Oleic, linoleic and linolenic acids increase ros production by fibroblasts via NADPH oxidase activation

Abstract

The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47 (phox) phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47 (phox) mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts.

Figure 1. Analysis of membrane integrity and DNA fragmentation.

Confluent 3T3 Swiss fibroblasts were treated with vehicle control (0) or oleic, linoleic or γ-linolenic acid (10, 20, 50, 100 or 200 μM). The results are presented as the mean ± standard error of at least three experiments that were performed in duplicate.

Figure 2. Comparative effect of fatty acids on the kinetics of superoxide production by fibroblasts.

Cells (2.5×10⁶ cells per mL) were treated with oleic, linoleic or γ-linolenic acid (100 μM) plus β-NADH (10 µM) in the presence of lucigenin (1 mM).

Figure 3. Dose response treatment with the fatty acids.

Superoxide anion levels in the incubation media for the Rat 1 (A, B and C) and 3T3 Swiss fibroblasts (D, E and F) (2.5×10⁶ cells per mL) containing β-NADH (10 µM), as measured by the lucigenin assay in the absence and presence of different concentrations of oleic (A and D), linoleic (B and E) or γ-linolenic (C and F) acid (0, 5, 50, 100 or 200 μM). The results are presented as the mean ± SEM of at least three experiments performed in triplicate. Presented as the effect of the fatty acids compared with the control, *p<0.05, ** p<0.01 and *** p<0.001.

Figure 4. The involvement of NADPH oxidase in fatty acid-induced ROS production.

Effect of DPI (200 µM), an inhibitor of NADPH, on the chemiluminescence of fatty acid-treated (100 µM) 3T3 Swiss fibroblasts. Presented as the effect of the fatty acids compared with the control, *p<0.05, ** p<0.01 and *** p<0.001.

Figure 5. Phosphorylation of NADPH oxidase component p47^phox by fatty acids.

Effect of oleic, linoleic and γ-linolenic acids on the phosphorylation of the NADPH oxidase component p47^phox in Rat 1 fibroblasts. After incubating the cells at 37°C in the absence (0 min) or presence of fatty acids (100 µM – for 5, 10 or 15 min), they were homogenized in extraction buffer and prepared for Western blot analysis. Western blotting was performed using a rabbit anti-p47^phox antibody. Similar results were obtained from three to four independent experiments. Presented as the effect of the fatty acids compared with the control, *p<0.05, ** p<0.01 and *** p<0.001.