清肝活血方下调胆汁酸-FGF19轴治疗酒精性肝纤维化中的胆汁淤积

Alcoholic liver disease (ALD) as a representative of non infectious liver diseases will replace the viral hepatitis and become of major cause of chronic liver diseases in the future.Stop and reverse hepatic fibrosis (ALF) is an important node in the control of ALD development. In clinical work, we found that ALD patients often accompanied by symptoms of cholestasis, But the pathogenesis is still not clear.Fibroblast growth factor 19 (FGF19) metabolism in the liver lipid regulation by bile acid (BA), but it,s role in ALF is still not clear. The research group put forward "damp heat and blood stasis" as the core pathogenesis of ALD based on clinical epidemiology and data mining technology. Multicentre clinical and experimental studies have confirmed that Qinggan Huoxue Recipe can obviously improve the ALD and symptoms of cholestasis. preliminary experiments suggest that the BA-FGF19 axis not through the farnesoid X receptor (FXR) involved in the ALF regulation. Qinggan Huoxue Recipe can down regulate the expression of bile acid. Therefore, we put forward the hypothesis: BA -FGF19 axis independently involved in ALF regulation, which is one of the mechanism of Qinggan Huoxue Recipe in the treatment of ALF cholestasis. In order to verify the hypothesis, we would apply the experimental techniques such as preparing of alcoholic liver disease rat and cell model, FXR-/-liver cell model, comprehensive application of Pathology, biochemistry, medicinal chemistry and molecular biology experimental technique. We would apply above techniques from molecular, cell, tissue and whole animal level to elucidate the key value of the BA -FGF19 axis in the regulation of ALF formation and Qinggan Huoxue Recipe regulation mechanism in the development of alcoholic liver fibrosis. This research results will not only lay the foundation for the improvement of the pathogenesis of ALF cholestasis, but also provide potential targets and new strategies for the treatment of ALF.

酒精性肝病(ALD)是未来肝病的主要病因之一，临床发现ALD患者常伴有胆汁淤积症状,而其机制不明。纤维母细胞生长因子19(FGF19)通过胆汁酸(BA)代谢参与了的肝内糖脂代谢，但对酒精性肝纤维化(ALF)的作用尚不清晰。课题组提出“湿热瘀血”为ALD的核心病机，依此拟定的清肝活血方经多中心研究证实对ALD疗效肯定。预实验提示BA-FGF19轴可不通过法尼酯X受体（FXR）独立参与ALF胆汁淤积的调控，清肝活血方可下调胆汁酸的表达。因此假设：BA -FGF19轴独立参与了ALF的调控，这也是清肝活血方治疗ALF胆汁淤积的机制之一。为验证假说，本课题拟制备FXR-/-肝细胞模型和ALF大鼠模型，综合应用病理学、生物化学和分子生物学等实验技术，从多水平探讨BA -FGF19轴介导ALF形成及清肝活血方治疗ALF胆汁淤积的作用机制，这将完善ALF的发病机制，也为其治疗提供新靶点和新策略

酒精性肝病是目前除病毒性肝病外第二大肝病，酒精性肝纤维化的预后较差，并有30%左右可能转为肝癌。但目前对其机制欠清，药物疗效欠佳。本项目研究了胆汁酸-FGF19/15轴在酒精性肝纤维化发展过程中的作用，后用清肝活血方进行干预，观察该方是否通过该通路发挥其防治酒精性肝纤维化的作用。.研究发现，胆汁酸-FGF19/15轴在酒精性肝纤维化发生发展过程中具有重要作用。用200uM甲醛浓度处理肝细胞复制成功ALF模型，检测显示ALF模型中α-SMA、胶原蛋白I 、CYP7A1表达升高，FGFR4、β-Klotho表达减低，在用FGF19干预后，得到了逆转的结果，α-SMA、胶原蛋白I 、CYP7A1表达较模型组明显降低，FGFR4、β-Klotho表达较模型组明显升高。在体外研究中和病理结果同步，也出现了类似结果，由此我们推测，胆汁酸-FGF19/15轴在酒精性肝纤维化发生发展过程中具有重要作用。在研究胆汁酸-FGF19/15轴在酒精性肝纤维化发生发展过程中是否必须要通过FXR的问题中，我们采用RNAi的方法对FXR进行干扰，观察FGF19与FXR之间的相互关系。研究发现肝细胞在进行FXR RNAi后，α-SMA和I型胶原有非常明显的升高，在用FGF19干预后正常肝细胞ALF模型后，发现有明显的降低α-SMA和I型胶原的作用，在用FGF19干预FXR RNAi的ALF模型时，α-SMA和I型胶原的表达较正常肝细胞ALF模型有明显提高，但提高的程度明显低于FXR RNAi ALF，因此我们推测，胆汁酸-FGF19/15轴在酒精性肝纤维化发展过程中，FXR在其中起到了一定的降低肝纤维化的作用，FGF19与FXR在酒精性肝纤维化的发展中的作用关系是相关，而不是依靠。在用清肝活血方干预酒精性肝纤维化的过程中，我们观察到清肝活血方有明确的防治肝纤维化的作用。在对胆汁酸生成以及α-SMA、胶原蛋白I、FGFR4、β-Klotho的表达方面，体现出清肝活血方的作用效果与FGF15的有无相关，由此我们推测，清肝活血方可通过胆汁酸-FGF19/15轴起到抗酒精性肝纤维化的作用。.该项目的研究为酒精性肝纤维化的防治增加了一种策略，有望在临床推广使用。