Network pharmacology analysis combined with experimental verification of the molecular mechanism of Xihuang pill in treating liver cancer

Background: Xihuang pill is a kind of traditional Chinese medicine, which has been widely used in the treatment of kinds of cancer. However, there is still a lack of systematic understanding of the molecular mechanism of Xihuang pill in the treatment of liver cancer. In this work, we aim to explore the molecular mechanism of Xihuang pill in treating liver cancer. Methods: The functional components in Xihuang pill were collected from Traditional Chinese Medicine Database and Analysis Platform. The target genes of these components were also collected using Traditional Chinese Medicine Database and Analysis Platform. The target genes of liver cancer were predicted using GeneCards database. The intersecting genes were then analyzed with Venn diagrams. Kyoto Encyclopedia of Genes and Genomes and Database for Annotation, Visualization, and Integrated Discovery were used to analyze the pathway. Then, cell counting kit-8 was used to measure the half-maximal inhibitory concentration of Xihuang pills. The living dead cell staining method was used to observe the survival of cells. HepG2 cell apoptosis was tested by flow cytometry with fluorescein isothiocyanate/propidium iodide double staining method, and then the mitochondrial damage was also detected by flow cytometry. The expression of target genes was detected by quantitative real-time polymerase chain reaction. Results: A total of 130 compounds and 198 genes were identified as potential active ingredients and putative liver cancer-related targets. We obtained 1,899 disease targets and 297 transcriptome targets from the database. Six drug-disease intersecting genes, CCNB1, BIRC5, TOP2A, ESR1, IGF2 and IGFBP3 were obtained. They are enrichment in apoptosis, PI3K-AKT signaling pathway, MAPK signaling pathway, pathways in cancer and p53 signaling pathway. Besides, it was found that the apoptosis rate of the HepG2 cells in Xihuang pill treated group was significantly higher than that of the control group. And the apoptosis rate gradually increased in a dose dependent manner of Xihuang pill treatment. Xihuang pill also induced the mitochondrial membrane potential damage. Compared with the control group, the expression level of CCNB1 and BIRC5 was induced, while the expression level of IGF2 was reduced after Xihuang pill treatment. Conclusion: Xihuang pill may act on six proteins (CCNB1, BIRC5, TOP2A, ESR1, IGF2 and IGFBP3) and cover multiple pathways to form a therapeutic network to treat liver cancer.