泽泻醇B 23-乙酸酯促肝再生作用的分子机制和作用靶点

Partial hepatectomy has been the most effective treatment for many patients suffering from liver cancer and end-stage liver disease by now. The survival rate and prognosis in patients mainly depend on the residual liver regeneration. However, most of the patients with hepatic resection have poor prognosis or even die, due to their low liver regeneration. There is an urgent demand for patients to be treated with drugs to improve residual liver regeneration. Now have the drugs with effect of promoting liver regeneration been few, therefore there is an urgent need to develop such drugs. Alisol B 23-acetate is a natural triterpene compound. Our previous studies have demonstrated for the first time that alisol B 23-acetate can promote liver regeneration and hepatocyte proliferation in mice. Based on the previous work, this research project is performed (1) to investigate promoting liver regeneration of alisol B 23-acetate in vivo and at cellular level; (2) to determine the gene and protein involved in hepatocyte proliferation by Real-time PCR and Western blot in order to elucidate the molecular mechanisms underlying alisol B 23-acetate liver regeneration promotion; (3) to further determine the targets for alisol B 23-acetate by molecular docking software and Dual-Luciferase Reporter assay system. This project is expected to provide not only logical proofs for alisol B 23-acetate development and utilization, but also opportunities and perspectives for discovery of innovative Chinese medicine for liver regeneration treatment.

肝部分切除术目前为治疗肝癌和终末期肝病最有效的方法，术后患者的存活率和预后主要取决于残余肝脏的再生能力。然而临床上大部分行肝切除术的患者由于肝脏再生能力低下，预后不佳甚至死亡，患者急需药物治疗以提高残肝再生能力。目前已上市的促肝再生药物极少，因此迫切需要研发此类药物。泽泻醇B 23-乙酸酯是一种天然三萜类化合物，前期工作中首次发现其具有促进小鼠肝再生及肝细胞增殖的作用。本项目拟在前期工作的基础上进行：（1）体内和细胞实验评价泽泻醇B 23-乙酸酯的促肝再生作用；（2）Real-time PCR和Western blot方法研究调控肝细胞增殖过程的基因和蛋白，阐明此化合物促肝再生作用的分子机制；（3）分子对接模拟和双荧光素酶报告基因实验深入研究此化合物促肝再生作用的靶点。本项目不仅有望为泽泻醇B 23-乙酸酯的开发和利用提供理论依据，还可为肝再生治疗的创新中药新药的发现提供新的契机和视角。

环境污染物、毒物和过量药物常可造成肝脏损伤，若未得到恰当的治疗，将可能发展为肝脏纤维化、肝硬化，最终发展为肝癌。目前，肝切除术是肝癌或终末期肝病常用的治疗方法。然而，大部分行肝切除术的患者，往往由于残余肝脏再生能力低下，预后不佳。本项目主要研究了天然三萜类化合物泽泻醇B 23-乙酸酯（alisol B 23-acetate，AB23A）的促小鼠肝再生作用及分子机制、抗胆汁淤积的保护作用及分子机制、抗CCl4所致肝损伤作用及分子机制、抗雌激素所致胆汁淤积性肝损伤作用及分子机制。结果表明，（1）AB23A具有显著的促肝再生作用，其作用机制为AB23A通过上调肝细胞增殖相关蛋白FoxM1b，Cyclin D1和Cyclin B1表达，剂量依赖性地促进了小鼠肝细胞增殖；AB23A通过抑制胆汁酸合成限速酶Cyp7a1，诱导胆汁酸外排转运体Bsep表达，降低了肝内胆汁酸水平、发挥出肝保护作用；AB23A对上述基因表达的影响、促肝再生及肝保护作用被FXR拮抗剂GS所阻断；体外实验表明AB23A对FXR靶基因的调控作用被FXR基因沉默所逆转；双荧光素酶报告基因实验和分子对接实验进一步证实了AB23A对FXR的激活作用。因此，AB23A 的促肝再生作用是通过激活核受体FXR而发挥出来的。（2）AB23A具有良好的抗胆汁淤积作用，AB23A通过下调肝脏胆汁酸摄取转运体Ntcp，上调外排转运体Bsep，Mrp2和Mdr2的表达，减少了肝脏对胆汁酸的摄取，增加了肝脏对胆汁酸的外排；通过抑制胆汁酸合成酶Cyp7a1和Cyp8b1的表达，降低了肝内胆汁酸的合成。（3）AB23A通过调节参与肝细胞DNA复制及有丝分裂的重要基因FoxM1b，Cyclin D1和Cyclin B1的表达，促进了肝细胞的增殖；通过下调肝内胆汁酸摄取转运体和合成酶的表达，降低了肝内胆汁酸水平，发挥出抗CCl4所致肝损伤作用。（4）AB23A对雌激素所致肝损伤的保护作用是由于FXR介导的转运体和酶的表达变化而实现的。泽泻醇B 23-乙酸酯促肝再生、抗胆汁淤积、CCl4、雌激素所致肝损伤作用和分子机制的阐明，不仅为该化合物的开发和利用提供理论依据，而且有助于解决治疗肝病药物缺乏的难题，同时也为开发具有促肝再生和肝保护作用的药物提供了新的契机和视角。