To Investigate The Intervention Mechanism Of Ze-Qi-Tang On Lung Adenocarcinoma Based On PI3K/AKT Signaling Pathway

Objective:Aiming at the target genes of tumors and the complex intervention characteristics of traditional Chinese medicine,a suitable research model is constructed under the guidance of bioinformatics and network pharmacology technology to form a research model that conforms to the advantages of traditional Chinese medicine and reflects the advantages and value of natural drugs against tumors.Oriented research methods and strategies can further realize the prediction and mechanism analysis of specific anti-tumor targets of traditional Chinese medicine.In this study,the effects of Ze-Qi-Tang on the proliferation,apoptosis,invasion and cycle of lung cancer A549 cells were analyzed by in vitro cell experiments.The mechanism of action of LUAD)was discussed,which provided effective evidence of traditional Chinese medicine for the clinical treatment of LUAD.Methods:1.Construction of "drug-target" network under the guidance of network pharmacology Based on ADME parameters(absorption,distribution,metabolism,excretion)provided by TCMSP,Ze-Qi-Tang was pre-screened for active ingredients to set the standard of oral bioavailability(OB)>30%,drug similarity(DL)>0.18,Caco-2 permeability(Caco-2)>-0.14.Then,the target genes of these constituent compounds were predicted by TCMSP,Pub Chem and Swiss Target Prediction databases.In order to further analyze the interaction relationship between target genes,based on the STRING database and Cytoscape software,the construction and visualization of the proteinprotein interaction(PPI)network between Ze-Qi-Tang-related target genes was realized.In addition,We performed GO and KEGG enrichment analysis on the predicted Ze-Qi-Tang target genes using the DAVID database to gain insight into the biological significance behind the massive gene data to better understand the relevant functions and enrichment of target genes.2.Construction of lung adenocarcinoma "target-pathway" database under the guidance of bioinformatics Based on the TCGA database,the original transcriptome data and clinical data related to lung adenocarcinoma were obtained,and the perl language and the Ensembl online database were used to preliminarily organize the original data and standardize the annotation of genes.Subsequently,the differential analysis of lung adenocarcinoma-related genes was performed by R language and edge R package,and p < 0.05 and|log FC|> 2 were set as the screening criteria for differentially expressed genes in lung adenocarcinoma,and then the differentially expressed genes were analyzed based on the database.Network construction and GO and KEGG functional enrichment analysis.Combined with the clinical data of lung adenocarcinoma,the prognosis and mutation correlation analysis of key pathway-related genes was performed,and the functions of key pathways and pathway-enriched genes were further explored.3.Experimental study of Ze-Qi-Tang interfering with LUAD A549 cell line via PI3K/AKT signaling pathway The effect of Ze-Qi-Tang on the cell viability of LUADA549 cell line was observed by CCK-8 method.Cell scratch assay and Transwell assay were used to observe the effects of different concentrations of Ze-Qi-Tang on cell migration and cell invasion.The effects of Ze-Qi-Tang on A549 cell cycle and apoptosis were further examined by flow cytometry analysis.Finally,Western Blot and qRT-PCR experiments were performed to detect the protein expressions of PI3 K,AKT,p-AKT,Cyclin D1 and the m RNA expression levels of PI3 K and AKT in the cells.4.Intervention of the main components of Ze-Qi-Tang by molecular docking technology Chem Draw Ultra 12.0 software constructs the structure of the component small molecule,and optimizes it in Sybyl X;extracts the protein crystal structure of the docked gene from the RCSB database and saves it in the "PDB Format" format for future use.Then use Surflex-Dock program to perform molecular docking of components and protein molecules and obtain Surflex-Dock score(total score)to evaluate the molecular docking results,and finally use Py MOL software to further optimize,observe and analyze hydrogen bonds and their binding sites.Results:1.According to TCMSP,a total of 13 ingredients in Ze Lacquer were obtained,2 