S-1-propenylmercaptocysteine protects murine hepatocytes against oxidative stress via persulfidation of Keap1 and activation of Nrf2

Abstract The onion-derived metabolite, S -1-propenylmercaptocysteine (CySSPe), protects against oxidative stress and exhibits anti-inflammatory effects by modulating cellular redox homeostasis. We sought to establish whether CySSPe activates nuclear factor erythroid 2–related factor 2 (Nrf2) and whether activation of Nrf2 by CySSPe involves modification of the Kelch-like ECH-associated protein-1 (Keap1) to manifest these effects. We found that CySSPe stabilized Nrf2 protein and facilitated nuclear translocation to induce expression of antioxidant enzymes, including NQO1, HO-1, and GCL. Moreover, CySSPe attenuated tert-butyl hydroperoxide-induced cytotoxicity and dose-dependently inhibited reactive oxygen species production. Silencing experiments using Nrf2-siRNA confirmed that CySSPe conferred protection against oxidative stress by activating Nrf2. CySSPe enhanced cellular pool of reduced glutathione (GSH) and improved GSH:GSSG ratio. Pretreatment of cells with l -buthionine-S,R-sulfoximine (BSO) confirmed that CySSPe increases de novo synthesis of GSH by upregulating expression of the GSH-synthesizing enzyme GCL. Treatment of cells with CySSPe elevated hydrogen sulfide (H 2 S) production. Inhibition of H 2 S-synthesizing enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS), by pretreating cells with propargylglycine (PAG) and oxyaminoacetic acid (AOAA) revealed that H 2 S production was partially dependent on a CSE/CBS-catalyzed β-elimination reaction with CySSPe that likely produced 1-propenyl persulfide (RSSH). Depleting cells of their GSH pool by exposure to BSO and diethylmaleate attenuated H 2 S production, suggesting a GSH-dependent formation of H 2 S, likely via the reduction of RSSH by GSH. Finally, treatment of cells with CySSPe persulfidated Keap1, which may be the mechanism involved for the stabilization of Nrf2 by CySSPe. Taken together, our results showed that attenuation of oxidative stress by CySSPe is associated with its ability to produce H 2 S or RSSH, which persulfidates Keap1 and activates Nrf2 signaling. This study provides insights on the potential of CySSPe as an onion-derived dietary agent that modulates redox homeostasis and combats oxidative stress.