TLR4在LPS诱导的大鼠肝内胆管组织损伤中的作用及机制

Role of TLR4 in LPS-induced hepatic bile duct injury in rats

目的探讨TOLL样受体4(TLR4)在脂多糖(LPS)诱导的大鼠肝内胆管组织损伤中的作用及机制。方法将48只SD大鼠随机均分为四组,LPS+siRNA转染组和LPS+阴性病毒组经尾静脉分别注入siRNA-TLR4纯化腺病毒、阴性对照病毒,对照组和LPS组均经尾静脉注射等量生理盐水。给药后24 h,LPS组、LPS+siRNA转染组和LPS+阴性病毒组均经胆总管内缓慢注入LPS,对照组注入等量生理盐水。制模后48、72 h各组分别取6只大鼠,采集肝门部包含胆管的肝组织标本。采用HE染色观察肝门部肝内胆管组织炎性细胞浸润情况及胆管形态等病理变化。采用免疫组化法检测胆管上皮细胞TLR4、细胞角蛋白19(CK19)、波形蛋白(Vimentin)蛋白表达,采用RT-PCR法检测胆管组织TLR4、CK19、Vimentin mRNA表达。结果 LPS组造模48、72 h时胆管周围炎性细胞浸润且胆管间质水肿,伴周围纤维结缔组织轻度增生;LPS+阴性病毒组表现与LPS组相似;LPS+siRNA转染组造模48 h时胆管周围仅有少量炎性细胞,造模72 h时胆管周围炎性细胞稍增多,但较LPS组显著减轻。与对照组比较,其他三组造模48、72 h时胆管上皮细胞CK19蛋白及mRNA表达均降低,TLR4、Vimentin蛋白及mRNA表达均升高,组间比较P均<0.01;LPS+siRNA转染组造模48、72 h时胆管上皮细胞CK19蛋白及mRNA表达均高于LPS组、LPS+阴性病毒组,TLR4、Vimentin蛋白及mRNA表达均低于LPS组、LPS+阴性病毒组,组间比较P均<0.01。结论 LPS诱发肝内胆管组织发生炎症反应及纤维化损伤过程中产生大量TLR4,TLR4可导致肝胆管上皮细胞发生上皮间质转化,从而加重纤维化损伤程度。

Objective To investigate the effect and mechanism of Toll-like receptor 4 （TLR4） on hepatic bile duct injury induced by lipopolysaccharide （LPS） in rats. Methods Forty-eight SD rats were randomly divided into four groups. The LPS ＋ siRNA transfection group and the LPS ＋ negative virus group were injected with the siRNA-TLR4 puri- fied adenovirus and the negative control virus, respectively. The control group and the LPS group were injected with the same amount of normal saline. After 24 h, we injected LPS into the common bile ducts of the rats in the LPS group, LPS ＋ siRNA transfection group, and LPS ＋ negative virus group; the control group was injected with the same amount of normal saline. After the molding of 48 and 72 h, we collected the liver tissue samples from 6 rats in each group. HE staining was used to observe the infiltration of inflammatory cells and the morphological changes of bile duct. Immunohistochemical method was used to detect the protein expression of TLR4, cytokeratin 19 （CK19）, and Vimentin in hepatic duct epithelial cells, and the mRNA expression of TLR4, CK19, and Vimentin were detected by RT-PCR. Results In the LPS group, inflammatory cell infiltration and interstitial edema of the bile duct were found at 48 h. At 72 h, a large number of inflam- matory ceils infiltrated around the lumen, accompanied by mild hyperplasia of fibrous connective tissue. In the LPS ＋ siR- NA group, only a small number of inflammatory cells were found around the bile duct at 48 h, and the inflammatory cells in the bile duct were slightly increased at 72 h after modeling, but were significantly lower than those in the LPS group. Corn-pared with the control group, the protein and mRNA expression of CK19 in the epithelial cells decreased, and the protein and mRNA expression of TLR4 and Vimentin increased in the other three groups at 48 and 72 h （ all P 〈0.01 ）. The ex- pression of CK19 protein and mRNA in bile duct epithelial ceils of the LPS ＋ siRNA transfection group was higher than that of the LPS group and LPS ＋ negative group, while the protein and mRNA expression of TLR4 and Vimentin was lower than that in the LPS group and LPS ＋ virus negative group at 48 and 72 h （ all P 〈 0.01 ）. Conclusion LPS can promote the occurrence of inflammatory reaction and fibrosis injury of liver and biliary duct tissues and induce TLR4 production, which may lead to the occurrence of EMT and thus increase the degree of liver fibrosis.