二甲双胍对H_(2)O_(2)诱导的TM3小鼠睾丸间质细胞氧化应激和细胞凋亡的影响

Effects of metformin on oxidative stress and apoptosis of TM3 mouse Leydig cells induced by H_(2)O_(2)

目的探讨二甲双胍对H_(2)O_(2)诱导的TM3小鼠睾丸间质细胞损伤的作用。方法将TM3小鼠睾丸间质细胞随机分为4组,分别为正常对照组、二甲双胍组、H_(2)O_(2)模型组以及H_(2)O_(2)+二甲双胍组。各组细胞培养24 h后,利用CCK-8法检测细胞存活率,ELISA检测TM3细胞睾酮的分泌情况,实时定量PCR检测睾酮合成酶StAR、P450scc、17β-HSD的mRNA表达情况,采用比色法检测超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性及丙二醛(MDA)含量,Western blot检测TM3细胞内凋亡相关蛋白Cleaved-caspase3的表达情况。结果与正常对照组相比,H_(2)O_(2)模型组TM3细胞的睾酮水平显著降低,睾酮合成酶StAR、P450scc和17β-HSD的mRNA表达水平显著降低,细胞内SOD和CAT活性显著降低,细胞存活率亦显著降低(P均<0.05),MDA含量及Cleaved-caspase3表达水平显著升高(P<0.05)。与H_(2)O_(2)模型组相比,H_(2)O_(2)+二甲双胍组TM3细胞的睾酮水平显著升高,睾酮合成酶的mRNA表达水平显著升高,SOD和CAT活性显著升高,细胞存活率显著提高(P均<0.05),MDA含量及Cleaved-caspase3表达水平显著降低(P<0.05)。结论二甲双胍可减轻H_(2)O_(2)诱导的TM3小鼠睾丸间质细胞功能损伤。

Objective:To explore the effects of metformin on TM3 mouse Leydig cells damage induced by H_(2)O_(2).Methods:TM3 mouse Leydig cells were randomly divided into 4 groups:control group,metformin group,H_(2)O_(2)model group,and metformin+H_(2)O_(2)group.After 24 hours culture of the cells,TM3 cell viability was detected by a CCK-8 method.Testosterone was detected by ELISA kit.The mRNA expression of StAR,P450scc,and 17β-HSD were determined by real time-PCR.Colorimetric method was performed to detect the activity of superoxide dismutase(SOD)&catalase(CAT)and the content of malondialdehyde(MDA).Western blot was used to detect the expression of apoptosis related protein cleaved-caspase3 in TM3 cells.Results:Compared with the control group,the viability and testosterone levels of TM3 cells were significantly decreased,the mRNA levels of StAR,P450scc and 17β-HSD were significantly decreased,and the activity of SOD and CAT was also significantly decreased in the H_(2)O_(2)model group(all P<0.05).However,MDA content and cleaved-caspase3 expression were significantly increased(P<0.05).Compared with the H_(2)O_(2)model group,the viability and testosterone levels of TM3 cells were significantly increased,the mRNA expression level of testosterone synthase was significantly increased,and the activity of SOD and CAT was also significantly increased in the metformin+H_(2)O_(2)group(all P<0.05).However,the content of MDA and the expression of cleaved-caspase 3 were significantly decreased(P<0.05).Conclusions:Metformin can reduce TM3 mouse Leydig cell damage induced by H_(2)O_(2).