基于细胞代谢组学-血清药理学-分子生物学的京大戟、芫花致肝毒性机制、物质基础和炮制前后肝毒性变化的研究

In order to enhance new technology related with detection and diagnosis, the research concentrated on the cell metabonomic at the direction of Chinese Medical Theory. Coupled with metabonomics, the pharmacology of serum and molecular biology, the hepatotoxicity, the toxic mechanism and toxic basis were investigated on raw and processed herbs, using Radix Euphorbiae Pekinensis and Genkwa Flos as model herbs.Pharmaceutical analysis, phytochemistry, pharmacology, molecular biology and informatics were integrated to create pharmacological models in vitro and vivo.Using UPLC-MS, a group biomarkers related to hepatotoxicity can be screened out through the combination of the expression profiles of cell metabolomics and biological metabolomics.Through multi-dimensional analysis, linked with chemical, biological,gene and protin indicators, histopathology, serum pharmacology, cell factor and enzyme activity assay, the toxicity mechanism of liver induced by Radix Euphorbiae Pekinensis and Genkwa Flos was clarified; the targets of toxic effects or toxic components of herbs were determined. Comparing the results of the two herbs, the laws of hepatotoxicity induced by the herbs were explored, which provide scientific support and theoretical basis for the establishment of biomarkers of liver toxicity in monitoring and prevention. The difference and causality of the hepatotoxicity-biomarkers-the processing of Chinese herbal medcine between raw and processed herbs were discussed to prove the processing theory of Chinese herbal medcine scientific and reasonable. The investigation explores the new method to resesrch hepatotoxicity induced by Chinese herbal medicine on cellular and molecular level, and provide useful information for the quality and safety assessment of Chinese herbal medcines.

以加强与人类健康相关的检测与诊断新技术、新方法研究为目的，中医药理论为指导，细胞代谢组学为主线，综合应用生物代谢组学、血清药理学、分子生物学多学科理论与分析技术，以集成应用性创新思路，在京大戟、芫花大鼠肝毒性作用机制、物质基础和炮制前后肝毒性变化研究中发挥作用。建立肝毒性评价体内外药理模型，应用液-质技术，将细胞代谢组学与生物代谢组学表达谱形成互补,确认一组与肝毒性相关生物标记物，结合生化指标、病理切片、血清药理学，分子生物学对中毒肝组织、血清的特定酶、细胞因子等基因和蛋白表达水平测定，从分子水平探讨中药肝毒性作用机制。以两味药研究结果互相验证，经多维分析，阐明京大戟、芫花致肝毒性作用机制，确定毒性物质基础，探讨炮制前后毒性作用及由此引起生物标记物变化的因果关系，验证炮制理论科学性。从细胞分子水平探索中药致肝毒性研究的新方法，为建立临床肝毒性标记物监测和安全性评价提供科学支撑和新的理论依据

本项目以细胞代谢组学为主线，综合应用生物代谢组学、血清药理学、分子生物学多学科理论与分析技术，对芫花、京大戟研究结果互相验证、多维分析。验证了炮制可减轻芫花和京大戟毒性，阐明了致肝毒性作用机制，确定了毒性物质基础。.以体内外肝细胞模型为指导，筛选出芫花、京大戟致肝毒性部位分别为95%乙醇提取物的氯仿和正丁醇萃取物，从毒性部位中得到芫花和京大戟中各7种单体成分。采用UPLC-MS/MS对芫花、京大戟致肝毒性血清、尿液进行生物代谢组学研究，确认了芫花致肝毒性血清、尿液生物标记物10和13个；其氯仿萃取物血清和尿液生物标记物各为8个；京大戟致肝毒性血清、尿液生物标记物12和9个；其正丁醇萃取物血清和尿液生物标记物为10和6个。芫花、京大戟血清和尿液样品生品与炮制组得到了良好的区分，炮制组与健康对照组接近，炮制后药材降低了毒性，生品能够扰乱健康动物血清和尿液中内源性代谢物，进而影响其正常生理状态。采用UPLC-MS/MS对芫花和京大戟致肝损伤HL-7702细胞裂解液代谢组学进行了研究，以含药血清培养基孵育细胞，测试细胞增殖抑制率和氧化损伤指标、细胞炎症因子和细胞内ATP酶水平，获得芫花、京大戟致肝毒性HL-7702细胞裂解液生物标记物各8个。代谢组学研究推测致肝损伤机制可能与氧化应激、氨基酸代谢、肠道菌群代谢、鞘酯代谢和磷脂代谢等生物途径相关。采用酶联免疫测定参与芫花肝毒性磷脂酶A2/溶血卵磷脂（LPA2/LPC）、鞘氨醇激酶/1-磷酸鞘氨醇（Sphk/S1P）代谢通路活性物质与关键酶，表明神经酰胺与1-磷酸鞘氨醇平衡紊乱、脂肪酸代谢异常参与了芫花致肝细胞损伤过程。.采用Q Exactive Focus UPLC-MS方法，对芫花和京大戟含药血清与HL-7702细胞亲和性成分鉴定。芫花、京大戟亲和性成分7个和11个。经细胞毒理学评价，芫花、京大戟中5和6个成分具备细胞毒性。采用LC-MS/MS测定芫花和京大戟给药后血清、尿液中与肝毒性显著性相关的10个内源物动态变化趋势。血清中CA，5-OP，GSA和3-IS等4种指标灵敏于传统指标AST和ALT，本研究为发掘灵敏快速肝毒性早期诊断做出了贡献。