Timosaponin B II as a novel KEAP1-NRF2 inhibitor to alleviate alcoholic liver disease:Receptor structure-based virtual screening and biological evaluation

Oxidative stress induced by excess ethanol is an important factor in the progression of alcoholic liver disease (ALD). In recent years, inhibiting Kelch-like ECH-associated protein 1 (KEAP1) to activate the antioxidant regulator Nuclear factor erythroid 2-related factor 2 (NRF2) has been considered an effective strategy for treating oxidative stress-related diseases, but its application in ALD remains insufficiently explored. This study aims to discover high-affinity inhibitors targeting the KEAP1 receptor. We conducted virtual screening of a compound library based on a structure-based pharmacophore model, ultimately identifying the candidate compound Timosaponin B II (TBII). Subsequently, we established ALD models in AML-12 cells and C57BL/6 mice, and evaluated the therapeutic effects and mechanisms of TBII on ALD using methods including Immunofluorescence, Western blotting, RT-qPCR, Biochemical assays, and histological staining. Results indicate that TBII significantly improved ethanol-induced liver injury, inhibited the elevation of serum Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Total Cholesterol (T-CHO), and Triglycerides (TG) levels, and reduced lipid droplet accumulation in liver tissues. Furthermore, TBII treatment enhanced the antioxidant capacity of AML-12 cells and mouse liver, increasing Glutathione (GSH) and Superoxide Dismutase (SOD) levels while reducing Malondialdehyde (MDA) and Reactive Oxygen Species (ROS) levels. Mechanistic studies indicated that TBII inhibited the ethanol-induced increase in KEAP1 and reversed the ethanol-induced changes in NRF2 and its downstream targets. In conclusion, this study suggests that TBII may become a potential therapeutic agent for ALD by modulating the KEAP1-NRF2 pathway to alleviate oxidative stress and lipid metabolism abnormalities.