Activation of Nrf2‐mediated protective pathways by nature‐inspired hybrids improves proteostasis in retinal pigment epithelial cells

References: Antioxidant systems are essential in many elderly diseases, including age-related macular degeneration (AMD). Oxidative stress, inflammation, protein degradation impairment, are well-described in AMD retinal pigment epithelial (RPE) cells. Nrf2 (NF-E2-related factor 2), master regulator of antioxidant defense, is linked to AMD, autophagy, inflammation. We previously showed that nature-inspired hybrids (NIH1-3) induce Nrf2-mediated protection against AMD-related pro-oxidant stimuli in RPE cells. Purpose: In human RPE cells we aimed to further investigate NIH1-3 for capability to modulate Nrf2 pathway and provide cytoprotection against stress caused by dysfunction of protein homeostasis. Methods: ARPE-19 cells were treated with 5µM NIH 1-3, NIH4 (the derivative unable to activate Nrf2) or vehicle (dimethyl sulfoxide; control) for a minimum of 3 hours. Proteasome inhibitor MG-132 (5µM) and autophagy inhibitor Bafilomycin-A1 (50nM) were added after 24h NIH pre-treatment. Levels of Nrf2, its target HO-1 and SQSTM1/p62, the autophagy flux marker LC3-II, were analyzed by Western blotting, whilst IL-8 release by ELISA, respectively. Cell survival was also assessed. Results: NIH1-3 activate Nrf2-pathway and increase HO-1 and SQSTM1/p62 expression, in turn exerting beneficial effects on intracellular redox balance, without modification of the autophagy flux. NIH1-3 treatments predispose ARPE-19 cells to a better response to the following exposure to proteasome and autophagy inhibitors, as revealed by the higher cell survival and decreased secretion of the pro-inflammatory IL-8 compared to NIH-untreated cells. Interestingly, also NIH4 compound, through an Nrf2-independent pathway, exerts pro-survival and anti-inflammatory effects, although to a less extent than NIH1-3. Conclusions: Our findings suggest that all NIHs are interesting for their cytoprotective properties and confirm Nrf2 as a valuable pharmacological target in contexts characterized by oxidative stress.