香加皮对斑马鱼发育毒性及心脏毒性的影响

Effects of Cortex Periplocae on zebrafish developmental toxicity and cardiac toxicity

目的探讨香加皮水提液对斑马鱼的发育毒性及不同暴露时间药物对心脏毒性的影响。方法参照经济合作与发展组织推荐的方法,将受精后6小时(6 hour-post-fertilization,6 hpf)胚胎暴露在含有不同浓度香加皮水溶液的24孔板中,分别在24、48、72、96 hpf阶段测定胚胎/幼鱼的自主抽动次数、心率、孵化率、死亡率等指标。随后,使用不同浓度的香加皮水溶液处理发育至48、72、96 hpf的斑马鱼24小时,通过死亡率确定不同暴露阶段对斑马鱼心脏毒性的影响。结果各暴露组24 hpf自主抽动次数增加、48 hpf心率以及72 hpf孵化率显著降低,与正常对照组相比均有显著性差异(P<0.05);24 hpf低浓度给药组胚胎发育迟缓、眼点未发育、头部偏小或未发育,高浓度组发育畸形或停留在体节期;48 hpf胚胎卵黄囊畸形、黑色素生成抑制、尾芽未脱落;72 hpf幼鱼出现身体弯曲、心包水肿、卵黄囊畸形等中毒症状。不同发育阶段斑马鱼急性毒性暴露实验结果表明:香加皮对48、72、96 hpf斑马鱼的半数致死浓度(LC_(50))分别为:0.866 mg/m L、0.693 mg/m L和0.843mg/m L。结论香加皮对斑马鱼具有明显的发育毒性和心脏毒性,且48 hpf斑马鱼非常适合进行心脏毒性药物筛选。

Objective To study the toxic effects of Cortex Periplocae on zebrafish embryo development, and the effects of different exposure duration on the heart toxicity.Methods The methods recommended by Organization for Economic Co-operation and Development was refered, zebrafish embryos of 6 hour-post-fertilization were exposed to 24-well plates with aqueous extraction of Cortex Periplocae of various concentrations.Spontaneous movements, heart rate, hatching rate, mortality rate in 24 hpf and 48 hpf were used to assess the effects of Cortex Periplocae on the zebrafish development under the microscope.Zebrafish at 48 hpf, 72 hpf, 96 hpf were treated with aqueous extraction of cortex periplocae with various concentrations for 24h to determine the effects of different exposure duration on the heart toxicity.Results Compared with the normal control group, the number of spontaneous movements of 24 hpf, heart rate of 48 hpf and the hatching rate of 72 hpf were significantly lower in the exposed group,the results had significant difference （P〈0.05）.Embryos of 24 hpf exposed to low concentration showed immature development of head and eyes spot, and embryos in high concentration group were malformed or stayed in the segmentation period.Embryos of 48 hpf showed malformation of visceral yolk sac, inhibition of melanin and tail bud was not fallen off.After 72 hpf, the abnormality of zebrafish was observed for curved body, pericardial edema, yolk sac edema.Experimental result of acute toxicity exposure at different developmental stages of zebrafish showed that the median lethal concentration（LC50） were 0.866 mg/mL, 0.693 mg/mL, 0.843 mg/mL respectively.Conclusion Cortex Periplocae had significant toxic effect on the development of zebrafish embryos and heart.Moreover, 48hpf zebrafish is well suited for drug screening of cardiac toxicity.