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-1ß. 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.
ABSTRACT
Pathogenic bacteria were isolated from the uterine lavage fluid of a mare with endometritis. After identification and purification, the pathogenic bacteria were injected into the uterus of rabbits to induce endometritis. Then, anatomical, blood routine, chemical examination, and histopathological examinations were performed on the rabbits. Rabbit uterus was collected, and quantitative polymerase chain reaction (qPCR) was used to detect the mRNA expression of inflammatory factors including IL-1ß, IL-6, and TNF-α in the rabbit uterus. In addition, enzyme-linked immunosorbent assay (ELISA) was used to detect the uterine concentrations of the inflammatory factors IL-1ß, IL-6, and TNF-α. Western Blot was used to detect the protein expressions of NF-kB, IkBα, and TNF-α in the NF-kB pathway. An antibiotic treatment group was also set up to verify the accuracy of the results. The clinical examination results showed that there was a significant increase of leukocytes in the blood of the rabbits in the model group (P < 0.01). The uterus was congested, enlarged, and purulent. The integrity of the uterine lining was destroyed, and there was a significant increase of lymphocytes in the uterus (P < 0.01). The qPCR and ELISA results showed that the expressions of the inflammatory factors IL-1ß, IL-6, and TNF-α in the uterus of rabbits were significantly increased (P < 0.01). Western blot results showed that the inflammatory factors IL-1ß, IL-6, and TNF-α play a role in promoting inflammation through the NF-kB pathway. The results of the test provide a simple, economical, and reliable means of studying the occurrence, development, prevention, and treatment of equine endometritis.