基于有害结局路径“Top-Down”策略的中药毒性进程动态机制研究：以补骨脂致肝毒性为例

The aristolochic acid event has aroused widespread concern in the world in 2017, revealing two key problems for the toxicity study of traditional Chinese medicines (TCMs). Firstly, the toxicological evaluation indicators are lack of process effect indicators. Secondly, the initiating factors of toxicity are not clear either. In order to resolve the above mentioned problems, this project uses the new method called adverse outcome pathway (AOP) framework for international toxicology research. Taking Psoralea Fructus as an example, given full consideration to the particularity of TCMs, we establish a new method for the research of dynamic mechanism of toxicity progress of TCMs based on the three key elements of AOP: molecular initiation events, critical events and adverse outcomes (toxicity). By "top-down" strategy, starting with the adverse outcome, the lower-level key events were identified step by step and associated with the specific molecular initiation event. First of all, we confirmed the adverse outcomes of Psoralea Fructus by whole animals. Secondly, a “core target” map was constructed using metabolomics and quantitative proteomics techniques to identify key events related to adverse outcomes. Then molecularly-initiated events associated with adverse outcomes and critical events were confirmed using Click-Chemistry as a tool. Finally AOP framework of hepatotoxicity induced by Psoralea Fructus was established. The successful completion of this project will put forward a suitable modular method for the toxicity mechanism study of TCMs and promote TCM internationalization and modernization.

2017年马兜铃酸致肝癌事件引起海内外广泛关注，暴露中药毒性基础研究存在两个关键问题：一是毒性评价缺少进程效应指标；二是毒性产生的始动因素亦不清楚。为了化解以上难题，本项目借助国际毒理学研究新方法-有害结局路径（AOP），从AOP三个关键要素-即分子起始事件、关键事件（毒性进程拐点）及有害结局（毒性）出发，以补骨脂为例，充分考虑中药特殊性，建立中药毒性进程动态机制研究的新方法。采取“top-down”策略，从有害结局开始，逐级确定低层次关键事件并与特定分子起始事件相关联。首先从整体动物确定补骨脂致肝毒性有害结局；随后采用代谢组学和定量蛋白质组学技术构建“核心靶标”群，发现并验证关键事件；再以点击化学为工具，“钓取”毒性成分直接靶标，确定分子起始事件；最后建立补骨脂致肝毒性AOP框架。本项目的顺利完成将为中药毒性进程动态机制研究提出一条适宜新方法，推动中药毒理学的发展。

按照申请书内容，本项目计划取得如下进展：（1）考察了补骨脂致肝毒性的“量-时-毒”关系，明确补骨脂有害结局路径。（2）利用代谢组学和定量蛋白质组学技术关联分析，发现差异蛋白与小分子代谢物密切相关，并与有害结局关联分析，确定其关键事件。（3）采用化学生物学技术，制备补骨脂毒性成分的分子探针，利用点击化学和DARTS技术“钓取”靶点，初步确定小分子起始事件和潜在毒性机制。（4）应用“top-down”的“有害结局-关键事件-分子起始事件”的中药毒性进程动态机制研究新策略，最后建立补骨脂致肝毒性AOP框架。本项目的顺利完成不仅仅深入阐释补骨脂致毒机制，更为中药毒性研究提出一条新的适宜模块化研究方法，有利于中药国际化及现代化进程的推进。发表SCI论文4篇，中文文章6篇，已接收中文文章2篇，另有2篇SCI论文在投,正在申请专利1项。