1.(B)GluR-1.(C)Phosphorylationof4E-BP1level.(D)phosphorylatedAktnormalizedtototalAkt.
图6.快速抗抑郁对大鼠抑郁模型PFC中mTOR相关活性及AMPA/NMDA受体表达影响的作用模式。
Figure6.Aworkingmodeloftheeffectsoffast-actingantidepressantsonmTORrelatedactivityandAMPA/NMDAreceptorexpressionsinthePFCofarodentmodelofdepression.
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