Effect of EPA:DHA formulations on cell viability, proliferation and reactive oxygen species generation under inflamatory and oxidative stress conditions

Abstract

Purpose: Following AREDS results on omega 3, we decided to test the effect of different mixtures of EPA:DHA on human retinal epithelial (ARPE-19) and monkey choroidal endothelial (RF6A) cell lines under inflammatory and oxidative stress conditions

Methods: Ten supplements (Sp) consisting of various proportions of EPA:DHA and different formulations for the mixture (ethylesters (EE), triglycerides (TG) and phospholipids (PL)) were tested. There were 4 Sp in TG formulation, 1 in EE and 5 in a mixture of TG and PL. Cells were subjected to standard (serum-free), inflammatory (induced by lipopolysaccharides, LPS) or oxidative stress (hydrogen peroxide, H₂O₂) conditions and supplemented with the different Sp. Cell viability by MTT colorimetric assay, proliferation by BrdU incorporation colorimetry and dichlorofluorescein detection as an indicator for reactive oxygen species (ROS) were evaluated. Results were statistically analyzed with SPSS 20.0 software

Results: Seven out of 10 Sp (4 TG and 3 TG+PL) significantly increased (p<0.05) the ARPE-19 cell viability under standard conditions. When ARPE-19 cells were exposed to H₂O₂, 8 Sp (3 TG and 5 TG+PL) significantly increased (p<0.05) the cell viability. Under inflammatory conditions, 8 Sp (3 TG and 5 TG+PL) presented a significant increase (p<0.05) in cell viability. The ARPE-19 cell proliferation assay showed an increase after Sp under standard conditions with 4 TG Sp. After H₂O₂-induced oxidative damage, 7 Sp (2 TG, 1 EE and 4 TG+PL) reduced the anti-proliferation effect of H₂O₂. When ARPE-19 cells were exposed to LPS 2 Sp (2 TG) mitigated the antiproliferation effects of LPS. ROS measurements showed a highly significant (p<0.001) decrease in all cases of serum-free cell culture treated with the Sp in both cell lines. The Sp that showed the best results was a 40:20 mixture of EPA:DHA in TG form while the least effective was the same mixture in EE formulation

Conclusions: Use of EPA:DHA supplements increased cell viability and proliferation and reduced the amount of ROS under oxidative stress and inflammatory conditions. Our study suggest that, unlike EPA:DHA EE used in AREDS formulation, which is almost not effective, a mixture of 40:20 TG (EPA:DHA) could be useful in retinal pathologies where inflammatory and/or oxidative stress conditions play an important role, such as Age-related macular degeneration