Microbial Metabolites of Gallotannins Suppress Inflammation in RAW 264.7 Macrophages Through the Modulation of the AMPK/NF-kb Axis

Abstract

Abstract Objectives Obesity has been positively correlated with alterations in adipose tissue such as increased production of pro-inflammatory molecules and high content of adipose tissue macrophages. In previous in vitro studies, we have shown that microbial metabolites of gallotannins (GT), including gallic acid (GA) and pyrogallol (PG), possess anti-inflammatory activities in cancer cells, as well as anti-lipogenic activities in adipocytes. In this study, we explored the molecular mechanisms of microbial metabolites of GT by investigating the effect of GA and PG on the inflammatory cytokines expression, AMP-activated protein kinase (AMPK) and NF-kb signaling pathways in RAW 264.7 macrophages. Methods RAW 264.7 macrophages were pre-treated with GA or PG (2.5 and 5 mg/L). Afterwards, inflammation was induced by 1 mg/mL lipopolysaccharide (LPS) along with the previous treatment. In a separate experiment, RAW 264.7 cells were pre-treated with or without 10 mM Compound C, an AMPK activity inhibitor, along with GA or PG (5 mg/L) and incubated with 1 mg/mL LPS. Analyses of gene and protein expression of inflammatory cytokines, AMPK and NF-kb were performed using qPCR and Western blot. Results mRNA and protein expressions of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were significantly decreased in LPS-treated RAW 264.7 macrophages by GA and PG. Additionally, GA and PG inhibited LPS-induced inflammation through the up-regulation of AMPK and sirtuin 1 (Sirt1) activities, and the down-regulation of NF-kb activity. AMPK inhibition by Compound C in RAW 264.7 macrophages partially blocked LPS-induced inflammatory signaling. As a result, the inhibitory effects of GA and PG on LPS-induced inflammation were weakened. Conclusions GA and PG attenuate inflammation in RAW 264.7 macrophages at least in part through the activation of the AMPK pathway and the suppression of NF-kb activity. Overall, microbial metabolites of GT might possess therapeutic potential in the prevention of obesity-related adipose tissue inflammation. Funding Sources COALS's Nutrition Obesity Strategic Fellowship at Texas A&M University.