Effect Of Wnt Classic Pathway In Hepatic Stellate Cells And Intervention Mechanismof Ganfukang

Objective:Liver fibrosis is a repair process of the body to liverinjury,and the characteristic change in the liver cells is excessive depositionof extracellular matrix (ECM).The occurrence of liver fibrosis based onhepatic stellate cells(HSC) activation,the activated HSCs transform intomyofibroblast phenotype,proliferate,express ECM,smooth muscle actin(α-smooth muscle actin, α-SMA) and various cytokines and inflammatoryfactors.Many studies found Wnt signal pathway,especially the classicone,also called Wnt/β-Catenin signaling pathway contact with hepaticfibrosis.β-Catenin is a multifunctional cytoplasmic proteinthe,is the key ofthe classic Wnt signaling pathway,it can mediate Wnt signaling transduction.Glycogen synthase kinase (GSK-3β) participate in the beta-cateninphosphorylation and its subsequent degradation,maintaining the quiescentstate of the canonical Wnt pathway.SB216763is an inhibitor of GSK-3β,which can promote β-Catenin in the cytoplasm into the nucleus,therebySB216763can activate the Wnt/β-Catenin signaling pathway.Theexperiment applied inhibitors of GSK-3β to inhibit its activity in culturedrat HSC cells,explore the mechanism of the canonical Wnt signalingpathway in rat HSC by measuring mRNA expression of β-catenin levels ofWnt target genes.In recent years,traditional Chinese medicine treatment of liver fibrosisgets great progress,Ganfukang is summarized from the traditional Chinesemedicine after years of laboratory experiments.The experiment culturedhepatic stellate cells and applied SB216763and Ganfukang to explore theantifibrotic mechanism of Ganfukang through the Wnt/β-Catenin signaling pathway.Methods:1.Cell culture:Rat HSC-T6cells in DMEM culture mediumwith10%fetal bovineserum,at37℃,incubator5%CO2gas mixture.When the cells acheieve a goodcondition,all the cells in serum-free DMEM starved for24hours afterpacket dosing.Cells were randomly divided into five groups,concentrationof SB216763was5,10,20,30,40uM,and after cell proliferation of12hours,detected the cell proliferation by Microtitre tetrazolium assay,analyze the data and selected the best concentration of SB216763is10uM,20uM.The cells were randomly divided into the following five groups:controlgroup（10%normal control group in DMEM); the SB21676310uM group;SB21676320uM group;Ganfukang group(10%Ganfukang drug serum inDMEM);SB21676320uM plus Ganfukang group(SB216763plus10%Ganfukang drug serum).2.MTT:Detection of the proliferation of rat hepatic stellate cells.3.Quant Reverse Transcriptase(RT)-polymerase chain reaction (PCR):Detection of mRNA expression of GSK-3β,β-Catenin, CollagenI/Ⅲ,α-SMA, Wnt1, Wnt3a,Wnt10b in the rat hepatic stellate cells.4.Western blot:Detection of protein expression of β-Catenin in rat hepatic stellatecells.5.Results of the experiment used SPSS13.0statistical analysissoftware.Results:1.MTT assay results:The proliferation of hepatic stellate cells in group SB21676310uM andgroup SB21676320uM are significant compared with the controlgroup.Compared with the control group,the proliferation of hepatic stellatecells in ganfukang drug serum group decreased,,the difference wasstatistically significant (P<0.05).At the same time, cell proliferation of ganfukang drug serum plus SB21676320uM group was decreasedsignificantly compared to the SB216763group, the difference wasstatistically significant (P<0.05).2.RT-RCR results:Hepatic stellate cells of SB21676310uM and SB21676320uM groupwere significantly activated.MRNA expression levels of CollagenI,a-SMA,β-Catenin,Wnt1, Wnt3a, Frizzled1, Frizzled2increasedsignificantly,while the expression in hepatic stellate cells of Ganfukangdrug serum group decreased obviously,compared with the control group, thedifference had statistical significance (P<0.05).Compared with SB21676320uM group, the SB21676320uM plus Ganfukang drugs group can bettersuppress these indicators expression.Compared with the control group,themRNA expression of GSK-3β in SB21676320uM group decreasedsignificantly, the difference had statistical significance(P<0.05).3.Western blot assay results:The control group had a low expression of β-Catenin in rat hepaticstellate cells.Compared with the control group cells,β-Catenin expression ofSB21676310uM group and20uM group was significantly increased.Whilein ganfukang drug serum group,the expression was decreased significantlycompared to the control group (P<0.05);The expression of β-Catenin inSB21676320uM plus Ganfukang drug serum group was decreased,comparedwith SB21676320uM group,the difference was significant (P<0.05).Conclusion:1. Inhibition of GSK-3β activity, can activate the classic Wnt signalingpathway of HSC-T6s in the rat,raised the HSC-T6s activity.2.Chinese medicine Ganfukang provide a theoretical basis for thetreatment of liver fibrosis by blocking Wnt classic signal pathway,inhibiting the cell activity of rat hepatic stellates.