Effect of nitro-oleic acid on the oxidative stress and glial reactivity in Müller Glial Cells

MARIA VICTORIA VAGLIENTI; MAGALI E. RIDANO; PAULA SUBIRADA; MARIA CONSTANZA PAZ; PABLO F. BARCELONA; GUSTAVO BONACCI; MARIA CECILIA SANCHEZ

Congreso; Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO) 2019; 2019

Purpose : Metabolic changes that occur in Diabetes result in alterations of BRB, 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). Antioxidant genes expression such as HO-1 was measured by WB assays 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 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