Epicatechin Down-regulates Inflammatory Genes in Adipocytes Co-cultured With Endotoxin-activated Macrophages

Objectives: Chronic low-grade inflammation has been suggested to associate with insulin resistance and atherosclerotic changes, and periodontal disease could be one such inflammatory disease. It is well known macrophages were integrated into adipose tissues to interact with adipocytes, thereby exacerbating inflammatory responses. Recent studies suggested cocoa flavonol (epicatechin: EC) intake appears to reduce the risk of cardiovascular events, suggesting that it exhibits anti-inflammatory effects. In this study, we evaluated the influence of EC on gene expression in adipocytes co-cultured with endotoxin-stimulated macrophages by using microarray technique, and evaluated its ability to inhibit inflammation.

Methods: RAW264.7 mouse macrophage cell line and differentiated 3T3-L1 mouse preadipocytes were co-cultured. The cells were pre-treated either with or without EC, followed by the stimulation with bacterial endotoxin. Total RNA was isolated from adipocytes at 4, 8, 12, and 24h after endotoxin stimulation. Isolated RNA were subjected to microarray analysis. Heat map was constructed and clustering analysis was performed. To confirm the results of microarray analysis, real time PCR and ELISA were performed.

Results: Pre-treatment of the co-cultures of adipocytes and macrophages by EC resulted in dynamic changes in adipocyte gene expression following endotoxin stimulation. The expression of the genes encoding inflammatory cytokine and chemokine such as IL-6 was markedly down-regulated. The most dynamic changes were observed in NF-κB pathways. Further, we examined whether target cells of EC were macrophage or adipocytes. The target cell appeared to be mainly macrophages, as it markedly suppressed macrophage inflammatory signals and adipocyte inflammatory signals were only down-regulated when the cells were co-cultured with macrophages.

Conclusions: These results suggest that EC inhibits inflammatory responses in adipose tissue up-regulated by gram-negative bacterial infection. Thus, EC may be a favorable candidate supplement for the subjects with severe periodontitis.