目的 以对乙酰氨基酚(APAP)为例,通过优化其对斑马鱼幼鱼肝毒性评价的影响因素和参数,确立基于斑马鱼幼鱼模型的肝毒性快速评价方法及药物肝损伤分级方法。方法 利用不同浓度APAP处理斑马鱼幼鱼,在实验终点(药物暴露后48 h/24 h)统计不同处理组斑马鱼的死亡率,并绘制“死亡率—浓度”效应曲线,计算最大耐受浓度(LC0)和半数致死浓度(LC50);考察并优化斑马鱼幼鱼在APAP毒性研究中的相关影响因素,包括斑马鱼鱼龄 [3 dpf、4 dpf(受精后天数:dpf)]、给药暴露时间(48 h、24 h)和孔板(6孔板、12孔板;6 mL、4 mL),并从以上3个水平设计正交实验,建立斑马鱼肝毒性模型的最佳实验条件参数。另择3 dpf斑马鱼幼鱼[3 dpf-48 hpe-12孔板(暴露后小时:hpe))]与选定条件(4 dpf-24 hpe-12孔板)进行比较,以表型评价、组织病理学评价、体内凋亡评价、生化指标及肝脏区域面积作为系统生物学评价指标,确认斑马鱼整体肝毒性评价的方法和损伤参照标准。结果 以实验所得各组LC50为指标,通过正交实验优化得到建立毒性模型的最佳实验条件参数为:选择4 dpf的斑马鱼幼鱼置于12孔板中给药暴露24 h;与对照组相比,各暴露组斑马鱼幼鱼肝脏透明度下降、颜色变暗且卵黄囊吸收延迟;肝脏组织结构松散、肝细胞空泡化严重;吖啶橙染色可见黄绿色荧光,提示存在肝细胞凋亡;谷丙转氨酶(ALT)和谷草转氨酶(AST)水平均呈显著性升高;肝脏面积表现出剂量依赖性的减小,以此确认方法的合理性。相关性分析结果证明以斑马鱼幼鱼肝面积的变化值(δ)评价斑马鱼肝损伤的可靠性,并以该指标初步建立了斑马鱼幼鱼的肝损伤评级。结论 考察并优化得到最佳斑马鱼幼鱼急性毒性评价参数,确认斑马鱼幼鱼肝毒性评价方法并建立斑马鱼幼鱼的肝损伤评级,为基于斑马鱼幼鱼模型快速评估药物肝毒性提供了参考。
Abstract
OBJECTIVE To establish a rapid evaluation method of hepatotoxicity and a grading method of drug liver injury based on zebrafish larvae model taking acetaminophen (APAP) as an example by optimizing the influencing factors and parameters for evaluation of its hepatotoxicity on zebrafish larvae. METHODS Zebrafish larvae were treated with different concentrations of acetaminophen (APAP). The mortality of zebrafish in different treatment groups was counted at the end of the experiment (48 h/24 h after drug exposure), and the mortality-concentration effect curve was drawn, and 0% lethal concentration (LC0) and half lethal concentration (LC50) were calculated. The relevant influencing factors of zebrafish larvae in APAP toxicity study were investigated and optimized, including zebrafish age [3 dpf, 4 dpf (day post fertilization:dpf)], exposure time (48 h, 24 h) and well plate (6 well plate, 12 well plate, 6 mL, 4 mL), and orthogonal experiments were designed from the above three levels to establish the best experimental conditions and parameters of the zebrafish hepatotoxicity model. In addition, 3 dpf larval zebrafish (3 dpf-48 hpe-12 well plate) was selected for comparison with the selected conditions (4 dpf-24 hpe-12 well plate)(hour post exposure: hpe). Phenotypic evaluation, histopathological evaluation, apoptosis evaluation in vivo, biochemical indexes and liver area were used as system biological evaluation indicators to confirm the method and damage reference standard for overall hepatotoxicity evaluation of zebrafish. RESULTS Taking the LC50 of each group obtained from the experiment as the index, the optimal experimental parameters for establishing the hepatotoxicity model were obtained through orthogonal experiment optimization: 4 dpf larval zebrafish was selected to be exposed to the drug for 24 h in a 12 well plate. The RESULTS showed that compared with the control group, the liver transparency of zebrafish larvae in each exposure group was decreased, the color became dark, and the absorption of yolk sac was delayed. The structure of livertissue was loose, the vacuolization of liver cells was severe; and acridine orange staining showed yellow green fluorescence, indicating the existence of hepatocyte apoptosis. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were increased significantly. The liver area showed a dose-dependent reduction, which confirmed the rationality of the method. The RESULTS of correlation analysis proved the reliability of using the change value of liver area (δ) to evaluate the liver injury of zebrafish larvae, and the liver injury rating of zebrafish larvae was preliminarily established based on this index. CONCLUSION The best acute toxicity evaluation parameters of zebrafish larvae are investigated and optimized, the liver toxicity evaluation method of zebrafish larvae is confirmed, and the liver injury rating of zebrafish larvae is established, which provides a reference for rapid evaluation of drug hepatotoxicity based on the zebrafish larvae model.
关键词
对乙酰氨基酚 /
斑马鱼 /
评价标准 /
药物肝毒性
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Key words
acetaminophen /
zebrafish /
evaluation criteria /
drug hepatotoxicity
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中图分类号：
R965.1
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参考文献
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脚注
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基金
国家重点研发计划项目资助(2019YFC1711502);北京中医药大学重点攻关项目资助(2020-JYB-ZDGG-035)
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