调控微小RNA-17-92表达对血吸虫病大鼠肝纤维化的抑制作用

Inhibitory effect of modulating microRNA-17-92 expression on liver fibrosis in schistosomiasis rats

目的探讨调控微小RNA(miRNA)-17-92表达对血吸虫病大鼠肝纤维化的抑制作用及可能机制。方法在40只SD健康雄性大鼠中随机选取10只作为正常组,其余30只大鼠用于建立血吸虫病模型。将建模成功后的30只大鼠随机分为血吸虫病组、下调组、上调组各10只。分别给予下调组、上调组大鼠经尾部静脉注射miRNA-17-92模拟物、miRNA-17-92抑制物,给予其余组大鼠经尾部静脉注射生理盐水,共2次。注射结束后24 h,检测各组大鼠血清肝功能指标(γ-谷氨酰转移酶、ALT、AST)、肝纤维化指标[透明质酸、Ⅲ型前胶原(PCⅢ)、层粘连蛋白]、氧化应激指标[活性氧簇、8-羟基脱氧鸟苷(8-OHdG)]、炎症反应指标[白细胞介素(IL)-2、IL-22]水平。取各组大鼠肝组织标本观察病理变化情况,并检测miRNA-17-92及相关蛋白[转化生长因子β受体Ⅲ(TGFβRⅢ)、Smad2、Smad3及磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)]的相对表达量。结果(1)正常组、上调组、血吸虫病组、下调组大鼠的血清γ-谷氨酰转移酶、ALT、AST、透明质酸、PCⅢ、层粘连蛋白、活性氧簇、8-OHdG、IL-2、IL-22水平均依次升高(均P<0.05)。(2)血吸虫病组大鼠肝组织细胞排列杂乱且疏松,出现部分细胞坏死、结构损坏、纤维结缔组织增生、炎性细胞浸润状况;下调组上述情况更为严重,而上调组明显减轻。(3)正常组、上调组、血吸虫病组、下调组大鼠的肝组织中miRNA-17-92的表达量依次降低,而TGFβRⅢ、Smad2、Smad3、PI3K、Akt、mTOR蛋白表达量均依次升高(均P<0.05)。结论上调miRNA-17-92表达能够改善血吸虫病大鼠的肝功能和肝纤维化程度,其作用机制可能是调控miRNA-17-92能够减轻肝组织氧化应激反应和炎症反应,并靶向调控TGF-β/Smad通路、PI3K/Akt通路蛋白从而抑制肝纤维化及肝组织细胞凋亡。

Objective To explore the inhibitory effect and possible mechanism of modulating microRNA(miRNA)-17-92 expression on liver fibrosis in schistosomiasis rats.Methods Among 40 healthy male SD rats,10 rats were randomly selected as normal group,and the remaining 30 rats were used to establish schistosomiasis models.The 30 rats with successful modeling were randomly divided into schistosomiasis group(n=10),down-regulation group(n=10)and up-regulation group(n=10).Rats in the down-regulation group and rats in the up-regulation group were injected with miRNA-17-92 mimics and miRNA-17-92 inhibitor via tail vein twice,respectively,while rats in the remaining groups were given a normal saline injection via tail vein twice.Twenty-four hours after the end of the injection,the multiple indices of rats were detected in each group,including serum levels of liver function indicators(gamma-glutamyltransferase,ALT,AST),liver fibrosis indicators(hyaluronic acid,typeⅢprocollagen[PCⅢ],laminin),oxidative stress indicators(reactive oxygen species,8-hydroxy-2′-deoxyguanosine[8-OHdG]),and inflammatory reaction indicators(interleukin[IL]-2,IL-22).The specimens of liver tissues were harvested from the rats in each group to observe the pathological changes,and the relative expression of miRNA-17-92 and related proteins(transforming growth factorβreceptor typeⅢ[TGFβRⅢ],Smad2,Smad3,phosphatidylinositol-3-kinase[PI3K],protein kinase B[Akt],mammalian target of rapamycin[mTOR])was detected.Results(1)Rats′serum levels of gamma-glutamyltransferase,ALT,AST,hyaluronic acid,PCⅢ,laminin,reactive oxygen species,8-OHdG,IL-2,and IL-22 increased in the normal group,up-regulation group,schistosomiasis group and down-regulation group successively(all P<0.05).(2)In the schistosomiasis group,cells in rat liver tissues presented a tanglesome and sparse arrangement,some of them manifested as necrosis,damaged structure,fibrous connective tissue hyperplasia,and inflammatory cell infiltration;the aforementioned conditions were even more severe in the down-regulation group,but were substantially milder in the up-regulation group.(3)Rats′expression of miRNA-17-92 in liver tissues decreased while the expression of TGFβRⅢ,Smad2,Smad3,PI3K,Akt,and mTOR proteins increased in the order of the normal group,the up-regulation group,the schistosomiasis group and the down-regulation group(all P<0.05).Conclusion Up-regulating the expression of miRNA-17-92 can improve the liver function and the liver fibrosis degree in schistosomiasis rats,and the mechanism of action may be as follow:the modulation of miRNA-17-92 can ameliorate oxidative stress response and inflammatory reaction in liver tissues and targetedly modulate TGF-β/Smad pathway and PI3K/Akt pathway proteins,thus inhibiting liver fibrosis and apoptosis in liver tissues.