NR1D1 activation alleviates inflammatory response through inhibition of IL-6 expression in bovine endometrial epithelial cells

Endometritis, an inflammatory disease affecting dairy cattle, causes substantial economic losses in the dairy industry. Conventional treatment using uterine infusion of antibiotics often results in bacterial resistance and antibiotic residues in milk. Thus, identifying novel, effective therapeutic targets for endometritis in dairy cows is necessary. Nuclear receptor subfamily 1 group D member 1 (NR1D1) activation attenuates inflammatory responses in various diseases through transcriptional repression; however, its role in treating bovine endometritis remains unclear. This study investigated the role and underlying mechanisms of NR1D1 in endometritis using a bovine endometrial epithelial cell line (BENDs) and primary bovine endometrial epithelial cells, both induced with Escherichia coli lipopolysaccharide (LPS). Immunofluorescence staining revealed the predominant nuclear localization of NR1D1 in endometrial epithelial cells. LPS treatment (1 μg/mL for 12 h) significantly increased the expression levels of NR1D1 and proinflammatory cytokines (IL-6, IL-1β, IL-8, and CCL5) in BENDs. Immunohistochemical staining showed elevated NR1D1 expression in uterine tissues of cows with endometritis. Deletion of NR1D1 significantly increased IL-6 mRNA expression; NR1D1 overexpression substantially repressed IL-6 expression in BENDs. NR1D1 agonist SR9009 attenuated LPS-induced mRNA expression of proinflammatory cytokines (IL-6, IL-1β, CCL5) in both BENDs and primary endometrial epithelial cells. Additionally, SR9009 treatment attenuated LPS-induced inflammatory responses in the endometrium of mice. Dual-luciferase reporter assays and real-time monitoring via luminescence assays showed that NR1D1 overexpression significantly repressed luciferase activity driven by the IL-6 promoter region, which was abolished by deletion of the retinoic acid receptor-related orphan receptor-responsive element (−473 to −479) within the IL-6 promoter fragment. In summary, NR1D1 activation alleviates the inflammatory response of BENDs by repressing the expression of proinflammatory cytokines, at least partly via IL-6, suggesting NR1D1 is a promising therapeutic target for endometritis prevention and treatment.