Melatonin Alleviates Lipopolysaccharide-Induced Endometritis by Inhibiting the Activation of NLRP3 Inflammasome through Autophagy

Simple Summary Bovine endometritis is characterized by reduced milk production and high infertility rates, resulting in substantial economic losses for the dairy farming sector. Melatonin, an amine hormone produced in the mammalian pineal gland, has been widely studied for its anti-inflammatory effects. In this work, we aimed to investigate whether melatonin inhibits Lipopolysaccharide (LPS)-induced endometritis and explore its anti-inflammatory mechanism. In LPS-induced bovine endometrial epithelial cell lines (BEND cells), melatonin promotes autophagy to inhibit the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation and thus exerts anti-inflammatory effects. In a mouse model of LPS-induced endometritis, melatonin inhibited the expression of inflammatory factors and alleviated pathological changes. These findings demonstrate that melatonin inhibition of LPS-induced inflammation in vivo and in vitro may be a novel treatment for endometritis. 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-1 & beta;. 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.