Study on the Mechanism of Acacetin in Treating Hepatocellular Carcinoma Based on Network Pharmacology, Molecular Docking, Molecular Dynamics Simulation, and in Vitro Experiments

Background Hepatocellular carcinoma (HCC) was one of the most prevalent and aggressive cancers worldwide, marked by a high mortality rate. Previous clinical treatments predominantly relied on chemotherapeutic agents, which are often associated with severe side effects and a high risk of drug resistance. Therefore, the development of natural compounds for HCC treatment has emerged as a promising therapeutic strategy. Acacetin, a natural flavonoid found in various plants, possesses potent anti-inflammatory and anticancer properties. However, its mechanisms of action remain poorly understood. Objective An integrated strategy combining network pharmacology, molecular docking, molecular dynamics simulation, and in vitro experiments were employed to elucidate the potential targets and underlying mechanisms of acacetin against HCC. Methods Firstly, we identified acacetin-related targets and HCC-related targets from public databases and determined their overlapping genes. Secondly, network pharmacology and enrichment analyses were performed to predict the potential biological functions and mechanisms underlying the anti-HCC effects of acacetin. Subsequently, molecular docking and molecular dynamics simulation were conducted to evaluate the stability and reliability of the interactions between candidate targets and acacetin. Finally, a series of in vitro experiments were performed to validate the computational predictions. Results Network pharmacology analysis identified 125 targets of acacetin, 2228 HCC-related targets, and 77 overlapping targets. The protein-protein interaction (PPI) network construction dentified six core targets: TP53, AKT1, EGFR, CASP3, ESR1, and MMP9. GO and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed significant involvement of negative regulation of apoptosis, the PI3K-Akt signaling pathway, and the p53 signaling pathway. Molecular docking and molecular dynamics simulation confirmed stable binding between acacetin and the core targets. In vitro experiments demonstrated that acacetin inhibited the proliferation and induced apoptosis of Hep-G2 cells. In addition, acacetin inhibited the expression of MMP9. Conclusion This study demonstrated the multi-target and multi-pathway nature of acacetin in the treatment of HCC. The proposed mechanism involves inhibition of key genes MMP9, suppression of the PI3K-AKT signaling pathway, reduced cancer cell proliferation, and induction of apoptosis. These findings offered a comprehensive theoretical basis for the therapeutic potential of acacetin in HCC treatment.