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M. A. Reinoso, P. K. Mukherjee, P. Gjorstrup, N. G. Bazan; Oxidative Stress-Induced Apoptosis and Pro-Inflammatory Cox-2 Expression Are Down-Regulated by Resolvins in Retinal Pigment-Epithelial (arpe-19) Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2438.
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Resolvins and neuroprotectin D1 are distinct and highly stereospecific lipid mediators with strong anti-inflammatory bioactivity and, in addition, have immunoregulatory properties at picomolar to nanomolar concentrations. Our goal in this study was to test the effect of resolvins on apoptotic cell death induced by oxidative stress and on Il-1β induced pro-inflammatory COX-2 gene expression in ARPE-19 cells.
72 h-grown cells in 6 well plates were serum starved for 8 h, and then oxidative stress was induced with TNF-α/H2O2 (600 µM) for 16 h. Cells were incubated with different concentrations of RvE1, Rx-10008 (RvE1 analog), and NPD1. Apoptotic cell death was scored by Hoechst positive cells. COX-2 (-830) promoter construct, linked to luciferase reporter gene, was used to transfect ARPE-19 cells by Fugene-6. A β-galactosidase plasmid was co-transfected as transfection control. Transfected cells were serum starved, induced by Il-1β, and incubated with different concentrations of resolvins. Luciferase assays were performed using luciferin as substrate.
The results demonstrated that resolvins inhibit oxidative stress-induced apoptosis in a concentration dependent manner. Of the three concentrations of resolvins used (10, 30, and 50 nM), highest inhibition was achieved at 50 nM (40-46%), lowest at 10 nM (1.5-2%), and intermediate at 30 nM (28-32%). The inhibition of apoptosis by resolvins was further enhanced upon the addition of NPD1. Combined inhibition by resolvin and NPD1-mediated enhanced cell survival is 54-68%. The expression of pro-inflammatory COX-2 was also inhibited by resolvins, and with NPD1 showing the stronger inhibition.
The inhibitory effect of resolvins on oxidative stress-induced apoptosis and COX-2 expression demonstrates strong anti-inflammatory bioactivity of resolvins in an oxidative-stress environment. The data suggest that resolvins target signaling mechanisms critical for cell survival, and further suggest their potential as therapeutic intervention in diseases where protecting the RPE photoreceptor integrity is supported.
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