ingredients in Iwami Chuan,5 ingredients in ginger,18 ingredients in ginseng,34 ingredients in Scutellaria baicalensis,4ingredients in Guizhi There are 10 ingredients in Pinellia,2 ingredients in Baiqian and 87 ingredients in Licorice.After removing the repeated ingredients,a total of 156 active ingredients related to Ze-Qi-Tang were obtained.After removing duplicates,a total of 530 target genes related to the action of Ze-Qi-Tang components were predicted from the Swiss Target Prediction database and TCMSP database.The functional enrichment analysis with p<0.05 as the screening cutoff value found that the predicted target genes of Ze-Qi-Tang were mainly enriched in 178 KEGG pathways,and the top3 pathways ranked by Count included Pathways in cancer,PI3K/AKT signaling pathway and Chemical carcinogenesis-receptor activation.2.The LUAD-related transcriptome data obtained in the TCGA database included a total of 594 samples,including 535 cancer samples and 59 normal tissue samples.After standardized annotation of the Ensembl database and differential analysis of the edge R package,with p<0.05 and |log FC|> 2 as the screening cut-off conditions,a total of 1421 LUAD-related differentially expressed genes were obtained,including 1096 up-regulated genes and 325 down-regulated genes.The functional enrichment analysis with p<0.05 as the screening cutoff value found that 14 KEGG pathways were mainly enriched in LUAD-related differentially expressed genes,including PI3K/AKT signaling pathway,Cell cycle and ECM-receptor interaction,etc.Kaplan-Meier survival analysis of 28 genes enriched in the PI3K/AKT signaling pathway found that 9 genes(CCNE1,COL6A6,COL11A1,EFNA3,FGF19,IL7 R,VEGFD,SPP1,and VWF)were closely associated with survival in patients with LUAD,p <0.05 means the difference is statistically significant.3.The results of CCK-8 experiment showed that Ze-Qi-Tang could inhibit the proliferation of A549 cells,and the inhibitory effect was dose-and time-dependent(p<0.05).Cell scratch experiments showed that Ze-Qi-Tang could significantly inhibit the migration ability of A549 cells in a concentration-dependent manner(p<0.05).The results of the invasion assay showed that Ze-Qi-Tang could significantly reduce the number of A549 cells passing through Matrigel,and statistical analysis found that the effect of Ze-Qi-Tang at high concentration was more significant than that at low concentration,indicating that Ze-Qi-Tang intervention The invasive ability of A549 cells was significantly decreased,and it was affected by the concentration of Ze-Qi-Tang in a concentration-dependent manner(p<0.05).The results of flow cytometry detection of cell cycle and apoptosis showed that Ze-Qi-Tang could promote cell apoptosis,affect cell cycle,and induce G0/G1 phase arrest in A549 cells.Western Blot experiments showed that the Ze-Qi-Tang group,like the PI3 K specific inhibitor LY294002group(10nmol/L),significantly reduced the protein expression levels of PI3 K,AKT,p-AKT and Cyclin D1 in A549 lung cancer cells compared with the control group(p<0.05),and qRT-PCR detection showed that the m RNA expression levels of PI3 K and AKT in cells decreased significantly after Ze-Qi-Tang intervention for 24 h.4.The docking results showed that multiple Ze-Qi small molecules had good binding effects with PI3Kα,PI3Kβ,PI3Kγ,PI3Kδ,AKT1,AKT2,and AKT3,and there were stable binding sites in the molecular docking model.The component 7-Angelyl-9-echimidinylheliotridine binds well to PI3Kα,PI3Kβ,PI3Kδ and related factors AKT1,AKT2,AKT3,and can form multiple hydrogen bonds.The component beta-sitosterol binds well to PI3Kγand PI3Kδ.The components euphoscopin B and PI3Kα,and the components euphoscopin D and PI3Kβ also have good binding.Conclusion:With the support of bioinformatics and network pharmacology technology,this study visualized the multi-component and multi-target compound characteristics of Ze-Qi-Tang and the complex mechanism of LUAD multi-gene and multi-pathway involvement based on biomolecular big data.And through experimental analysis,it was found that Ze-Qi-Tang can inhibit the proliferation of lung cancer A549 cells,promote the apoptosis of A549 cells,and induce the G0/G1 phase arrest of A549 cells,and the mechanism of action may be related to Ze-Qi-Tang’s intervention in PI3K/AKT signaling pathway.