基于Ⅲ型PI3K-Beclin1通路研究加味茵芍散含藥血清對非酒精性脂肪肝的體外調(diào)控作用

基于Ⅲ型PI3K-Beclin1通路研究加味茵芍散含藥血清對非酒精性脂肪肝的體外調(diào)控作用摘要：本研究旨在探究基于Ⅲ型PI3K/Beclin1通路的加味茵芍散含藥血清對非酒精性脂肪肝的體外調(diào)控作用。首先，我們將建立一套體外模擬脂肪肝環(huán)境的實(shí)驗(yàn)系統(tǒng)，并收集非酒精性脂肪肝患者和正常人群的原代肝細(xì)胞以及HepG2細(xì)胞，分別接種在含不同濃度加味茵芍散的培養(yǎng)基中，以探究不同劑量加味茵芍散對脂肪肝細(xì)胞凋亡、氧化應(yīng)激、代謝紊亂等方面的影響。同時，我們將利用Westernblotting和實(shí)時熒光定量PCR等技術(shù)，檢測Ⅲ型PI3K/Beclin1通路相關(guān)蛋白的表達(dá)水平，闡明該通路在加味茵芍散對脂肪肝的影響中的作用。最后，我們還將利用LC-MS/MS技術(shù)鑒定加味茵芍散中可能對脂肪肝治療具有潛在效應(yīng)的化合物，并與藥效學(xué)指標(biāo)進(jìn)行相關(guān)性分析，為臨床治療提供科學(xué)依據(jù)。

關(guān)鍵詞：基于Ⅲ型PI3K/Beclin1通路；加味茵芍散；非酒精性脂肪肝；體外實(shí)驗(yàn)；代謝紊亂；化合物篩選

Abstract:ThisstudyaimstoexploretheinvitroregulatoryeffectofmodifiedYinzhiSancontainingserumonnon-alcoholicfattyliverdisease(NAFLD)basedontheIII-typePI3K/Beclin1pathway.Firstly,wewillestablishaninvitroexperimentalsystemsimulatingthefattyliverenvironment,andcollectprimarylivercellsfromNAFLDpatientsandnormalpopulationsaswellasHepG2cells.CellswillbeculturedinmediumcontainingdifferentconcentrationsofmodifiedYinzhiSan,andtheeffectsofdifferentdosesofmodifiedYinzhiSanonapoptosis,oxidativestress,andmetabolicdisordersofNAFLDcellswillbeexplored.Further,wewilluseWesternblottingandqRT-PCRtoexaminetheexpressionlevelsofproteinsrelatedtotheIII-typePI3K/Beclin1pathwayandclarifytheroleofthispathwayintheeffectsofmodifiedYinzhiSanonNAFLD.Finally,wewilluseLC-MS/MStoidentifycompoundsthatmayhavepotentialeffectsonNAFLDtreatmentinmodifiedYinzhiSanandperformcorrelationanalyseswithpharmacologicalindicestoprovidescientificbasisforclinicaltreatment.

Keywords:III-typePI3K/Beclin1pathway;modifiedYinzhiSan;non-alcoholicfattyliverdisease(NAFLD);invitroexperiments;metabolicdisorders;compoundscreeningNon-alcoholicfattyliverdisease(NAFLD)isametabolicdisordercharacterizedbytheaccumulationoffatintheliver,whichcanleadtoliverinflammationandeventuallyliverdamage.TheIII-typePI3K/Beclin1pathwayhasbeenidentifiedasakeyplayerinthedevelopmentofNAFLD.Specifically,activationofthispathwaypromotesautophagy-mediatedlipiddegradation,therebypreventinglipidaccumulationinhepatocytes.

PreviousstudieshaveshownthatmodifiedYinzhiSan,atraditionalChinesemedicineformula,caneffectivelyalleviateNAFLD.However,themechanismunderlyingitstherapeuticeffectsisnotfullyunderstood.Inthisstudy,weaimtoinvestigatewhethertheIII-typePI3K/Beclin1pathwayisinvolvedintheeffectsofmodifiedYinzhiSanonNAFLD.

Tothisend,wewilluseinvitroexperimentstoexaminetheeffectsofmodifiedYinzhiSanontheactivationoftheIII-typePI3K/Beclin1pathwayinhepatocytes.Specifically,wewillmeasuretheexpressionlevelsofkeyproteinsinthispathway,includingPI3K,Beclin1,andLC3-II,inhepatocytestreatedwithorwithoutmodifiedYinzhiSan.WewillalsoperformlipidstainingtoassesstheeffectsofmodifiedYinzhiSanonlipidaccumulationinthesecells.

Inaddition,wewillusecompoundscreeningwithLC-MS/MStoidentifypotentialbioactivecompoundsinmodifiedYinzhiSanthatmayhavetherapeuticeffectsonNAFLD.WewillthenperformcorrelationanalysestodeterminetherelationshipbetweenthesecompoundsandpharmacologicalindicesrelatedtoNAFLDtreatment.

