Berberine alleviates LPS-induced inflammatory response by targeted activation of NR1D1 in bovine endometrial...

Endometritis, a prevalent inflammatory disease in dairy cattle, causes substantial economic losses to the dairy industry. Conventional therapies mainly rely on antibiotic treatment, but this often leads to bacterial resistance and antibiotic residues in milk. Thus, it is essential to identify novel and effective therapeutic methods for endometritis treatment in dairy cows. The activation of nuclear receptor subfamily 1 group D member 1 (NR1D1), an important circadian clock component, potently attenuates inflammatory responses in various diseases through transcriptional repression of its downstream target genes. In spite of the growing evidence for NR1D1 regulation of inflammatory diseases, its involvement in the curing of bovine endometritis remains largely unidentified. Recently, berberine (BBR), a natural isoquinoline alkaloid, has been identified as a natural NR1D1 agonist. The present study was aimed to investigate the role of NR1D1 in bovine endometritis, and to elucidate the potential therapeutic effect of BBR on endometritis treatment and its underlying mechanisms by targeted activation of NR1D1, using bovine endometrial epithelial cell line (BENDs) and mouse model treated by Escherichia coli lipopolysaccharide (LPS). In comparison with their control groups, NR1D1 expression was elevated in uterine tissues from cows with endometritis and in BENDs treated with 1 μg/mL LPS for 12 h, accompanying by the upregulation of IL-6, IL-1β, IL-8, and CCL5 expression. Activation of NR1D1 by SR9009 (a synthetic NR1D1 agonist) or lentiviral overexpression of NR1D1 markedly reduced the levels of proinflammatory cytokines (IL-6, IL-1β, and CCL5) and critical NF-κB pathway proteins (p-p65 and p-IκB), whereas NR1D1 inhibition or knockout conversely resulted in a pronounced upregulation of proinflammatory cytokines and NF-κB pathway proteins. Subsequently, BBR treatment attenuated LPS-induced inflammatory response in both BENDs and the mouse endometrium; however, this anti-inflammatory effect was abolished in NR1D1-deficient BENDs. In addition, western blotting detected robust circadian rhythmicity of NR1D1 in the uterus of mouse model, and BBR treatment at zeitgeber time (ZT) 8 showed the better therapeutic efficacy on attenuating the mouse endometrial inflammatory response than at ZT20, displaying a time-dependent treatment manner. In conclusion, BBR alleviates LPS-induced inflammatory response by targeted activation of NR1D1 in BENDs, and that the anti-inflammatory efficacy of berberine in a mouse model of endometritis is time-dependent. These findings suggest that NR1D1 is a promising therapeutic target for bovine endometritis, and its natural agonist BBR potentially act as an anti-inflammatory compound for the prevention and treatment of bovine endometritis.