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<0.0001). Conclusion: A positive modulation of Nrf2-pathway by NIHs is protective in RPE cells, encouraging further studies on their potential use in pathologies featured by oxidative stress. Layman Abstract (optional) Retina is subjected to stressors (such as free radicals) for the entire life span, and endogenous protective factors are essential to preserve this tissue. In particular, the Nuclear factor E2-related factor 2 (Nrf2) pathway is a master regulator of the stress response in the retina, and especially in the retinal pigment epithelium (RPE). Nrf2 pathway is impaired in aging and in some ocular pathologies, such as Age-related Macular Degeneration (AMD), and molecules able to stimulate Nrf2 are interesting as new potential drugs. We studied some new nature-inspired hybrids that activate Nrf2 pathway, and we found they can protect RPE cells in vitro from AMD-related stressors. Our findings encourage further studies on the potential use of these molecules in AMD and other pathologies featured by oxidative stress.