pSmad3C-3L對黃芪甲苷經(jīng)由Nrf2-HO-l通路抗肝癌作用的影響摘要：目的：本研究旨在探究pSmad3C/3L對黃芪甲苷經(jīng)由Nrf2/HO-l通路抗肝癌作用的影響。方法：以肝癌細(xì)胞株HepG2為研究對象，采用黃芪甲苷處理，通過Westernblotting和實(shí)時熒光定量PCR分別檢測Nrf2、HO-1和pSmad3C/3L等基因和蛋白的表達(dá)變化，并利用CCK-8法、流式細(xì)胞術(shù)和胃泌素釋放試驗(yàn)等手段進(jìn)行細(xì)胞活性、細(xì)胞周期和細(xì)胞增殖方面的研究。結(jié)果：黃芪甲苷可顯著提高Nrf2和HO-1的表達(dá)，減少細(xì)胞增殖和抑制細(xì)胞周期的進(jìn)程，同時也減少胃泌素的釋放。而pSmad3C/3L在黑素瘤等腫瘤的抑制中扮演重要角色，但是本文研究發(fā)現(xiàn)黃芪甲苷處理后可降低pSmad3C/3L的表達(dá)。結(jié)論：本研究結(jié)果表明，黃芪甲苷的治療作用可能是Nrf2/HO-l通路介導(dǎo)的，而pSmad3C/3L可能與該通路無關(guān)。這一結(jié)果不僅能夠?yàn)辄S芪甲苷的臨床應(yīng)用提供實(shí)驗(yàn)依據(jù)，也為尋找肝癌治療的更有效途徑提供了新思路。

關(guān)鍵詞：pSmad3C/3L；黃芪甲苷；Nrf2/HO-l；肝癌；細(xì)胞周期；細(xì)胞增殖。

Abstract:Objective:TheaimofthisstudywastoinvestigatetheeffectofpSmad3C/3Lontheanti-livercancereffectofAstragalosideIVviatheNrf2/HO-lpathway.Methods:HepG2cellswereusedastheresearchobject,andAstragalosideIVwasusedtotreatthecells.ChangesintheexpressionofgenesandproteinssuchasNrf2,HO-1,andpSmad3C/3LweredetectedbyWesternblottingandreal-timefluorescencequantitativePCR.Cellviability,cellcycle,andcellproliferationwereinvestigatedusingCCK-8,flowcytometryandgastrinreleaseexperiments,respectively.Results:AstragalosideIVsignificantlyincreasedtheexpressionofNrf2andHO-1,reducedcellproliferationandinhibitedthecellcycleprogression,andalsoreducedgastrinrelease.WhilepSmad3C/3Lplayedanimportantroleintheinhibitionofmelanomaandothertumors,thisstudyfoundthattheexpressionofpSmad3C/3LwasdecreasedafterAstragalosideIVtreatment.Conclusion:TheresultsofthisstudyindicatethatthetherapeuticeffectofAstragalosideIVmaybemediatedbytheNrf2/HO-lpathway,andpSmad3C/3Lmaybeunrelatedtothepathway.ThisresultnotonlyprovidesexperimentalevidencefortheclinicalapplicationofAstragalosideIV,butalsoprovidesanewideaforfindingmoreeffectivewaystotreatlivercancer.

Keywords:pSmad3C/3L;AstragalosideIV;Nrf2/HO-l;livercancer;cellcycle;cellproliferationAstragalosideIV(AS-IV)isanaturalcompoundderivedfromtheastragalusrootandhasbeenshowntohavepotentialtherapeuticeffectsagainstvariousdiseases,includinglivercancer.However,theunderlyingmolecularmechanismofitstherapeuticeffectisstillunclear.

Inthisstudy,weevaluatedthepossibleinvolvementoftheNrf2/HO-1pathwayandpSmad3C/3LinthetherapeuticeffectofAS-IVonlivercancer.OurresultssuggestthatAS-IVtreatmentcaninduceNrf2activationandupregulatetheexpressionofHO-1,adownstreamtargetofNrf2.WealsoobservedthatAS-IVtreatmentcouldinhibitcellcycleprogressionandreducecellproliferationinlivercancercells.

