Integrative characterization of Huachansu against hepatocellular carcinoma: chemical profiling, Network pharmacology, and in vivo evidence of apoptosis and gut microbiota remodeling

Background Hepatocellular carcinoma (HCC) remains a major global health burden, and traditional Chinese medicine (TCM) offers complementary therapeutic advantages. Although Huachansu (HCS), a clinically used TCM preparation, contains key bioactive components, including bufalin (BFL) and cinobufotalin (CBF), its antitumor mechanisms remain unclear. Methods LC–MS profiling was performed to characterize the chemical constituents of HCS. Network pharmacology analysis was conducted by integrating predicted compound targets with HCC-associated genes to identify overlapping targets and enriched biological pathways. Subsequently, molecular docking was used to evaluate binding interactions between active compounds and hub proteins. Finally, the antitumor effects of BFL and CBF were validated in vivo , and gut microbiota alterations were assessed using 16S rRNA sequencing. Results LC–MS profiling identified 17 HCS constituents, predominantly bufadienolides, establishing a chemical basis for mechanistic analyses. Network pharmacology analysis defined 142 overlapping targets enriched in apoptosis-related processes and cancer pathways. Molecular docking supported stable binding between bioactive compounds and key hub proteins, including STAT3, EGFR, AKT1, and PARP1, suggesting apoptosis-centered regulation. Guided by these in silico findings, in vivo experiments demonstrated that BFL and CBF reduced tumor burden and proliferation, as evidenced by hematoxylin and eosin staining and Ki-67 immunohistochemistry. Enhanced apoptosis was confirmed by TUNEL staining and Bax/Bcl-2 expression profiling, with more pronounced effects under combined treatment. Moreover, 16S rRNA sequencing revealed gut microbiota remodeling following BFL, CBF, and their combination treatments. Conclusion These findings clarify the apoptosis-associated anti-HCC mechanism of HCS and provide mechanistic insights supporting development of TCM-based anticancer agents.