Overall,thisstudyaimstoclarifytheroleoftheIII-typePI3K/Beclin1pathwayinthetherapeuticeffectsofmodifiedYinzhiSanonNAFLD.ThefindingsofthisstudymayprovideascientificbasisfortheclinicaltreatmentofNAFLDusingtraditionalChinesemedicineInaddition,thisstudywillalsoinvestigatethepotentialmechanismsunderlyingtheregulationoftheIII-typePI3K/Beclin1pathwaybymodifiedYinzhiSan.Onepossiblemechanismmightinvolvethemodulationofautophagyflux,whichisacriticalprocessinmaintaininghepaticlipidmetabolismandpreventinglipidaccumulationinhepatocytes.Autophagyisalysosomaldegradationpathwaythateliminatesdamagedorganellesandmisfoldedproteinstomaintaincellularhomeostasis.IthasbeenshownthatimpairedautophagyfluxcontributestothepathogenesisofNAFLD,astheaccumulationofdamagedmitochondriaandlipiddropletsinhepatocytesleadstooxidativestressandmetabolicdysfunction.Therefore,promotingautophagyfluxmayrepresentapromisingapproachforNAFLDtreatment.

ModifiedYinzhiSanhasbeenreportedtoenhanceautophagyfluxandreducelipidaccumulationintheliverofNAFLDratsbyactivatingtheAMPK/mTORpathway.Thispathwayisakeyregulatorofcellularenergymetabolismandautophagyflux,anditsdysregulationhasbeenimplicatedinthedevelopmentofNAFLD.ByactivatingAMPK,modifiedYinzhiSancanstimulateautophagyandinhibitlipogenesis,whichreducestheproductionoffattyacidsandtriglyceridesinhepatocytes.Inaddition,modifiedYinzhiSancanalsoincreasetheexpressionofPGC-1α,atranscriptionalcoactivatorthatpromotesmitochondrialbiogenesisandfattyacidoxidation,furthersupportingthetherapeuticeffectsofmodifiedYinzhiSanonNAFLD.

AnotherpotentialmechanismunderlyingthetherapeuticeffectsofmodifiedYinzhiSanonNAFLDistheregulationofgutmicrobiota.Thegutmicrobiotaplaysacrucialroleinregulatingenergymetabolismandregulatingtheimmunesystem,anditsdysbiosishasbeenassociatedwiththedevelopmentofmetaflammationandmetabolicdisorders,includingNAFLD.ModifiedYinzhiSanhasbeenshowntoregulatethecompositionofgutmicrobiotainNAFLDrats,byincreasingtherelativeabundanceofbeneficialbacteria(suchasBifidobacteriumandLactobacillus)anddecreasingtheabundanceofharmfulbacteria(suchasEscherichiacoli).Thismodulationofgutmicrobiotamaycontributetothereductionofinflammationandoxidativestressintheliver,whicharekeypathologicalprocessesinNAFLD.

Inconclusion,thisstudyaimstoelucidatethetherapeuticmechanismsandactivecompoundsofmodifiedYinzhiSaninthetreatmentofNAFLD,withafocusontheIII-typePI3K/Beclin1pathwayanditsupstreamanddownstreamsignalingpathways.ThefindingsofthisstudymayprovideinsightintothedevelopmentofeffectiveandsafetherapiesforNAFLD,andmaycontributetotheintegrationoftraditionalChinesemedicineandmodernpharmacologyNon-alcoholicfattyliverdisease(NAFLD)hasbecomeaglobalhealthproblem,asitisassociatedwithobesity,type2diabetes,andcardiovasculardiseases.NAFLDischaracterizedbytheaccumulationoffatintheliver,whichcanleadtoinflammation,fibrosis,andinseverecases,cirrhosisandhepatocellularcarcinoma.TherearecurrentlynoapprovedtherapiesforNAFLD,andthedevelopmentofeffectivetreatmentsisurgentlyneeded.

TheIII-typePI3K/Beclin1pathwayhasbeenidentifiedasakeyregulatorofautophagy,whichplaysacrucialroleinthemaintenanceofhepaticlipidhomeostasis.Autophagyisacellularprocessthatinvolvesthedegradationofdamagedorganellesandproteins,aswellastherecyclingofintracellularcomponents.DysfunctionalautophagyhasbeenimplicatedinthepathogenesisofNAFLD,asitcanleadtotheaccumulationoflipidsandoxidativestressinhepatocytes.

ModifiedYinzhiSanisatraditionalChinesemedicineformulathathasbeenusedtotreatliverdiseasesforcenturies.RecentstudieshaveshownthatmodifiedYinzhiSancanimprovehepaticsteatosisandinflammationinanimalmodelsofNAFLD.TheactivecompoundsofmodifiedYinzhiSanincludeflavonoids,terpenoids,andpolysaccharides,whichhavebeenshowntopossessantioxidant,anti-inflammatory,andimmunomodulatoryactivities.

InadditiontotheIII-typePI3K/Beclin1pathway,modifiedYinzhiSanmayalsotargetothersignalingpathwaysinvolvedinthepathogenesisofNAFLD,suchastheAMP-activatedproteinkinase(AMPK)pathway,