2,3,7,8-四氯二苯并对二噁英诱导原代培养大鼠卵巢颗粒细胞的氧化应激和凋亡

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces oxidative stress and apoptosis of ovarian granulosa cells in rats in vitro

目的探讨2,3,7,8-四氯二苯并对二噁英(2,3,7,8-Tetrachlorodibenzo-p-dioxin,TCDD)对体外原代培养大鼠卵巢颗粒细胞的氧化损伤和促进凋亡作用,探究氧化应激介导TCDD诱导卵巢颗粒细胞凋亡的机制,为临床防治提供基础应用性依据。方法体外原代培养未成熟大鼠卵巢颗粒细胞,0.1、1、10和100 nmol/L TCDD染毒24 h后采用AnnexinV-FITC/PI双染流式检测细胞凋亡率;检测10nmol/L TCDD染毒0.5、1、5和24 h后细胞内活性氧(reactive oxygenspecies,ROS)、丙二醛(Malondialdehyde,MDA)、谷胱甘肽(Glutathione,GSH)水平;并添加超氧化岐化酶(Superoxidedismutase,SOD)联合处理,检测10 nmol/L TCDD联合不同剂量SOD处理后的ROS水平和100 IU/ml SOD联合处理1、3、6、18和24 h的细胞凋亡率。结果大鼠卵巢颗粒细胞暴露于1 nmol/L TCDD 24h早期凋亡发生显著增加,10、100 nmol/L染毒24h诱导晚期凋亡和细胞死亡增加明显,与对照组差异有统计学意义(P<0.05)。10 nmol/L TCDD处理不同时间均诱导颗粒细胞ROS生成、MDA增加和GSH损耗,与对照组比较差异有统计学意义(P<0.05),具有剂量依赖关系。SOD联合处理明显抑制TCDD诱导卵巢颗粒细胞的氧化应激作用和凋亡的发生。结论 TCDD诱导大鼠卵巢颗粒细胞的氧化损伤和细胞凋亡作用明显,氧化应激途径是TCDD诱导卵巢颗粒细胞凋亡的早期机制,是TCDD介导的生殖毒性机制之一。

Objective To investigate whether 2,3,7,8-Tetrachlorodibenzo-p-dioxin（TCDD） induces oxidative stress and apoptosis in rat granulosa cells（GCs）,and to further evaluate whether TCDD is capable of inducing apoptosis in this cell type.Methods GCs were obtained from preovulatory follicles in immature rats and cultured in Vitro.AnnexinV-FITC /PI was used to determine the percentage of apoptosis of granulosa cells following the exposure to 0.1% DMSO,0.1,1,10,100nmol /L TCDD for 24 h,as well as 10nmol /L TCDD co-treated with 100 IU /ml SOD for various times.Intracellular reactive oxygen species（ROS） level,Malondialdehyde（MDA） and Glutathione（GSH） content were measured respectively after GCs exposed to 10 nmol/L TCDD for 0.5,1,5,24 h intervals and co-cultured with superoxide dismutase（SOD） for 5 h.Results Results indicated that early apoptotic events in GCs,induced by treatment with 1 nmol /L TCDD for up to 24 h,were remarkably increased when compared to controls.In addition,the percentage of later stages of cell apoptosis and cell death increased were observed to be significantly greater with the 10,100 nmol /L dose of TCDD for 24 h,compared with controls.Moreover,TCDD increased cell apoptosis in dose-dependent manners.The levels of ROS and MDA formation significantly increased in GCs after 10 nmol /L TCDD treatment for various times.Further,we observed significant decreases in intracellular GSH production compared with controls.Further investigation indicated that the addition of SOD to the culture media almost abolished the effect of TCDD on oxidative stresses and apoptosis in rat GCs in vitro.Conclusion Our findings added further support to the theme indirectly that ROS formation was a significant determinant factor in mediating the induction of apoptosis in rat granulosa cells in vitro exposed to TCDD andthat the TCDD-induced oxidative stress and DNA damage may,in part,contribute to TCDD-induced endocrine-toxicity effects.