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L. Li, P. K. Mukherjee, N. G. Bazan; Lipoxin Inhibits Apoptotic Cell Death and Pro-Inflammatory Gene Expression Induced by Oxidative-Stress in Retinal Pigment Epithelial (ARPE-19) Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5928.
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© ARVO (1962-2015); The Authors (2016-present)
In vitro studies show that oxidant treated RPE cells undergo apoptosis, a possible mechanism of developing AMD and other eye diseases. NPD1 is one of endogenous lipid mediators compromise this process. Lipoxins(LX) are endogenous anti-inflammatory lipids which signals the resolution of inflammation in a range of pathophysiologic processes. In this report, we elucidate the role of lipoxin A4 (LX A4) and its analog15-epi (LX A4-15-epi) on the oxidative-stress induced cell survival in ARPE-19 cells.
72 hour grown ARPE-19 cell were serum starved for 8 hour before oxidative-stress was introduced for 16 hour with TNF-α(10 ng/ml) and H2O2 (600 uM). Treated cells were then challenged with LX A4 or LX A4-15-epi either alone, or together with NPD1 at different concentrations . Cell apoptosis was detected with Hoechst stain. COX-2 promotor construct (-830) linked with luciferase was transfected to RPE cells by Fugene-6. Transfected cells were induced with IL-1β then treated with lipoxins at different concentrations. Cell homogenates were prepared and luciferase assays were carried out using luciferin as substrate.
Oxidative-stress induced apoptotic cell death was inhibited by lipoxins in concentration dependent manner. Highest inhibition (70%) was detected at 500 nM. and lowest (5-8%) at 10 nM. Intermediate inhibition was achieved at 50 (28%) and 100 nM (40%). Lipoxins themselves did not cause any apoptosis. Lipoxins mediated inhibition of apoptosis further enhanced (85-90%) when NPD1 at low concentration (30nM) added together. Lipoxins and NPD1 mediated inhibition of apoptosis was additive. IL-1β induced activation of COX-2 in RPE cells dramatically inhibited by LX A4 and LX A4 -15-epi.
LX A4 and LX A4 -15-epi are anti-inflammatory lipid mediators in oxidative stress challenged RPE cells. We found that lipoxins inhibit cell apoptosis and pro-inflammatory COX-2 induction. The additive effect of lipoxins and NPD1 in cell survival suggest the involvement of different cell signaling pathways.
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