Elucidating the Molecular Mechanisms of Angelica sinensis Against Hepatocellular Carcinoma Through Network Pharmacology, in silico Molecular Docking, and in vitro Validation.

Abstract

Background: Hepatocellular carcinoma (HCC) is a lethal liver malignancy associated with substantial morbidity and mortality and remains a major socioeconomic burden worldwide. Given the high risk associated with HCC, novel therapeutic approaches are urgently needed. Angelica sinensis is a traditional Chinese herbal remedy that contains bioactive phytochemicals with promising anticancer properties, making it a potential candidate for evaluation in HCC.

Objectives: This study integrated computational network pharmacology, molecular docking, and in vitro cellular assays to elucidate the mechanisms by which A. sinensis exerts anti-HCC effects in Hep-G2 cells.

Methods: Phytochemicals were identified using the Traditional Chinese Medicine Systems Pharmacology database and filtered according to drug-likeness and oral bioavailability criteria. Key compounds (β-sitosterol, alpha-cephalin, sitogluside, and stigmasterol) were further analyzed using the SuperPred Target Prediction tool to predict potential targets. HCC-related targets were curated from GeneCards and refined using the GeneCards Inferred Functionality Score. The overlap between compound-related and disease-specific targets was used to construct a protein-protein interaction network using STRING, which was subsequently visualized in Cytoscape to identify hub genes. Molecular docking between stigmasterol and the top 3 hub genes was evaluated using the CB-Dock2 online server, and all preparations were performed in BIOVIA Discovery Studio. In vitro, Hep-G2 cells were treated with varying concentrations (0, 25, 50, 100, and 200 µg/mL) of A. sinensis extract. Cell viability, clonogenic potential, apoptosis induction, and migratory capacity were evaluated using the MTT assay, clonogenic assay, Annexin V/PI staining, and Transwell migration assay, respectively. Western blotting was used to assess the expression of key hub proteins in Hep-G2 cells.

Results: In silico analysis identified an initial pool of 126 phytochemicals, which was refined to 4 key compounds yielding 139 unique targets. Intersection with HCC-related targets produced 123 common targets, generating a network comprising 119 nodes and 520 edges. STAT3, NFKB1, and TLR4 were identified as pivotal hub genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses of the hub genes demonstrated significant enrichment in HCC-related pathways and biological processes. Molecular docking indicated a strong binding affinity of stigmasterol to STAT3, NFKB1, and TLR4, with binding energies of -7.8, -6.9, and -6.9 kcal/mol, respectively. In vitro assays showed a dose-dependent reduction in Hep-G2 cell viability and colony formation, significant induction of apoptosis, and marked inhibition of migration. Western blotting confirmed significant downregulation of STAT3, NFKB1, and TLR4 expression at higher extract concentrations.

Conclusions: This study concluded that A. sinensis herbal extract was predicted to exert potent antiproliferative effects against HCC through the possible modulation of key signaling pathways and the targeting of hub genes, supporting its potential for further therapeutic development.