基于质谱成像代谢组学的中药肝、肾毒性分析新方法发展及其应用研究

Drug-induced hepatotoxicity and nephrotoxicity has become a major bottleneck restricting the development of traditional Chinese medicine (TCM). An accurate and sensitive method for characterizing drug-induced hepatotoxicity and nephrotoxicity is in great needed for elucidating metabolic mechanism and change rules of hepatic and renal toxicity of TCM. Here, we proposed a new strategy to analyze the TCM-induced hepatotoxicity and nephrotoxicity by combining mass spectrometry imaging technology with metabolomics method. The aristolochic acid (AA)-induced liver and kidney damage animal models were first built for MSI-based metabolomics study at the level of whole animal and tissues, respectively. The toxicity biomarkers of drug hepatotoxicity and nephrotoxicity were screened, and the space↔time↔dose variations of the screened biomarkers were also explored. Furthermore, in vitro hepatotoxicity and nephrotoxicity cell models were established to verify the in vivo toxicity biomarkers. On this basis, the metabolic pathways of the toxicity markers were analyzed, and the key metabolic enzymes and transcriptional mRNA of the liver and kidney toxicity markers were also characterized. The spatial distribution of metabolites, metabolic enzymes and mRNA were matched to perform correlation analysis. A "bottom-up" strategy, from metabolite to metabolic enzyme and transcriptional mRNA, for the explanation of TCM hepatotoxicity and nephrotoxicity was realized in this study. This will provide new perspective and analytical method for the evaluation of hepatotoxicity and nephrotoxicity effect of TCM, and facilitate the explanation of toxic mechanism.

药物诱导产生的肝、肾毒性是当前制约中药发展的一大瓶颈问题，发展准确、敏感的药物肝、肾毒性表征新方法对于阐明中药肝、肾毒性代谢机制和变化规律非常关键。本项目拟采用质谱成像技术与代谢组学相结合的思路开展中药肝、肾毒性分析新方法及其应用研究。首先以马兜铃酸诱导肝、肾损伤动物模型，分别从整体动物、组织水平开展基于质谱成像技术的代谢组学研究，寻找药物肝、肾毒性原位标志物，并探究其空间↔时间↔剂量变化规律；进一步建立体外肝、肾毒性细胞模型，从细胞水平验证体内肝、肾毒性标志物；在此基础上，对毒性标志物进行代谢通路分析，对其上游调控关键代谢酶、mRNA进行原位表征，并将三者的空间分布进行匹配关联，从代谢物→靶向代谢酶→靶向mRNA三个水平“自下而上”可视化地揭示药物肝、肾毒性发生发展规律，为中药肝、肾毒性评价、毒性代谢机制阐释提供新的研究视角和分析手段。

中药诱导所产生的肝毒性和肾毒性是中药被迫撤出市场或限制使用的重要原因之一，已经成为制约中医药持续发展、走向世界的一大瓶颈问题。本项目旨在通过开发准确、敏感的药物肝、肾毒性表征新方法，揭示中药肝、肾毒性代谢机制和变化规律。首先，采用可对生物组织中代谢物进行原位表征且在检测覆盖范围上具有显著“互补性”的AFAI与MALDI质谱成像技术，整合代谢组学的整体性优势，建立代谢物高灵敏、高覆盖、高特异的质谱成像代谢组学方法。进一步，以肝、肾毒性明确的马兜铃酸为对象开展了质谱成像代谢组学研究，发现谷胱甘肽、油酸、亚油酸、花生四烯酸、苯丙氨酸、精胺、亚精胺等一系列代谢物在中药诱导的肝肾毒性反应中发生显著的异常改变。更进一步地，建立了人原代肝细胞、人原代肾细胞药物毒性体外评价模型，分析马兜铃酸对人原代肝细胞、人原代肾细胞代谢轮廓的改变情况，筛选细胞水平的毒性代谢标志物，并将其与动物水平的肝、肾毒性代谢标志物进行了比较，结合代谢酶和转录组学分析，确定谷胱甘肽代谢、脂肪酸生物合成、磷脂代谢、多胺代谢、苯丙氨酸代谢通路的异常改变与中药肝肾毒性的发生密切相关。相关研究方法和结论为中药肝、肾毒性分析提供新的分析手段和研究策略。经过课题组成员的共同努力，目前相关研究成果在Anal.Chem., Theranostics, Anal.Chim.Acta, J.Chromatogr.A等药物分析领域权威期刊发表SCI论文6篇，其中中科院1区TOP期刊4篇；授权发明专利4项；参加学术会议8人次；培养博士研究生1名，硕士研究生2名。