3β-羟类固醇脱氢酶对邻苯二甲酸二-(2-乙基己)酯处理细胞后诱导型一氧化氮合酶和蛋白激酶C信号通路的影响

Effect of 3β-hydroxysteroid dehydrogenase gene on inducible nitric oxide synthase/protein kinase C signal pathway induced by di(2-ethylhexyl)phthalate

目的探讨邻苯二甲酸二-(2-乙基己)酯(di-(2-ethylhexyl)phthalate,DEHP)对MCF-7细胞中诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和蛋白激酶C(protein kinase C,PKC)信号通路相关基因及蛋白表达的影响以及3β-羟类固醇脱氢酶(3β-hydroxysteroid dehydrogenase,3β-HSD)基因在此过程中的作用。方法用浓度为0.00~0.40 mmol/L的DEHP分别染毒MCF-7细胞、3β-HSD沉默细胞和3β-HSD高表达细胞24 h,检测细胞中iNOS/PKC相关基因表达水平的变化;应用信号通路抑制剂处理后再进行DEHP染毒,荧光定量PCR和免疫印迹法检测MCF-7细胞中iNOS/PKC相关基因和凋亡基因表达水平的变化。结果DEHP染毒MCF-7细胞,iNOS、PKCα在基因和蛋白表达水平均高于对照组;与同一染毒剂量的MCF-7细胞比较,3β-HSD沉默细胞中iNOS、PKCαmRNA表达水平降低;而3β-HSD高表达细胞中iNOS、PKCαmRNA表达水平升高。应用iNOS抑制剂(NG-nitro-L-arginine methyl ester,L-NAME)50μmol/L处理后,DEHP染毒组iNOS、Bax、Caspase-3、Caspase-8表达下调;PKCα抑制剂(chelerythrine chloride,CHE)0.20μmol/L处理后,DEHP染毒组PKCα表达下调;而各抑制剂处理组中与对照组比较无明显降低的基因,其变化趋势与未加抑制剂处理的染毒组无显著差异。结论DEHP引起的毒性作用可能与iNOS/PKC信号通路有关,3β-HSD对iNOS/PKC信号通路相关基因的表达有一定作用。

Objective To explore the effect of di-(2-ethylhexyl)phthalate(DEHP)on the expression of genes and proteins related to signal pathway of inducible nitric oxide synthase(iNOS)and protein kinase C(PKC)in MCF-7 cells and the role of 3β-hydroxysteroid dehydrogenase(3β-HSD)gene in these processes.Methods MCF-7 cells,3β-HSD silencing cells and 3β-HSD over-expressed cells were exposed to DEHP at the concentration of 0.00-0.40 mmol/L for 24 hours.The changes in the expression levels of iNOS/PKC signal pathway-related genes were detected.After treatment with the inhibitors of iNOS/PKC signal pathway,the cells were exposed to DEHP at different concentration,and then the changes in the expression levels of iNOS/PKC signal pathway-related genes and apoptosis genes were detected by quantitative PCR(q-PCR)and Western blot.Results The gene and protein expression levels of iNOS and PKCαin the groups of MCF-7 cells exposed to different concentration of DEHP were higher than those in the control group.Compared with MCF-7 cells exposed to the same dose of DEHP,the expression levels of iNOS and PKCαmRNA in 3β-HSD silencing cells decreased,but the expression levels of iNOS and PKCαmRNA in 3β-HSD over-expressed cells increased.After treatment with the iNOS inhibitor NG-nitro-L-arginine methyl ester 50μmol/L,the expression of iNOS,Bax,Caspase-3 and Caspase-8 in DEHP-treated group was down-regulated.After treatment with PKCαinhibitor chelerythrine chloride 0.20μmol/L,the expression of PKCαin DEHP-treated group was down-regulated.No significantly decreased expression of genes was found in each group treated with inhibitor as compared with the control group,and also no significant differences were observed in the changing trends of genes between the DEHP-exposure groups treated and untreated with inhibitor.Conclusions DEHP-induced toxicity may be related to iNOS/PKC signal pathway,and 3β-HSD may regulate the expression of iNOS/PKC signaling pathway-related genes.