Unveiling the molecular mechanisms of Zhuang medicine in liver cancer treatment: From Active ingredient screening to diosgenin-mediated targeted regulation

• Network pharmacology was utilized to screen active components and effects of Zhuang medicine on hepatocarcinoma. • A network was established linking active compounds, their presumed targets, and associated pathways. • Diosgenin demonstrated strong binding affinity with CAT, NR1I2, and NR3C2—potential therapeutic targets in LC. • Diosgenin inhibits Hepa1-6 cell proliferation and migration. Zhuang medicine has long been used for the clinical management of liver cancer (LC); however, its underlying therapeutic mechanisms remain largely unelucidated. This study aimed to identify bioactive compounds within Zhuang medicine formulations and investigate their potential mechanisms of action against LC. Differentially expressed genes (DEGs) in LC were identified using the Gene Expression Omnibus (GEO) database. High-frequency, robust herbs employed in LC treatment were screened utilizing established network pharmacology approaches, and their primary active ingredients were retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. LC-associated gene datasets were integrated from GeneCards, Online Mendelian Inheritance in Man (OMIM), PharmGKB, Therapeutic Target Database (TTD), and DrugBank databases. Key candidate genes were pinpointed through the integration of Weighted Gene Co-expression Network Analysis (WGCNA) and Protein-Protein Interaction (PPI) network analysis. Molecular docking simulations using AutoDock software assessed potential interactions between the identified active ingredients and their putative targets. The therapeutic effects of diosgenin, a prioritized compound, were further validated through in vitro experiments utilizing Hepa1-6 murine hepatoma cells. Screening identified ten frequently prescribed Zhuang herbs and eight core active ingredients. Molecular docking analyses revealed that diosgenin exhibits favorable binding affinity with key targets CAT, NR1I2 (PXR), and NR3C2 (MR). In vitro validation demonstrated that diosgenin dose-dependently and significantly inhibited the proliferation and migration of Hepa1-6 cells. Furthermore, Western blot and immunofluorescence analyses confirmed that diosgenin treatment significantly reduced the expression levels of metastasis-associated proteins MMP2 and vimentin, as well as the proliferation marker PCNA. This study provides a systematic investigation into the potential active ingredients and molecular mechanisms underpinning Zhuang medicine's efficacy against liver cancer. Our findings strongly implicate diosgenin as a promising therapeutic candidate for LC and establish a crucial theoretical foundation for the further development of anticancer agents derived from Zhuang medicinal resources.