Anti-Hepatocellular Carcinoma Effects of Phillygenin: Integrated Network Pharmacology, Molecular Modeling, and Experimental Validation.

Abstract

PURPOSE: Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with limited therapeutic options and poor prognosis. Phillygenin, a bioactive lignan isolated from Forsythia suspensa, exhibits anti-inflammatory, hepatoprotective, and antitumor properties; however, its anti-HCC activity and associated molecular regulators remain incompletely understood. This study aimed to investigate the potential anti-HCC effects of Phillygenin and explore the regulatory molecules involved in its biological activity. METHODS: An integrated strategy combining network pharmacology, bioinformatics analysis, molecular docking, molecular dynamics simulations, and experimental validation was employed to investigate the anti-HCC effects of Phillygenin. Potential targets and related signaling pathways were identified through network pharmacology and enrichment analyses. Molecular docking and molecular dynamics simulations were performed to evaluate the predicted interactions between Phillygenin and candidate targets. The effects of Phillygenin on proliferation, migration, invasion, colony formation, and apoptosis were evaluated in HepG2 and Huh-7 HCC cell lines. The expression levels of ESR1 and EP300 were examined using immunofluorescence staining and qRT-PCR, and their functional involvement was further assessed through ESR1 knockdown and EP300 overexpression experiments. RESULTS: Network pharmacology analysis identified 185 overlapping targets between Phillygenin-associated targets and HCC-related genes, with ESR1 and EP300 identified as candidate regulatory molecules. Enrichment analysis suggested that these targets were mainly associated with cancer-related signaling pathways. Molecular docking and molecular dynamics simulations suggested potential interactions and conformational stability of Phillygenin with ESR1 and EP300. Cellular experiments demonstrated that Phillygenin significantly inhibited proliferation, migration, invasion, and colony formation while promoting apoptosis in both HepG2 and Huh-7 cells. Phillygenin increased ESR1 expression and decreased EP300 expression at both mRNA and protein levels. Functional validation further indicated that ESR1 knockdown attenuated, whereas EP300 overexpression partially reversed, the inhibitory effects of Phillygenin on HCC cell malignant behaviors. In the mouse safety assessment, no statistically significant differences were observed in organ indices, serum hepatic and renal function parameters, or hematological indices compared with the control group. Histopathological examination also revealed no obvious abnormalities in the major organs examined under the tested conditions. CONCLUSION: Phillygenin exhibits potential anti-HCC activity by suppressing malignant cellular behaviors and promoting apoptosis, with ESR1 and EP300 implicated in its cellular effects. Preliminary in vivo safety evaluation revealed no obvious treatment-related toxicity in the major organs or the blood-related parameters assessed at the tested doses. These findings support further investigation of Phillygenin, including its in vivo antitumor efficacy, pharmacokinetics, and comprehensive safety profile.

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