Objective  To investigate the effects of silibinin on proliferation, apoptosis, migration, invasion and ferroptosis of liver cancer HepG2 cells, and to verify the regulatory mechanisms of ferroptosis based on network pharmacology and experimental validation.

Methods  Following silibinin intervention in HepG2 cells, cell viability was assessed using CCK-8 assay, apoptosis levels were measured using Hochest staining, cell migration and invasion capabilities were evaluated through scratch assays and Transwell assays, ROS level was measured using DCFH-DA probe, mitochondrial membrane potential was assessed using JC-1 staining, and Fe²⁺ levels were detected by FerroOrange probe. Online databases were used to obtain silibinin, liver cancer, and ferroptosis targets, with subsequent analysis of intersecting targets through PPI network analysis, GO and KEGG enrichment analysis. After intervention with silybin alone and treatment with GN44028, Western Blot was used to detect the protein expression levels of hypoxia-inducible factor 1α (HIF-1α) and glutathione peroxidase 4 (GPX4).

Results  The semi-inhibitory concentration of silibinin for inhibiting HepG2 cell was 87.97 μmol/L, with a dose-dependent inhibitory effect. Compared to control HepG2 cells, silibinin promoted cell apoptosis, significantly inhibited cell scratch closure ability (P<0.05), reduced the number of migrating and invading cells (P<0.05), elevated intracellular ROS and Fe²⁺ levels, and induced mitochondrial membrane potential polarization. The intersecting targets of silibinin, liver cancer, and ferroptosis were identified as HIF-1α, mTOR, SRC, NRAS, PIK3CA, TERT, CA9, MAPK14, PARP-1, and PGD. Compared to control HepG2 cells, silibinin significantly increased the expression of HIF-1α in cells and decreased the expression of GPX4 (P<0.05). GN44028 can reverse the increase in Fe²⁺ concentration, the reduction in GPX4 expression, and the decrease in cell viability induced by silibinin in HepG2 cells.

Conclusion  Silibinin can inhibit the proliferation, migration, and invasion of HepG2 cells, induce apoptosis and ferroptosis, which may be related to its activation of HIF-1 α and subsequent inhibition of GPX4 expression.

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