Abstract
Bovine endometritis is characterized by reduced milk production and high rates of infertility. Prior research has indicated that melatonin may possess anti-inflammatory and antioxidant properties that can counteract the progression of inflammatory diseases. In this research, we attempted to elucidate the protective effects of melatonin on LPS-induced endometritis. The results obtained from enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (qRT-PCR) revealed that melatonin effectively reduced the production and release of pro-inflammatory cytokines in an LPS-induced bovine endometrial epithelial cell line (BEND cells). Furthermore, western blotting demonstrated that melatonin treatment reduced the expression levels of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome-related proteins, including NLRP3, activated caspase-1, and cleaved IL-1β. Importantly, we further demonstrated that the anti-inflammatory effect of melatonin on BEND cells was related to autophagy by western blotting. Moreover, we used western blotting to detect autophagy-related proteins, MitoSOX to detect mitochondrial reactive oxygen species production (mtROS), and mitochondrial membrane potential (MMP) assay to detect mitochondrial membrane potential. The administration of melatonin demonstrated a significant enhancement in autophagy within BEND cells, leading to the effective elimination of impaired mitochondria. This process resulted in a reduction in the generation of reactive oxygen species within the mitochondria, restoration of mitochondrial membrane potential, and inhibition of the NLRP3 inflammasome activation. Moreover, in a mouse model of LPS-induced endometritis, melatonin treatment repressed the expression of pro-inflammatory cytokines by ELISA and qRT-PCR, alleviated pathological changes by hematoxylin-eosin staining (H&E), and inhibited myeloperoxidase (MPO) activity. In conclusion, our study showed that melatonin inhibited the activation of the NLRP3 inflammasome in BEND cells through autophagy, which may provide a novel therapeutic strategy for bovine endometritis.