马兜铃酸对斑马鱼的耳毒性及肾毒性研究

目的探讨马兜铃酸对斑马鱼的耳毒性及肾毒性。方法①使用不同浓度的马兜铃酸处理受精后5天(5 dpf)的AB系斑马鱼,探索马兜铃酸对斑马鱼的最大非致死浓度(maximum non-lethal concentration,MNLC)。②依据MNLC选取4个浓度(MNLC、1/2MNLC、1/4MNLC及1/8MNLC)为实验组,2.5μg/ml庆大霉素为阳性对照组,溶剂组[1.0%二甲基亚砜(DMSO)]为阴性对照组,每组分别处理30尾斑马鱼。24 h后,每组取10尾斑马鱼用DASPEI染料对斑马鱼身体侧线毛细胞进行荧光染色,分析马兜铃酸对毛细胞的影响。每组剩余的20尾斑马鱼继续饲养至72 h,观察肾包膜的情况。结果①实验所用马兜铃酸浓度无法得出其MNLC,取其中未出现斑马鱼死亡的最大浓度(3.4μg/ml)为MNLC。②庆大霉素组毛细胞的损伤率为88.5%,与阴性对照组比较,差异有统计学意义(P<0.01);马兜铃酸MNLC组、1/2MNLC组、1/4MNLC组及1/8MNLC组毛细胞损伤率分别为-7.2%、6.9%、-0.4%、12.4%,与阴性对照组比较,差异均无统计学意义(P>0.05)。③继续处理斑马鱼至72 h后,马兜铃酸MNLC组斑马鱼全部死亡;1/2MNLC组10%的斑马鱼死亡,余下11.1%的斑马鱼出现严重的肾包膜水肿;1/4MNLC组、1/8MNLC组未观察到肾包膜水肿。结论本实验所用浓度的马兜铃酸对斑马鱼有肾毒性,无耳毒性。

The Ototoxicity and Nephrotoxicity of Aristolochic Acid in Zebrafish

Objective To investigate the ototoxicity and nephrotoxicity of aristolochic acid in zebrafish.Methods Five different concentrations of aristolochic acid were used to determine the maximum non-lethal concentration(MNLC) of aristolochic acid in zebrafish. Four concentrations( MNLC, 1/2 MNLC, 1/4 MNLC and 1/8 MNLC) were selected to evaluate ototoxicity(hair cell loss) and nephrotoxicity(edema of renal capsule). 2.5 μg/mL gentamicin was used as a positive control, 1% DMSO served as a negative control. Thirty zebrafish were treated for 24 hours in each group, and 10 of them were stained with fluorescent vital dye for ototoxicity evaluation. The rest zebrafish in each group were continue treated until 72 hours for nephrotoxicity evaluation.Results ①MNLC could not be determined from the concentration selected in our study. As a matter of experience, the greatest concentration(3.4 μg/ml) in which there was no death was taken as the MNLC. ②The hair cell loss percentage of zebrafish in the gentamicin group was 88.5%. When compared to the negative control group, the differences of hair cell loss were statistically significant(P<0.001). Zebrafish treated with aristolochic acid at concentrations of MNLC, 1/2 MNLC, 1/4 MNLC and 1/8 MNLC showed hair cell loss of-7.2%,6.9%,-0.4% and 12.4%, respectively. When compared to the negative control group, the differences were not statistically significant. ③At concentration of MNLC, all zebrafish were dead after treated for 72 hours. At concentration of 1/2 MNLC, 10% of zebrafish were dead, and 11.1% of the rest were observed edema of renal capsule. There was no edema of renal capsule in zebrafish treated with concentration of 1/4 MNLC and 1/8 MNLC.Conclusion Aristolochic acid presented nephrotoxicity but not ototoxicity in zebrafish.