Melatonin alleviates oxidative stress and inflammation of Leydig cells of Min pig through SIRT1 pathway.

Inflammation responses and oxidative stress adversely affect testicular function, reducing fertility. Melatonin exhibits anti-inflammatory and antioxidant properties. However, the molecular mechanism by which melatonin alleviates inflammation and oxidative stress in Leydig cells of the Min pig testis remains unclear. To investigate this, primary Leydig cells were isolated from 7-day-ld Min pigs' testes and treated with LPS, HO and melatonin, respectively. The results showed that co-treatment with melatonin and LPS significantly decreased the expression of TLR4, NF-κB, IL-6 and IL-1β compared to LPS group. Co-treatment with melatonin and HO significantly mitigated reactive oxygen species and malondialdehyde levels. Melatonin also enhanced glutathione and superoxide dismutase levels and upregulated the mRNA expression levels of Nrf2, Keap1, HO-1 and NQO1. In the co-treatment group of melatonin, LPS, and SIRT1 inhibitor, the secretion of IL-6 and IL-1β and the mRNA expression levels of TLR4 and NF-κB were elevated significantly compared to the control group and the melatonin-LPS co-treatment group. In the combined treatment group of melatonin, HO and a SIRT1 inhibitor, ROS levels increased significantly, while the expression of Nrf2, Keap1, HO-1 and NQO1 were decreased significantly compared to the control group and the melatonin-HO co-treatment group. Furthermore, mRNA expression levels of testosterone synthesis-related genes StAR, CYP11A1, 3β-HSD, CYP17A1 and 17β-HSD significantly decreased following HO treatment, which was alleviated by co-treatment with HO and melatonin, but not by the addition of SIRT1 inhibitor. In conclusion, melatonin exhibits the capability to ameliorate inflammatory responses, oxidative stress and testosterone secretion in Leydig cells via the SIRT1 pathway.