褪黑素对小鼠leydig细胞睾酮合成抑制作用及机制-inhibitory effect and mechanism of melatonin on testosterone synthesis in leydig cells of mice.docxVIP

褪黑素对小鼠 Leydig 细胞睾酮合成的抑制作用及机制 英文摘要 Inhibitory effects and mechanism of melatonin on testosterone synthesis in mouse Leydig cells Abstract Objective: To observe inhibitory effect of melatonin on testosterone synthesis of Leydig cells, and to study functional mechanism of the melatonin effect. Methods: (1) The primary culture of Leydig cells isolated from C57BL/6J mouse was used as the experimental model. By Type I collagenase digestion method, Leydig cells were filtered and purified, using 400(30μm) mesh stainless steel, and stained with trypan blue. The cells were identified with 3β-HSD specific staining and observed with a microscope. (2) In cultured Leydig cells, different concentrations (0, 10-6, 10-8, 10-10 and 10-12 mol/L) of melatonin, and/or melatonin receptor antagonist Luzindole (1 μmol/L)were added. Then the CCK-8 method was used to detect cell viability and proliferation, flow cytometry to detect cell cycle, cell mitochondrial membrane potential and levels of reactive oxidative species(ROS) and testosterone levels were measured by ELISA. (3) The cells were divided into three groups: Control group, melatonin group and melatonin + Luzindole group. Immunofluorescence assay was used to detect the protein expression and distribution of melatonin receptors Mel1a and testosterone biosynthesis related genes GATA-4, StAR and 3β-HSD. Results: (1) In vitro cultured Leydig cells were spindle in morphology and constant in proliferation. Trypan blue staining showed a cell viability of above 90%. 3β-HSD specific staining showed a blue-black cytoplasm. The result indicated a successful establishment of the primary cultured mouse Leydig cells. (2) Compared with the control group, the cells proliferation was inhibited in melatonin group, with a decreased testosterone level and reduced mitochondrial membrane potential, and increased ROS. The inhibitory effect of melatonin on testosterone synthesis in Leydig cells was blocked by Luzindole. (3) The melatonin treatment resulted