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

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 mu M) irreversibly inhibited CASP3 activities, and subsequently decreased the cleavage of PARP and cell apoptosis. Finally, pre-administration of UA (20-80 mg center dot 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.

Automated summary

This study utilized an alkynyl-modified UA probe to identify CASP3 as the primary target protein of UA associated with liver protection. Through molecular docking analysis, it was found that the epoxy group of UA metabolite reacted with Cys-163 of CASP3, implying the inhibition mechanism of CASP3 by UA. Additionally, the study demonstrated that UA could alleviate alcohol-induced liver injury by inhibiting CASP3 in a dose-dependent manner.