Anti-Inflammatory Action of Epigallocatechin-3-Gallate at a Physiological Concentration through the 67-KDA Laminin Receptor in Lipopolysaccharide-Stimulated Raw264.7 Cells

Abstract

It has been reported that epigallocatechin-3-gallate (EGCG), major active polyphenol of green tea, can modulate the production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and interleukin (IL)-8, in lipopolysaccharide (LPS)-stimulated macrophage cells. Generally, high concentrations of EGCG (10–50 μM) have been used for investigating a variety of biological activities although the peak plasma concentration of EGCG is less than 1 μM. Recently, we have identified the 67-kDa laminin receptor (67LR) as a cell-surface receptor for EGCG that mediates the anti-cancer activity of EGCG at a physiologically achievable concentration (0.1 μM). However, the relationship of 67LR and anti-inflammatory activity of EGCG in LPS-stimulated macrophage cells still remained unclear. To explore whether the effects of EGCG on cellular responses to the LPS stimulation are mediated by the 67LR, RAW264.7 cells were stably transfected with short hairpin RNA (shRNA) expression vector to reduce the 67LR expression. Western blot analysis showed that RAW264.7 cells expressed the 67LR, and this expression has been shown to be lowered in RAW264.7 cells transfacted with shRNA for 67LR. In the 67LR-downregulated cells, the suppressive effects of physiologically relevant EGCG (1 μM) on LPS-stimulated inflammatory mediators, such as TNF-α, IL-6, nitric oxide, nitric oxide synthase and cyclooxygenase-2 were prevented. These results suggest that 67LR plays an important role in mediating anti-inflammatory actions of EGCG at a physiologically achievable concentration. This is the first evidence providing the molecular basis for regulation of macrophage activation by physiologically relevant EGCG.