Integrated network pharmacology and experimental validation reveal Coreopsis tinctoria Total Flavonoids (CTFs) as potential therapeutic agents against hepatocellular carcinoma

Objective: To validate the anti-hepatocellular carcinoma (HCC) efficacy of Coreopsis tinctoria Total Flavonoids (CTFs) and explore its underlying mechanisms using a comprehensive approach integrating network pharmacology and experimental verification, thereby supporting its potential as a multi-target therapeutic agent for liver cancer.Methods: Potential targets of CTFs were retrieved from Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database, while HCC-related targets were collected from GeneCards, OMIM, and DrugBank.Common targets were identified using VENNY2.1, and protein-protein interaction (PPI) networks were constructed via STRING.Functional Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using DAVID.A "CTFs-HCC-target-pathway" network was built with Cytoscape to identify key components and core targets.Molecular docking was performed using Autodock Vina.The Differential expression of key targets between HCC and normal tissues was visualized using boxplots, and prognostic relevance was evaluated by Kaplan-Meier survival analysis.In vitro assays, including CCK-8, live/dead staining, colony formation, flow cytometry, qPCR, were used to evaluate proliferation, viability, reactive oxygen species (ROS) levels, cell cycle distribution, and gene expression.A zebrafish xenograft model was established to determine the minimum toxic concentration (MTC) and evaluate tumor inhibition through fluorescence imaging and HE staining.Results: Network analysis identified 27 bioactive components and 318 putative targets of CTFs, with 32 associated with HCC.Core targets included Caspase-3, P53, MAPK1, Bcl-2 and Bax, primarily interacting with quercetin, (-)-Epigallocatechin (EGCG), fisetin, acacetin, luteolin, and kaempferol.Molecular docking confirmed strong binding affinities between these compounds and core targets.Pro-apoptotic genes (Bax, Caspase-3, P53) were upregulated in HCC tissues, and low expression of Bax/Caspase-3 correlated with poor survival.CTFs treatment further enhanced expression of Bax, p53 and Caspase-3, suppressed Bcl-2 while increased the Bax/Bcl-2 ratio.In vitro, CTFs inhibited HepG2 proliferation, promoted LO2 growth, induced ROS production, G 2 /M and S-phase arrest and apoptosis.In vivo, CTFs significantly suppressed tumor growth in zebrafish xenografts.Conclusion: CTFs exert anti-HCC effects through multi-target regulation of apoptosis-related genes and multiple signaling pathways, effectively inhibiting cancer cell proliferation.