雷公藤多苷促进斑马鱼肝脏损伤和氧化应激的实验研究

Experimental Study on the Effects of Tripterygium Glycosides on Liver Damage and Oxidative Stress in Zebrafish

目的研究雷公藤多苷诱导斑马鱼肝脏损伤及氧化应激的作用。方法随机选取正常3 dpf野生型AB品系斑马鱼,分为7组:正常对照组、溶剂对照组(0.5%DMSO)、雷公藤多苷300、350、400、450和500μg/ml浓度处理组,每组30尾斑马鱼。雷公藤多苷处理斑马鱼直至120 hpf(3 dpf～5 dpf),观察记录每个实验组斑马鱼的死亡情况并计算死亡率,用Origin 8.0统计学软件绘制最佳的"浓度-死亡率"效应曲线并分别计算雷公藤多苷对斑马鱼的MNLC和LC_(10)。另随机选取正常3 dpf野生型AB品系斑马鱼,分为5组:正常对照组、对乙酰氨基酚(8 mM)组、雷公藤多苷片42.5、128和383μg/ml浓度组,每组60尾斑马鱼。药物处理48 h后,每组随机选取30条斑马鱼置于4%多聚甲醛固定,固定后将斑马鱼转移到70%乙醇中,进行脱水、包埋、切片、HE染色和封片,在显微镜下对染色后的斑马鱼切片进行肝脏病理学分析;在雷公藤多苷处理48 h后,每组随机取30尾斑马鱼收集并处理检测样本,检测过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。结果雷公藤多苷对斑马鱼的MNLC为383μg/ml,LC_(10)为417μg/ml。HE染色结果提示:对乙酰氨基酚对斑马鱼有明显肝毒性,随着雷公藤多苷片浓度增加,斑马鱼肝脏组织结构破坏程度增大。对乙酰氨基酚组、雷公藤多苷片42.5、128和383μg/ml浓度组的斑马鱼体内CAT、SOD活力以及MDA含量,与正常对照组比较,差异无统计学意义(P>0.05)。结论雷公藤多苷可通过诱导氧化应激促进斑马鱼的肝损伤。

Objective To study the effects of tripterygium glycosides on liver damage and oxidative stress in zebrafish. Methods Normal 3 dpf wild type AB strain zebrafish were randomly selected and divided into 7 groups: normal control group, solvent control group (0.5% DMSO), tripterygium glycosides 300, 350, 400, 450 and 500 μg/ml concentration treatment group, each group of 30 zebrafish. Tripterygium wilfordii treatment of zebrafish up to 120 hpf (3 dpf^5 dpf), observe the death of zebrafish in each experimental group and calculate the mortality, and use the Origin 8.0 statistical software to draw the best "concentration-mortality" The effect curves were calculated and the MNLC and LC10 of zebrafish were calculated for tripterygium glycosides. The normal 3 dpf wild type AB strain zebrafish were randomly selected and divided into 5 groups: normal control group, acetaminophen (8 mM) group, tripterygium glycosides 42.5, 128 and 383 μg/ml concentration group, each group 60 zebrafish. After 48 h of drug treatment, 30 zebrafish were randomly selected from each group and fixed in 4% paraformaldehyde. After fixation, the zebrafish were transferred to 70% ethanol for dehydration, embedding, sectioning, HE staining and sealing. The liver pathological analysis of the stained zebrafish sections was performed under microscope. After 48 hours of treatment with tripterygium glycosides, 30 zebrafish were randomly selected from each group to collect and process the test samples to detect catalase (CAT) and superoxide. Dismutase (SOD) activity and malondialdehyde (MDA) content. Results Tripterygium glycosides had a MNLC of 383 μg/ml for zebrafish and 417 μg/ml for LC10. The results of HE staining indicated that acetaminophen had obvious hepatotoxicity to zebrafish. With the increase of the concentration of tripterygium glycosides, the damage of liver tissue structure of zebrafish increased. The CAT, SOD activity and MDA content of zebrafish in the acetaminophen group and tripterygium glycosides group 42.5, 128 and 383 μg/ml were not significantly different from those in the normal control group (P>0.05). Conclusion Tripterygium glycosides can promote liver damage in zebrafish by inducing oxidative stress.