摘要
采用斑马鱼幼鱼模型探讨闹羊花对肝脏的毒性作用,根据闹羊花不同浓度暴露处理下受精后4 d斑马鱼幼鱼的存活数计算死亡率,并拟合其量毒曲线初步评估闹羊花的毒性大小,从而通过苏木精-伊红(hematoxylin-eosin,HE)染色法和吖啶橙(aeridine orange,AO)染色法对比观察闹羊花提取物亚致死浓度处理组和阴性对照组的肝脏表型,并测定对比不同处理组谷丙转氨酶(alanine aminotransferase,ALT)和谷草转氨酶(aspartate aminotransferase,AST)的活性以确认闹羊花的肝脏毒性,进一步采用实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)及蛋白质印迹法检测斑马鱼幼鱼体内基因及蛋白的表达水平,并通过气相色谱时间飞行质谱联用(GC-TOF-MS)进行非靶向代谢组学检测,以探讨其作用机制。结果显示,闹羊花对斑马鱼幼鱼的毒性大小呈剂量依赖性,全部致死浓度为1100μg·mL^(-1),亚致死浓度为448μg·mL^(-1);在亚致死浓度下,闹羊花暴露处理能够引起斑马鱼幼鱼肝脏组织结构紊乱,肝细胞凋亡、变性;实验终点的斑马鱼幼鱼体内ALT和AST水平显著提高,且呈剂量依赖性,而闹羊花给药组的斑马鱼幼鱼体内凋亡相关基因及蛋白的表达水平与阴性对照组相比皆显著上升。通过非靶向代谢组学发现闹羊花致肝毒性相关的重要代谢物主要富集于丙氨酸、天冬氨酸和谷氨酸代谢等途径。综上推测闹羊花对斑马鱼幼鱼有一定肝毒性,其作用机制或与细胞凋亡相关。
This study used the zebrafish model to explore the hepatotoxicity of Rhododendri Mollis Flos(RMF).The mortality was calculated according to the number of the survival of zebrafish larvae 4 days after fertilization under different concentration of RMF,and the dose-toxicity curve was fitted to preliminarily evaluate the toxicity of RMF.The liver phenotypes under the sublethal concentration of RMF in the treatment group and the blank control group were observed by hematoxylin-eosin(HE)staining and acridine orange(AO)staining.Meanwhile,the activities of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were determined to confirm the hepatotoxicity of RMF.Real-time quantitative polymerase chain reaction(real-time PCR)and Western blot were used to determine the expressions of genes and proteins in zebrafish larvae.Gas chromatography time-of-flight mass spectrometry(GC-TOF-MS)was used to conduct untargeted metabolomics testing to explore the mechanism.The results showed that the toxicity of RMF to zebrafish larvae was dose-dependent,with 1100μg·mL^(-1)of the absolute lethal concentration and 448μg·mL^(-1)of sublethal concentration.The hepatocyte apoptosis and degeneration appeared in the zebrafish larvae under the sublethal concentration of RMF.The content of ALT and AST in zebrafish larvae at the end of the experiment was significantly increased in a dose-dependent manner.Under the sublethal concentration,the expressions of genes and proteins related to apoptosis in zebrafish larvae were significantly increased as compared with the blank control group.The results of untargeted metabolomics showed that the important metabolites related to the he-patotoxicity of RMF were mainly enriched in alanine,aspartic acid,glutamic acid,and other pathways.In conclusion,it is inferred that RMF has certain hepatotoxicity to zebrafish larvae,and its mechanism may be related to apoptosis.
作者
陈美琳
李芝奇
范琦琦
郭思敏
蔡琼
林瑞超
吴嘉瑞
赵崇军
CHEN Mei-lin;LI Zhi-qi;FAN Qi-qi;GUO Si-min;CAI Qiong;LIN Rui-chao;WU Jia-rui;ZHAO Chong-jun(Beijing Key Laboratory for Quality Evaluation of Chinese Materia Medica,School of Chinese Materia Medica,Bejing University of Chinese Medicine,Beijing 102488,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2023年第1期140-147,共8页
China Journal of Chinese Materia Medica
基金
国家“重大新药创制”科技重大专项(2018ZX09735005)
国家中医药管理局公益性中医药行业科研专项(201507004)
北京中医药大学新教师基金项目(2020-JYB-XJSJJ-009)
国家中医药传承创新团队子项目(ZYYCXTD-C-202005-10)。
关键词
闹羊花
斑马鱼
肝毒性
细胞凋亡
Rhododendri Mollis Flos
zebrafish
hepatotoxicity
apoptosis
作者简介
通信作者:赵崇军,博士,助理研究员,主要从事中药品质评价与安全性研究,E-mail:1014256537@qq.com;通信作者:林瑞超,博士,教授,主要从事中药品质评价、中药及天然药物活性成分及安全性评价、中药标准及标准物质研究,E-mail:linrch307@sina.com;通信作者:吴嘉瑞,博士,教授,主要从事临床药物上市后再评价研究,E-mail:exogamy@163.com;陈美琳,硕士研究生,E-mail:erdongXD@163.com。
