α-硫辛酸对热应激小鼠睾酮分泌失调的保护作用

Protective Effect of Alpha Lipoic Acid on Heat Stress-induced Serum Testosterone Dyssecretosis in Mice

目的:研究α-硫辛酸对热应激引起小鼠睾酮分泌失调的保护作用。方法:将性成熟雄性SPF昆明小鼠60只,随机分为3组,即对照组、热应激组和α-硫辛酸+热应激处理组。分别采用酶联免疫吸附测定法(ELISA)检测各组小鼠血清睾酮含量,石蜡切片免疫组化染色法(IHC)分析各组小鼠非折叠蛋白反应信号通路的关键信号转导调节分子肌醇需求酶1(IRE1)在睾丸中的表达定位,以及蛋白免疫印迹法(Western blot)检测各组小鼠IRE1和促凋亡基因半胱氨酸蛋白酶-3(Caspase-3)的蛋白表达量变化。结果:与对照组相比,热应激组的血清睾酮含量显著下降(P<0.01),睾丸组织中IRE1和Caspase-3蛋白表达量显著增加(P<0.01,P<0.01);与热应激组相比,α-硫辛酸+热应激处理组的血清睾酮含量显著升高(P<0.05),在睾丸生精小管和间质细胞中均可观察到IRE1蛋白的阳性免疫染色,但IRE1蛋白阳性细胞数量显著减少(P<0.05),IRE1和Caspase-3的蛋白表达量均显著降低(P<0.05,P<0.05)。结论:热应激能够抑制小鼠睾丸睾酮的分泌,降低血清睾酮的含量;在本试验条件下,添加60 mg/(kg·d)的α-硫辛酸能够有效增加热应激状态下小鼠的血清睾酮含量,其发挥保护作用的机制可能与α-硫辛酸抑制IRE1介导的非折叠蛋白反应信号通路激活、降低凋亡促进基因Caspase-3的蛋白表达量有关。

Objective:To explore the protective effect of alpha-lipoic acid on testosterone dyssecretosis induced by heat stress in mice.Methods:The 60 sexually mature male SPF Kunming inbred strain mice were randomly distributed into three groups:control group,heat stress group and heat stress+alpha lipoic acid group.The enzyme-linked immunosorbent assay(ELISA)of mice among each group was performed to analyze the content of serum testosterone,immunohistochemical staining(IHC)was used to determinate the localization of IRE1,a key signal regulatory molecule which is responsible for the transduction of unfolded protein response signaling,and the protein expression of Caspase-3 and a proapoptotic gene was detected by western blot.Results:After treatment with heat stress,the serum testosterone concentration in the heat stress group was obviously lower than that in the control group(P<0.01),and the protein expression of IRE1 and Caspase-3 was both significantly increased in testis(P<0.01,P<0.01).Compared with the heat stress group,the serum testosterone concentration in the alpha lipoic acid group was significantly increased(P<0.05),the positive immunostaining of IRE1 protein was observed in testicular spermatogenic tubules and leydig cells,but the number of IRE1 protein-positive cells was significantly reduced intestis(P<0.05),and the protein expression levels of IRE1 and Caspase-3 were both significantly decreased(P<0.05,P<0.05)in testis.Conclusion:Heat stress can inhibit the secretion of testosterone resourced from testis,and decrease serum testosterone levels in mice.60 mg/(kg·d)alpha lipoic acid can significantly increase serum testosterone levels in heat-stressed mice under these experimental conditions.This protective role of alpha lipoic acid may be resulted from its inhibiting effect including the suppression of the activation of IRE1-mediated unfold protein response signal pathway,and the reduction of expression levels of Caspase-3 protein that is a proapoptotic gene in testis.