Abstract
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.
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Acknowledgements
This research was funded by a grant from the National Key R&D Program of China (No. 2018YFC1704500), Guangxi Innovation-driven Development 20 Special Foundation Project (Guike No. AA18118049), and Guangxi Collaborative Innovation Center for Functional Ingredients Study of Agricultural Residues (No. CICAR 2019-Z3).
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GB and YYH designed the study and drafted and edited the manuscript; XYM performed the experiments and acquired and analyzed the data; MZ and GF performed CASP3 protein purification; CJC analyzed the mass spectrum data; MKW performed the molecular dynamic simulation and docking experiments. MZ and YMH assisted with the experiments. XTH and EWH contributed to the data discussion and review of the manuscript.
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Ma, Xy., Zhang, M., Fang, G. et al. Ursolic acid reduces hepatocellular apoptosis and alleviates alcohol-induced liver injury via irreversible inhibition of CASP3 in vivo. Acta Pharmacol Sin 42, 1101–1110 (2021). https://doi.org/10.1038/s41401-020-00534-y
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DOI: https://doi.org/10.1038/s41401-020-00534-y
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