丹参经TET调控Nrf2/BSEP通路及其对利福平致肝损伤的保护机制研究

基于文献meta分析可知，中药在保肝领域的效果具有积极的临床意义。丹参作为活血化瘀中药的代表，在保肝领域的临床应用已证明其重要性；然而，对其保护机理的研究一直处于空白。为此，本项目围绕丹参是否通过影响Nrf2/ARE-MARE信号通路，继而诱导BSEP的表达，减少胆汁酸在肝脏中的蓄积，从而实现对利福平致肝损伤的保护作用展开。研究工作采用报告基因系统、染色质免疫共沉淀等方法，揭示Nrf2调控BSEP的机理；应用DNA甲基化免疫沉淀、测序和液质联用技术探索丹参对于肝细胞中Nrf2的表观遗传调控机制，即基于TET的主动去甲基化途径，并深入到影响Nrf2基因甲基化的位点；结合基因敲除的动物模型，宏观呈现利福平联用丹参对小鼠肝功能的影响。研究结果将加深对Nrf2在药理学和毒理学研究领域的认识，为探索丹参减轻利福平肝毒性的分子机制，及两者的联用提供科学依据，也为中西药配伍使用解毒机制的研究开阔思路。

A meta-analysis of the data published in the literatures showed that some Chinese medicines could significantly protect the liver function from prescription drugs. Radix Salviae Miltiorrhizae, a typical Chinese medicine for improvement of blood circulation, has been proven clinically active in prevention of liver damage. However, the underlying mechanism has not been well elucidated. Based on our previous work and publications, Nrf2 is found to be able to regulate the expression of BSEP, an efflux transporter responsible for secretion of bile acids. Thus, this project is aim to determine the effect of Radix Salviae Miltiorrhizae on the reduction of cholestasis by inducing the expression of BSEP via Nrf2/ARE-MARE pathway. Therefore, Radix Salviae Miltiorrhizae may cause the reduction of the accumulation of bile acids in hepatocytes resulting in a protective effect of cholestatic liver injury. The reporter gene system, chromatin immunoprecipitation will be used to reveal the mechanism of regulation BSEP by Nrf2. DNA methylation immunoprecipitation and sequence techniques to explore the induction of TETs activity by Radix Salviae Miltiorrhizae in human hepatocytes, which in turn will demethylate the promoter of Nrf2. The exact CpG island site in Nrf2 promoter will also be identified. In addition, the protective effect of Radix Salviae Miltiorrhizae will be verified using an in vivo animal model using both regular and knockout mice treated with rifampicin. The current research will enhance understanding the role of Nrf2 in pharmacology and toxicology research area. It can provide the scientific evidence for alleviation of hepatic toxicity by concomitant administration of Radix Salviae Miltiorrhizae with rifampicin. These findings can broaden the research ideas in the study of herb-drug combination in reducing side effect.