LPS-TLR4信号通路在慢性酒精摄入大鼠模型肝纤维化发生中的作用及其机制

目的: 通过检测慢性酒精摄入大鼠门静脉血中内毒素脂多糖(LPS)水平,探讨LPS-Toll样受体4(TLR4)通路在慢性酒精摄入大鼠模型肝纤维化发生中的作用及意义。方法: 24只雄性SD大鼠随机分为酒精组和对照组,分别喂养等热量的Lieber-Decarli酒精饲料和对照饲料,10周后采集大鼠门静脉血,留取肝组织标本。采用HE染色和Masson染色评估肝脏的组织学特点,分光光度法检测大鼠门静脉血浆内LPS水平,免疫组织化学法检测肝组织纤维母细胞特异蛋白1(FSP-1)和波形蛋白(vimentin)以识别肝星状细胞(HSCs),原位杂交方法检测肝组织中TLR4 mRNA表达水平。应用计算机图像分析系统检测FSP-1阳性HSCs和TLR4 mRNA阳性细胞的积分光密度值(IA)。采用RT-PCR法检测肝组织中白细胞介素1(IL-1)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)和转化生长因子β1(TGF-β1) mRNA表达水平。结果: 与对照组比较,酒精摄入10周末酒精组大鼠肝脏出现中央静脉周围及肝血窦周边纤维化,肝脏TLR4 mRNA表达水平及FSP-1阳性激活HSC均有明显增加(P<0.01),大鼠门静脉血浆LPS水平明显升高(P<0.01)。与对照组比较,酒精组大鼠肝脏致炎症因子IL-1、IL-6、TNF-α及TGF-β1 mRNA表达水平明显升高(P<0.01)。 Bivariate相关分析,10周末酒精组大鼠肝脏TLR4 mRNA表达水平与门静脉血浆LPS水平呈正相关关系(r=0.856,P<0.05)。结论: 慢性酒精摄入可引起大鼠肝脏TLR4信号通路活化,通过释放致炎因子和纤维源性因子在肝纤维化发病过程发挥重要的作用。

Effect of LPS-TLR4 pathway in hepatic fibrogenesis of rats with chronic alcohol intake

Objective To determine the levels of lipopolysaccharide (LPS) in portal vein blood from the chronic intake rats,and to explore the mechanism and significance of LPS-Toll-like receptor 4 (TLR4) pathway in hepatic fibrogenesis in the rat models of chronic alcohol intake. Methods Twenty-four male Sprague-Dawley rats were randomly divided into alcohol group and control group,and the rats were fed with isocaloric Lieber-Decarli alcohol liquid diets and control diets for 10 weeks.The histological characteristics of liver tissue of the rats were evaluated by HE staining and Masson Trichrome staining.The plasma LPS levels in portal veins of the rats were determined by spectrophotometric method. The fibroblast specific protein 1(FSP-1) and vimentin were determined for identification of hepatic stellate cells (HSCs) by immunohistochemistry.The TLR4 mRNA expression levels in rat liver tissue were examined by in situ hybridization method.Computer image analysis was performed to measure the integrated optimal density (IA) of FSP-1-positive HSCs or TLR4 mRNA-positive cells,respectively.The expression levels of interleukin 1(IL-1),interleukin 6(IL-6),tumor necrosis factor α(TNF-α) and transforming growth factor beta 1(TGF-β1) mRNA in liver tissue were detected by RT-PCR. Results Compared with control group, the liver tissue of the rats in alcohol group showed a mild perisinusoidal and periportal fibrosis;the expression level of TLR4 mRNA and the number of FSP-1-positive HSCs were increased at the end of 10 weeks (P<0.05).At the end of 10 weeks,the plasma LPS level in portal veins of the rats in alcohol group was significantly higher than that in control group (P<0.01).Compared with control group,the expression levels of IL-1,IL-6,TNF-α and TGF-β1 mRNA in liver tissue of the rats in alcohol group were increased significantly(P<0.01).Bivariate correlation analysis showed that there was a positive correlation between the expression intensity of TLR4 mRNA in liver tissue and the levels of portal vein plasma LPS of the rats in alcohol group (r=0.856,P<0.05). Conclusion The activation of TLR4 pathway triggered by chronic intake plays an important role in the rat hepatic fibrogensis through up-regulation of profibrogenic and proinflammatory cytokine release.