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


OBJECTIVE
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.
Babcock
Tricia A
TA
Department of Surgery, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Helton
W Scott
WS
Anwar
Khandaker N
KN
Zhao
You-Yang
YY
Espat
N Joseph
NJ
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000276:immunology / Q000494:pharmacology*; D002460:Cell Line; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000276:immunology / Q000494:pharmacology*; D015232:Dinoprostone / Q000008:administration & dosage / Q000494:pharmacology; D004357:Drug Synergism; D015525:Fatty Acids, Omega-3 / Q000276:immunology / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007783:Lactones / Q000276:immunology / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology; D008565:Membrane Proteins; D016328:NF-kappa B / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D013450:Sulfones / Q000276:immunology / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000187:drug effects
D000894:Anti-Inflammatory Agents, Non-Steroidal; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D015525:Fatty Acids, Omega-3; D007783:Lactones; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D016328:NF-kappa B; D013450:Sulfones; D014409:Tumor Necrosis Factor-alpha; C116926:rofecoxib; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone