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。

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.