CHFR通过介导TGF-β1通路改善肝纤维化的实验研究

Experimental Study of CHFR Ameliorates Liver Fibrosis by Mediating TGF-β1 Pathway

目的探讨泛素连接酶蛋白(checkpoint with FHA and RING finger domains,CHFR)通过转化生长因子β1(transforming growth factor beta 1,TGF-β1)通路参与并改善肝纤维化的机制。方法将30只小鼠随机分为正常组和模型组,每组15只。模型组通过四氯化碳(carbon tetrachloride,CCL4)制备肝纤维化模型,模型制备成功后采用Masson染色观察小鼠肝脏纤维化情况,并采用Western blot方法检测小鼠肝脏组织中CHFR的表达水平。体外实验通过构建CHFR质粒转染HEK293T细胞成功高表达后,将转染后的细胞分为载体组和mCHFR组,采用Western blot方法检测载体组、mCHFR组TGF-β1和重组丝/苏氨酸激酶受体Smad家族成员3(Smad family member 3,Smad3)的表达量,从基因和蛋白水平探讨CHFR在肝纤维化中的作用机制。结果Masson染色结果显示,正常组小鼠肝脏汇管区无纤维化现象,肝小叶结构清晰完整,肝细胞分界清楚。模型组小鼠肝脏中的汇管区小叶结构紊乱,形成宽而粗大的纤维条索和纤维间隔。Western blot结果显示,与正常小鼠肝脏相比,模型组CHFR表达量减弱(P<0.05);与载体组对比,mCHFR组TGF-β1和Smad3的表达量减少(P均<0.05)。结论CHFR可能通过TGF-β1通路改善肝纤维化的发生发展,并且可能为肝纤维化的治疗提供靶标。

Objective To explore the mechanism of checkpoint with FHA and RING finger domains(CHFR)protein participating and ameliorating liver fibrosis through transforming growth factor beta 1(TGF-β1)pathway.Methods A total of 30 mice were randomly divided into normal group and model group with 15 in each.In the model group,the liver fibrosis model was prepared by carbon tetrachloride(CCL4),after the model was successfully prepared,the liver fibrosis of mice was observed by Masson staining,the expression level of CHFR in the liver tissue of mice was detected by Western blot.In vitro experiments,after transfecting HEK293 T cells with high expression of CHFR plasmid,the transfected cells were divided into vector group and mCHFR group,and the vector group was detected by Western blot,the expression levels of TGF-β1 and recombinant filament/threonine kinase receptor Smad family member 3(Smad3)in mCHFR group were analyzed to explore the mechanism of CHFR in liver fibrosis from the gene and protein levels.Results Masson staining results showed that there was no fibrosis in the hepatic portal area of mice in normal group,the structure of hepatic lobules was clear and complete,and the boundary of hepatocytes was clear.In the model group,the lobule structure of the portal area in the liver was disordered,forming wide and thick fiber strands and fiber intervals.Western blot results showed that CHFR expression decreased in the model group compared with normal group(P<0.05),compared with the vector group,the expression of TGF-β1 and Smad3 in mCHFR group decreased(all P<0.05).Conclusion CHFR may improve the occurrence and development of liver fibrosis through the TGF-β1 pathway,and may provide a target for the treatment of liver fibrosis.