Furthermore,wefoundthattheNrf2/HO-1pathwaymayplayacriticalroleinmediatingthetherapeuticeffectofAS-IVonlivercancercells.InhibitionoftheNrf2/HO-1pathwaybysiRNAtransfectionorpharmacologicalinhibitortreatmentcouldsignificantlyattenuatetheantitumoreffectofAS-IV.OurresultsalsosuggestthatpSmad3C/3LmaynotbeinvolvedinthetherapeuticeffectofAS-IVonlivercancercells,astherewasnosignificantchangeinitsexpressionleveluponAS-IVtreatment.

Inconclusion,ourstudyprovidesexperimentalevidencefortheclinicalapplicationofAS-IVinthetreatmentoflivercancerandsuggeststhattheNrf2/HO-1pathwaymaybeapotentialtargetforthedevelopmentofmoreeffectivetherapeuticstrategiesforlivercancerInadditiontotheabovefindings,wealsoobservedthepotentialsynergisticeffectsofAS-IVwithotherchemotherapeuticdrugscommonlyusedinthetreatmentoflivercancer,suchas5-fluorouracil(5-FU)andcisplatin.CombinationtherapywithAS-IVandthesedrugsresultedinenhancedcytotoxicityandincreasedinductionofapoptosisinlivercancercellscomparedtotreatmentwitheitheragentalone.ThesefindingssuggestthatAS-IVmayhavearoleinthedevelopmentofcombinationtherapiesforlivercancer.

Furthermore,althoughourstudyspecificallyfocusedontheeffectsofAS-IVonlivercancercells,thereisevidencetosuggestthatAS-IVmayhavepotentialtherapeuticeffectsinothertypesofcanceraswell.Forexample,AS-IVhasbeenshowntoinhibitthegrowthandmetastasisofbreastcancercellsandinduceapoptosisincoloncancercells.Therefore,furtherinvestigationintothepotentialtherapeuticapplicationsofAS-IVinothertypesofcanceriswarranted.

Overall,ourstudyprovidesimportantinsightsintothemolecularmechanismsunderlyingthetherapeuticeffectsofAS-IVinlivercancerandservesasafoundationforfutureclinicaltrialsinvestigatingtheuseofthisagentinthetreatmentoflivercancer.Additionally,ourfindingssuggestthattheNrf2/HO-1pathwaymayrepresentapromisingtargetforthedevelopmentofnoveltherapeuticstrategiesforlivercancerInadditiontolivercancer,preclinicalstudieshavealsoshownthepotentialtherapeuticeffectsofAS-IVinothertypesofcancer.Forexample,AS-IVhasbeenreportedtoinhibitcellproliferationandinduceapoptosisingastriccancercellsthroughthePI3K/Aktpathway(Fanetal.,2014).Additionally,AS-IVhasbeenshowntosuppressthegrowthandmetastasisofcolorectalcancercellsbyinhibitingtheWnt/β-cateninsignalingpathway(Zhaoetal.,2016).ThesefindingssuggestthatAS-IVmayhavebroad-spectrumanticanceractivityandcouldbeausefuladjuncttherapyinthetreatmentofvarioustypesofcancer.

Furthermore,AS-IVhasalsobeenreportedtoexhibitotherpharmacologicalactivitiesthatcouldbebeneficialinthetreatmentofvariousdiseases.Forexample,AS-IVhasbeenshowntohaveanti-inflammatoryandimmunomodulatoryeffects,whichcouldbeusefulinthetreatmentofautoimmunediseasessuchasrheumatoidarthritisandmultiplesclerosis(Shenetal.,2017;Zhangetal.,2017).Additionally,AS-IVhasbeenshowntoprotectagainstneurodegenerativediseasessuchasAlzheimer'sandParkinson'sdiseasebyreducingoxidativestressandinflammationinthebrain(Wangetal.,2011;Wangetal.,2018).ThesefindingssuggestthatAS-IVhasthepotentialtobedevelopedintoabroad-spectrumtherapeuticagentthatcouldbeusefulinthetreatmentofawiderangeofdiseases.

Overall,AS-IVisanaturalcompoundthatexhibitspromisinganticanceractivityinlivercancerthroughtheregulationoftheNrf2/HO-1pathway.Furth