BMP-7/Smads信号通路在胆总管结扎大鼠肝纤维化组织中的表达及意义

Expression and Significance of BMP-7/Smads Signaling Pathway in Hepatic Fibrosis Tissues induced by Common Bile Duct Ligation in Rats

目的探讨BMP-7/Smads信号通路在胆总管结扎大鼠肝纤维化组织中的表达及意义。方法将雄性Wistar大鼠简单随机化分组成假手术组,胆总管结扎7天组、14天组、28天组、42天组,采用胆总管结扎方法制备大鼠胆汁淤积肝纤维化模型。苏木精-伊红染色、Msaaon染色观察各组大鼠肝组织形态及胶原沉积情况,实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)测各组大鼠肝脏中α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、纤连蛋白(fibronectin,FN)等肝纤维化指标mRNA的表达变化,免疫组化法明确肝脏胶原蛋白Ⅰ(collagenⅠ)沉积情况,Western blot法检测各组肝组织内FN、collagenⅠ、胶原蛋白Ⅲ(collagenⅢ)、α-SMA和金属基质蛋白酶组织抑制因子1(tissue inhibitor of matrix metalloproteinase,TIMP1)等蛋白的表达,同时用免疫组化法及Western blot法检测各组肝组织内BMP-7及p-Smad1/5/8蛋白定位及表达量的变化。结果与假手术组比较,胆总管结扎组随着胆总管结扎时间延长,肝纤维化程度逐渐加重,肝内胶原增生明显,形成粗大的纤维间隔,肝组织内FN、α-SMA、collagenⅠ、collagenⅢ及TIMP1蛋白的表达明显增多,且差异有统计学意义(P<0.05),在肝纤维化组织内,BMP-7及p-Smad1/5/8蛋白在肝纤维化早期显著升高,随着纤维化程度加重,其表达逐渐减少,与假手术组比较,差异有统计学意义(P<0.05)。结论BMP-7/Smads信号通路在大鼠肝纤维化早期明显激活,作为一种保护性措施发挥内源性保护作用,而随着肝纤维化的进展,BMP-7/Smads信号通路的激活程度逐渐减弱,这一作用逐渐被削弱。

Objective To investigate the expression and significance of BMP7/Smads signaling pathway in hepatic fibrosis tissues induced by common bile duct ligation in rats.Methods Male Wistar rats were simple randomizationally divided into sham operation group,common bile duct ligation group for 7days group,14days group,28days group and 42days group.Common bile duct ligation method was used to establish the cholestatic hepatic fibrosis animal model.hematoxylin-eosin staining and Masson staining were used to observe the hepatic tissues morphology and collagen deposition of the rats in each group.Real-time quantitative polymerase chain reaction(RT-qPCR)technology was used to detect the mRNA expression of hepatic fibrosis indicators,such asα-smooth muscle actin(α-SMA)and fibronectin(FN)in each group.Immunohistochemistry was used to determine the deposition of collagenⅠin the hepatic tissues,Western blot was used to detect the proteins expression of FN,collagenⅠ,collagenⅢ,α-SMA and tissue inhibitor of matrix metalloproteinase(TIMP1)in the hepatic tissues.The expression and localization of BMP-7 and p-Smad1/5/8 proteins in the hepatic tissues were detected by Western blot and Immunohistochemistry.Results Compared with the sham operation group,with the extension of common bile duct ligation time,the degree of hepatic fibrosis gradually increased,the intrahepatic collagen hyperplasia was obvious,and thick fibrous septum were obviously formed in the hepatic issues of BDL groups.The expression of FN,α-SMA,collagenⅠ,collagenⅢand TIMP1 proteins in the BDL hepatic tissues were significantly increased,the difference were statistically significant(P<0.05).In the hepatic fibrosis tissue,the expression of BMP-7 and p-Smad1/5/8 proteins were significantly increased in the early stage of hepatic fibrosis,while which were decreased with the aggravation of hepatic fibrosis.Compare with the sham operation group,the difference was statistically significant(P<0.05).Conclusion The BMP-7/Smads signaling pathway is significantly activated in the early stage of hepatic fibrosis in rats,which exerted protective effect on bile duct ligation-induced hepatic fibrosis.However,the protective effect of activation of BMP-7/Smads signaling pathway was inhibited with the progression of hepatic fibrotic severity.