基于胆红素代谢酶和转运体研究何首乌诱导高胆红素血症的物质基础及预测方法

It is reported that Polygoni multiflori radix and other similar traditional Chinese medicine (TCM), as well as their preparations have hepatotoxicity in their clinic use. The adverse drug reactions including hyperbilirubinemia, jaundice, liver injury could be induced by those TCM, with the mechanism and material basis remaining unclear. This project proposes that: the metabolism regulation of active ingredients in those TCM is similar to bilirubin, and their in vivo glucuronidation process may competitively inhibit the activity of bilirubin metabolizing enzyme and transporters, and thereby inhibit the metabolism and excretion of bilirubin, then induce different levels of liver injury. Thus, the influence of the active ingredients in those TCM on bilirubin metabolism and the risk of hyperbilirubinemia induced by them can be predicted by studying the inhibition effect of those TCM and their active ingredients on bilirubin metabolizing enzyme and transporters through the in vivo and in vitro research, in combination of the drug plasma concentrations and activity of metabolism enzyme and transporters in liver. Therefore, an in vitro screening model of natural inhibitors (active ingredients) from Polygoni multiflori radix on bilirubin metabolizing enzyme and transporters will be established based on enzyme incubation and cell transfer experiments in this project. The correlation between the glucuronidation process of the active ingredients and their inhibition effects on bilirubin metabolism will be investigated, combined with the pharmacokinetic and the pharmacodynamic results of Polygoni multiflori radix and its active ingredients in different physiological and pathological rat modes. In addition, the material basis, mechanism and prediction method of hyperbilirubinemia induced by Polygoni multiflori radix and its ingredients will be explored, which could provide a new idea and method for evaluating the safety and adverse reaction of TCM.

何首乌等中药及其制剂在临床上被报道具有一定的肝毒性，可引发高胆红素血症、黄疸或肝损伤等不良反应，但其物质基础和作用机制尚不明确。本项目提出以下假说：中药中某些活性成分与胆红素体内代谢规律相似，其活性成分体内葡萄糖醛酸化过程可能会竞争性抑制胆红素代谢酶和转运体的活性，从而抑制胆红素代谢与排泄，造成不同程度的肝损伤。通过研究此类中药及活性成分体内外对胆红素代谢酶和转运体的抑制作用，结合血药浓度、肝脏代谢酶和转运体活性，可预测这类中药对胆红素代谢的影响，以及诱导高胆红素血症的风险。本项目以酶孵育和细胞转运实验建立何首乌中胆红素代谢酶和转运体“天然抑制剂”（竞争性活性成分）的体外筛选模型，结合不同生理病理模型大鼠中药代动力学和药效学实验，研究其活性成分葡萄糖醛酸化过程与其抑制胆红素代谢的相关性，阐明何首乌诱导高胆红素血症的物质基础、作用机制及预测方法，探索中药安全性评价新思路和不良反应预警方法。

本课题以酶孵育和细胞转运实验建立了何首乌中胆红素代谢酶和转运体“天然抑制剂”（竞争性活性成分）的体外筛选模型，结合不同生理病理模型大鼠中药代动力学和药效学实验，研究其活性成分葡萄糖醛酸化过程与其抑制胆红素代谢的相关性，明确其诱导胆红素升高的关键因素，建立相应预测方法，为中药安全性评价提供新的思路和方法，进一步指导临床合理用药。.研究结果表明：生何首乌药材的主要化学成分为二苯乙烯苷（TSG）、大黄素、大黄素甲醚、大黄素-8-O-β-葡萄糖苷以及大黄素甲醚-8-O-β-葡萄糖苷。其水提液和醇提液中二苯乙烯苷含量最高，分别为2.38%、3.28%，其次为大黄素、儿茶素、大黄素-8-O-β-葡萄糖苷以及大黄素甲醚-8-O-β-葡萄糖苷。醇提液中各成分含量高于水提液。肝毒性评价结果显示，连续灌胃60天，水提液未见明显肝毒性，高剂量组醇提液（50.4 g生药/kg，相当于人临床剂量80倍）存在一定肝毒性，主要引起肝药酶和胆红素水平的升高。何首乌诱导大鼠肝毒性过程中，肝组织中转运蛋白OATP1A1、OATP1A2、Bsep和代谢酶UGT1A1是其作用的关键靶点。在肝损伤大鼠模型中，何首乌表现出一定的保肝作用，未见明显毒性作用。药代动力学结果表明何首乌中大黄素、TSG、芦荟大黄素和白藜芦醇等化合物体内主要以葡萄糖醛酸化和硫酸化产物为主要代谢物。在正常、ANIT和CCl4大鼠体内的药代动力学行为存在明显差异，TSG和芦荟大黄素的AUC值与UGT1A1、BSEP、OATP1A4、OCT1、NTCP、MRP2和MDR1的表达水平呈显著的负相关（p <0.01）。大黄素、大黄素甲醚和白藜芦醇的AUC值与OATP1A2、NTCP和MRP2的表达水平呈正相关（p<0.05）。何首乌中主要化学成分及其代谢物对胆红素代谢酶UGT1A1、转运体OATP1B1、OATP1B3和MRP2体外抑制评价结果胆红素代谢酶UGT1A1是何首乌抑制胆红素代谢的主要靶点。并参考目前对化学药物诱导高胆红素血症的体内安全预测参数的计算方法，进行何首乌安全性评价。结果显示：当In vivo Fi>0.46和R>3.1时，何首乌存在诱导肝毒性的风险。

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