基于“CYP450-UGT”双重模式的吴茱萸致肝毒性和甘草炮制减毒的物质基础及机理研究

Evodiae Fructus (EF), having slight toxicity, needs to be processed with licorice juice, which is recorded in the Chinese pharmacopoeia. Some research showed that hepatotoxicity resulted from EF was connected with associated protein expression. But present attempts lack metabolic process of poisonous substance in vivo and in vitro. And there is little study on the mechanism of toxicity attenuated by Evodiae Fructus processed (EFP) with licorice juice. Recently, our studies showed that hepatotoxicity of EFP was remarkably decreased, while bioavailability of main alkaloids from EFP had significant increase compared with those from EF. We speculated that toxicity of EF could be related to its metabolites in vivo. Meanwhile, there are constituents inducing activities of UGTs in licorice. So we first proposed the idea of studies on material basis and mechanism-based “CYP450-UGT” hepatotoxicity resulted from EF and toxicity attenuated by EFP: 1. research the effect of hepatotoxicity and pharmacokinetic resulted from metabolism activation of EF in rats ; monitor the relationship of the levels of GSH, concentration of EF and hepatotoxicity in cellular level; 2. identify the metabolites of EF and the related enzymes；3. investigate the processes of inhibiting CYP450s and inducing UGTs of licorice. We will employ various technologies and methods such as drug metabolism, toxicology chemistry and molecular biology technologies. The study will provide strongly scientific evidence and reference for the study of toxicity attenuation of the same processing of TCM. Clarifying mechanism of toxicity attenuated by Chinese crude drug processing is the scientific problem of the fair use of TCM, which needs to be solved promptly.

吴茱萸有小毒，《中国药典》规定用甘草炮制。一些研究表明吴茱萸致肝毒性与相关蛋白表达有关，但目前的努力缺乏毒性物质体内、外代谢过程的支撑，且甘草制吴茱萸减毒机理不明。我们研究表明，甘草炮制吴茱萸肝毒性明显降低，吴茱萸主要生物碱类在大鼠体内生物利用度明显增加，推测吴茱萸肝毒性与体内代谢物密切相关。同时，甘草中有诱导II 相代谢酶的成分。因此，我们首次提出基于“CYP 450-UGT”双重模式的吴茱萸致肝毒性和甘草炮制减毒的物质基础和机理研究：1. 从整体动物层面研究吴茱萸在大鼠体内代谢活化对肝毒性及药动学的影响；从细胞层面监测GSH水平-吴茱萸浓度-肝毒性的相关性；2. 鉴定吴茱萸的代谢物和相关的酶；3. 考察甘草抑制P450酶和诱导UGT的代谢过程。本课题融合药物代谢学、毒理化学和分子生物学等技术，为同类中药炮制减毒研究提供模式和借鉴，阐明炮制减毒机理是中药合理使用亟待解决的科学问题。

吴茱萸收载于中国药典，有小毒，需要用甘草炮制，其肝毒性及炮制减毒的物质基础和机制不清楚。本研究通过在动物和细胞水平建立了吴茱萸致肝毒性模型，考察了CYP3A的诱导剂/抑制剂对吴茱萸致肝毒性的影响，表明诱导剂增强了吴茱萸致小鼠肝毒性和L02细胞毒性，抑制剂降低了其毒性，首次阐明了基于代谢活化的吴茱萸致肝毒性的机制。通过建立吴茱萸指纹图谱，利用OPLS法建立谱-毒相关性，找到了吴茱萸中5种主要生物碱与肝毒性呈正相关、柠檬苦素与肝毒性负相关。基于LC-MS/MS建立了吴茱萸中5种主要生物碱类成分在SD大鼠体内有/无P450酶诱导剂/抑制剂的药动学分析法，表明地塞米松诱导组加快了5种生物碱在SD大鼠体内的代谢，酮康唑抑制组和柠檬苦素组均抑制了生物碱的代谢，从药动学度角度进一步验证了吴茱萸经代谢活化的肝毒性的物质基础和机制。.基于UPLC-Q-Exactive-MS建立吴茱萸及其体内外代谢物的分析方法和靶向代谢组学分析法，全面表征吴茱萸成分及体内外代谢物，分析鉴定了吴茱萸在大鼠血浆、胆汁、尿液和粪便及肝微粒体孵育的成分，吴茱萸生物碱类成分主要发生羟基化、脱甲基、脱氢、葡萄糖醛酸化和硫酸化反应，推导了其在体内的代谢途径，并捕获到吴茱萸碱和吴茱萸次碱的GSH结合物。探讨了吴茱萸生物碱对P450酶和UGT的抑制作用，阐明了吴茱萸致肝毒性的生化机制。.甘草汁炮制吴茱萸后能够显著降低吴茱萸的肝毒性，基于P450酶和UGT酶建立探针底物法，比较了甘草不同提取部位及其肠菌代谢液对P450酶的抑制作用，结合建立的甘草指纹图谱，证明甘草中以甘草素和异甘草素为母核的黄酮类在肠菌作用下代谢成苷元，发挥对P450 3A的抑制作用，从而减少吴茱萸生物碱代谢活化而具有减毒作用。同时，甘草能够抑制吴茱萸生物碱与GSH结合物的产生，增加吴茱萸体内II相代谢物的种类和含量，促进毒性活化中间体与葡萄糖醛酸结合，加速有毒成分排出体外，阐释了甘草减毒的物质基础和机制。.本研究丰富了中药炮制减毒的科学内涵，拓宽本领域研究思路，使中药致毒和炮制减毒的物质基础和机制研究取得新进展，具有重要的科学意义和应用价值，为同类中药研究提供新的研究方法、模式和借鉴。