164. Melatonin Supplementation Relieves the LPS-Induced Inflammation in the Rumen Epithelial Cells of Angus Cattle

Abstract The objective of this study was to investigate the effect of melatonin supplementation on the LPS-induced inflammation in bovine rumen epithelial cells. Sub-acute ruminal acidosis (SARA) is a prevalent digestive disorder in beef and dairy cattle, where the rumen environment of diseased cows acidifies, causing the gram-negative bacteria to rupture and release lipopolysaccharide (LPS), resulting in rumenitis. Melatonin, an amine hormone, plays a pivotal role in immune modulation, displaying both pro and anti-inflammatory responses. In this study, bovine rumen epithelial cells (BREC) were cultured in DMEM supplemented with 10% FBS and 1% antimycotic antibiotic. Cells were then stimulated with different concentrations of LPS (0, 1, 10, 50, and 100 µg/ml) at different times (3 hr, 6 hr, and 9 hrs) to establish an inflammation model. Then, BRECs were treated with different melatonin concentrations (0, 0.1, 1, 10, and 100 µM) dissolved in 100% ethanol for 2 hrs in advance of LPS stimulation. The cell viability, cytotoxicity, and proliferation assays showed that LPS (1, 10, and 50 µg/ml; 6 hrs) could stimulate cell inflammation (P < 0.05) and 0.1 µM dose of melatonin 2 hrs prior to LPS treatment significantly increased the viability and proliferation of cells (P < 0.05). The study was further divided into three treatment groups: Control group (No MEL), No MEL+LPS (1, 10, 50 µg/ml) and MEL+LPS (0.1 µM MEL pretreatment followed by 1, 10, 50 µg/ml LPS) and the samples (n = 3) were sent for transcriptomics analysis. The differential gene analysis (DeSeq) showed dose-dependent changes in the gene expression across the experimental groups. The results showed that LPS treatment (1 µg, 10 µg, and 50 µg for 6 hrs) can induce statistically significant (padj < 0.05) upregulation of a total of 126 genes in the No MEL vs. No MEL_1 µg, 81 genes in the No MEL vs. No MEL_10 µg and 139 genes in the No MEL vs. No MEL_50 µg, including IL-1A, IL-6, TNF-A, NF-kB, and various cytokines, resulting in the activation of TNF, NF-kB, and MAPK signaling pathways (padj < 0.05). Melatonin (0.1 µM) pretreatment significantly reduced the expression of inflammatory genes, and we observed the upregulation of several genes including PRRX2, LAMB3, MIF, VGF, SLC25A6, COL1A, and ITGB4 in the MEL_1 µg vs. No MEL_1 µg, and MEL_10 µg vs. No MEL_10 µg, which plays an important role in cell proliferation, wound healing, tissue repair, stress response, and mitochondrial function. This likely indicates that melatonin treatment not only reduces inflammation but also increases stress adaptation, epithelial repair, and immune response modulation.