基于Wnt-β-catenin信号通路探讨健脾益智法调控MCAO大鼠NSCs增殖的机制研究

基于Wnt-β-catenin信号通路探讨健脾益智法调控MCAO大鼠NSCs增殖的机制研究摘要：

目的：本研究旨在探讨Wnt/β-catenin信号通路对健脾益智法对MCAO大鼠神经干细胞（NSCs）增殖的调控作用及其机制。

方法：将60只雄性SD大鼠随机分为正常组、模型组、健脾益智法组和哥林立德盐酸盐组。采用中颈动脉永久性闭塞（MCAO）方法制备脑局部缺血再灌流（IR）模型。健脾益智法及哥林立德盐酸盐分别通过胃管灌饮给予，每天一次，持续4周。通过CCK-8试验、流式细胞术和Westernblotting法检测NSCs增殖及Wnt/β-catenin信号通路相关蛋白表达情况。

结果：与模型组相比，健脾益智法组NSCs增殖显著增加，且明显高于哥林立德盐酸盐组（P<0.05）。Westernblotting实验发现健脾益智法组β-catenin、TCF4、CyclinD1和c-myc蛋白表达均显著高于模型组和哥林立德盐酸盐组（P<0.05），TLE1、GSK3β和Axin蛋白表达均显著低于模型组和哥林立德盐酸盐组（P<0.05）。流式细胞术结果进一步证实了健脾益智法组NSCs增殖显著高于模型组和哥林立德盐酸盐组（P<0.05）。

结论：健脾益智法能够通过激活Wnt/β-catenin信号通路，促进MCAO大鼠NSCs增殖，可能是其为治疗脑缺血和神经损伤提供保障的机制之一。

关键词：Wnt/β-catenin信号通路；健脾益智法；MCAO大鼠；神经干细胞；增殖；缺血再灌流模型

Abstract:

Objective:ThestudyaimstoinvestigatetheeffectoftheWnt/β-cateninsignalingpathwayontheregulationofNSCsproliferationinMCAOratsbytheuseofthetraditionalChinesemedicineformula-JianpiYizhiFang,anditsunderlyingmechanisms.

Methods:SixtymaleSDratswererandomlydividedintonormalgroup,modelgroup,JianpiYizhiFanggroupandGalantaminehydrobromidegroup.Themiddlecerebralarteryocclusion(MCAO)wasusedtoestablishthefocalcerebralischemiareperfusion(IR)model.JianpiYizhiFangandGalantaminehydrobromideweregivenbygavageonceadayfor4weeks.CCK-8assay,flowcytometry,andWesternblottingwereusedtodetectNSCsproliferationandtheexpressionofWnt/β-cateninsignalingpathway-relatedproteins.

Results:NSCsproliferationintheJianpiYizhiFanggroupwassignificantlyhigherthanthatinthemodelgroup,andsignificantlyhigherthanthatintheGalantaminehydrobromidegroup(P<0.05).Westernblottingshowedthattheexpressionofβ-catenin,TCF4,CyclinD1andc-mycproteinsintheJianpiYizhiFanggroupwassignificantlyhigherthanthatinthemodelgroupandtheGalantaminehydrobromidegroup(P<0.05),whiletheexpressionofTLE1,GSK3βandAxinproteinswassignificantlylowerthanthatinthemodelgroupandtheGalantaminehydrobromidegroup(P<0.05).FlowcytometryresultsfurtherconfirmedthattheNSCsproliferationintheJianpiYizhiFanggroupwassignificantlyhigherthanthatinthemodelgroupandtheGalantaminehydrobromidegroup(P<0.05).

Conclusion:JianpiYizhiFangcanpromoteNSCsproliferationinMCAOratsbyactivatingtheWnt/β-cateninsignalingpathway,whichmaybeoneofthemechanismsforitsprotectiveeffectsinthetreatmentofcerebralischemiaandnerveinjury.

Keywords:Wnt/β-cateninsignalingpathway;JianpiYizhiFang;MCAOrats;NSCs;proliferation;IRmodeCerebralischemiaandnerveinjuryareconsideredtobesomeofthemostcommonneurologicaldisorders,whichcanleadtoseverecognitiveimpairmentanddisability.Therefore,itisessentialtoexploreeffectivetherapeuticoptionstocounterthenegativeeffectsofcerebralischemiaandnerveinjury.JianpiYizhiFangisatraditionalChinesemedicinalformulathathasbeenwidelyusedtotreatneurologicaldisorders,includingcerebralischemiaandnerveinjury.

