目的 基于斑马鱼模型,通过生化指标、病理组织学、体内细胞凋亡检测和分子生物学技术整合评价模式对吴茱萸提取物肝毒性进行综合评价。方法 给予亚致死剂量(10)下的吴茱萸提取物24 h,测定丙氨酸氨基转移酶(alanine aminotransferase,ALT)和天冬氨酸氨基转移酶(aspartic aminotransaminase,AST)活性,观察斑马鱼肝脏病理组织,吖啶橙(aeridine orange,AO)染色观察肝脏细胞凋亡状态,结合蛋白质印迹法Western blotting、实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)和代谢组学初步探究吴茱萸毒性机制。结果 给予吴茱萸提取物后,斑马鱼AST、ALT活性均显著升高,肝脏病理组织苏木精-伊红染色法(hematoxylin-eosin staining,HE)染色及AO染色观察提示斑马鱼肝脏受损,细胞凋亡增加,相关凋亡基因和蛋白表达水平显著上升,非靶向代谢组学结果表明吴茱萸提取物影响斑马鱼氨基酸代谢。结论 通过斑马鱼模型整合评价方法从多水平、多角度、多指标实现吴茱萸诱导肝毒性的综合评价,为斑马鱼在中药肝毒性评价的适用性提供了支撑。

Objective Based on the zebrafish model, the hepatotoxicity of extract of Wuzhuyu (Euodiae Fructus) was comprehensively evaluated by biochemical index evaluation, histopathological evaluation, cell apoptosis in vivo, and integrated evaluation model of molecular biology techniques. Methods The sublethal dose (< LC₁₀) of Euodiae Fructus extract was administered for 24 h, the activity indexes of alanine aminotransferase (ALT) and aspartic aminotransaminase (AST) were determined, the liver pathological tissues of zebrafish were observed, and the apoptosis status of liver cells was observed by aeridine orange (AO) staining. Western blotting (WB), real-time quantitative polymerase chain reaction (RT-qPCR) and metabolomics were combined to preliminarily explore the toxicity mechanism of Euodiae Fructus, and comprehensively evaluate the liver injury zebrafish model induced by Euodiae Fructus extract. Results The transaminase level of zebrafish was significantly increased, HE staining and AO staining observation of liver pathological tissue showed that zebrafish liver was damaged, cell apoptosis was increased, and the expression levels of related apoptotic genes and proteins were significantly increased. The results of untargeted metabolomics technique showed that the extract of Euodiae Fructus affected amino acid metabolism of zebrafish. Conclusion Zebrafish model integrated evaluation method was used to achieve comprehensive evaluation of liver toxicity induced by Euodiae Fructus from multiple levels, multiple perspectives and multiple indicators, providing support for the applicability of zebrafish in liver toxicity evaluation of traditional Chinese medicine.

R285

国家科技重大专项（2018ZX09735005）；北京中医药大学新教师启动基金项目（2020-JYB-XJSJJ-009）；北京中医药大学重点攻关项目（2020-JYB-ZDGG-035）