Ursolic acid reduces hepatocellular apoptosis and alleviates alcohol-induced liver injury via irreversible inhibition of CASP3 in vivo

Xiao-yao Ma1, Man Zhang1, Ge Fang1, Chuan-jing Cheng1, Mu-kuo Wang1, Yi-man Han1, Xiao-tao Hou2, Er-wei Hao2, Yuan-yuan Hou1, Gang Bai1
1 State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300000, China
2 Guangxi Collaborative Innovation Center for Functional Ingredients Study of Agricultural Residues, Guangxi Key Laboratory of Efficacy Study on Chinese Materia Medica, Guangxi University of Chinese Medicine, Nanning 530200, China
Correspondence to: Yuan-yuan Hou:,
DOI: 10.1038/s41401-020-00534-y
Received: 20 June 2020
Accepted: 10 September 2020
Advance online: 7 October 2020


Alcoholic liver disease (ALD) is one of the pathogenic factors of chronic liver disease with the highest clinical morbidity worldwide. Ursolic acid (UA), a pentacyclic terpenoid carboxylic acid, has shown many health benefits including antioxidative, anti- inflammatory, anticancer, and hepatoprotective activities. We previously found that UA was metabolized in vivo into epoxy- modified UA containing an epoxy electrophilic group and had the potential to react with nucleophilic groups. In this study we prepared an alkynyl-modified UA (AM-UA) probe for tracing and capturing the target protein of UA from liver in mice, then investigated the mode by which UA bound to its target in vivo. By conducting proteome identification and bioinformatics analysis, we identified caspase-3 (CASP3) as the primary target protein of UA associated with liver protection. Molecule docking analysis showed that the epoxy group of the UA metabolite reacted with Cys-163 of CASP3, forming a covalent bond with CASP3. The binding mode of the UA metabolites (UA, CM-UA, and EM-UA) was verified by biochemical evaluation, demonstrating that the epoxy group produced by metabolism played an important role in the inhibition of CASP3. In alcohol-treated HepG2 cells, pretreatment with the UA metabolite (10 μM) irreversibly inhibited CASP3 activities, and subsequently decreased the cleavage of PARP and cell apoptosis. Finally, pre-administration of UA (20–80 mg· kg−1 per day, ig, for 1 week) dose-dependently alleviated alcohol-induced liver injury in mice mainly via the inhibition of CASP3. In conclusion, this study demonstrates that UA is a valuable lead compound for the treatment of ALD.
Keywords: ursolic acid; metabolite; CASP3; covalent binding; alcoholic liver disease

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