The Study Of Combination Therapy Against Sd Rat Hepatic Fibrosis Through MST4、Glud1 And Hdgf Relatede Rgulation Signal Pathways

ObjectiveTo investigate thefunction of Hdgf,MST4 and Glud 1 in combination therapy against hepatic fibrosis in rats,and furtherly analyizesthe moleculer mechanism whether it is through relevant signal pathways or not,which provides a preliminary pharmacological basis for preclinical study of combination therapy,and provides aids for the treatment of liver fibrosis.Methods1.The function of MST4 、 Glud1 and Hdgf in the treatment of liver fibrosis by combination therapy.(1)The subjects of HSC-T6 cells were divided into transfection silencing group,negative control group and blank control group.Using viral vectors to interfere with the MST4、Glud1and Hdgf genes in HSC-T6 cells,and the cell with optimal jamming efficiency were selected for the next experiment by real-time qPCR and western blot validation.(2)The growth of HSC-T6 cells were observed by CCK-8 method after silent MST4、Glud1 and Hdgf genes,analysing the effects of MST4、Glud1 and Hdgfon the proliferation of HSC-T6 cells.(3)The inhibition rate of all silent cells was detected by CCK-8 after combination therapy intervention,analyzing the effect of combination therapy on HSC-T6 proliferation after silent MST4、Glud1and Hdgf genes.(4)Flow cytometry detected the cell cycle and apoptosis of HSC-T6 after silent Hdgf gene.2.Mechanism of MST4 、 Glud1 and Hdgf in the regulation of correlated signaling pathway against hepatic fibrosis.⑴ MST4 gene:Western blot analysis the expression of ERK,p-ERK and TGF-β1 in cells aftersilent MST4 gene.⑵Glud1 gene: Western blot analysis the expression of STAT3,p-STAT3 and TGF-β1 in cells aftersilent Glud1 gene.⑶Hdgf gene:Western blot analysis the expression of ERK,Akt,MST4,p-ERK,p-Akt,p-MST4,and TGF-β1 in cells aftersilent Hdgf gene.Results1.Effects of MST4、Glud1 and Hdgf on biological behavior of HSC-T6.(1)Silent efficiency of MST4 、 Glud1 and Hdgf gene: Lv-stk26-2 、Lv-Glud1-1 and Lv-Hdgf-3 cell were selected for the further experiments through western blot and real-time qPCR validation.(2)The effects of MST4、Glud1 and Hdgf gene on HSC-T6 proliferation: The proliferation were slowed down after silent MST4 、 Glud1 、 Hdgf gene.(3)The effect of combination therapy on HSC-T6 proliferation aftert silencing MST4、Glud1 and Hdgf gene:The effect was decreased after silent Glud1 gene,while MST4 and Hdgf gene was no significant change.(4)Effects of combination therapy on the cycle and apoptosis of HSC-T6 after silent Hdgf gene.(1)Compared with the control group,the percentage of S period in the group of combination therapy increased,the percentage of G1 and G2 period decreased,and the percentage of S period in silent group decreased,the percentage of G1 period increased,the percentage ofG2 period was not significantly changed;silence + combination therapy group,the percentage of S phase increased.the percentage of G1 period decreased,the percentage of G2 period was not significantly changed.(2)Compared with the control group,the apoptosis of the combination therapy group increased significantly,but the apoptosis of silent group was not significantly changed.In addition,compared with the combination therapy group,the apoptosis of the silence + combination therapy group decreased significantly.2.The mechanism of MST4 、 Glud1 and Hdgf in the regulation of related signaling pathway against hepatic fibrosis.⑴Silent MST4 gene :(1)Compared with control group,the expression of p-ERK protein decreased insilent group,and the expression of p-ERK protein in silent +combination therapy group also decreased,and what’s more,combination therapycould furtherly reduce the expression of p-ERK protein with slient MST4 gene,and the expression of ERK protein decreased correspondingly.(2)The expression of TGF-β1 decreasedafter silent MST4 gene,in addition,combination therapycould furtherly reduce the expression of TGF-β1 protein with slient MST4 gene.⑵Silent Glud1 gene :(1)Compared with control group,the expression of p-STAT3 protein in silent group and silent+combination therapy group decreased(p<0.05),at the same time,the expression of STAT3 protein in silent group reduced(p<0.05).(2)The expression of TGF-β1 protein increasedafter silent Glud1 gene,and the combination therapy could decrease TGF-β1 expression.⑶Silent Hdgf gene :(1)Compared with the control group(HSC-T6 group),the expression of p-ERK protein in the silent group group decreased,the difference was not statistically significant,the p-ERK protein in the combined drug group decreased and the difference was statistically significant.besides,compared with the combineddrug group,the expression of p-ERK protein in the silent + combination therapy group was lower,while the expression of total ERK protein remained unchanged.However,compared with the control group,the expression of p-MST4 protein in the silent group decreased,and the expression of MST4 protein increased.(2)Compared with the control group,the combination therapy group can decrease the p-Akt and Akt protein expression,but the silent group p-Akt was no significant changes.(3)The expression of TGF-β1 protein increasedafter silent Hdgf,and the combined drugs could reduce TGF-β1 expression in silent group.Conclusion1.After silent MST4 、 Glud1 and Hdgf gene,the proliferation of HSC-T6 was slowed down,and the silent of Glud1 gene could enhance the inhibitory effect of combination therapy on the proliferation of HSC-T6,suggesting that the Glud1 protein may play an important role in resisting liver fibrosis by inhibiting cell proliferation,which is the key protein,while the function of Hdgf and MST4 needs further study.2.The important part of the molecular mechanism of anti hepatic fibrosis by combined drugs may be realized by the common regulation of ERK and STAT3 signaling pathways in rat hepatic stellate cells through MST4 and Glud1 gene.