新型苯甲酰胺类化合物16-4通过抑制CYP7A1缓解胆汁淤积性肝病的效果和机制研究

Cholestatic liver disease is a common clinical disease and difficult to treat, its main characteristics is high concentration bile retention in liver, resulting in multiple type of hepatic cells injury, causing liver fibrosis, cirrhosis and other liver diseases. And its clinical therapeutic selection is limited at present, waiting to be solved with new drug research and development urgently. CYP7A1 is a key enzyme in bile acid synthesis process, and a popular target for drug development. Previous data showed a novel benzamide compound 16-4, synthesized by us, remarkably improved cholestasis, anti hepatic fibrosis in vitro and in vivo, suggests that 16-4 might have a good potential in cholestatic liver diseases treatment. it can inhibit the transcription of CYP7A1, through FXR independent mechanism which is unknown in detail. Based on those data, this project intends to study the effect and regulation mechanism of 16-4 in suppression of CYP7A1 transcription, explore its direct target protein, validate the interaction by immunoprecipitation and Biacore assays etc., and clarify the details of the interaction of 16-4 and its target protein. Then study the molecular mechanism in primary hepatic parenchymal cells, and use lentiviral vector to over express or knock down the target protein in mouse liver to further verify the efficacy and explore the in vivo mechanism of 16-4. The final goal is to clarify the pharmacological and molecular mechanisms of 16-4, which might provide experimental basis for its usage in clinical.

胆汁淤积性肝病是一种临床上常见且难治的疾病,主要特点是肝脏内高浓度胆汁潴留，导致肝脏内多种细胞损伤，引起肝纤维化、肝硬化等肝脏病变，目前临床用药效果十分有限，亟待新药的研发。CYP7A1是胆汁合成的关键酶，是目前研发的热点药靶。在前期研究中我们自主合成的新型苯甲酰胺类化合物16-4被发现具有明显的体内外改善胆汁淤积、抗肝纤维化作用，提示其在胆汁淤积性肝病的治疗应用中有良好的潜力；16-4通过非FXR途径抑制CYP7A1的转录，具体机制未明。以此为基础本项目拟进一步研究16-4抑制CYP7A1转录的分子机理，寻找其直接作用靶蛋白，运用免疫共沉淀、BIACORE等技术进行验证，并分析16-4与其靶蛋白的作用机制，然后在原代肝实质细胞中进行分子机制验证，再用慢病毒载体构建靶蛋白肝脏高表达或敲除小鼠模型进一步探索16-4的体内作用机制。最终阐明16-4的药效与分子机制，为其推向临床提供实验依据。

胆汁淤积性肝病是一种临床上常见且难治的疾病,主要特点是肝脏内高浓度胆汁潴留，导致肝脏内多种细胞损伤，引起肝纤维化、肝硬化等肝脏病变，目前临床用药效果十分有限，亟待新药的研发。本研究中，我们发现自主研发的新型小分子化合物16-4灌胃给药可以降低大鼠血清转氨酶、总胆汁酸水平，纠正脂质代谢紊乱，抑制肝纤维化发生发展。机制研究发现，16-4灌胃给药后可以通过抑制胆汁酸合成关键酶CYP7A1从而降低大鼠血清总胆汁酸水平，深入的通路分析表明16-4主要通过下调HNF4α来起到抑制CYP7A1的作用。此外我们还发现16-4可以通过抑制大鼠肝脏组织以及肝星状细胞系LX-2及鼠原代肝星状细胞中STAT3上游激酶JAK2的磷酸化从而抑制STAT3的磷酸化，进一步分析表明16-4处理后可以降低磷酸化的STAT3和Smad3的入核量，从而阻断两者在细胞核内的相互作用，抑制下游有促纤维化相关基因的转录。其作用均强于JAK2抑制剂鲁索利替尼。此外，还发现其可以改善肝实质细胞的内质网应激状态，并且对于PPARγ具有一定的激动作用。总之，16-4可能的作用机制主要包括以下三个方面：首先，16-4可通过下调HNF4α抑制CYP7A1改善肝内胆汁淤积；其次，通过抑制JAK2-STAT3信号通路阻断肝星状细胞的活化以及减轻肝脏炎症反应。最后，可通过上调并激动PPARγ改善肝实质细胞的内质网应激状态和脂质代谢异常，从而减轻肝脏损伤。通过上述三种机制协同发挥抗胆汁淤积性肝纤维化活性。