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Background: Mitogen-activated protein kinase (MAPK) cascades represent a major signal system to transduce extracellular signals into cellular responses. Overactivity of MAPK has been implicated in the development of many diseases, including cancer and sepsis. This study investigated the hypothesis that fish oil altered the membrane phospholipid composition and modulated MAPK activity., Methods: RAW 264.7 cells, a mouse macrophage (Mphi) cell line, were grown in eicosapentaenoic acid (EPA)-rich media (114 micromol/L) for 48 hours. Mphi were washed and exposed to Escherichia coli lipopolysaccharide (LPS; 1 microg/mL) for 10 minutes. Both total and activated (phosphorylated) portions of MAPK (P44 and P42) were determined by Western blot assays. AP-1 transcription factor activity was determined by electrophoretic mobility gel shift assays (EMSA). Mphi tumor necrosis factor (TNF) mRNA expression was measured by Northern blot assays., Results: LPS stimulation induced RAW cell phosphorylation of P44/P42. In contrast, RAW cells grown in EPA-rich media had less P44/P42 activation in the presence of LPS. Total P44/P42 were not affected by EPA or LPS. Similarly, EPA also inhibited AP-1 activity. Inhibition of P44/P42 activity with PD98059 reduced both AP-1 activity and TNF mRNA expression of LPS-stimulated Mphi., Conclusions: Our data suggest that fish oil regulates macrophage proinflammatory gene activation, at least in part, by modulating the MAPK activity.

Background: Fish oil-supplemented diets have anti-inflammatory and immunomodulating effects. Although fish oil is readily incorporated into the cell membrane and influences the production of eicosanoids, the exact mechanism is not clear. This study was designed to investigate the effects of eicosapentaenoic acid (EPA), a major component of fish oil, on macrophage (Mphi) cyclooxygenase (COX) gene expression induced by LPS., Methods: RAW 264.7 cells, a mouse Mphi cell line, were grown in EPA-rich media for 24 h. Mphi were washed and exposed to Escherichia coli LPS (10 microg/ml). Membrane lipid profile was determined by gas chromatographic analysis. COX-1 and COX-2 mRNA expressions were determined by Northern blot assays with mouse-specific cDNA probes. PGE(2) production of Mphi was measured by ELISA. Mphi production of COX-2 protein was determined by Western blot assays with an anti-COX-2 antibody., Results: Incubation in EPA-rich media increased membrane EPA and decreased arachidonic acid (AA) composition. COX-2 mRNA expression was induced by EPA and further augmented by LPS stimulation. EPA also augmented Mphi production of COX-2 protein. In comparison, COX-1 mRNA expression was not affected by either LPS stimulation or EPA incubation. EPA reduced PGE(2) production by LPS-stimulated Mphi. To further support that COX-2 mRNA was regulated by COX product, exogenous PGE(2) was added to Mphi prior to LPS stimulation. PGE(2) reduced COX-2 mRNA of LPS-stimulated Mphi., Conclusion: EPA displaces AA and reduces PGE(2) production by LPS-stimulated Mphi. Fish oil inhibition of Mphi PGE(2) production induces COX-2 mRNA expression through a COX-2 product-mediated feedback mechanism., (Copyright 1999 Academic Press.)

Background: Fish oil-supplemented diets have anti-inflammatory and immunomodulating effects, though the exact mechanism(s) are unknown. This study investigated the effects of eicosapentanenoic acid (EPA), a major component of fish oil, on transcriptional regulation of tumor necrosis factor (TNF) gene in lipopolysaccharide (LPS)-stimulated macrophages (MO)., Methods: RAW 264.7 cells, a mouse MO cell line, were grown in EPA-rich media for 24-48 h. MO were washed and exposed to Escherichia coli LPS (1 microg/ml) for 2 h. TNF mRNA expression was measured by Northern blot assays. Total nuclear extracts were harvested for the measurement of NF kappa B with electrophoretic mobility shift assays. Supershift assays were performed with anti-P50 or anti-P65 antibodies to show components of NF kappa B dimers. TNF production was determined by L929 bioassays., Results: LPS stimulated RAW cell TNF mRNA expression and NF kappa B activity. In contrast, RAW cells grown in EPA-rich media had less TNF mRNA expression and an altered composition of the NF kappa B subunits (P65/P50 dimers) in the presence of LPS. TNF production by LPS-stimulated MO was reduced by EPA., Conclusions: The inhibitory effect of EPA on LPS-stimulated MO TNF gene transcription and protein elaboration is, in part, mediated through altering NF kappa B activation by reducing the P65/P50 dimers., (Copyright 1999 Academic Press.)

Liver ischemia and reperfusion injury is mediated by oxygen free radicals, cytokines, and prostanoids produced by Kupffer cells and infiltrating neutrophils. Fish oil-supplemented diets alter membrane phospholipid composition and modify prostanoids and cytokine production in response to ischemia and reperfusion. This study tested the hypothesis that a fish oil-supplemented diet would attenuate warm liver ischemia and reperfusion injury in the rat. Male Sprague-Dawley rats were fed Vital HN supplemented with either fish oil (FO) or corn oil (CO) by the continuous duodenal infusion for 5 days. Total dietary fat (26% of total calories), caloric intake (70 cal/day), and volume (60 ml/day) were identical between two groups. Plasma eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) levels increased significantly in rats fed fish oil (0 to 16.3% for EPA and 2 to 12% for DHA). Liver histology was similar in both groups before ischemia. On Day 6, rats were subjected to 60 min of reversible hepatic ischemia. Plasma TNF levels, 1 and 24 hr after reperfusion, were not different between FO and CO rats. Liver injury assessed by bile flow, histology, plasma ALT, and bile glutathione efflux did not differ between groups. We conclude that our fish oil-supplemented enteral diet does not attenuate warm liver ischemia and reperfusion injury in rats.