| Objective:Shugan jianpi huoxue decoction is the clinical treatment of liver fibrosis,but the mechanism of its regulation pathway lack of research.In this study,TGF-β1 was used to induce HSC-T6 as a model of activated hepatic stellate cells.The mechanism of anti-fibrosis effect from the level of molecular biology of Shugan jianpi huoxue decoction,clear anti-hepatic fibrosis Shugan jianpi huoxue decoction on the target or link,and provide the basis for the development of traditional Chinese medicine in the treatment of liver fibrosis.Methods:1.With different concentrations of Shugan jianpi huoxue decoction given orally to rats to prepare the serum containing drug serum,cultured in vitro HSC-T6 cells by CCK-8 assay.Shugan jianpi huoxue decoction with different concentrations of serum on the proliferation of HSC-T6,to determine the most effective blood drug concentration.2.The rat Smad4 gene to construct shRNA recombinant lentivirus transfection of cultured rat hepatic stellate cells(HSC-T6),using microscopy of green fluorescent protein(green fluorescent,protein,GFP)expression,analysis of transfection efficiency;by using real-time quantitative PCR(QRT-PCR)detection of rat hepatic stellate cells(HSC-T6)the expression of Smad4 gene were screened,effectively inhibit the expression of Smad4 gene of recombinant lentivirus.3.The selected Smad4 RNAi HSCs-T6 cells and non transfected HSCs-T6 cells were inoculated into cell culture flasks and were randomly divided into two groups.The change was detected by ELISA type of rat hepatic stellate cells and collagen content;detection of rat liver by RT-PCR Smad2,Smad3,Smad4,Smad7,ERK1 and ERK2 mRNA expression;the expression of p-Smad2/3,p-MEK1/2,p-ERK1/2 protein was detected in cells of Western Blot.Results:1.There was no significant difference in the proliferation of HSC-T6 cells(P>0.05)There was no significant difference between the two groups(P>0.05).Compared with N group and T group,Z group had some inhibitory effect on HSC-T6 cells,the difference was not statistically significant(P>0.05);different concentrations of Shugan Jianpi Huoxue prescription serum group(L group,M group,H group)had significant inhibitory effect on HSC-T6 cells,the difference was statistically significant(P<0.05).(P>0.05).Compared with N group and T group,the serum levels of HSC-T6 cells in HSC-T6 cells were significantly higher than those in the control group(P<0.05),but there was no significant difference between the two groups The inhibitory effect was statistically significant(P<0.05).2.Recombinant lentivirus was successfully transfected into hepatic stellate cells and transfected into target gene-related recombinant lentivirus group(except 824 group).Compared with transfection-free recombinant lentiviral group and blank control group,Smad4 gene expression(P<0.05).There was no significant difference between the transfection group and the blank control group(P>0.05).3.Compared with model group,the serum containing hepatocytes of Smad4 RNAi HSC-T6 and untransfected HSC-T6 cells were significantly higher than those in model group(P<0.01 or P<0.05).The expression of Smad2,Smad3,Smad4,Smad7,ERK1,ERK2 mRNA and p-ERK1/2 protein was decreased in the cells(P<0.01 or P<0.05).Conclusion:1.The serum concentration of HSC-T6 induced by TGF-β1 was significantly inhibited in the time and concentration-dependent manner,and the concentration of 40%Drug serum on the inhibition rate of the most obvious cells.2.The recombinant lentivirus was successfully transfected into the rat hepatic stellate cells by RNAi technique.The recombinant lentivirus was screened by the transfection of Smad4 mRNA.The recombinant lentivirus was screened.The experimental effect.3.The mechanism of anti-fibrosis in the serum ofShugan jianpi huoxue decoction may inhibit the expression of Smad2,Smad3,Smad4,ERK1 and ERK2 mRNA,inhibit the expression of ERK1/2 and prevent the expression of Smad and ERK transduction pathway in the key cytokine signaling,and thus play a better role in anti-hepatic fibrosis. |
PDF Full Text Request