Recovering of inflammatory and oxidative stress damage in retinal epithelial and endothelial cells: synergistic action of anti-oxidant and vitamin D

Abstract

Purpose : We aimed to test the possible synergistic effect of the Vitamin D and nutritional complex containing anti-oxidants, macular pigments and omega 3 (Nutrof Total®, Laboratoires Thea, France) combination in human retinal epithelial (ARPE-19), human retinal endothelial (HREC) and primate choroidal endothelial (RF6A) cells under inflammation and oxidative stress.

Methods : Three groups; N, nutritional complex (Nutrof® Total); D, 1,25-Dihydroxyvitamin D and their combination (ND) were tested in ARPE-19 (RPE), HREC and RF6A cells under inflammatory (lipopolysaccharide, 20 µg/ml, 24 h) or oxidative stress (H₂O₂ 800 µM, 3h or sodium iodate, 750 µM, 24 h) conditions. N and ND groups’ doses were adjusted to D concentration (1 nM, 1 h). Vitamin D receptors and enzymes were confirmed by PCR. Cell viability (MTT) and proliferation (BrdU) were tested. Interleukin (IL)6, 8, 12p70, Interferon (IFN)-g, monocyte chemoattractant protein (MCP)-1, vascular endothelial growth factor (VEGF) and 8OHdG were assayed by ELISA. Cell integrity (ZO1) and apoptosis (TUNEL) were evaluated by immunofluorescence confocal microscopy. Results were analyzed by ANOVA or Kruskal-Wallis and post hoc test or U Mann-Whitney (SPSS v. 20.0)

Results : Vitamin D synthesizing components were reported in 3 cell lines. MTT was reduced in RF6A cells treated with ND (p<0.05), while it was not affected in HREC and RPE. Under oxidative stress, all the treatments reduced BrdU in RF6A (p<0.05), increasing in RPE (p<0.001) and HREC (p<0.05) cells in the ND group only. Cell integrity was restored and MTT and TUNEL were improved in HREC cells. Also, N and ND treatments reduced 8OHdG (p<0.05). Under inflammation, ND treatment showed a reduction in IFN-g, MCP1 and IL6 in RPE (p<0.05) and HREC (p<0.05) cells. Interestingly, VEGF and IL8 levels were restored to control in RPE cells and IL12p70 was exclusively reduced by ND treatment in HREC cells (p<0.05)

Conclusions : Combination of Vitamin D and nutritional complex showed a protective effect in the human retinal cells and an additive beneficial effect over the nutritional complex alone for the RPE cells and the retinal endothelial cells, recovering of inflammatory and oxidative stress damage. Moreover, Nutrof Total® reduced the proliferation of choroidal endothelial cells suggesting a favourable effect in choroidal neovascularization prevention

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.