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,Beijing University of Chinese Medicine Beijing 102488, China.
Zhongguo Zhong Yao Za Zhi. 2023 Jan;48(1):140-147. doi: 10.19540/j.cnki.cjcmm.20220914.401.
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 1 100 μg·mL(-1) of the absolute lethal concentration and 448 μg·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.
本研究采用斑马鱼模型探讨闹羊花（RMF）的肝毒性。根据不同浓度RMF作用下受精后4天斑马鱼幼鱼的存活数量计算死亡率，并拟合剂量-毒性曲线以初步评估RMF的毒性。通过苏木精-伊红（HE）染色和吖啶橙（AO）染色观察处理组和空白对照组中RMF亚致死浓度下的肝脏表型。同时，测定丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）的活性以确认RMF的肝毒性。采用实时定量聚合酶链反应（real-time PCR）和蛋白质免疫印迹法测定斑马鱼幼鱼中基因和蛋白质的表达。运用气相色谱-飞行时间质谱（GC-TOF-MS）进行非靶向代谢组学检测以探究其作用机制。结果表明，RMF对斑马鱼幼鱼的毒性呈剂量依赖性，绝对致死浓度为1 100 μg·mL(-1)，亚致死浓度为448 μg·mL(-1)。RMF亚致死浓度下的斑马鱼幼鱼出现肝细胞凋亡和变性。实验结束时，斑马鱼幼鱼中ALT和AST的含量呈剂量依赖性显著增加。在亚致死浓度下，与空白对照组相比，斑马鱼幼鱼中与凋亡相关的基因和蛋白质表达显著增加。非靶向代谢组学结果显示，与RMF肝毒性相关的重要代谢物主要富集在丙氨酸、天冬氨酸、谷氨酸等途径中。综上所述，推测RMF对斑马鱼幼鱼具有一定的肝毒性，其机制可能与凋亡有关。
