Omega-3 fatty acids decrease oxidative stress and inflammation in macrophages from patients with small abdominal aortic aneurysm

Abstract

Abdominal aortic aneurysm (AAA) is associated with inflammation and oxidative stress, the latter of which contributes to activation of macrophages, a prominent cell type in AAA. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been reported to limit oxidative stress in animal models of AAA. The aim of this study was to evaluate the effect of the n-3 PUFA docosahexaenoic acid (DHA) on antioxidant defence in macrophages from patients with AAA. Cells were obtained from men with small AAA (diameter 3.0-4.5 cm, 75 ± 6 yr, n = 19) and age- matched male controls (72 ± 5 yr, n = 41) and incubated with DHA for 1 h before exposure to 0.1 µg/mL lipopolysaccharide (LPS) for 24 h. DHA supplementation decreased the concentration of tumour necrosis factor-α (TNF-α; control, 42.1 ± 13.6 to 5.1 ± 2.1 pg/ml, p < 0.01; AAA, 25.2 ± 9.8 to 1.9 ± 0.9 pg/ml, p < 0.01) and interleukin-6 (IL-6; control, 44.9 ± 7.7 to 5.9 ± 2.0 pg/ml, p < 0.001; AAA, 24.3 ± 5.2 to 0.5 ± 0.3 pg/ml, p < 0.001) in macrophage supernatants. DHA increased glutathione peroxidase activity (control, 3.2 ± 0.3 to 4.1 ± 0.2 nmol/min/ml/μg protein, p = 0.004; AAA, 2.3 ± 0.5 to 3.4 ± 0.5 nmol/min/ml/μg protein, p = 0.008) and heme oxygenase-1 mRNA expression (control, 1.5-fold increase, p < 0.001). The improvements in macrophage oxidative stress status serve as a stimulus for further investigation of DHA in patients with AAA.

Figure 1

Concentration of 8-isoprostane in supernatants of monocyte-derived macrophages obtained from control participants and AAA patients. Monocytes were isolated from whole blood, grown in culture and, following spontaneous differentiation into macrophages, exposed to a 24 h incubation with 0.1 μg/ml lipopolysaccharide (LPS) prior to collection of the cells and supernatants. LPS-stimulated 8-isoprostane production (pg/ml), plotted against total number of circulating monocytes present in whole blood samples, was lower in AAA (B) compared to control macrophage supernatants (A). DHA decreased LPS-stimulated 8-isoprostane production in a dose-dependent manner in control participant macrophages. A non-significant trend for decreased 8-isoprostane production was observed in macrophages from AAA patients.

Figure 2

Cytokine concentrations in macrophage supernatants obtained from control participants and AAA patients. Control DHA-supplemented non-stimulated macrophages (20 μM n = 5–9; 80 μM, n = 7–10) produced lower levels of tumour necrosis factor alpha (TNF-α; A), interleukin-6 (IL-6; C), interleukin-1 β (IL-β; E) and interleukin-10 (IL-10; I) compared to non-supplemented macrophages. DHA supplementation of AAA non-stimulated patient macrophages (20 μM n = 6–7; 80 μM, n = 8–10) decreased the production of TNF-α (B), IL-6 (D) and IL-10 (J). A trend toward decreased levels of IL-β (F; 20 μM n = 7; 80 μM n = 10) was observed. Transforming growth factor-β (TGF-β) levels in both control (G) and AAA (H) macrophages were unaffected by DHA supplementation. *p < 0.05.

Figure 3

Antioxidant enzyme activity levels in control participant and AAA patient monocyte-derived macrophage lysates. Macrophage catalase activity was unaffected by DHA supplementation in control participants (A) and AAA patients (B). DHA supplementation increased glutathione peroxidase (GPx) activity in non-stimulated macrophages from control participants (C) and patients with AAA (D). Catalase and GPx activity and expression levels were unaffected by supplementation with OA. *p < 0.05.

Figure 4

Increase in HO-1 transcripts and decrease in 8-isoprostane concentration in monocyte-derived macrophages from control participants. Real-time quantitative PCR, with raw data normalised to GAPDH, indicated increased HO-1 expression levels in DHA-supplemented non-stimulated macrophages and a trend for increase in LPS-stimulated macrophages exposed to DHA (A). In parallel, significant decreases were observed in 8-isoprostane concentrations in supernatants from hemin- and LPS-stimulated macrophages (B). Values are mean ± SEM of duplicate determinations (n = 5). *p < 0.05.

Figure 5

8-Isoprostane levels in non-stimulated and LPS-stimulated U937 cells incubated in the presence and/or absence of DHA (20 μM; 80 μM), the FFA4 antagonist AH7614 and the HO-1 inhibitor SnPP. Supplementation of cells with DHA 80 μM decreased 8-isoprostane production (A), a finding that was not reversed with AH7614 treatment (B). In LPS-stimulated cells treated with SnPP (C), no significant effect was observed for DHA at either of the concentrations tested. Data are expressed as mean ± SEM and are representative of at least six independent experiments. *p < 0.05.

Figure 6

Free radical scavenging activity of quercetin 50 μM, BHT 227 μM, DHA 80 μM, EPA 80 μM and OA 80 μM as measured by colorimetric 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Quercetin and BHT were included as positive controls. Absorbance values for the diluent (BSA) were subtracted from all data sets. Data are expressed as mean ± SEM and are representative of four independent experiments.