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

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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.

Introduction

An increasing emphasis is being placed on the potential for diet and dietary supplements to serve as modulators of the host response to disease, injury, and infection. One such supplement under investigation as an anti-inflammatory agent is eicosapentaenoic acid (EPA) an omega-3 fatty acid (ω-3 FA), which is a primary component of deep water marine fish oils. Clinically these polyunsaturated ω-3 FA are recognized to be essential for normal growth and development and have also been demonstrated to be of benefit in a number of inflammation-associated disease states including artherosclerosis, auto-immune disorders, malignancy, and sepsis [1, 2, 3]. However, in order for widespread clinical use of ω-3 FA to occur, the underlying mechanism for the suggested biological activities of fish oil need to be defined. In these studies a pharmaceutical grade ω-3 FA lipid emulsion (Omegaven; Fresinius-Kabi, Bad-Homburg, Germany) and a formulated dose equivalent ω-6 FA lipid emulsion (Lipovenos, Fresinius-Kabi) were used to facilitate experiments in vitro that more adequately represent ω-3 FA effects compared to previously used reagents containing EPA salts and EPA–albumin complexes that might themselves alter the inflammatory response in vitro.

Previously, in a lipopolysaccharide (LPS)-stimulated macrophage (MΦ) models treated with this ω-3 FA lipid emulsion has demonstrated an alteration in the inflammatory response at least in part through their ability to decrease tumor necrosis factor-alpha (TNF-α) and prostaglandin E2 (PGE2) production [4, 5, 6]. However, the mechanism(s) for these alterations appears to occur through a series of complex events including the displacement of arachidonic acid in the cellular membrane leading to the production of less metabolically active compounds including PGE3 instead of PGE2 and leukotriene B5 instead of leukotriene B4 [7]. Additionally, ω-3 FA lipid emulsion treatment leads to significant alterations in nuclear transcription factor activation, particularly nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) [8], both of which are primary regulators of the inflammatory response in experimental MΦ models.

While TNF-α is significantly decreased by ω-3 FA, this reduction only accounts for approximately 45–50% of the overall anti-inflammatory effects observed experimentally, suggesting that other factors are likely involved [9]. Nitric oxide (NO) may be one such factor that is potentially altered by ω-3 FA, subsequently resulting in a decreased pro-inflammatory milieu.

NO is a short-lived free radical and internal messenger that mediates a variety of functions including vascular homeostasis, neurotransmission, and host defense. NO is synthesized from l-arginine by nitric oxide synthase (NOS). Three isoforms of NOS have been identified including: endothelial NOS, neuronal NOS, and inducible NOS (iNOS) [10]. In the MΦ, iNOS is the primary regulator of NO production, a known principal mediator of macrophage bactericidal and tumoricidal activities [11, 12]. However, when there is excess or prolonged production of NO, the potential for inflammation-associated tissue damage is present [13, 14], suggesting a potential role for targeted attenuation of iNOS-mediated NO production.

Regulation of iNOS gene expression is through transcriptional regulation, particularly focused by NF-κB activation [15, 16]. In the mouse, iNOS gene promoter contains two NF-κB binding sites, both of which need to be bound to get full induction of iNOS by LPS stimulation. However, in addition to NF-κB, there are also binding sites for CCAAT/enhancer binding protein, AP-1, and cAMP response element-binding; all are thought to be involved in regulating iNOS production [17, 18].

ω-3 FA lipid emulsions have been demonstrated to significantly decrease NF-κB activation in the LPS-stimulated MΦ model [8]. Additionally, ω-3 FA lipid emulsions have also altered the activation of the mitogen-activated protein kinase pathway components as well as attenuation of AP-1 activation. [9] Furthermore, since iNOS/NO production is at least in part regulated by these nuclear transcription factors, in combination, these observations support the potential for ω-3 FA to modify NO production.

In the present studies, we hypothesized that OmegavenR, an ω-3 FA lipid emulsion, pretreatment would decrease the production of NO in LPS-stimulated macrophages and that this effect would occur through alterations in iNOS.

Section snippets

Materials

RAW 264.7 cells (murine macrophage cell line) were purchased form American Type Tissue Culture Collection (ATCC, Rockville, MD). Escherichia coli 0111:B4 LPS, β-actin antibody, and the Greiss reagent were purchased from Sigma (St. Louis, MO). Omegaven® (ω-3 FA lipid emulsion) and Lipovenos® (ω-6 FA lipid emulsion) were purchased from Fresinius-Kabi. Endotoxin-free serum was purchased from Hyclone (Logan, UT). Western blot detection reagents were purchased from Cell Signaling (Beverly, MA). iNOS

Effects of ω-3 Lipid on TNF-α Production

In non-LPS-stimulated MΦs there is minimal baseline TNF-α production, control (DMEM, 62.1 ± 1.37 pg/m), ω-3 FA (59.6 ± 13.6 pg/mL), and ω-6 FA (182.4 ± 32.3 pg/mL) with a significant baseline alteration observed for ω-6 FA without LPS stimulation (P < 0.05). In the presence of 24 h LPS stimulation there is a significant increase in TNF-α production. DMEM+LPS (11,390 ± 496.4 pg/mL) and ω-6 FA (11,595 ± 203.6 pg/mL) -pretreated cells demonstrated similar amounts of TNF-α. However, ω-3 FA

Discussion

NO is a ubiquitous messenger in biological systems, participating in the regulation of a variety of biological processes when it is released by ΜΦ in response to noxious stimuli (endotoxin, pro-inflammatory cytokines, etc). A significant portion of NO is released several hours after an initial stimulation; the magnitude of this response is determined by the up-regulation of iNOS gene transcription. Under homeostatic conditions, NO participates in a host-beneficial system to destroy pathogens,

Acknowledgment

This work was supported in part by NIDDK-1 K08 DK DK60778 (N.J.E.).

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