miR-29a通过调控PTEN/AKT信号通路对CCl4诱导的肝纤维化大鼠的影响

Effects of miR-29a on CCl4-induced hepatic fibrosis in rats by regulating PTEN/AKT signaling pathway

目的探究miR-29a对四氯化碳(CCl 4)诱导的肝纤维化大鼠的保护作用及机制。方法通过腹腔注射40%的CCl 4橄榄油溶液构建大鼠肝纤维化模型,将造模成功的30只大鼠随机分为3组:肝纤维化组(B组)、NC-miRNA+肝纤维化组(C组)和miR-29a+肝纤维化组(D组),每组10只,采用胰岛素注射器通过尾静脉注射注入质粒。另取8只大鼠设为正常组(A组)。采用HE染色评估大鼠肝组织的损伤情况。酶联免疫法(ELISA)检测各组血清中天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)的含量。采用实时定量PCR法(Real-time PCR)检测大鼠肝脏组织中miR-29a mRNA、张力蛋白同源第10染色体丢失的磷酸酶基因(PTEN)mRNA苏氨酸激酶(AKT)mRNA表达水平,采用Western blot法检测PTEN和磷酸化蛋白激酶B(p-AKT)蛋白的变化情况。结果B、C组大鼠肝细胞坏死及空泡变性,有炎性细胞浸润,明显病理性改变。D组大鼠肝小叶内偶见肝细胞坏死,有少量炎性细胞浸润。D组ALT、AST水平、p-AKT的蛋白表达水平明显低于B、C组,而miR-29a mRNA、PTEN mRNA和PTEN的蛋白表达水平明显高于B、C组,差异均有统计学意义(P均<0.05)。结论miR-29a过表达可抑制肝纤维化大鼠的肝损伤,其机制可能与调控PTEN/AKT信号转导通路有关。

Objective This paper aims to investigate the protective effect and mechanism of miR-29a on carbon tetrachloride(CCl 4)induced hepatic fibrosis in rats.Methods The rat liver fibrosis model was established by intraperitoneal injection of 40%CCl 4 olive oil solution,and the successfully modeled 30 rats were randomly divided into the liver fibrosis group(Group B),the NC-miRNA+liver fibrosis group(Group C),and the miR-29a+liver fibrosis group(Group D),ten in each group.They were injected with plasmids through the tail vein using an insulin injector.Another 8 rats were selected as the normal group(Group A).Hematoxylin-eosin(HE)staining was used to evaluate liver tissue injuries.The contents of AST and ALT in the serum of each group were detected by ELISA.The expression of miR-29a mRNA and the missing phosphatase gene(PTEN)mRNA and the thonine kinase(AKT)mRNA in the 10th chromosome of the tonin isotopic chromosome were detected by real-time quantitative PCR(Real-time PCR),while the changes of PTEN and p-AKT protein were detected by Western blot.Results Hepatic cell necrosis and vacuolar degeneration were observed in Groups B and C,with inflammatory cell infiltration.There was occasional necrosis of hepatic cells in hepatic lobule in Group D with a small amount of inflammatory cell infiltration.The expressions of ALT,AST,PTEN mRNA,AKT mRNA,and p-AKT protein in Group D were significantly lower than those in Groups B and C,while the expression of miR-29a mRNA,PTEN mRNA and PTEN protein were significantly higher than that in Groups B and C,with statistically significant differences(P<0.05).Conclusion The miR-29a overexpression can inhibit liver injury in liver fibrosis rats,whose mechanism may be related to the regulation of PTEN/AKT signal transduction pathway.