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

Abstract

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<0.001). To determine whether NO₂-OA could be beneficial for retinal cells against oxidative stress, we pre-treated MIO-M1 cells with or without NO₂-OA before PMA and LPS stimulus. Both, PMA (1µM) and LPS (1µg/ml) significantly increased ROS in MIO-M1 cells compared with control (p<0.001) and 5µM NO₂-OA prevented the increase in ROS levels induced by LPS or PMA stimuli (p>0,05). On the other hand, α₂M induced glial reactivity mediated by the significantly increase of GFAP and vimentin expression (p<0.001). In addition to this effect, α₂M also increased ROS levels compared to control (p<0.05) and the pre-treatment with NO₂-OA reduced α₂M induction of GFAP and ROS levels to the control level (p>0,05).

Conclusions : These results indicate that NO₂-OA may act as an antioxidant protecting retinal cells from oxidative and α₂M damage.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.