药物肝肾损伤的细胞器代谢组深度分析及代谢传递介导的互作网络解析

以靶器官整体作为研究对象开展代谢组学研究极易因“拉平现象”造成漏筛或误导。为解决这一问题，本项目提出基于细胞器代谢传递和互作网络的药物器官毒性作用机制研究新思路，并针对“细胞器制备分离”、“代谢物深度分析”和“互作网络解析”三个关键分析技术瓶颈开展系统探索：建立基于蛋白标签重组膜蛋白的细胞器免疫纯化分离新方法，实现多细胞器同时、快速、有效分离；建立整合“配对衍生化”、“拟靶标检测”和“一测多评”的LC/MS分析新方法，实现细胞器代谢组宽覆盖、高灵敏定量分析；建立代谢传递介导的细胞器互作网络解析新策略，从代谢物“交换”和“转化”角度解析细胞器间互作关系；利用所建立的方法从细胞器层面识别对乙酰氨基酚肝损伤和顺铂肾损伤毒性，并从代谢传递介导的互作网络角度阐释毒性机制。本项目的开展将建立以细胞器为微观研究单元的药物毒性识别和评价方法学体系，为药物早期成药性评价研究、临床安全用药提供技术支撑。

"Organelle isolation", "depth analysis of metabolites" and "interaction network analysis" are the three key technical bottlenecks in organelle metabolomics research on drug-induced organ toxicity. In this project, a new method of organelle immunopurification based on epitope-tagged recombinant membrane protein was developed to realize simultaneous, rapid and effective separation of multiple organelles; "twin derivatization strategy", "pseudo-targeted detection strategy" and " quantitative analysis of multi-components by single-marker strategy " were used in combination with LC-MS to realize the detection of trace amounts of metabolites in organelles with high sensitivity and wide coverage; network analysis of organelle interactions mediated by "metabolite exchange" and "metabolite conversion" was based on metabolic flux analysis. Based on metabolomics analysis data of organelles, establishment of a static-dynamic network model and identification of key metabolic nodes and pathways of organ toxicity were achieved, during which metabolic flow analysis technology was used to analyze the interaction between organelles from the perspective of "metabolite exchange" and "metabolite transformation". Furthermore, these new methods were applied to identify the toxicity of acetaminophen induced liver injury and cisplatin induced kidney injury from the organelle level, and to reveal the critical molecular initiation events, toxic metabolic pathways and related mechanism of toxicity from the perspective of interaction networks mediated by metabolic delivery. The implementation of this project will improve the research ideas and methods of pharmaco-metabonomics, and promote the development of research level of drug-induced organ toxicity metabolomics from organ and tissue to cells and organelle. The new drug toxicity identification system and individualized evaluation methodology with organelles as microscopic research units can provide technical support for evaluation of early druggability and medication safety in clinical practice.