Capsaicin acts as a novel NRF2 agonist to suppress ethanol induced gastric mucosa oxidative damage by directly disrupting the KEAP1-NRF2 interaction

Excessive drinking poses serious health risks and is closely associated with oxidative damage. The KEAP1-NRF2-ARE axis serves as the primary antioxidant system. However, the existing small molecule inhibitors are all covalently bound to KEAP1, meaning that once bound, they are not easily dissociated, while continuous inhibition of KEAP1 exhibits severe side effects. In this study, BLI, CETSA, Pull-down, Co-IP and HDX-MS assay analysis were conducted to detect the KEAP1 binding behavior of natural product, capsaicin (CAP), bothin vitroand in GES1 cells. The ethanol-induced acute gastric mucosal damage rat model was also established to determine the therapeutic effect of CAP. We demonstrated that CAP ameliorated mitochondrial damage, facilitated the nuclear translocation of NRF2, thereby promoting the expression of downstream antioxidant response elements, HO-1, Trx, GSS and NQO1 in GES1 cells. Subsequently, CAP could directly bind to KEAP1 and inhibit the interaction between KEAP1 and NRF2. While in the KEAP1-knockout 293T cells, CAP failed to activate NRF2 expression. It was also found that CAP non-covalently bound to Kelch domain and allosterically regulated three regions of KEAP1: L342-L355, D394-G423 and N482-N495. To enhance drug solubility and delivery efficiency, we designed IR-Dye800 modified albumin coated CAP nanoparticle. The nanoparticles significantly alleviated the gastric mucosal inflammation and activated the NRF2 downstream genesin vivo. Our work provided new insights that CAP is a safe and novel NRF2 agonist by allosterically regulating KEAP1, which may contribute to the development of lead drugs for oxidative stress-related illness, e.g. aging, cancer, neurodegenerative and cardiovascular diseases.