Abstract
The anti-inflammatory properties of DHA have been largely demonstrated in vitro and in vivo but research gaps remain regarding the contribution of its oxygenated metabolites also called oxylipins. We aimed to investigate the anti-inflammatory properties and potential mechanisms of action of different types of DHA-derived oxylipins including Neuroprostanes (NeuroP), Protectin DX (PDX) as well as Neuroprotectin D1 (NPD1/PD1) and its isomer 10S,17S-diH n-3 DPAEEZ. Human peripheral blood mononuclear cells were isolated from healthy donors by Ficoll density gradient centrifugation. Monocytes were differentiated into resting macrophages (RM) for 7 days. RM were exposed to the different types of oxylipins (i.e. 14-A4-NeuroP, 4(RS)-4-F4t-NeuroP, PDX, PD1 and 10S,17S-diH n-3 DPAEEZ) at 3 different doses (i.e. 0.1, 1 and 10 μM) during 30 min. The inflammatory response was then induced with LPS (100 ng/mL) for 6 hours. Preliminary results of gene expression analysis (qPCR) show that IL-6, MCP-1, COX-2, TNFα or CCL3 mRNA were significantly lower in macrophages preexposed to 10μM 14-A4-NeuroP (-84%, -57%, -29%, -41% and -23% respectively). Significant but less pronounced effects on IL-6 and MCP-1 were also observed with 10μM 4(RS)-4-F4t-NeuroP (-25% and -25% respectively). Reduced levels of TNFα protein secretion (ELISA) were found in macrophages pre-exposed to 10μM 4(RS)-4-F4t-NeuroP (-12% p<0.05) while measurable but less pronounced effects were observed with 14- A4-NeuroP, PDX, PD1 or 10S,17S-diH n-3 DPAEEZ (-9%, -22%, -10% and -15% ns, respectively). Abundance and phosphorylation of IκBα (Western Blot) suggest that 14-A4- and 4(RS)-4-F4t-NeuroPs could exert their antiinflammatory effects through the inhibition of IκBα phosphorylation. Finally, cotransfection of luciferase reporter vector with human PPARγ expression vector performed in Cos-7 cells suggests that 14-A4- and 4(RS)-4-F4t-NeuroPs probably act independently of PPARγ. In conclusion, these results suggest that the anti-inflammatory properties of DHA could be mediated, at least in part, by oxylipins, and bring new insights into their mechanism of action