基于体内外代谢产物及UGT1A1抑制作用探索何首乌肝毒性机制及毒效物质基础

The hepatotoxicity induced by Polygonum multiflorum has been widely confirmed. But the hepatotoxicity mechanism and hepatotoxic substances has not been clarified. Because the liver damage caused by Polygonum multiflorum after long term use was usually characterized of jaundice and hyperbilirubinemia which was attributed to the bilirubin metabolism impediment, we proposed that the inhibition of UGT1A1-mediated glucuronidation of bilirubin was the main reason of the hepatotoxicity. In this project the experimental model determining UGT1A1 activity in vivo and in vitro is going to be established to investigate the inhibition of Polygonum multiflorum and its composition on bilirubin metabolism, simultaneously corresponded with animal toxicity experiments in order to demonstrate the hepatotoxicity mechanism. On the other hand, the potential hepatotoxic substances from main components and the metabolites of Polygonum multiflorum will be affirmed through combining multivariate data of metabolism with the inhibition of UGT1A1. This reseach may demonstrate the hepatotoxicity mechanism and hepatotoxic substances from molecular level as well as Ⅰ and Ⅱ phase metabolism reaction. The results will supply a new method to study the traditional Chinese medicine induced liver injury. Meanwhile, it will provide new scientific basis for the safe, effective application of TCM with hepatotoxicity.

何首乌肝毒性客观存在，但致毒作用机制和毒性物质尚不明确。本课题针对长期应用何首乌所引发的肝毒性，常有胆红素代谢障碍导致的黄疸等临床表现，提出对胆红素代谢酶UGT1A1的抑制可能是何首乌致肝毒性的重要原因。拟通过建立体内、外对UGT1A1酶介导的胆红素葡萄糖醛酸结合抑制的实验模型，同时结合毒理实验，确定何首乌主要成分及其代谢产物的肝毒性作用机制；并尝试将何首乌原型成分及代谢产物多变量数据与UGT1A1抑制情况相结合，探索何首乌致肝毒性的物质基础。本课题在分子水平，通过同时起动Ⅰ、Ⅱ两相代谢反应，尝试揭示何首乌肝毒性作用机制和毒性物质基础。本课题研究结果将为中药肝毒性研究提供新的研究思路和实验方法，对中药临床安全、有效应用具有重要的理论价值和临床意义。

何首乌导致肝毒性问题逐渐凸显，虽然针对此问题研究广泛，但何首乌致肝毒性作用机制及毒性物质尚存争议。本研究从何首乌致肝毒性的临床特点出发，即长期口服含何首乌制剂后出现胆红素异常升高,并伴有黄疸等症状，且停药后多数患者自行恢复；由于UDP-葡萄糖醛酸转移酶1A1（UGT1A1）酶为胆红素唯一代谢酶，可将胆红素代谢为胆红素葡萄糖醛酸结合物，经胆汁排泄，完成解毒过程。因此本研究以胆红素体内代谢环节为切入点，提出：“何首乌致肝毒性作用机制可能为抑制了UGT1A1介导的胆红素代谢”的科学假说。拟通过体外（肝微粒体）实验，首先系统考察何首乌及单体成分对UGT1A1酶的影响，初步推测其可能的肝毒性机制及潜在毒性成分；其次，采用2D，3D细胞从人源层面证实待测物的肝毒性作用；在体外实验的基础上选择抑制作用明确的单体成分及70%何首乌醇提物进行大鼠体内毒性实验，进一步验证课题假说；最后，采用计算机分子对接手段，从蛋白结构及分子结构角度出发，明确单体成分与UGT1A1酶的作用靶点，作用强弱，以期综合阐释何首乌致肝毒性作用机制，及毒性物质。本研究最终证实70%何首乌醇提物、其主要单体成分及其代谢产物对UGT1A1酶存在抑制作用，存在引发胆红素原型堆积，继而产生肝毒性作用的风险；并推测潜在毒性物质为二蒽酮类 ：trans-emodin dianthrones、cis-emodin dianthrones；二蒽酮糖苷类 ：polygonumnolides C1-4；单蒽酮类：大黄素型单蒽酮；蒽醌类 ：大黄素-8-O-β-D-葡萄糖苷及大黄素甲醚。本研究所建立的肝毒性评价方法为毒性中药体内、外研究提供了新的思路，同时也是建立肝毒性中药的研究体系的重要探索。