Zhao Yun-Tao, Qi Ya-Wei, Hu Chuan-Yin, Chen Shao-Hong, Liu You
Modern Biochemistry Center, Guangdong Ocean University, Zhanjiang, Guangdong 524088, P.R. China.
Institute of Plastic Surgery, Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong 524001, P.R. China.
Int J Mol Med. 2016 Aug;38(2):659-65. doi: 10.3892/ijmm.2016.2645. Epub 2016 Jun 16.
Diabetes severely impairs male reproduction. The present study assessed the effects and mechanisms of action of advanced glycation end products (AGEs), which play an important role in the development of diabetes complications, on testosterone secretion by rat Leydig cells. Primary rat Leydig cells were cultured and treated with AGEs (25, 50, 100 and 200 µg/ml). Testosterone production induced by human chorionic gonadotropin (hCG) was determined by ELISA. The mRNA and protein expression levels of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD), which are involved in testosterone biosynthesis, were measured by reverse transcription-quantitative PCR and western blot analyssi, respectively. Reactive oxygen species (ROS) production in Leydig cells was measured using the dichlorofluorescein diacetate (DCFH-DA) probe. The expression levels of endoplasmic reticulum stress-related proteins [C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78)] in the Leydig cells were measured by western blot analysis. We found that the AGEs markedly suppressed testosterone production by rat Leydig cells which was induced by hCG in a concentration-dependent manner compared with the control (P<0.01). The mRNA and protein expression levels of StAR, 3β-HSD and P450scc were downregulated by the AGEs in a dose-dependent manner compared with the control (P<0.01). The antioxidant agent, N-acetyl‑L‑cysteine (NAC), and the endoplasmic reticulum stress inhibitor, tauroursodeoxycholic acid (TUDCA), reversed the inhibitory effects of AGEs. In addition, the content of ROS in Leydig cells treated with AGEs increased significantly. The expression levels of CHOP and GRP78 were markedly upregulated by the AGEs in the Leydig cells. From these findings, it can be concluded that AGEs inhibit testosterone production by rat Leydig cells by inducing oxidative stress and endoplasmic reticulum stress.
糖尿病严重损害男性生殖功能。本研究评估了晚期糖基化终末产物（AGEs）对大鼠睾丸间质细胞睾酮分泌的影响及其作用机制，AGEs在糖尿病并发症的发生发展中起重要作用。培养原代大鼠睾丸间质细胞，并用AGEs（25、50、100和200μg/ml）进行处理。采用酶联免疫吸附测定法（ELISA）测定人绒毛膜促性腺激素（hCG）诱导的睾酮生成。分别通过逆转录定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹分析，检测参与睾酮生物合成的类固醇生成急性调节蛋白（StAR）、胆固醇侧链裂解酶（P450scc）和3β-羟基类固醇脱氢酶（3β-HSD）的mRNA和蛋白表达水平。使用二氯荧光素二乙酸酯（DCFH-DA）探针检测睾丸间质细胞中活性氧（ROS）的产生。通过蛋白质免疫印迹分析检测睾丸间质细胞中内质网应激相关蛋白[C/EBP同源蛋白（CHOP）和葡萄糖调节蛋白78（GRP78）]的表达水平。我们发现，与对照组相比，AGEs以浓度依赖性方式显著抑制hCG诱导的大鼠睾丸间质细胞睾酮生成（P<0.01）。与对照组相比，AGEs以剂量依赖性方式下调StAR、3β-HSD和P450scc的mRNA和蛋白表达水平（P<0.01）。抗氧化剂N-乙酰-L-半胱氨酸（NAC）和内质网应激抑制剂牛磺熊去氧胆酸（TUDCA）可逆转AGEs的抑制作用。此外，用AGEs处理的睾丸间质细胞中ROS含量显著增加。AGEs显著上调睾丸间质细胞中CHOP和GRP78的表达水平。从这些研究结果可以得出结论，AGEs通过诱导氧化应激和内质网应激抑制大鼠睾丸间质细胞睾酮生成。
