基于组合质谱技术与代谢组学方法的中药何首乌体内物质作用组群与肝毒性研究

The hepatotoxicity mechanism caused by taking the decoction of Polygonum multiflorum (PM) has not been clearly elucidated so far, although dozens of cases of the adverse reactions had been reported. Our preliminary study showed that the extracts of PM can cause the liver cell damage and the hepatotoxicity mechanism may cause by the main components of PM. Yet the contradictory conclusions have been made between the vivo and the vitro hepatotoxicity experiments of some components, suggesting that the hepatotoxicity may be caused by some metabolic abnormalities of the PM constituents. .Based on the well understanding of the fragmentation pathways of the main constituents in PM, the present study mainly focused on analysis the metabolites of the PM in rats. A method will be developed based on UHPLC coupled with combined LTQ-Oribtrap mass spectrometry, as well as multiple statistical analysis technique, for further investigating the general material basic of in-vivo hepatotoxicity caused by PM. The project would be also designed to characterize the metabolomic differences of biological samples of rats between the normal groups and PM-taking groups by pattern recognition methods. Combined with the in-vivo component analysis and hepatotoxicity results, the global body fluids and organs metabolic profiles would be constructed to screen the potential biomarkers and to identify the metabolic pathways. It will be provided a new methodological cue for studying the potential toxicity mechanism of PM. Our study could also provide useful information for further comprehensively understandding the pharmacological activity and potential toxicity of the roots of PM in the clinic.

何首乌肝毒性不良反应的报道屡见不鲜，但其肝毒性机制以及引起肝毒性的体内物质基础目前还缺乏系统研究。我们前期对何首乌及其单体成分的体内外肝毒性研究结果显示，何首乌中确实存在可致肝细胞损伤的成分，有些成分虽然在体外实验中肝细胞毒性不明显，但体内实验却表现出明显肝毒性，这可能与何首乌的体内异常代谢产物有关，所以要全面了解何首乌的肝毒性，必须结合其体内代谢来研究。本项目在对何首乌主要成分的质谱裂解规律已有认识的基础上，利用组合质谱的技术优势，研究体内微量成分的质谱裂解规律与结构信息，分析何首乌吸收入血后的代谢物质基础与代谢规律研究。同时采用代谢组学研究手段，结合何首乌成分分析和毒性特点，寻找与肝毒性相关的生物标志物，构建代谢图谱的变化特征，揭示何首乌引起肝毒性作用的体内物质基础和机制，为何首乌临床安全用药提供科学依据。

背景：何首乌是我国临床常用的中药品种，近年来大量临床证实其具有肝损伤作用，但引起肝损伤的作用物质基础和存在形式仍然未可知，相关中毒机制仍需进一步证实。.目的：利用组合质谱技术分析何首乌体内代谢成分，同时采用组学方法探讨何首乌肝损伤的潜在机制。.方法：采用UHPLC-LTQ-Orbitrap MS分别对何首乌单体成分，何首乌醇提物及大鼠体内的化代谢成分进行定性与定量分析，获得其化学物质基础和体内代谢物质基础；并利用代谢组学技术分析大鼠血液、尿液代谢谱变化，利用iTRAQ标记的蛋白质组学分析大鼠肝组织蛋白谱表达变化，探讨其毒性机制。.结果：建立了快速定性定量分析何首乌体外化学成分和体内代谢成分的鉴定策略和检测方法，完善了何首乌主要成分的高分辨多级质谱裂解途径，并总结了其质谱裂解规律和亚结构特征离子，在线鉴定何首乌化学成分50余种，比较不同来源和炮制何首乌的主要成分的区别，构建了何首乌主要成分的代谢通路，基本阐明何首乌大鼠体内代谢物质基础。代谢组学研究证实口服何首乌改变了大鼠花生四烯酸代谢通路，甘油磷脂代谢通路等相关代谢谱的变化。肝脏组织蛋白质组学研究共鉴定差异性蛋白108个，其中上调蛋白45个，下调蛋白63个，通路富集分析表明这些差异蛋白主要参与MAPK信号通路、谷胱甘肽代谢、亚油酸代谢通路、视黄醇代谢等通路，.结论：何首乌体内代谢成分主要以大黄素、大黄素甲醚的I、II代谢产物为主，说明何首乌致肝损伤的成分可能与蒽醌苷元密切相关；代谢组学与蛋白质组学研究结果初步表明，何首乌致肝损伤可能为对肝细胞的直接损伤，相关蛋白参与了肝细胞的正常生长、分化和凋亡等调控过程，其干预可能与细胞氧化应激导致线粒体功能变化有关，该研究仍需进一步实验证实。