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

Abstract

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 HO1 mRNA total level with respect to control cells (n=3, p<0.05); ii) a long-lasting (up to 48 hrs) increase of HO1 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).

Conclusions : 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.

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