Mechanism of action of Pulsatilla chinensis (Bunge) Regel compounds in hepatocellular carcinoma (HCC) treatment: An integrated analysis combining network pharmacology, molecular docking, molecular dynamics simulations and luciferase reporter gene assay.

ETHNOPHARMACOLOGICAL RELEVANCE

Hepatocellular carcinoma (HCC) is a primary malignancy originating from hepatocytes in the liver parenchyma. Pulsatilla chinensis (Bunge) Regel(P. chinensis) (verified via http://www.theplantlist.org, accessed April 5, 2025), a perennial herb of the Ranunculaceae family, contains multiple bioactive constituents with demonstrated pharmacological effects, including antitumor, anti-inflammatory, antibacterial, antiviral, and immunomodulatory activities.

AIM OF THE STUDY

To investigate the mechanisms of action and pharmacodynamic material basis of active compounds from P. chinensis against HCC cells.

MATERIALS AND METHODS

Active compounds of P. chinensis were screened using the HERB database. Potential drug targets were predicted via the SwissTargetPrediction database. HCC-related targets were retrieved from GeneCards, OMIM, and TTD databases, followed by Venn diagram analysis to identify shared drug-disease targets. STRING database was employed for protein-protein interaction (PPI) network analysis and core target screening. DAVID platform was used for Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Cytoscape software constructed a compound-target-pathway network to identify key active components and potential mechanisms. Molecular docking simulations validated the binding affinity between core targets and Pulsatilla's active compounds. A luciferase reporter gene system was established, generating A549-TP53 monoclonal cell lines stably expressing p53-NLuc, followed by functional validation. Traditional Chinese medicine (TCM) monomer compounds were screened using A549-TP53 cells. Flow cytometry and Western blot assessed their effects on apoptosis in 7402 and 7721 human HCC cells. Molecular dynamics simulations validated the binding interactions between the compounds and TP53.

RESULTS

HERB database identified 29 active compounds from P. chinensis. SwissTargetPrediction predicted 606 potential drug targets. GeneCards, OMIM, and TTD yielded 1095 disease-related targets, with 163 overlapping targets identified via Venn analysis. PPI analysis using the STRING database revealed the top five core targets: TP53, GAPDH, AKT1, EGFR, and STAT3. Cytoscape analysis identified 14 core active compounds from P. chinensis. Molecular docking results revealed that among these 14 core active compounds from P. chinensis, the top five with the highest binding affinity to TP53 protein were: Pulchinenoside C, Pulsatilla Saponin D, Pulsatilloside B, Qingdainone and Sitogluside. The recombinant retroviral vector pQCXIP-p53-NLuc was successfully constructed, and luciferase activity assays confirmed A549-TP53 as a stable NanoLuc (Nluc)-expressing cell line regulated by TP53. Luciferase assays demonstrated that Pulchinenoside C (20-80 μM), Pulsatilla Saponin D (5-80 μM), and Qingdainone (20-80 μM) significantly modulated A549-TP53 transcriptional activity. Flow cytometry revealed that Pulsatilla Saponin D and Pulchinenoside C markedly induced apoptosis in 7402 and 7721 cells.Western blot revealed Pulchinenoside C significantly elevated cleaved caspase-3 in both 7402 and 7721 cells, whereas Pulsatilla Saponin D markedly increased it in 7402 cells. Molecular dynamics simulation results indicate that Pulsatilla Saponin D and Pulchinenoside C may exert their biological effects by inhibiting the TP53 target protein.

CONCLUSION

The active compounds Pulchinenoside C and Pulsatilla Saponin D significantly promoting apoptosis in HCC cells via TP53 targeting.