Network Pharmacological Analysis And Experimental Study Of Cucurbitacin B On Oral Squamous Cell Carcinoma

Objective: Oral squamous cell carcinoma(OSCC)is a malignant tumor with high morbidity and poor prognosis.Currently,there is a lack of ideal therapeutic drugs.Cucurbitacin B(Cu B)is a tetracyclic triterpenoid small molecule compound with a variety of biological functions,especially in anti-tumor.In this study,the effects and underlying mechanisms of Cu B on OSCC were explored through network pharmacology,animal experiments and cell experiments.Methods: Part I: Network pharmacological analysis of cucurbitacin B on oral squamous cell carcinoma: CTD database,HERB database,ETCM database and Pharm Mapper database were used to predict related targets of Cu B;Gene Cards,OMIM database were used to collect potential therapeutic targets of OSCC;we obtained the intersection of Cu B and OSCC by Venn diagram Target;a protein-protein interaction(PPI)network was constructed based on the STRING platform and visualized by Cytoscape software to screen core genes;GO functional enrichment analysis and KEGG signaling pathway enrichment analysis were performed based on Metascape platform to screen core pathways;Autodock vina 1.1.2 software was used for molecular docking of Cu B and core genes was performed to verify the binding force between drugs and small molecules.Part II: In vitro cell experimental study on the effect of cucurbitacin B on oral squamous cell carcinoma:Cu B acts on SCC25 and CAL27 cells to validate the results of network pharmacology.The inhibitory effect of Cu B on cell growth was examined by CCK-8 method at 24 h,48h and 72 h,respectively;the effect of Cu B on cell clone formation ability was detected by colony formation assay;the effect of Cu B on cell migration ability was detected by cell scratch assay;Cytometry was used to detect the effect of Cu B on cell cycle and apoptosis;Western blot was used to detect the effect of Cu B on the protein expression levels of key pathway signaling molecules.Part III: In vivo study of cucurbitacin B in an animal model of oral squamous cell carcinoma induced by 4NQO: 4NQO drinking water was used to induce the development of oral squamous cell carcinoma of the tongue in C57BL/6 mice.At the same time,the mice were intraperitoneally injected with Cu B for 20 consecutive weeks,and the drug was discontinued and observed for 4 weeks,for a total of 24 weeks.By recording the water intake and body weight of the mice,the effect of the drug on the drinking water and the overall condition of the mice was observed;the mice were dissected,and the tongue tissue was visually observed and histopathologically diagnosed to study the inhibitory effect of Cu B on OSCC.Results: Network pharmacology analysis results: 1.Cu B predicted 317 targets,OSCC predicted 1584 related therapeutic targets,and a total of 134 common targets were obtained after the intersection of Cu B and OSCC.2.PPI analysis of the intersection targets showed that PIK3R1,SRC,STAT3,AKT1 and MAPK1 were the core target proteins.3.GO functional enrichment analysis showed that biological process(BP)was enriched to 1805 entries;cellular component(CC)was enriched to 88 entries;molecular function(MF)was enriched to 137 entries;KEGG pathway enrichment results showed that a total of 188 pathways were enriched,and PI3K-AKT was the most critical pathway.4.Molecular docking results showed that the binding forces between Cu B and PIK3R1,SRC,STAT3,AKT1 and MAPK1 were all less than-6 kcal/mol,indicating that the drug and small molecules were well combined,and Cu B may act on OSCC through core targets.In vitro cell experiment results: 1.The CCK8 assay found that Cu B could inhibit the growth of SCC25 and CAL27 cells in a concentration-and time-dependent manner.Colony formation assays showed that Cu B had an inhibitory effect on the clonogenic ability of SCC25 and CAL27 cells.All these indicate that Cu B can inhibit the proliferation of cancer cells.2.Flow cytometry showed that compared with the untreated group,the cell cycle of SCC25 and CAL27 cells after Cu B treatment was arrested in the G2 phase,and the apoptosis of SCC25 and CAL27 cells in the Cu B treatment group was increased,and the difference was statistically significant.significance.All these indicate that Cu B can inhibit the proliferation of cancer cells by arresting the cell cycle and inducing apoptosis.3.Cell scratch experiments showed that compared with the untreated group,the migration of SCC25 and CAL27 cells after Cu B treatment was significantly reduced,with almost no migration.This indicated that Cu B could inhibit the migration ability of OSCC cells.4.Western blot results showed that Cu B could inhibit the phosphorylation of p-PI3 K,p-AKT and p-m TOR related signaling molecules of PI3K-AKT pathway in SCC25 cells.This indicates that Cu B may inhibit OSCC growth by regulating the core pathway PI3K-AKT.In vivo animal experiment results: 1.There was no statistical difference in the amount of water drinking among the mice in each group that drank 4NQO water,indicating that 4NQO had the same inducing effect on mice in each group.2.At the end of the experiment,the weight of the mice in the Cu B treatment group was higher than that in the model group,and the difference was statistically significant,indicating that the mice were in better overall condition and heavier after the Cu B treatment.3.Through visual observation and histopathological analysis,it was found that the mice were less ill after Cu B treatment,indicating that Cu B could inhibit the progression of 4NQO-induced oral cancer in mice.Conclusion: 1.The network pharmacology analysis showed that Cu B had a total of 134 targets on OSCC,among which PIK3R1,SRC,STAT3,AKT1 and MAPK1 were the key ones.The core pathway of Cu B acting on OSCC is the PI3K-AKT pathway.2.In vitro cell experiments showed that Cu B could inhibit the proliferation and migration of SCC25 and CAL27 cells,arrest the cell cycle of SCC25 and CAL27 cells in G2 phase,and induce apoptosis.Western Blot experiments showed that Cu B could regulate the expression of PI3K-AKT pathway.3.in vivo animal experiments show that Cu B has inhibitory effect on 4NQO-induced oral mucosal carcinogenesis in C57BL/6 mice.