Peng_2026_J.Ethnopharmacol_370_121964

ETHNOPHARMACOLOGICAL RELEVANCE: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease globally, yet effective therapeutic options remain limited. Dysregulated lipid metabolism plays an important role in MASLD progression, making it a promising target for intervention. In Malaysia, the resin extract of Agathis dammara (Lamb.) Rich. & A.Rich. (AD) is traditionally used as a health supplement to regulate metabolism and treat inflammatory diseases. Araucarone (AO) is the most abundant monomeric component in AD. However, the therapeutic efficacy of AD and AO against MASLD, as well as their underlying mechanisms of action, remains unknown. AIM OF THE STUDY: The study aimed to clarify the inhibitory effects of AD and AO against MASLD, elucidate their mechanisms of action, and identify their primary molecular target, thereby providing a novel and effective strategy for the treatment of MASLD. METHODS: We evaluated the anti-steatotic effects of AD and AO using in vitro (oleic acid-induced steatosis in LO2 cells and primary mouse hepatocytes) and in vivo (high-fat diet [HFD]- and methionine-choline-deficient diet [MCD]- induced MASLD mice) models. Oil red O staining and lipid quantification were applied to evaluate the severity of hepatic steatosis, while Western blotting and qPCR analyses were used to detect changes in protein or gene expression within the metabolism-related pathways. Proteomics, molecular docking, surface plasmon resonance (SPR), and cellular thermal shift assays (CETSA) were employed to identify the direct target of AO. Functional validation was performed via siRNA knockdown. RESULTS: AD and AO significantly attenuated hepatic steatosis in both in vitro and in vivo models by inhibiting lipid synthesis (via LXRalpha/SREBP-1c downregulation) and promoting fatty acid oxidation (via PPARalpha activation). Mechanistically, AO directly bound to carboxylesterase 2 (CES2), enhancing its protein stability and enzymatic activity. CES2 knockdown abolished the lipid-lowering effects of AO, confirming CES2 as the primary functional target. Furthermore, AO increased intracellular free fatty acids (FFAs), which acted as signaling molecules to modulate both the LXRalpha and PPARalpha pathways. CONCLUSION: This study identifies AO as the first small-molecule CES2 agonist capable of ameliorating MASLD, highlighting its potential as a novel therapeutic strategy and providing a foundation for further drug development.