氰戊菊酯对大鼠睾丸Leydig细胞睾酮合成的影响及其机制

Effect and mechanism of fenvalerate on testosterone synthesis in Leydig cells of rats testis

目的探讨氰戊菊酯(Fen)对大鼠睾丸Leydig细胞睾酮合成的影响及其可能机制。方法采用差速贴壁法分离提纯SD大鼠睾丸Leydig细胞。用0、25、50和100μmol/L Fen处理Leydig细胞1、12和24 h,采用ELISA法检测睾酮水平。设置空白对照组(加入0.1%DMSO处理)、Fen暴露组(加入100μmol/L Fen处理)、Fen+NAC组(加入100μmol/L Fen和5 mmol/L NAC处理)、Fen+CsA组(加入100μmol/L Fen和2 mmol/L CsA处理),给药后继续培养24 h。采用流式细胞仪检测活性氧(ROS)和线粒体膜电位变化,ELISA法检测睾酮水平及谷胱甘肽(GSH)、cAMP含量,化学发光法检测ATP含量,Western blotting检测超氧化物歧化酶(SOD)、类固醇激素合成急性调节蛋白(StAR)、3β-羟类固醇脱氢酶(3β-HSD)和细胞色素P450胆固醇侧链裂解酶(CYP11A1)的表达。结果选择100μmol/L Fen处理24 h进行实验。与空白对照组比较,Fen暴露组Leydig细胞睾酮合成水平,GSH、SOD、ATP、cAMP含量,线粒体膜电位,以及StAR、3β-HSD、CYP11A1蛋白相对表达量明显降低(P<0.01),ROS含量明显升高(P<0.01);与Fen暴露组比较,Fen+NAC组、Fen+CsA组Leydig细胞睾酮合成水平,GSH、SOD、ATP、cAMP含量,线粒体膜电位,以及StAR、3β-HSD、CYP11A1蛋白相对表达量明显升高(P<0.05或P<0.01),ROS含量明显降低(P<0.01)。结论Fen可能通过诱导氧化应激引起大鼠睾丸Leydig细胞线粒体损伤,致使ATP和cAMP合成受阻,从而抑制依赖cAMP/PKA信号通路的睾酮合成相关蛋白和酶的表达,最终导致Leydig细胞睾酮合成障碍。

Objective To investigate the effect and mechanism of fenvalerate(Fen)on testosterone synthesis in Leydig cells of rats testis.Methods Leydig cells of SD rat testis were isolated and purified by differential adhesion method,then treated with 0,25,50 and 100μmol/L Fen for 1,12 and 24 h,and the level of testosterone was detected by ELISA.Set blank control group(treatment with 0.1%DMSO),Fen exposure group(treatment with 100μmol/L Fen),Fen+NAC group(treatment with 100μmol/L Fen and 5 mmol/L NAC),Fen+CsA group(treatment with 100μmol/L Fen and 2 mmol/L CsA),and cultured for 24 h after administration.The changes of cellular reactive oxygen species(ROS)and mitochondrial membrane potential were detected by flow cytometry,the levels of testosterone,glutathione(GSH)and cAMP were detected by ELISA,and the content of ATP was detected by chemi-luminescence,Western blotting was used to detect the expressions of superoxide dismutase(SOD),steroidogenic acute regulatory protein(StAR),3β-hydroxysteroid dehydrogenase(3β-HSD)and cytochrome P450 cholesterol side-chain cleavage(CYP11A1).Results 100μmol/L Fen treatment for 24 h was selected in the experiments.Compared with blank control group,the testosterone synthesis level,the contents of GSH,SOD,ATP and cAMP,mitochondrial membrane potential,and the relative expression levels of StAR,3β-HSD and CYP11A1 decreased significantly in Leydig cells of Fen exposure group(P<0.01),the ROS content increased significantly(P<0.01).Compared with Fen exposure group,the testosterone synthesis level,the contents of GSH,SOD,ATP and cAMP,mitochondrial membrane potential,and the relative expression levels of StAR,3β-HSD and CYP11A1 increased significantly in Leydig cells of Fen+NAC group and Fen+CsA group(P<0.05 or P<0.01),the ROS content decreased significantly(P<0.01).Conclusion Fen may cause mitochondrial damage in Leydig cells by inducing oxidative stress,resulting in the inhibition of ATP and cAMP synthesis,thereby inhibiting the expression of testosterone synthesis related proteins and enzymes dependent on cAMP/PKA signaling pathway,and ultimately leading to testosterone synthesis disorder in Leydig cells.