基于Wnt-β-catenin信號(hào)通路探討健脾益智法調(diào)控MCAO大鼠NSCs增殖的機(jī)制研究摘要：

目的：本研究旨在探討Wnt/β-catenin信號(hào)通路對(duì)健脾益智法對(duì)MCAO大鼠神經(jīng)干細(xì)胞（NSCs）增殖的調(diào)控作用及其機(jī)制。

方法：將60只雄性SD大鼠隨機(jī)分為正常組、模型組、健脾益智法組和哥林立德鹽酸鹽組。采用中頸動(dòng)脈永久性閉塞（MCAO）方法制備腦局部缺血再灌流（IR）模型。健脾益智法及哥林立德鹽酸鹽分別通過(guò)胃管灌飲給予，每天一次，持續(xù)4周。通過(guò)CCK-8試驗(yàn)、流式細(xì)胞術(shù)和Westernblotting法檢測(cè)NSCs增殖及Wnt/β-catenin信號(hào)通路相關(guān)蛋白表達(dá)情況。

結(jié)果：與模型組相比，健脾益智法組NSCs增殖顯著增加，且明顯高于哥林立德鹽酸鹽組（P<0.05）。Westernblotting實(shí)驗(yàn)發(fā)現(xiàn)健脾益智法組β-catenin、TCF4、CyclinD1和c-myc蛋白表達(dá)均顯著高于模型組和哥林立德鹽酸鹽組（P<0.05），TLE1、GSK3β和Axin蛋白表達(dá)均顯著低于模型組和哥林立德鹽酸鹽組（P<0.05）。流式細(xì)胞術(shù)結(jié)果進(jìn)一步證實(shí)了健脾益智法組NSCs增殖顯著高于模型組和哥林立德鹽酸鹽組（P<0.05）。

結(jié)論：健脾益智法能夠通過(guò)激活Wnt/β-catenin信號(hào)通路，促進(jìn)MCAO大鼠NSCs增殖，可能是其為治療腦缺血和神經(jīng)損傷提供保障的機(jī)制之一。

關(guān)鍵詞：Wnt/β-catenin信號(hào)通路；健脾益智法；MCAO大鼠；神經(jīng)干細(xì)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