促卵泡激素通过活性氧途径调控卵巢癌细胞Nrf2蛋白的表达

目的 通过观察促卵泡激素(FSH)、活性氧(ROS)及其特异性抑制剂对卵巢癌细胞中红系衍生的核因子2相关因子2(nuclear factor-erythroid 2-related factor-2,Nrf2)表达的影响,探讨FSH是否通过ROS途径调控卵巢癌细胞Nrf2的表达.方法 (1)采用免疫组织化学方法检测Nrf2在卵巢癌良恶性组织中的表达.(2)用不同浓度的FSH刺激卵巢癌Hey细胞不同时间,采用蛋白质印迹法检测细胞内Nrf2蛋白的表达.(3)采用ROS检测试剂盒检测80 mIU/ml的FSH刺激后不同时间Hey细胞内ROS的生成情况.(4)Hey细胞经不同浓度的H2O2刺激48 h后,或经ROS特异性抑制剂N-乙酰半胱氨酸(NAC)预处理后加入150 mmol/L的H2O2处理48 h,观察细胞内Nrf2的表达.(5)Hey细胞经不同浓度的NAC预处理1h,加入80 mIU/ml的FSH共孵育48 h,观察细胞内Nrf2表达的变化.结果 (1)10例良性卵巢囊肿组织中仅3例有Nrf2蛋白的弱阳性表达,而64例卵巢癌组织中42例(65.6％)有Nrf2蛋白的阳性表达,两组差异有统计学意义(P=0.042).(2)FSH可上调Hey细胞内Nrf2的表达,并呈一定的剂量、时间依赖效应.(3) FSH可促进Hey细胞ROS的产生.(4) H2O2可诱导Hey细胞Nrf2蛋白的表达,且其作用可被NAC阻断.(5)NAC可阻断FSH所诱导的Nrf2蛋白的表达.结论 FSH可促进卵巢癌Hey细胞中ROS产生,上调Nrf2蛋白表达,抑制ROS途径可阻断FSH诱导的Nrf2高表达.提示FSH可能通过ROS途径调控卵巢癌Nrf2的表达,进而促进肿瘤发生.

ROS production is required for follicle-stimulating hormone-induced Nrf2 activation in human epithelial ovarian cancer cells

ObjectiveTo observe the effects of follicle-stimulating hormone (FSH), reactive oxygen species (ROS) and its specific inhibitor on expression of Nrf2, so as to study the possible role of ROS signal pathway in FSH-mediated Nrf2 expression in Hey ovarian cancer cells.MethodsThe expression of Nrf2 in benign and malignant ovarian tissues was determined by immunohistochemical method. Ovarian cancer Hey cells were treated with different concentrations of FSH for different periods, and Western blotting analysis was used to examine Nrf2 protein expression. The generation of ROS was evaluated in Hey ovarian cancer cells by reactive oxygen species assay kit after stimulation with FSH (80 mIU/ml) for different periods. Hey cells were treated with different concentrations of H2O2 stimulation for 48 h or 150 mmol/L H2O2 after pretreatment with specific ROS inhibitors NAC for 48 h, then the expression of Nrf2 was examined by Western blotting analysis. Hey cells were pretreated with different concentrations of NAC for 1 h, and then incubated with FSH(80 mIU/ml) for 48 h and Nrf2 expression was observed.Results(1) Three of 10 benign ovarian cyst tissues had weak expression of Nrf2 protein; 42 (65.6%) of the 64 ovarian cancer tissues had Nrf2 protein expression (P=0.042). FSH up-regulated Nrf2 expression in a dose- and time-dependent manner. FSH promoted the generation of ROS in ovarian cancer cells. H2O2 promoted Nrf2 expression, which could be blocked by NAC. FSH-induced Nrf2 protein expression could be inhibited by NAC.ConclusionFSH can promote ROS generation and Nrf2 protein expression in ovarian cancer cells. Block of ROS pathway could inhibit FSH-induced overexpression of Nrf2, indicating FSH may regulate Nrf2 protein by ROS pathway, contributing to the development of ovarian cancer.