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

SUBIRADA CALDARONE PAULA; BONACCI GR; VAGLIENTI MV; MC PAZ; SANCHEZ MC; RIDANO MAGALI E; BARCELONA PF

Vancuver

Congreso; ARVO Annual MEETING 2019; 2019

Association for Research in Vision & Ophthalmology

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 retinal blood barrier, which allows the extravasation of plasma proteins. α2-macroglobulin (α2M) is a proteinase inhibitor and a cargo protein of growth factors and cytokines in the blood and in the extracellular space. 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 24h, 48h and 72h of NO2-OA (0,1 to 10 µM) treatment by MTT assays(N=3). MIO-M1 cells were treated with or without different doses of NO2-OA, and antioxidant genes expression such as HO-1 was measured by WB at 8 and 16 h post-stimulus (N=3). Six hours after NO2-OA treatment, MIO-M1 cells were challenged by PMA or LPS for 30 min, 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 6 h, 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.Results: MIO-M1 cells exposed to vehicle (methanol), 0.1, 1, 2.5, 5 or 10 μM NO2-OA during 24 to 72 h showed no significant reduction in cell viability (p>0,05) compared to untreated cells (medium). MIO-M1 strongly increased Nrf2 target genes expression such as HO-1 after 8h with 5µM NO2-OA stimulus (p< 0.001). To determine whether NO2-OA could be beneficial for retinal cells against oxidative stress, we treated MIO-M1 cells with or without NO2-OA before PMA and LPS stimulus. PMA (1µM) and LPS (1µg/ml) significantly increased ROS in MIO-M1 cells compared with control (p< 0.001). Pre-treatment with 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 increased ROS levels compared to control (p< 0.05). The pre-treatment with NO2-OA before the 6 h with α2M reduced the 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.