New nature-inspired hybrids targeting the Nrf2-HO1 pathway protect retinal pigment epithelial cells under stress conditions.

Purpose: An impairment in the Nuclear factor E2-related factor 2 (Nrf2) pathway, a master regulator of stress response in retinal pigment epithelium (RPE), is suggested to contribute to the Age-related Macular Degeneration (AMD) pathogenesis; Nrf2-activators may be of interest in AMD and other pathologies featured by oxidative stress. We tested in vitro in ARPE19 cells some novel Nature-Inspired Hybrids (NIHs) for their capability to activate Nrf2 pathway and to provide protection from AMD-related stressors. Methods: Four NIHs (5μM) were individually tested for their pharmacological activity and SAR in ARPE-19 cells. Control cells (CTR) were treated with the solvent DMSO. The Nrf2 activator dimethyl-fumarate (DMF; 10μM) was used as a positive control. Nrf2 nuclear translocation was evaluated by Western blotting. Among Nrf2-target genes, the Heme-Oxygenase 1 (HO-1) expression was measured by real-time qPCR and Western blotting. The NIHs’ effects on ROS levels and cell viability were evaluated following either a short term exposure to H2O2 (0.3mM), or a long term exposure to MG132 (5μM) + Bafilomycin (50nM) (M+B, autophagy inhibitor) by fluorimetric assays. We performed ≥3 independent replicates for each experiment. Prism was used for statistical analysis (one-way or two-way ANOVA followed by Dunnett’s multiple comparisons test). Results: ARPE-19 cells well tolerate all NIHs (no cell viability changes after 48 hrs treatments; n=6). NIHs activate Nrf2 pathway with different times and intensities among molecules. The most promising NIH, lead compound (NIH1) provides: i) Nrf2 nuclear translocation after 3 hrs (+65.8±12.4% vs CTR), accompanied by a 4.5-fold increase of HO-1 mRNA total level with respect to control cells (n=3, p<0.05); ii) a long-lasting (up to 48 hrs) increase of HO-1 protein levels (+311.3±30% vs CTR; n=3, p<0.01); iii) a direct antioxidant effect against 4 hrs H2O2 treatment (n=4, p<0.0001); iv) an increase of cell viability under 48 hrs MG132+Bafilomycin (M+B) stress (CTR: 100±0.6%; M+B: 75.6±0.94%; NIH1+M+B: 90.1±1.35%; n=4-8, p