中药注射剂（茵栀黄）的反应性代谢谱及致敏潜因研究

In recent years, there are increasing clinical reports and social close attention on the anaphylaxis and organ toxicities associated with a globally increasing application of Chinese herbal injection to treat various chronic diseases and to promote health. New small-molecule-based approaches for detecting and characterizing chemically reactive metabolites (RMs) from herbal injection show a promising alternative method for interpreting the molecular mechanisms of the herb-induced toxicity. In this funding project, Yinzhihuang injection was selected as a representative herbal injection, and a new tag, D-γ-Glu-Cys-Lys (D-GSK),is used for chemoselective trapping of the RMs generated from the in vitro incubations of the complex injection in liver microsomes or cultured liver cells, followed by LC-MS targeted profiling of the tagged RMs from the complex mixtures. Based on the GSK tag for DESI-MS monitoring of RMs in cell-culture supernatant upon damage of the cell, a new analytical strategy termed "reactive metabolic profiling" is proposed by the integration of multi-level data of chemical structures, concentration distribution and cell viability for global screening and assessing of drug bioactivation-dependent anaphylaxis and toxicities by the herbal injection. Thus the study will provide a useful and rapid method for researching the untargeted metabolites as well as a potential key breakthrough in understanding the roles of low-abundant reactive metabolites of complicated Chinese Traditional Medicine in vitro/ex vivo.

近年来，中药注射剂致敏反应凸显多发性和普遍性趋势，对其成因与预测研究，尚无有效对策。课题从中药反应性代谢产物（RM）入手，提出复方制剂的代谢活化致敏潜因新思路，寻找可能的致敏诱因将助于进一步加深对中药毒副潜性物质基础的新认识。本项目以D 型γ-谷氨酰-半胱氨酰-赖氨酸（D-GSK）为分子捕获剂和质量标签，运用敞开式电离质谱直接检测和信息挖掘技术，对茵栀黄中药注射剂体外代谢活化体系可能产生的RM 分子进行同族体靶向轮廓分析。基于新型寡肽标签-在线质谱技术对活体细胞内外基质中RM 信号的快速捕识，同时整合应激细胞存活率监测数据，拟构建多组分中药"反应性代谢谱"分析策略，以系统获取茵栀黄RM分子组的整体多层次信息（结构、浓度、活化途径和细胞毒性），从而为深入探讨复方制剂致敏发生的分子机制、配伍减毒原理以及潜在致敏原的快速筛选提供新的线索、依据和手段。

基于药酶依赖反应性分子探索药物不良反应发生机制是一个新的研究视角；近年来，从活性小分子代谢物角度研究中药注射液致敏成因也逐渐成为热点。课题以茵栀黄注射液为研究对象，针对复方中药代谢体系中的反应性分子组分析及代谢活化表征，提出多组分中药“反应性代谢谱”分析策略，建立基于三肽Trp-Cys-Lys质谱探针的定量靶向代谢谱分析方法和功能食品/中草药活性小分子非目标广谱检测新型高分辨多级裂解质谱技术；构建复方中药茵栀黄质谱化学指纹、多组分血浆药动学轮廓与葡醛酸转移酶代谢谱；发展体外药酶活性评价与茵栀黄代谢-细胞应激存活检测体系。首次系统解析茵栀黄化学组分32种，鉴别出注射液中稳定存在但尚未见报道的11种化合物，为茵栀黄的科学精准质量控制和深入研究其疗效及致敏性提供重要物质基础。利用α-萘异硫氰酸酯（ANIT）诱导急性小鼠肝内胆汁淤积模型，结合中药代谢-暴露-效应参数，对茵栀黄组分调节胆酸代谢的靶向活性筛选，发现活性成分绿原酸和栀子苷具有显著的肝保护效果；进一步发现它们可逆转ANIT诱导的胆汁酸代谢调控（合成/转运）相关基因以及炎症相关基因mRNA及蛋白表达变化，并通过抑制STAT3、NFκB炎症信号通路。项目从胆汁酸代谢/致敏炎性信号通路角度探讨其抗胆汁淤积性肝损伤作用的新机制，为深入研究茵栀黄临床退黄疗效及致敏分子机理提供了新的线索。.课题资助期内已在国内外中药和药物代谢领域学术刊物上发表高质量科研论文13篇，其中SCI源刊论文11篇、特邀评述论文2篇、特邀封面论文1篇；SCI收录刊物总引达95次。研究成果被美国化学会新闻周刊(ACS News Service Weekly PressPac)及时报道和ScienceDaily等多家国际科技新闻网转载。项目申请国家发明专利3项，获批授权2项。