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

  • Phytomedicine. 2026 Jun 11:159:158413. doi: 10.1016/j.phymed.2026.158413.
Ze Zhang  1 ,  Xinnan Gu  1 ,  Guanghao Zhu  2 ,  Yu Meng  1 ,  Mengjuan Wei  3 ,  Xiayan Chu  2 ,  Yuxuan Luo  1 ,  Zhenlin Huang  4 ,  Guangbo Ge  5 ,  Lili Ji  6
Affiliations
  • 1. State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai, 201203, China; The MOE Key Laboratory for Standardization of Chinese Medicines, Shanghai Key Laboratory of Compound Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai, 201203, China.
  • 2. State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
  • 3. State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai, 201203, China; The MOE Key Laboratory for Standardization of Chinese Medicines, Shanghai Key Laboratory of Compound Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai, 201203, China; Shanghai Academy of International Standardization for Traditional Chinese Medicine, Shanghai, 201203, China.
  • 4. State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai, 201203, China; The MOE Key Laboratory for Standardization of Chinese Medicines, Shanghai Key Laboratory of Compound Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai, 201203, China. Electronic address: [email protected].
  • 5. State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China. Electronic address: [email protected].
  • 6. State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai, 201203, China; The MOE Key Laboratory for Standardization of Chinese Medicines, Shanghai Key Laboratory of Compound Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai, 201203, China; College of Traditional Chinese Medicine,Bozhou University, Bozhou, 236800, China. Electronic address: [email protected].
PMID: 42314351 DOI: 10.1016/j.phymed.2026.158413
Abstract

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.

Keywords
Cysteine residues modification; DILI; Keap1; Nrf2; Sal B.
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