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

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.

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。

利福(Rifampicin, RFP)是广谱抗生素类抗结核病治疗的首选药物。由于RFP需长期使用，故其出现药物不良反应的风险较大，其中以肝毒性最为严重，主要表现为肝内胆汁淤积现象。目前临床上对此类药源性胆汁淤积型肝损伤(Drug induced liver injury, DILI)的治疗和预防手段较为单一。胆汁淤积型肝损伤以肝内积聚胆汁酸(Bile acids, BAs)为特征，且肝脏胆汁酸转运障碍是引起该损伤的重要因素之一。胆汁酸外排转运体(Bile salt export pump, BSEP)与Na+/牛磺胆酸协同转运体(Sodium taurocholate cotransporting polypeptide, NTCP)分别是BAs肝肠循环的开端和终点。在DILI状态下，BSEP和NTCP的表达可能受到抑制，亦或是受到肝内和血液中蓄积的BAs反馈性上调。因此若能持续有效促进这两种转运体的表达，维持BAs肝肠循环通畅，是解决RFP引起的胆汁淤积型肝损伤的有效途径之一。. 前期的实验研究发现丹参酮IIA(Tanshinone IIA, TAN IIA)具有对抗药源性肝损伤的作用，且能显著诱导核因子E2相关因子2(Nuclear Factor E2-related factor 2, NRF2)表达的能力。在人源BSEP和NTCP的启动子区域均发现了NRF2结合位点的存在，因此本项目探索了NRF2与BSEP和NTCP之间的调控关系。利用RFP胆汁淤积模型小鼠考察了TAN IIA的预防作用，并利用Nrf2基因敲除小鼠证明了Nrf2-Bsep/Ntcp信号通路是TAN IIA实现预防RFP所致胆汁淤积型肝损伤的重要途径之一。同时，本项目研究了TAN IIA通过甲基胞嘧啶双加氧酶2(Ten-eleven translocation methylcytosine dioxygenase, TET2)发挥DNA去甲基化实现对NRF2的激活作用以及进一步研究TET2对NRF2-BSEP/NTCP信号通路激活的影响，更深层次地阐明TAN IIA预防RFP所致胆汁淤积型肝损伤的机制。. 本课题为中药用于预防DILI开阔了研究思路，也为解决RFP临床治疗的难题，即TAN IIA用于预防RFP引起的胆汁淤积型肝损伤奠定了理论基础。