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通过考察氯化镉(CdCl2)对斑马鱼胚胎发育的毒性效应及迷迭香酸(rosmarinic acid,RA)的干预作用,探讨镉的发育毒性机理。将受精1 h后的斑马鱼胚胎暴露于不同浓度的CdCl2或含不同浓度RA的CdCl2溶液中,观察胚胎死亡、孵化及幼鱼畸形的情况。采用吖啶橙染色,定性观察胚胎细胞凋亡情况;以单细胞凝胶电泳法检测胚胎细胞的DNA损伤;以活性氧(reactive oxygen species,ROS)荧光探针DCFH-DA染色法检测胚胎的ROS水平,硫代巴比土酸比色法测定胚胎脂质过氧化水平,5,5-二硫二硝基苯甲酸比色法测定胚胎的还原谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)水平;光泽精化学发光法检测胚胎还原型辅酶(nicotinamide adenine dinucleotide phosphate,NADPH)Ⅱ氧化酶(NADPH oxidase,Nox)活性。结果显示,RA浓度依赖性地抑制了CdCl2诱导的胚胎死亡和幼鱼畸型,同时提高了胚胎孵化率。RA可浓度依赖性地改善CdCl2诱导的胚胎ROS和MDA(malonic dialdehyde)水平升高、GSH/GSSG比值降低和Nox活性升高,抑制CdCl2诱导的胚胎细胞凋亡。RT-PCR结果显示,RA对CdCl2诱导的胚胎Cu/Zn-Sod基因表达上调和Bcl-2/Bax水平下调也有明显改善作用。结果说明,CdCl2对斑马鱼胚胎发育具有毒性,可活化Nox,促进ROS生成,造成胚胎细胞氧化胁迫损伤以至胚胎细胞凋亡,而RA可抑制Nox活化,改善氧化胁迫状态,从而对CdCl2的发育毒性起保护作用。
The toxic mechanism of cadmium chloride (CdCl2) on the embryonic development of zebrafish and the intervention of rosmarinic acid (RA) were investigated to explore the mechanism of Cd toxicity. The zebrafish embryos after 1 h of fertilization were exposed to different concentrations of CdCl2 or CdCl2 solution with different concentrations of RA to observe embryo death, hatching and juvenile deformity. Apoptosis of embryos was observed by acridine orange staining. DNA damage of embryonic cells was detected by single cell gel electrophoresis. ROS of embryos was detected by reactive oxygen species (ROS) fluorescent probe DCFH-DA staining Level, thiobarbituric acid colorimetric determination of the level of lipid peroxidation in embryos, 5 - Dithiaditrobenzoic acid colorimetric determination of embryonic reduction of glutathione (GSH) and oxidized glutathione (GSSG). The luciferase assay was used to detect the NADPH oxidase (NADPH) oxidase (Nox) activity. The results showed that RA inhibited CdCl2-induced embryonic death and juvenile malformations in a concentration-dependent manner, while increasing the rate of embryo hatching. RA could increase CdCl2-induced embryonic ROS and MDA (malonic dialdehyde) concentration, decrease GSH / GSSG ratio and increase Nox activity in a concentration-dependent manner, and inhibit CdCl2-induced embryonic cell apoptosis. The result of RT-PCR showed that RA could up-regulate the expression of Cu / Zn-Sod gene induced by CdCl2 and down-regulate the level of Bcl-2 / Bax significantly. The results showed that CdCl2 was toxic to embryonic development of zebrafish and activated Nox, promoted the production of ROS, induced oxidative stress injury of embryonic cells and apoptosis of embryonic cells, while RA inhibited the activation of Nox, and improved the oxidative stress, thus the developmental toxicity of CdCl2 Play a protective role.