迷迭香酸对氯化镉致斑马鱼胚胎发育毒性的保护作用  

Protective Effects of Rosmarinic Acid Against the Developmental Toxicity in Zebrafish Embryos Induced by Cadmium Chloride

作　　者：鲁疆[1] 贺靖 孙培环[1] 姚雯[1] 司婧[3] 甘露[3] 周鑫[3] 张红[3] 王振华[1,3,4] 

机构地区：[1]石河子大学药学院,新疆石河子832002 [2]库尔勒市第一人民医院药剂科,新疆库尔勒841000 [3]中国科学院近代物理研究所,兰州730000 [4]烟台大学生命科学学院,山东烟台264005

出　　处：《生物物理学报》2013年第5期338-350,共13页Acta Biophysica Sinica

基　　金：国家自然科学基金面上项目(11175222);教育部新世纪优秀人才支持计划项目(NCET-10-0967);兵团国际科技合作计划项目(2011BC006);兵团医药专项项目(2011BA039)~~

摘　　要：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。To investigate the developmental toxicity of cadmium（Cd） and intervention effects of rosmarinic acid（RA） to the embryonic stage of zebrafish fish,zebrafish embryos（1 hour post fertilization,1 hpf） were exposed to the solution of cadmium chloride（CdCl2） or which containing different concentrations of RA.The survival and hatching of embryos and the juvenile malformations were observed.Cell apoptosis in embryos was observed by the staining of acridine orange（AO）.Embryonic reactive oxygen species（ROS） level was measured by fluorescent probe of DCFH-DA.Lipid peroxidation degree in embyos was measured by the TBA colorimetric method,as well as levels of reduced glutathione（GSH） and oxidized glutathione（GSSG） were assayed by DTNB colorimetric method.Activity of triphosphopyridine nucleotide（NADPH） oxidase（Nox） in embryos was measured by lucigenin chemiluminescence method.The mRNA expression was detected by sybr greenⅠnucleic acid stain（SYBR） real time fluorescence quantitative real-time PCR（RT-PCR） method.Results showed that RA inhibited the embryos serious lethal and teratogentic effects,increased the hatched rate of embryos caused of CdCl2 in a concentration-dependent manner.RA inhibited the changes of ROS and malonic dialdehyde（MDA） level,GSH/GSSG rate and Nox activity caused of CdCl2 in zebrafish embryos,while inhibited the apoptosis of embryonic cell.The expression change of Cu/Zn-Sod mRNA,Bcl-2/Bax level in embryos caused by CdCl2 can be inhibited significantly of RA.Therefore,it can be concluded that the developmental toxicology of CdCl2 on zebrafish embryos may be associated with activated Nox,promoted ROS generation,caused oxidase stress damage of embryos and can lead to apoptosis of embryonic cell.The protective effect of RA by inhibiting Nox activation,improves oxidase stress state with developmental toxicity of CdCl2.

关 键 词：氯化镉 迷迭香酸 氧化胁迫 发育毒理学 斑马鱼 

分 类 号：R151[医药卫生—营养与食品卫生学]