Lipopolysaccharide-stimulated RAW 264.7 macrophage inducible nitric oxide synthase and nitric oxide production is decreased by an omega-3 fatty acid lipid emulsion

Abstract

Background: Omega-3 fatty acids (omega-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 E(2) and leukotriene B(4) 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 omega-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 omega-3 or omega-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, omega-3 FA pretreatment at 12 and 24 h produced significantly less NO (P < 0.05) compared to omega-6 FA or media-only conditions. omega-3 FA pretreatment at 12 and 24 h also had less iNOS protein expression compared to omega-6 FA or media-only conditions. Tumor necrosis factor-alpha production was significantly decreased with omega-3 FA treatment compared to omega-6 FA treatment (P < 0.05) after 24 h LPS stimulation.

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

Figure 1

Effect of ω-3 FA and ω-6 FA pretreatment on TNF-α production in LPS-stimulated MΦ. MΦ were pretreated with media-only (DMEM), Φ-3 FA (Omegaven®), or ω-6 FA (Lipovenos®) with or without LPS-stimulation for 24 hrs. Supernatants were collected and analyzed for TNF-α production by ELISA. The data represents mean ± SEM (n=3). * significant (p<0.05) increase from baseline DMEM and w-3 FA alone, ** significant (p<0.05) decrease from DMEM+LPS and ω-6 FA+LPS.

Figure 2

A and B Effects of ω-3 FA and ω-6 FA pretreatment on NO production in LPS-stimulated MΦ. In both experiments, MΦ were pretreated with media-only (DMEM), ω-3 FA (Omegaven®) or ω-6 FA (Lipovenos®) for 4 hrs. A) cells were washed and stimulated with or without LPS for 12 hrs. B) cells were washed and stimulated with or without LPS for 24 hrs. Supernatants were then collected and NO levels were measured by the Griess reagent. Each value represents the mean ±SEM of 3 assays. * significant (p<0.05) decrease in NO production compared to DMEM+LPS and ω-6 FA+LPS.

Figure 3

A and B Effect of ω-3 FA and ω-6 FA pretreatment on LPS-induced iNOS protein expression. MΦ were pretreated with media-only, ω-3 FA (Omegaven®) or ω-6 FA (Lipovenos®) for 4 hrs. A) cells were then stimulated with or without LPS for 12 hrs and β-actin was the internal loading control for this experiment. ω-3 FA were found to decrease iNOS protein expression after LPS-stimulation compared to DMEM+LPS and ω-6 FA+LPS. B) Ban intensity was measured using densitometry in which iNOS protein expression was normalized relative to the β-actin expression levels.

Figure 4

A and B Effect of ω-3 FA and ω-6 FA pretreatment on LPS-induced iNOS protein expression. MΦ were pretreated with media-only, ω-3 FA (Omegaven®) or ω-6 FA (Lipovenos®) for 4 hrs. A) cells were then stimulated with or without LPS for 24 hrs and β-actin was the internal loading control for this experiment. ω-3 FA were found to decrease iNOS protein expression after LPS-stimulation compared to DMEM+LPS and ω-6 FA+LPS. B) Ban intensity was measured using densitometry in which iNOS protein expression was normalized relative to the β-actin expression levels.