Abstract
BACKGROUND: Alcoholic liver disease (ALD) poses a major global health challenge, with limited effective interventions. The Dampness-Heat Regulating Formula (DRF), a traditional Chinese herbal tea composed of nine edible medicinal herbs, has shown promise in mitigating alcohol-induced liver injury. This study aimed to identify its core active components and elucidate underlying mechanisms. METHODS: Active compounds were retrieved from multiple databases and screened using chemical similarity, target prediction, and ADMET filtering. Disease-related targets were identified through public transcriptomic datasets. Three machine learning algorithms-random forest, support vector machine, and LASSO-were used to prioritize therapeutic targets. High-throughput molecular docking and virtual screening were combined with untargeted metabolomics to identify candidate compounds. The interaction between oleanolic acid (OA) and AKT1 was further verified by cellular thermal shift assay (CESTA). In vitro and in vivo assays were conducted to validate hepatoprotective effects. Additionally, the content of OA in DRF was quantified by HPLC to assess the relevance of experimental dosing. RESULTS: A total of 690 candidate compounds and 33 ALD-associated targets were identified. AKT1 emerged as the top-ranked hub target. OA showed strong binding affinity to AKT1, and CESTA confirmed their direct interaction. Functional assays demonstrated that OA alleviated ethanol-induced damage in hepatocytes and zebrafish models. HPLC analysis confirmed that DRF contained physiologically relevant concentrations of OA, supporting the translational relevance of the selected doses. CONCLUSION: This study reveals a potential AKT1-centered mechanism through which DRF protects against ALD and identifies oleanolic acid as a bioactive compound with dual computational and experimental validation. It offers a scientific basis for integrating traditional herbal formulas with modern drug discovery approaches in the prevention of alcohol-related liver injury.