Melatonin modulates lipopolysaccharides-induced inflammatory response and maintains circadian rhythm associated with histone H3 acetylation in bovine mammary epithelial cells

Abstract

Lipopolysaccharides (LPS) can disrupt the barrier function of mammary epithelial cells, leading to mastitis. The protective effects of melatonin (MT) have been demonstrated against various infections, but the underlying mechanism remains unclear in LPS-induced bovine mastitis. This study, therefore, aimed to investigate the modulatory effect of melatonin (MT) on LPS-induced inflammation in bovine mammary epithelial cells (BMECs), its influence on the disruption of circadian rhythms triggered by this inflammation, and the potential underlying molecular mechanisms. BMECs extracted from lactating cows underwent one of 4 treatments in triplicates for 12 h simultaneously: Control (CON), LPS (exposed to LPS at 1 μg/mL), MT (exposed to MT at 40 μmol/L), or LPS + MT (LMT, exposed to both LPS at 1 μg/mL and MT at 40 μmol/L). The flow cytometry results demonstrated that MT reduced the ratios of cells in the G0/G1 phase but increased those in the G2/M phase (P < 0.05). Additionally, MT inhibited p65 phosphorylation levels, leading to a decreased cellular inflammatory response (P < 0.05). Treatment with MT elevated the acetylation levels of histone H3 in BMECs compared to the LPS treatment (P < 0.05). The circadian rhythm analysis revealed a significant increase in the gene expression levels of BMAL1, PER1, CRY1, and CRY2 at the 12 h mark in LMT compared to the LPS treatment (P < 0.05). Additionally, this study assessed the fluctuations in clock-related genes PER2 and CLOCK over 24 h under various treatment conditions. The results demonstrated that BMECs exposed to LMT treatment exhibited a significant increase in the expression levels of CLOCK and PER2 genes from 4 to 24 h in comparison to the LPS treatment (P < 0.05). Moreover, cells in the LMT treatment displayed a more regular circadian rhythm cycle compared to those in the LPS treatment. Overall, the data suggest that MT could alleviate the inflammatory response in BMECs by upregulating histone H3 acetylation, thereby maintaining the physiological circadian rhythm of BMECs.