Wang Junjie, Chen Baoyi, Cheng Chaofan, Wang Qingqing, Yang Lili, Li Zeng, Lv Xiongwen
Anhui Prevention and Control Engineering Research Center for Fatty Liver Disease, Hefei, Anhui, 230032, PR China; The Key Laboratory of Anti-inflammatory and Immune Medicines, Ministry of Education, Hefei, PR China; Inflammation and Immune-Mediated Diseases Laboratory of Anhui Province, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, PR China.
Anhui Prevention and Control Engineering Research Center for Fatty Liver Disease, Hefei, Anhui, 230032, PR China; The Key Laboratory of Anti-inflammatory and Immune Medicines, Ministry of Education, Hefei, PR China; Inflammation and Immune-Mediated Diseases Laboratory of Anhui Province, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, PR China.
Chem Biol Interact. 2025 Feb 25;408:111390. doi: 10.1016/j.cbi.2025.111390. Epub 2025 Jan 23.
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.
过量乙醇诱导的氧化应激是酒精性肝病（ALD）进展的重要因素。近年来，抑制 Kelch 样 ECH 相关蛋白 1（KEAP1）以激活抗氧化调节因子核因子红细胞 2 相关因子 2（NRF2）被认为是治疗氧化应激相关疾病的有效策略，但其在 ALD 中的应用仍未得到充分探索。本研究旨在发现靶向 KEAP1 受体的高亲和力抑制剂。我们基于基于结构的药效团模型对化合物库进行虚拟筛选，最终确定候选化合物知母皂苷 B II（TBII）。随后，我们在 AML-12 细胞和 C57BL/6 小鼠中建立了 ALD 模型，并使用免疫荧光、蛋白质印迹、RT-qPCR、生化分析和组织学染色等方法评估了 TBII 对 ALD 的治疗效果和机制。结果表明，TBII 显著改善了乙醇诱导的肝损伤，抑制了血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、总胆固醇（T-CHO）和甘油三酯（TG）水平的升高，并减少了肝组织中的脂滴积累。此外，TBII 处理增强了 AML-12 细胞和小鼠肝脏的抗氧化能力，提高了谷胱甘肽（GSH）和超氧化物歧化酶（SOD）水平，同时降低了丙二醛（MDA）和活性氧（ROS）水平。机制研究表明，TBII 抑制了乙醇诱导的 KEAP1 增加，并逆转了乙醇诱导的 NRF2 及其下游靶点的变化。总之，本研究表明，TBII 可能通过调节 KEAP1-NRF2 途径减轻氧化应激和脂质代谢异常，从而成为 ALD 的潜在治疗药物。
