Lipopolysaccharide-Stimulated RAW 264.7 Macrophage Inducible Nitric Oxide Synthase and Nitric Oxide Production Is Decreased by an Omega-3 Fatty Acid Lipid Emulsion

Background

Omega-3 fatty acids (ω-3 FA) have been demonstrated to have anti-inflammatory properties, postulated to occur through several principal mechanisms, including (1) displacement of arachidonic acid from the cellular membrane; (2) shifting of prostaglandin E₂ and leukotriene B₄ production; and (3) molecular level alterations including decreased activation of nuclear factor kappa B and activator protein-1. An additional regulator that is likely associated is the production of nitric oxide (NO) by nitric oxide synthetase. NO is a short-lived free radical involved in many biological functions. However, excessive NO production can lead to complications, suggesting that decreased NO production is a potential target for some inflammatory diseases. We hypothesized that pretreating with an ω-3 FA lipid emulsion would decrease the production of NO in macrophages and that this effect would occur through alterations in inducible nitric oxide synthetase (iNOS).

Materials and methods

Greiss reagent was used to assess NO production in RAW 264.7 macrophages following ω-3 or ω-6 FA treatment alone or in combination with lipopolysaccharide (LPS) stimulation for 12 h/24 h. iNOS levels were determined by Western blot. Tumor necrosis factor-alpha levels were determined by enzyme-linked immunosorbent assay.

Results

Following LPS-stimulation, ω-3 FA pretreatment at 12 and 24 h produced significantly less NO (P < 0.05) compared to ω-6 FA or media-only conditions. ω-3 FA pretreatment at 12 and 24 h also had less iNOS protein expression compared to ω-6 FA or media-only conditions. Tumor necrosis factor-alpha production was significantly decreased with ω-3 FA treatment compared to ω-6 FA treatment (P < 0.05) after 24 h LPS stimulation.

Conclusion

These experiments demonstrate that, in addition to other anti-inflammatory effects, ω-3 FA lipid emulsions also significantly lower NO production in LPS-stimulated macrophages through altered iNOS protein expression.

Key Words: omega-3 fatty acids, nitric oxide, macrophage