Inthecurrentstudy,weaimedtoinvestigatethepotentialmechanismofJianpiYizhiFanginpromotingtheproliferationofNSCsinMCAOrats.OurfindingssuggestthatJianpiYizhiFangcanactivatetheWnt/β-cateninsignalingpathway,whichleadstothesignificantproliferationofNSCsinMCAOrats.Furthermore,thelevelofproliferationobservedintheJianpiYizhiFanggroupwassignificantlyhigherthanthatobservedinthemodelgroupandtheGalantaminehydrobromidegroup.

ActivationoftheWnt/β-cateninsignalingpathwayisknowntoplayacrucialroleinpromotingNSCsproliferationanddifferentiation.OurresultsdemonstratethatJianpiYizhiFangsignificantlyactivatesthispathway,whichmaybeoneofthepotentialmechanismsbywhichitexertsitsprotectiveeffectsagainstcerebralischemiaandnerveinjury.

Inconclusion,ourstudyprovidesnewinsightsintothepotentialtherapeuticmechanismofJianpiYizhiFanginpromotingNSCsproliferationinMCAOrats.OurfindingshighlighttheimportanceoftheWnt/β-cateninsignalingpathwayasapotentialtargetforthedevelopmentofnoveltherapeuticinterventionsforcerebralischemiaandnerveinjury.FurtherstudiesarewarrantedtoinvestigatethepotentialclinicalbenefitsofJianpiYizhiFanginthetreatmentofneurologicaldisordersMoreover,ourstudyalsoraisessomequestionsthatwarrantfurtherinvestigation.Forinstance,weonlyexaminedtheproteinlevelsofcertainmarkersinvolvedintheWnt/β-cateninsignalingpathway,anditremainsunclearwhetherthemRNAlevelsofthesemarkersarealteredintheMCAOratstreatedwithJianpiYizhiFang.Additionally,furtherstudiesshouldexaminetheeffectsofthisherbalformulaonothersignalingpathwaysinvolvedinNSCsproliferation,suchastheNotchsignalingpathway.Furthermore,thepotentialsideeffectsandtoxicityofJianpiYizhiFangshouldbethoroughlyexaminedinanimalmodelsandclinicaltrials.

Insummary,ourstudysuggeststhatJianpiYizhiFangpromotesNSCsproliferationthroughtheactivationoftheWnt/β-cateninsignalingpathwayinMCAOrats.Thesefindingsprovideapotentialtherapeutictargetforthetreatmentofcerebralischemiaandnerveinjury,andhighlightthepotentialofthisherbalformulaasafuturetherapeuticinterventionforneurologicaldisorders.FurtherresearchisnecessarytofullyelucidatetheunderlyingmechanismsandclinicalapplicationsofJianpiYizhiFangInadditiontopromotingNSCsproliferationthroughtheactivationoftheWnt/β-cateninsignalingpathway,JianpiYizhiFanghasbeenshowntohaveotherpotentialneuroprotectiveeffects.Forexample,onestudyfoundthattheherbalformulaimprovedcognitivefunctionandreducedcerebralinfarctsizeinMCAOratsbyregulatingtheexpressionofbrain-derivedneurotrophicfactor(BDNF)andapoptoticsignalingpathways(Zhaoetal.,2018).AnotherstudysuggestedthatJianpiYizhiFanghadaneuroprotectiveeffectonneuronsinvitrobyreducingoxidativestressandimprovingmitochondrialfunction(Liuetal.,2019).

WhilethesestudiesprovidepromisingevidenceforthepotentialofJianpiYizhiFangasatherapeuticinterventionforneurologicaldisorders,thereareseverallimitationstoconsider.Firstly,manyofthesestudieshavebeenconductedinanimalmodels,anditisunclearhowwelltheresultstranslatetohumans.Secondly,manystudieshavefocusedontheeffectsofindividualcomponentsoftheherbalformula,ratherthantheformulaasawhole,anditisunclearhowthevariouscomponentsinteractwitheachotherinvivo.Finally,thereisalackofstandardizedprotocolsforproducingandadministeringJianpiYizhiFang,whichmakesitdifficulttocomparetheeffectsofdifferentpreparations.

Despitetheselimitations,thepotentialbenefitsofJianpiYizhiFangaresignificant,especiallygiventhelackofeffectivetreatmentsformanyneurologicaldisorders.Furtherresearchisneededtodeterminetheoptimalprotocolsforproducingandadministeringtheherbalformula,aswell