Effect of NO2-OA on the oxidative stress and glial reactivity in Müller Glial Cells

VAGLIENTI, MV; RIDANO, ME; SUBIRADA, PV; PAZ, MC; BARCELONA, P; BONACCI, GR; SANCHEZ, MC

Vancuver

Congreso; ARVO Annual MEETING 2019; 2019

ARVO

Purpose: Inflammation, oxidative and nitrosative stress are involved in the pathogenesis of diabetic complications, including retinopathy. Metabolic changes that occur in Diabetes result in alterations of blood retinal barrier, which allows the extravasation of plasma proteins such as α2-macroglobulin (α2M). Previous work of our group has demonstrated that α2M induce glial reactivity in Müller Glial cells (MGCs) mediated by the increase of GFAP levels. Nitro-fatty acids (NO2-FA) are important electrophilic signaling mediators with anti-inflammatory and cytoprotective properties (Keap1/Nrf2 pathway). Our goal was to determine whether nitro-oleic acid (NO2-OA) could be beneficial for retinal cells against oxidative stress and glial reactivity in the human MGC line (MIO-M1).Methods: MIO-M1 cell viability was assessed after 24, 48 and 72h of NO2-OA (0,1 to 10µM) treatment by MTT assays (N=3). MIO-M1 cells were treated with or without NO2-OA, and antioxidant genes expression such as HO-1 was measured by WB at 8 and 16h post-stimulus (N=3). Six hours after NO2-OA treatment, MIO-M1 cells were challenged by PMA or LPS for 30min, ROS levels were determined by DCF assay (N=2). Moreover, MIO-M1 cells were treated with or without NO2-OA for 30 min before the α2M stimulus for 2, 4 and 6h, the protein levels of GFAP, Vimentin and HO-1 were measured by WB (N=2), and ROS levels were determined by DCF assay. GraphPad Prism program was employed for statistical analysis.Methods: MIO-M1 cell viability was assessed after 24, 48 and 72h of NO2-OA (0,1 to 10µM) treatment by MTT assays (N=3). Antioxidant genes expression such as HO-1 was measured by WB after NO2-OA treatment (8 and 16h) in MIO-M1 cells (N=3). ROS levels were determined by DCF probe, for that MIO-M1 cells were pre-treated or not with NO2-OA (6h) and stimulated with PMA or LPS for 30min (N=2). Moreover, glial reactivity was evaluated by protein expression levels of GFAP, Vimentin and HO-1 were measured by WB (N=2) in MIO-M1 cells treated or not with NO2-OA for 30 min before the α2M stimulus (2, 4 and 6h). GraphPad Prism program was employed for statistical analysis. Results: MIO-M1 cells exposed to vehicle (methanol) or 0.1 to 10 μM NO2-OA during 24 to 72h showed no significant reduction in cell viability (p>0,05) compared to untreated cells (medium). Under 5µM NO2-OA (8h) treatment, MGCs strongly increased Nrf2 downstream genes expression such us HO-1 (p