Abstract
Purpose :
The bioactivity of the novel derivatives of VLC-PUFAs n-3, ELVs, (Bhattacharjee S, et al. Sci Adv. 2017; Jun B, et al. Sci Rep. 2017) was compared with NPD1 to discern if they are additive or synergistic as protectors of RPE cells against uncompensated oxidative-stress (UOS) in human RPE cells. Moreover IL-1β transcription induction of COX-2 was also assessed.
Methods :
72h grown human primary RPE cultures were serum starved for 8h, stress by H2O2 (600µM) plus TNF- α (10ng/ml), and then challenged with 200nM of ELVs (32:6 and 34:6 Me) and NPD1(100) or together (ELVs 200nM and 30nM NPD1) for 16h. Apoptotic cell death was assessed by Hoechst staining using a Zeiss LSM 510 confocal microscope. Human RPE cells grown in 6-well plates were transfected with 5μg of a human COX-2–promoter (830bp) linked to luciferase as reporter gene by FUGENE 6. Four hours later, fresh medium was added, and the cells were further incubated for 8h at 37°C. Transfected cells were serum-starved for 4h and treated with IL-1β (10ng/ml) challenged with or without added ELVs or NPD1 for 8h. Cell homogenates were made, protein concentrations were adjusted, and luciferase assays were performed.
Results :
UOS cause 80-90% apoptosis. Interestingly, ELVs (200nM) or NPD1 (100nM) attenuated UOS mediated apoptosis (45-50%). This bioactivity was additive when UOS stressed RPE was challenged with 200nM ELVs plus 30nM NPD1. Also we found that IL-1β mediated upregulation of luciferase activity is inhibited by ELVs or NPD1. This result also supports the notion that ELVs (200nM) and NPD1 (30nM) mediated cell signaling is additive but not synergistic.
Conclusions :
ELVs inhibition of COX-2 transcriptional upregulation induced by IL-1β in human RPE uncovers this event as part of the inflammatory targeting of the novel mediators in their cyto-protective bioactivity. The ELVs- and NPD1- mediated cyto-protective additive effect suggest that the two lipid mediators follow different pathways in modulating inflammatory and RPE cell survival consequences after UOS. It is possible that they might work in a concerted manner under certain circumstances. UOS conditions likely recapitulate homeostatic disruptions in early stages and progression of age-related macular degeneration.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.