ROS和MAPK信号通路在镍致睾酮合成障碍中的调控机理研究

Nickel is a ubiquitous industrial and environmental pollutants. Previous studies showed that nickel could result in adverse effects on reproductive function by interfering with spermatogenesis, inducing the apoptosis and enhancing oxidative stress in testicular cells. The applicant study have demonstrated that excessive nickel could decrease the testosterone levels of serum and the amount of testosterone secretion of Leydig cells in male rats, but the underlying molecular mechanisms of nickel on the blocked synthesis of testosterone are unclear. According to current reports, ROS and MAPK signaling pathways play an important role in testosterone synthesis. To address this issue, the purified mouse Leydig cells treated by different concentration nickel at different times is used as analysis model, and ROS scavengers and MAPK inhibitors are used to explore the regulatory mechanisms of ROS and MAPK signaling pathways in modulating testosterone secretion of Leydig cells following exposure to nickel in vitro.. First, transmission electron microscope will be employed to determine if the ultrastructural of Leydig cells are damaged. Testosterone secretion of Leydig cells will be analysised by radioimmunoassay. Meanwhile, the expression levels of steroid biosynthesis related protein and mRNA, including StAR, P450scc, CYP17A1, 3β-HSD and 17β-HSD, will be detected by immunoblot and real-time fluorescence quantitative PCR to observe whether nickel could affect steroid biosynthesis. Furthermore, laser confocal microscope will be performed to measure the intracellular ROS content and the expression levels of MAPK related phosphorylated protein will be detected by immunoblot to explore the molecular mechanism of nickel on testosterone synthesis regulated by ROS and MAPK signaling pathways.. With Leydig cells model induced by nickel, we could perfectly address the issues that whether nickel could inhibit steroid synthetase and this effect is related to ROS and MAPK signaling pathways dependently. These findings will deepen our insights into the molecular mechanisms of testosterone biosynthesis in Leydig cell exposed to nickel and provide theoretical support for precaution of nickel on reproduction toxicity.

镍是一种普遍存在的工业和环境污染物。研究证实，镍可干扰精子发生并导致精子数量下降、诱发睾丸细胞凋亡及氧化应激增强等。申请人所在课题组前期研究发现镍还可引起大鼠血清睾酮降低和体外睾丸间质细胞睾酮分泌量降低，但有关镍对睾酮合成影响的分子机制尚不明确。据报道，ROS和MAPK信号通路在睾酮合成过程中发挥重要作用。因此，本研究拟以镍处理体外原代培养的小鼠Leydig细胞为模型，采用放射免疫、免疫荧光、免疫印迹和RT-PCR等技术检测Leydig细胞睾酮分泌量、ROS水平、睾酮合成关键酶和MAPK信号通路关键调控蛋白磷酸化水平的变化，旨在从ROS和MAPK信号通路两方面探讨镍对睾酮合成调控的分子机理，以期为镍生殖毒性的早期防治提供理论依据。

镍是普遍存在的工业和环境污染物。研究证实，镍可干扰精子发生并导致精子数量下降、诱发睾丸细胞凋亡及氧化应激增强等。睾酮参与调节雄激素依赖的器官分化发育及生精过程，并维持第二性征。睾丸间质细胞（Leydig cells）是合成睾酮场所。镍可通过损伤Leydig细胞的结构功能，致使睾酮合成减少，但目前有关镍对睾酮合成影响的分子机制尚不明确。据报道，ROS和MAPK信号通路在睾酮合成过程中发挥重要作用。虽然已知ROS/MAPKs信号途径（ERK1/2、p38 和JNK）参与睾酮合成过程，但是在不同的实验研究中得到了不一致的研究结果，而且它们是否在NiSO4诱导的睾酮合成障碍中发挥作用就不得而知了。因此，本研究拟以镍处理体外原代培养的大鼠Leydig细胞为模型，采用免疫荧光、免疫印迹和RT-PCR等技术检测Leydig细胞睾酮分泌量、ROS水平、睾酮合成关键酶和MAPKs信号通路关键调控蛋白磷酸化水平的变化情况，旨在从ROS和MAPK信号通路两方面探讨镍对睾酮合成调控的分子机制。研究结果显示，NiSO4可诱导Leydig细胞睾酮合成减少。究其原因，一方面，NiSO4可以诱导睾丸Leydig细胞产生过量ROS，而ROS清除剂NAC和Tempo可以通过减少ROS产生，并进一步提高睾酮合成酶StAR、CYP11A1、CYP17A1、3β-HSD和17β-HSD的mRNA及蛋白表达水平，从而提高睾酮分泌量。另一方面，NiSO4可以诱导ERK1/2、p38 和JNK MAPKs相关蛋白的磷酸化水平上调，从而激活MAPKs信号通路；再通过使用ERK1/2、p38 和JNK特异的抑制剂（U0126、SB203580和SP600125）预处理Leydig细胞后，观察NiSO4对Leydig细胞睾酮合成的影响，结果显示ERK1/2和p38 的抑制剂（U0126和SB203580）可显著提高睾酮合成酶StAR、CYP11A1、CYP17A1、3β-HSD和17β-HSD的mRNA及蛋白表达水平，从而促进睾酮合成的分泌。由此可见，ROS/MAPKs信号通路在镍致睾酮合成障碍中发挥重要的作用。