Salvianolic acid B attenuates drug-induced liver injury in an Nrf2-dependent mechanism via covalently modifying Keap1 at Cys395/Cys434.

BACKGROUND

Drug-induced liver injury (DILI) is a global health issue with limited treatment options. The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway plays a critical role in defending against DILI, making it a potential therapeutic target.

PURPOSE: This study aimed to investigate the hepatoprotective effect of salvianolic acid B (Sal B), a polyphenol derived from Salvia miltiorrhiza Bge., against DILI and to elucidate its underlying molecular mechanisms, particularly focusing on the Nrf2 pathway.

METHODS

Compounds screening was carried out to identify Sal B as a potent Nrf2 activator. Cellular thermal shift assay (CETSA), surface plasmon resonance (SPR), and microscale thermophoresis (MST) were employed to examine the direct interaction between Sal B and kelch-like ECH-associated protein-1 (Keap1), an Nrf2 inhibitor. Nrf2 knock-out (Nrf2-/-) mice and liver-specific Keap1 knockout (Hepcre-Keap1flox/flox) mice were used to assess the role of Nrf2 and Keap1 in Sal B-mediated hepatoprotection. Mass spectrometry-based chemoproteomic analysis and mutagenesis studies were performed to identify the specific cysteine residues modified by Sal B. Molecular dynamics simulations were used to analyze conformational changes in Keap1 following Sal B binding.

RESULTS

Sal B strongly activated Nrf2, and its hepatoprotective effects were significantly diminished by Nrf2 knock-out. Sal B directly bound to Keap1. In Hepcre-Keap1flox/flox mice, DILI was attenuated, and Sal B's protective effects were weakened. Sal B covalently modified Cys395 and Cys434 within the Keap1 double-glycine repeat (DGR) domain. Mutagenesis of these residues impaired Sal B-Keap1 interaction and abolished Nrf2 activation. Sal B's modification altered the conformation of the Keap1 kelch domain, loosening the Nrf2 binding pocket and facilitating Nrf2 activation.

CONCLUSION

Sal B exerts hepatoprotective effects against DILI by activating Nrf2 through covalent modification of Keap1 at Cys395 and Cys434. These findings provide mechanistic insights into the potential of Sal B as a novel therapeutic agent for DILI.