基于OATPs/UGT1A1/MRPs调控研究何首乌对胆红素代谢转运的影响及其肝毒性机理

The starting point of the project is to explore the scientific issues on cause and mechanism of bilirubin-related diseases (e.g. jaundice) or adverse reactions caused by traditional Chinese medicine (TCM). The research objects were bilirubin and model drug Polygonum multiflorum Thunb in the project. The experimental model, e.g. human OATPs/UGT1A1/MRPs transgenic MDCK-Ⅱcells and mice model, are established to study the influence of TCM and its constituents on OATP1B1/1B3-mediated uptake of bilirubin, UGT1A1-mediated glucuronidation of bilirubin, MRP2/1/3-mediated efflux of bilirubin at the level of molecular,cell and animal. The synergistic multi-target, multi-way regulatory effect of multicomponents of TCM on bilirubin metabolism will also be observed. Bilirubin was used as the main biomarker of hepatotoxicity in the study.Material basis and molecular mechanism of hepatotoxicity and attenuate toxicity by processing for TCM are investigated and interpreted from the perspective of regulation of enzymes and transporters. The research will demonstrate important theoretical value and clinical significance on revealing the causes and mechanisms of bilirubin-related diseases (e.g. jaundice)，material basis and molecular mechanism of liver toxicity, which are caused by TCM, as well as providing new scientific basis for the safe, effective application of TCM with hepatotoxicity. Meanwhile, the transgenic MDCK-Ⅱcells and mice models can provide a new tool for study on pharmacokinetics, toxicology and drug safety evaluation.

本课题从探寻中药引起黄疸等胆红素相关疾病或不良反应的原因和机制这一科学问题出发，以胆红素和模型药物何首乌为研究对象，构建人OATPs/UGT1A1/MRPs基因转染MDCK-Ⅱ细胞模型和转基因小鼠等实验模型，在分子细胞和整体动物水平上，研究中药及其组分对OATP1B1/1B3介导的胆红素摄取、UGT1A1介导的胆红素葡萄糖醛酸结合、MRP2/1/3介导的胆红素排泄的影响；考察中药多组分对胆红素代谢多靶点、多途径协同调控作用；并以胆红素为主要生物标志物，从代谢酶和转运体调控角度，研究并阐释中药肝毒性及炮制减毒的物质基础和分子机制。 本研究对于揭示中药引起的黄疸等胆红素相关疾病的发病原因和发病机制、肝毒性物质基础和分子机制具有重要的理论价值和临床意义，并为肝毒性中药安全、有效应用提供新的科学依据；所构建的基因转染细胞模型和转基因小鼠模型，可为药代动力学、毒理学研究和药物安全性评价提供新工具。

何首乌容易引起黄疸和肝损伤，其原因和机制一直不明。这可能与其抑制了OATP1B1、UGT1A1、MRP2等介导的胆红素代谢转运有关。本课题从转运体和代谢酶调控角度，研究了何首乌及其组分对OATP1B1介导的胆红素摄取、UGT1A1介导的胆红素代谢、MRP2介导的胆红素外排影响及其肝毒性。主要研究结果：（1）测定了生物介质中UCB、BMG1、BMG2、BDG浓度，发现人肝微粒体和UGT1A1介导胆红素葡萄糖醛酸结合符合米氏动力学。（2）同时测定了生首乌、制首乌中10种组分含量，构建了化学指纹图谱，发现12个产地及炮制前后何首乌主要组分含量有明显差异。（3）何首乌及其组分对人UGT1A1介导胆红素葡萄糖醛酸结合有强抑制作用（IC50:生首乌提取物31.16±0.23 μg/ml、制首乌提取物70.75±1.18μg/ml、大黄素1.53±0.17µM、EGCG 20.68±0.30µM）。（4）构建了大鼠原代肝细胞和“三明治培养”原代肝细胞模型；生首乌和制首乌提取物抑制胆红素肝细胞摄取，其IC50分别为211.1±12.27μg/mL和1331±128.93μg/mL，BEI分别为32.68%和37.80%，CL分别为0.0022min-1• kg-1和0.0033 min-1•kg-1。（5）构建了人OATP1B1、 MRP2基因单转染和双转染MDCKⅡ细胞模型；何首乌及其组分对胆红素转运有强抑制作用；大黄素（IC50 1.83±0.21 µM）、大黄素甲醚（IC50 6.67±0.42μM）、芦荟大黄素（IC50 8.71±1.25μM）和儿茶素（3.31±0.29µM）对OATP1B1介导胆红素摄取有显著抑制作用；大黄素甲醚（IC50 7.22±0.56μM）对MRP2介导胆红素外排有显著抑制作用。（6）给予小鼠何首乌提取物30d，血清肝毒性指标ALT、AST、GGT、LDH、TBIL、DBIL、IBIL等显著升高;有10个产地何首乌有明显肝毒性；何首乌炮制后毒性减弱。本课题揭示了何首乌引起黄疸等胆红素相关疾病是由于其所含蒽醌类成分抑制了OATP1B1、UGT1A1、MRP2等介导的胆红素代谢转运所致。该成果具有重要理论价值和临床意义，可为何首乌临床应用和黄疸防治提供新的理论依据和科学支撑；所构建细胞模型和动物模型为药动学、毒理学研究提供新工具。