Integrated network pharmacology, proteomics, molecular docking, and experiments in vivo and in vitro to explore the efficacy and potential mechanism of bufalin against hepatocellular carcinoma angiogenesis.

Ethnopharmacological Relevance: Bufalin is a potent bioactive compound extracted from the venom of toads such as Bufo gargarizans. It has rich pharmacological effects, and its traditional applications mainly include anti-cancer, anti-inflammatory and analgesic, especially in cancer treatment, which has been a hot topic of research. Prior research has suggested that bufalin may have anti-tumor angiogenic effects. However, the efficacy and mechanism of bufalin inhibiting hepatocellular carcinoma (HCC) angiogenesis have yet to be further investigated.

Aim Of The Study: An extensive detailed strategy via network pharmacology, proteomics, histopathological analysis, molecular docking, in vitro experiments, and in vivo magnetic resonance imaging (MRI) examinations were adopted to investigate the efficacy and mechanisms of bufalin against HCC angiogenesis.

Materials And Methods: Micro-vessel density (MVD) and intravoxel incoherent motion (IVIM) perfusion-related parameters based on magnetic resonance diffusion-weighted imaging were used to identify the effect of bufalin against HCC angiogenesis. Potential bufalin and HCC targets were gathered from appropriate databases. The STRING database was used to construct the target protein interaction networks. The "clusterprofiler" package (version 4.2.2) in R was applied to conduct the target-related Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis. Network pharmacology and proteomics were integrated to identify key targets and pathways related to bufalin against HCC angiogenesis. Molecular docking and Western Blot were utilized to validate the findings.

Results: Analysis through IVIM and MVD showed that bufalin could inhibit HCC angiogenesis in nude mice models. A total of 159 common targets of bufalin and HCC were identified by network pharmacology. GO analysis revealed that these targets focused on multiple angiogenesis-related biological processes, including endothelial cell proliferation and migration, sprouting angiogenesis, and regulation of angiogenesis. The KEGG enrichment results suggested that bufalin could regulate multiple signaling pathways to inhibit HCC angiogenesis, including VEGF, MAPK, PI3K-Akt, mTOR, and HIF-1 signaling pathways. MAPK1, MAPK14, PRKCA, EIF4E, and APEX1 might be critical targets in regulating the above pathways. The molecular docking and Western blot analysis verified the effects of bufalin on target proteins.

Conclusion: This study demonstrated that bufalin might inhibit HCC angiogenesis by regulating multiple targets and pathways. These findings offer theoretical insights and experimental foundations for the clinical application and commercial development of bufalin in the treatment of HCC.