Synergistic anti-inflammatory activity of omega-3 lipid and rofecoxib pretreatment on macrophage proinflammatory cytokine production occurs via divergent NF-kappaB activation

Abstract

Omega-3 lipid pretreatment significantly decreases TNF-alpha production in LPS-stimulated Mphis; however, this response is only a partial inhibition, suggesting that other nonsubstrate- (lipid) dependent mechanisms are involved. The cyclooxygenase (COX)-2 enzyme is principally responsible for lipid metabolism; thus, a selective COX-2 inhibitor (Rofecoxib) would clarify if it is an omega-3 lipid direct effect or a COX-2 enzyme-associated modulated reduction in TNF-alpha. Moreover, potential synergy between omega-3 lipids and selective COX-2 inhibition is postulated. HYPOTHESIS: Through divergent regulatory mechanisms, omega-3 lipids in combination with Rofecoxib will synergistically decrease the LPS-stimulated Mphi inflammatory response. METHODS: RAW 264.7 cells were pretreated with omega-3 lipids, Rofecoxib, or combination treatment and then washed and exposed to LPS. Supernatants were collected for ELISA, total proteins were obtained to determine COX-2 protein expression by Western blot, and nuclear extracts were isolated to determine NF-kappaB activation by electromobility shift assay. RESULTS: TNF-alpha and PGE2 production was significantly decreased with omega-3 and Rofecoxib pretreatment, and with combination treatment a further decrease in TNF-alpha production was observed. COX-2 protein expression was demonstrated to increase in omega-3, Rofecoxib, and combination groups stimulated with LPS. No alteration in NF-kappaB activation was observed with Rofecoxib or combination pretreatment compared with LPS-stimulated control cells. Repletion of prostaglandin (PGE2) in the Mphi model significantly decreased TNF-alpha in all groups. CONCLUSIONS: Omega-3 lipids and Rofecoxib independently decrease TNF-alpha and PGE2 production in LPS-stimulated Mphi, yet in combination a synergistic reduction in TNF-alpha production is observed. Although the anti-inflammatory effects observed from omega-3 lipids are known to occur partially through decreasing NF-kappaB activation, we demonstrated that Rofecoxib or even a combination of omega-3 and Rofecoxib does not alter NF-kappaB activation, as seen with omega-3 lipids alone. These data support that combination treatment may result in decreased Mphi inflammation, yet this occurs via divergent mechanisms.