Study On The Pharmacodynamic Material Basis And Mechanism Of Dysosma Versipellis In Intervention Of Small Cell Lung Cancer Under The Guidance Of Integrated Pharmacology Model

Objective:Based on the integrated pharmacology model of "bioinformatics,network pharmacology,and in vitro experimental verification",the research on the material basis and mechanism of the effect of the traditional Chinese medicine Dysosma versipellis on small cell lung cancer is explored.It provides theoretical support and mechanism explanation for the clinical application of Dysosma versipellis in the intervention of small cell lung cancer,and explores the anti-cancer research ideas and methods of traditional Chinese medicine that not only conform to the treatment concept of Chinese medicine but also can be connected with the principles of modern oncology.Methods:1.Under the guidance of network pharmacology,the prediction of "drug-target-pathway" of Dysosma versipellis was carried out.Retrieve the active ingredients of Dysosma versipellis through the TCMSP database,and combine with Uni Prot and Swiss Target Prediction databases to obtain the predictive targets of the active ingredients.KEGG and GO enrichment analysis of the predicted targets of the Dysosma versipellis through the DAVID database to study the active components of the Dysosma versipellis involved in the biological functions and pathway processes of the human body.2.Analysis of differential expressed genes related to small cell lung cancer diseases based on bioinformatics methods.Search the gene chip related to small cell lung cancer through the GEO database and sort and analyze it to obtain the differential expressed genes related to small cell lung cancer disease.Then use STRING database and Cytoscape software to visualize differential genes and perform Cyto NCA topology analysis;then perform Kaplan Meier Plotter survival analysis of key genes based on topological analysis;pass key genes that are meaningful to overall survival(p <0.05)through DAVID again KEGG and GO enrichment analysis were performed to screen out the key genes and pathways of Dysosma versipellis active ingredients that may act on small cell lung cancer.3.Research on the intervention of the effective components of Dysosma versipellis in small cell lung cancer by computer simulation of molecular docking technology.First obtain the PDB format file of the target crystal structure from the RCSB PDB protein database;directly download the corresponding active ingredient mol2 format file in the ZINC database.Then use the Surflex-Dock module of the SYBYL8.1 software to perform molecular docking techniques and obtain the Surflex-Dock score(total score),and use Py MOL software to further optimize it to obtain the most highly bound active ingredient of Dysosma versipellis for the next step cell experiment verification.4.Experimental study on the active ingredients of Dysosma versipellis and small cell lung cancer cell lines(NCI-H1339,NCI-H446 cells)and differential genes.MTT method was used to observe the effect of the active components of Dysosma versipellis on the cell viability of small cell lung cancer cell lines NCI-H1339 and NCI-H446.Cell migration and invasion experiments detect the changes in the migration and invasion ability of NCI-H1339 and NCI-H446 cells with different concentrations of the active ingredients of the Dysosma versipellis.Real-time fluorescent quantitative PCR experiment and Western Blot experiment(immunoblotting assay)were used to determine the expression of key genes in the small cell lung cancer cell lines NCI-H1339 and NCI-H446 with different concentrations of the active ingredients of Dysosma versipellis.Results:1.Through the TCMSP and Pub Chem database,we finally obtained 13 active ingredients and the corresponding SMILES structure of Dysosma versipellis.A total of 486 target genes were screened from Uni Prot,Swiss Target Prediction database and TCMSP database.DAVID database analysis revealed that target genes are involved in a variety of biological processes and a variety of cancer-related pathways.2.Three small cell lung cancer microarray gene chips were screened through the GEO database,including 49 healthy lung tissue samples and 28 primary small cell lung cancer tumor tissue samples.After crossing the 3 sets of differential genes on the Venn diagram,178 differential genes were obtained.After topological analysis and Kaplan Meier Plotter survival analysis,15 key genes were screened out: TP53,IL6,VEGFA,MYC,EGFR,TNF,MAPK3,CASP3,ESR1,JUN,CCND1,MAPK8,MMP9,CXCL8,PTEN and obtained the corresponding Survival analysis chart.Once again,the biological pathway enrichment analysis found that the target gene pathway analysis related to the active ingredients were similar to the previous analysis,and they were significantly enriched in cancer and related pathways.Further pathway analysis and literature review found that MAPK3 and MYC genes are located in the upstream and downstream of the MAPK signaling pathway,which are closely related to the disease process of small cell lung cancer and have important research significance.3.The two key genes of small cell lung cancer(MAPK3 and MYC)and the13 active ingredients of Dysosma versipellis were respectively subjected to molecular docking simulation,and it was concluded that the two key genes of MAPK3 and MYC were the best for docking simulation with podophyllotoxin.4.The results of MTT experiments showed that podophyllotoxin can inhibit the proliferation of human small cell lung cancer cells NCI-H1339 and NCI-H446;Migration and invasion experiments showed that podophyllotoxin significantly inhibited the migration and invasion of cells,and the difference was statistically significant;Real-time fluorescent quantitative PCR test results found that with the increase of podophyllotoxin concentration,it can inhibit the expression of MAPK3 and MYC in NCI-H1339 and NCI-H446 cells.Western Blot analysis showed that the key genes MAPK3 and MYC had a negative regulatory relationship at the protein level of NCI-H1339 and NCI-H446 cells.Conclusion:This study found that podophyllotoxin in Dysosma versipellis is a key active ingredient for small cell lung cancer,which can inhibit the proliferation,migration and invasion of human small cell lung cancer cells NCI-H1339 and NCI-H446;By interfering with the expression of MAPK3 and MYC genes on the MAPK signaling pathway,the regulation of small cell lung cancer tumor cells can finally be achieved.This result can provide a molecular basis for the clinical application of Dysosma versipellis in small cell lung cancer,and also shows the feasibility of finding anti-cancer research ideas and methods in traditional Chinese medicine that not only conform to the treatment concept of traditional Chinese medicine,but also can be connected with the principles of modern oncology.