Exploring the effect of Gupi Xiaoji Prescription on hepatitis B virus-related liver cancer through network pharmacology and in vitro experiments.
AIM AND OBJECTIVE
To study the effect of Gupi Xiaoji Prescription (GXP) on hepatitis B virus(HBV)-related liver cancer through network pharmacology coupled with in vitro experiments and explore their related mechanisms.


MATERIALS AND METHODS
Gupi Xiaoji Prescription's chemical constituents and the action targets of its six medicinal components were identified using several databases. These included the Traditional Chinese Medicine Systems Pharmacology Database （TCMSP), the Bioinformatics Analysis Tool for Molecular mechANism of TCM (BATMAN-TCM), and the Traditional Chinese Medicine Integrated Database (TCMID), while GeneCards and OMIM were used to compile relevant liver cancer disease targets. Pathway enrichment of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), analysis of potential targets, and analysis of the enriched pathways in literature were executed in R. The Hepatocellular carcinoma (HCC)-derived HepG2.2.15 cell line stably expresses and replicates HBV. In vitro experiments with HepG2.2.15 were used to verify GXP's effects on HBV-related liver cancer, while the human liver cancer cell line HepG2 was used as the control.


RESULTS
171 active ingredients and 259 potential drug targets were screened from GXP, involving 181 pathways in vitro. These assays identified Polyphyllin I as an effective GXP component. Notably, GXP inhibited cell proliferation and metastasis in a concentration-dependent manner (P < 0.01). In comparison with the vehicle group, the fluorescence intensity of each drug group was significantly weakened (P < 0.01), while the drug group Mitofusins 1(MFN1) and protein expression level of Mitofusins 2 (MFN2) increased significantly. The protein expression level of Mitochondrial fission protein 1 (FIS1) and Optic Atrophy 1 (OPA1) also showed significant decreases (P < 0.01). Molecular docking revealed Fructus saponins I's high affinity with FIS1, MFN1, MFN2, and OPA1.


CONCLUSION
The network pharmacology results indicate that Gupi Xiaoji Prescription may treat liver cancer by regulating mitochondrial division and fusion of key genes to disrupt liver cancer cells' energy metabolism. In vitro experiments also verified that GXP could inhibit the proliferation and migration of HepG2.2.15 cells by up-regulating MFN1 and MFN2, down-regulating the expression of FIS1 and OPA1 in addition to damaging mitochondria. Consistent with network pharmacology and molecular docking results, Polyphyllin I may be the most active compound of the formula's components. It also shows that Traditional Chinese medicine (TCM) plays a significant, targeted role in the treatment of HBV-related liver cancer.
['Yu|Shuxian|S|', 'Gao|Wenhui|W|', 'Zeng|Puhua|P|', 'Chen|Chenglong|C|', 'Zhang|Zhen|Z|', 'Liu|Zhuo|Z|', 'Liu|Jiyong|J|']
D000972:Antineoplastic Agents, Phytogenic D045744:Cell Line, Tumor D020558:GTP Phosphohydrolases D006509:Hepatitis B D006801:Humans D008113:Liver Neoplasms D008516:Medicine, Chinese Traditional D008565:Membrane Proteins D008928:Mitochondria D033681:Mitochondrial Membrane Transport Proteins D024101:Mitochondrial Proteins D062105:Molecular Docking Simulation D060066:Protein Interaction Maps
Gupi Xiaoji Prescription Hepatitis B virus-related liver cancer Network pharmacology HepG2.2.15 Molecular docking Mitochondrial fission and fusion