miR-146a介导PI3K/Akt信号通路抑制肝星状细胞诱导的肝纤维化

miR-146a Inhibits Hepatic Stellate Cell-induced Hepatic Fibrosis by Mediating PI3K/Akt Signaling Pathway

目的探讨微小RNA(miR)-146a在转化生长因子-β1 (TGF-β1)诱导的肝星状细胞活化中的作用,并通过磷脂酰肌醇3激酶(PI3K)/丝氨酸-苏氨酸蛋白激酶(Akt)通路观察其对肝纤维化的影响。方法采用四氯化碳(CC1_4)建立肝纤维化大鼠模型,检测大鼠血清透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原(PCⅢ)和Ⅰ型胶原(Ⅰ-C)水平,Masson染色观察肝组织病变,实时定量聚合酶链反应(Real-time PCR)检测肝组织miR-146a表达,蛋白免疫印迹实验(Westem blot)检测肝组织α-平滑肌肌动蛋白(α-SMA)、PI3K和磷酸化(P)-Akt蛋白表达。HSC (肝星状细胞)-T6细胞分为Normal组、TGF-β1组、TGF-β1+miR-NC组和TGF-β1+miR-M组。TGF-β1+miR-NC组和TGF-β1+miR-M组分别转染miR-146a mimics阴性对照物和miR-146a mimics,然后除Normal组外,其余各组采用TGF-β1诱导HSC-T6细胞。四甲基偶氮唑盐(MTT)法观察细胞增殖情况,Real-time PCR法检测miR-146a表达,Western blot法检测α-SMA、Ⅲ-C、Ⅰ-C、PI3K和p-Akt蛋白表达。结果与正常组比较,模型组大鼠肝组织胶原沉积明显增加,纤维增生显著;血清HA、LN、PCⅢ和Ⅳ-C水平显著升高;肝组织miR-146a表达明显降低;而PI3K和P-Akt蛋白表达明显增加。TGF-β1组和TGF-β1+miR-NC组HSC-T6细胞各指标差异均无统计学意义。与TGF-β1+miR-NC组比较,TGF-β1+miR-M组miR-14表达明显增加,细胞活性、α-SMA表达、Ⅲ-C和Ⅰ-C均明显降低,PI3K和P-Akt蛋白表达明显下调。结论 miR-146a能够抑制TGF-β1诱导的肝星状细胞活化,其抗肝纤维化机制可能与抑制PI3K/Akt信号通路有关。

Objective To investigate the role of microRNA(miR)-146a in the activation of hepatic stellate cells induced by transforming growth factor-β1,and to observe its effect on hepatic fibrosis through the phosphoinositide 3-kinase(PI3K)/serine-threonine kinase(Akt)signaling pathway.Methods A rat model of hepatic fibrosis was established using carbon tetrachloride(CCl4).The rat serum levels of HA,LN,PCⅢ and Ⅳ-C were then measured.Masson staining was used to observe the pathological changes of liver tissue.Real-time quantitative polymerase chain reaction(Real-time PCR)was used to detect the expression of miR-146a in liver tissue.α-smooth muscle actin(α-SMA),PI3K and phosphorylated(p)-Akt protein expressions in liver tissue were analyzed by western blot.HSC-T6 cells were divided into the normal group,TGF-β1 group,TGF-β1+miR-NC group and TGF-β1+miR-M group.TGF-β1+miR-NC group and TGF-β1+miR-M group were transfected with miR-146a mimics negative control and miR-146a mimics,respectively.Then all groups,except for the normal group,were treated with TGF-β1 to induce HSC-T6 cell activation.MTT assay was used to observe the cell proliferation.The expression of miR-146a was detected by real-time PCR.Western blot was used to detect the expressions of α-SMA,Ⅲ-C,Ⅳ-C,PI3K and p-Akt proteins.Results Compared with the normal group,collagen deposition in liver tissue of the model groups significantly increased,and fibroplasia was significant,also,serum HA,LN,PCⅢ and Ⅰ-C were significantly increased,and hepatic miR-146a expression was significantly decreased,while PI3K and p-Akt [(2.964±0.381)vs.(0.983±0.116),t =15.729,P <0.001] protein expressions were significantly increased.No significant difference was found between TGF-β1 group and TGF-β 1+miR-NC group.Compared with TGF-β 1+miR-NC group,the expression of miR-14 increased significantly in TGF-β1+miR-M group;cell activity,α-SMA expression,Ⅲ-C and Ⅰ-C contents reduced significantly in TGF-β1+miR-M group;PI3K and p-Akt protein expressions were significantly down-regulated in TGF-β1+miR-M group.Conclusion miR-146a can inhibit the activation of hepatic stellate cells,which induced by TGF-β1,and its anti-hepatic fibrosis mechanism might be related to the inhibition of PI3K/AKT signaling pathway.