Exploring The Mechanism Of Tongfu Liqi Decoction In Treating Gastric Cancer Based On Network Pharmacology And Experimental Validation

Objective:To explore the mechanism of Tongfu Liqi Decoction(TFLQ)in the treatment of gastric cancer(GC).Methods:Part I: Network Pharmacological Analysis of TFLQ on GCScreening the active components and targets of TFLQ through TCMSP database and HERB database;Obtain potential therapeutic targets for GC through Genecards database and OMIM database;Venn diagram to obtain TFLQ-GC intersection target;Use the software Cytascape 3.9.1 to draw a "traditional Chinese medicine-active ingredient-target" diagram;Using STRING to construct a protein protein interaction(PPI)network to analyze the target interaction between TFLQ and GC,and using Cytascape3.9.1 to screen and visualize core genes;Use Metascape database and Bio Ladder Bioinformatics cloud platform to conduct enrichment analysis on gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)of TFLQ treatment GC;Use Auto Dock 1.5.6 software and Py MOL software to perform molecular docking and visualization processing on TFLQ active components and core genes.Part II: In vitro and in vivo experiments of TFLQ acting on GCIn this study,TFLQ was applied to BGC823 cells and BALB/c nude mice subcutaneously inoculated with cells to verify network pharmacological analysis.The inhibitory effect of TFLQ on cell growth was examined by CCK8 experiment at 24 h,48h,and 72 h,respectively;Clonal formation experiments were conducted to detect the influence of TFLQ on the clonal formation ability of BGC823 cells;The ability of TFLQ to affect the migration of BGC823 cells was tested through scratch experiments;The antitumor effect of TFLQ in vivo was tested by subcutaneous tumor experiment in nude mice;Western blotting and immunohistochemistry were used to detect the PI3K/AKT/mTOR signaling pathway and related protein expression levels in the subcutaneous GC tissue of BALB/c nude mice.Results:Results of network pharmacological analysis:1.TFLQ predicted 289 corresponding targets,obtained 12261 targets related to GC treatment,and 158 potential targets for TFLQ to treat GC.2.The main active ingredients of TFLQ include quercetin,kaempferol,luteolin,stigmasterol β-Sitosterol,etc;PPI analysis shows that the key targets for TFLQ treatment of GC include TNF,VEGFA,IL6,and IL1 β and so on.3.GO enrichment analysis showed that 1604 entries were obtained by BP analysis,78 entries were obtained by CC analysis,and 161 entries were obtained by MF analysis;KEGG enrichment analysis showed that 202 signal pathways were enriched,and the main signal pathways for TFLQ treatment of GC were TNF,IL-17,and PI3K/AKT.In vitro and in vivo experimental results:1.CCK8 experiment showed that TFLQ inhibited the proliferation of BGC823 in vitro in a concentration dependent manner;Colony formation experiments showed that TFLQ had a concentration dependent inhibition on the long-term viability and clonal formation ability of cells;The scratch test found that the inhibitory effect of TFLQ on BGC823 cell migration was positively correlated with drug concentration and time.2.The subcutaneous tumor experiment in nude mice found that TFLQ inhibited the growth of subcutaneous tumors in BALB/c nude mice in a concentration dependent manner,and capecitabine and TFLQ had a synergistic effect;Western blot and immunohistochemistry experiments indicate that TFLQ inhibits the activation of PI3K/AKT/mTOR signaling pathway related proteins,promotes BGC cell apoptosis,and regulates the PI3K/AKT/mTOR signaling pathway,which is one of the mechanisms of TFLQ in the treatment of gastric cancer.Conclusion:TFLQ treats GC by means of multiple components,multiple targets and multiple pathways.PI3K/AKT/mTOR signal pathway is one of its mechanisms.