Melatonin Improves H₂O₂-Induced Oxidative Stress in Sertoli Cells Through Nrf2-Keap1 Signaling Pathway

Background: Oxidative stress in the testicles of male livestock can cause reduced fertility. Melatonin is a natural product with antioxidant effects, but its specific antioxidant mechanism is still unclear. This study used calf testicular Sertoli cells as materials to explore the mechanism by which melatonin alleviates the oxidative stress of Sertoli cells, laying a foundation for improving the fertility of bulls. Methods: The optimal treatment concentrations of H₂O₂ and melatonin (MLT) were screened out using a CCK8 kit and MDA kit. Then, the cells were divided into four groups for treatment: control group, H₂O₂ treatment group, MLT treatment group, and H₂O₂ and MLT co-treatment group, then the MDA, ROS, GSH, and SOD contents were detected. Real-time quantitative PCR analysis and Western blot analysis were used to detect genes and proteins related to the Nrf2-Keap1 pathway. Immunofluorescence staining was used to analyze changes in Nrf2. Results: Research results show that the MDA content of cells in the group treated with H₂O₂ and MLT combined was significantly lower than that in the group treated with H₂O₂ alone, but there was no difference from the control group. Compared with the control group, the ROS level of cells in the H₂O₂-treated group significantly increased, and the content of GSH and SOD significantly decreased. Compared with the H₂O₂-treated group, the ROS level of cells in the H₂O₂ and MLT co-treated group significantly decreased, and the content of GSH and SOD increased significantly, but no difference from the control group. Similarly, MTL can alleviate the changes in cellular Nrf2, Keap1, HO-1, and NQO1 expression caused by H₂O₂. Conclusions: Melatonin activates the Nrf2-Keap1 signaling pathway in Sertoli cells, elevating the expression of HO-1 and NQO1, and thereby exerting its antioxidant capabilities.