Abstract
Purpose: :
Docosahexaenoic acid (DHA) supplementation protects Neuro2A cells from serum starvation or staurosporine treatment via Akt signaling pathways. Highest concentrations of DHA are found in the rod photoreceptor outer segment (ROS), and when ROS are phagocytized by RPE cells, neuroprotectin D1 (NPD1) is synthesized. We have shown that DHA or NPD1 protect ARPE cells when applied during and prior to oxidative stress (ARVO 2011). Thus, the purpose of this study was to determine the mechanism by which DHA pretreatment protects ARPE-19 cells from subsequent oxidative stress.
Methods: :
ARPE-19 cells where cultured for 72 h to achieve ~100% confluencey. Cells were serum starved for 18 hours in 0.5% FCS, DMEM/F12 1:1 media. After which 200nM DHA or ETOH (equal volume) was added to the media. After 6 hours of incubation in 200nM DHA or ETOH, media was replaced with fresh 0.5% FCS, DMEM/F12 1:1 media for 24h. Cells were then challenged with increasing concentrations of H2O2 for 6 and 16 hours. Cells were collected for protein and lipid analysis. Cells also were fixed with neutral buffered formalin and stained with Hoestch 33258 in 1% triton X100. Cell death was assessed via nuclear chromatin condensation. (um2)
Results: :
Cells pretreated with 200 nM DHA for 6 h, 24 h prior to stress, exhibit protection as demonstrated (IOVS Knott et al. 2011). Western blot analysis of ARPE-19 cells treated with 200 nM DHA for 6h plus 24 hours DMEM/F12 resulted in a 2 fold increase in pMAPK, a 2 fold increase in pAKT, and a 3 fold reduction in BIM when compared to controls. Also, hydrolyzed phospholipid mass spectra analysis of deuterium labeled DHA demonstrates that 66% of supplemented DHA is incorporated into phospholipids.
Conclusions: :
Our results demonstrate that DHA pretreatment elicits protection of human-ARPE-19 cells, which involves increases pMAPK and pAKT ser 308; and a decrease in BIM. This protection is also characterized by an incorporation of DHA into phospholipids. This data further suggests protection via DHA-derived NPD1 synthesis, which is achieved via NPD1 mediated-PI3K/Akt signaling and regulation of anti- apoptotic protein BCL-xl in a PP2 a dependent manner. Thus, application of DHA/NPD1 could be used as a potential therapeutic mean for the prevention or attenuation of the initiation or early progression of retinal degenerative diseases.
Keywords: apoptosis/cell death • neuroprotection • retinal pigment epithelium