| WCA,a traditional Chinese medicine compound prescription mainly based on invigorating the spleen,is a clinical prescription of Professor Qiu Jiaxin,a famous Chinese medicine expert in China,a famous Chinese medicine expert in Shanghai and Oncology Department of Longhua Hospital.It has definite clinical efficacy in improving spleen deficiency syndrome of gastric cancer patients,prolonging survival period,reducing recurrence and metastasis after radical operation,and alleviating adverse reactions of chemotherapy.Experimental studies have shown that WCA based on the pathogenesis of spleen deficiency of gastric cancer has definite anti-mutagenic effect and can block the initiation and initiation of gastric cancer;inhibit the growth and metastasis of subcutaneous transplanted tumors in nude mice with human gastric cancer;inhibit the proliferation of gastric cancer cells,induce cell apoptosis,inhibit the invasion and metastasis of gastric cancer cells and epithelial-mesenchymal transformation.Regulating immunity and other functions.Based on the above studies,in order to explore the mechanism and target of WCA from a holistic point of view,this study uses network pharmacology and proteomics to explore the anti-gastric cancer mechanism of WCA and active compound Luteolin from multiple perspectives.Objective:From the overall point of view of the study of Chinese herbal compound prescription,the core chemical components,targets and signaling pathways of Fufang Weichangan were studied by network pharmacological method;proteomic technology was used to analyze the changes of differential proteins after the subcutaneous transplantation of human gastric cancer in nude mice;network pharmacological and proteomic combined analysis was used to screen the network of Fufang Weichangan.Potential targets and signaling pathways of synergistic effects,and the potential mechanism of luteolin,an important active compound in Compound Weichangan,against gastric cancer were also studied.Method: 1.Using network pharmacological research methods,we constructed multi-level and multi-level networks of compounds-targets,targets-diseases and targets-pathways.Using Cytoscape software,we constructed HIPPIE(Human Intermediate Protein Interaction Eference)protein interaction network.Gene Ontology(GO),KEGG signaling pathway(Kyoto encyclopedia of genes and genomes)were used for compound gastrointestinal security.Enrichment analysis of NES and genomes(KEGG),disease ontology(DO),medical subject headings(Me SH).2.By constructing the subcutaneous transplantation model of human gastric cancer in nude mice,proteomics technology was used to explore the effects of compound WCA on human gastric cancer in nude mice.The up-and down-regulated proteins were identified,and the gene ontology and signal pathway enrichment of the differential proteins were analyzed.3.According to the active compounds,core targets and differential proteins screened by proteomics,the target network of compound gastrointestinal safety was constructed.Cytoscape 3.6.0 software was used to calculate the network topological parameters,Betweenness Centrality(BC),Closeness Centrality(CC),Topological Coefficient(TC),Degree(DE),evaluate the role of the target in the network,find potential targets of overlapping network pharmacology and proteomics,and validate the predicted expression level of potential targets by Western-blot and Q-PCR in the subcutaneous transplantation model of human gastric cancer in nude mice treated with compound WCA.4.CCK8(Cell Counting Kit-8),Flow cytometry,Hoechst and Western-Blot were used to investigate the effects of luteolin on proliferation and apoptosis of gastric cancer cells.Result: 1.By network pharmacological analysis,136 active compounds,306 core targets and 151 targets related to gastric cancer were obtained.Through GO analysis of bioinformatics gene ontology,the results showed that 117 GO terms were significantly related to the level of molecular function(MF)and compound gastrointestinal security,and 72 GO terms were significantly related to gastric cancer.There are 1578 GO terms significantly related to the level of biological process(BP)and 1104 GO terms significantly related to gastric cancer.At the level of cell component(CC),there were 71 GO terms significantly related to compound gastrointestinal antagonist and 54 GO terms significantly related to gastric cancer.By KEGG signal pathway enrichment analysis,there were 8 signal pathways associated with gastric cancer,including HIF-1 signaling pathway,Endocrine resistance,EGFR tyrosine kinase inhibitor resistance,ERBB signaling pathway,FOXO signaling pathway,PI3K-Akt signaling pathway,Thyroid hormone signaling pathway,RAP1 signaling pathway.Using network analysis,eight potential targets were screened out in the network of network pharmacology,namely PTGS2,Bax,Cycs,RELA,ABCC1,HIF1 A,TOP1 and GSTP1.2.By proteomic analysis,3385 differentially expressed proteins were identified,of which 2856 had quantitative information,compared with the saline model group after the effect of Fufang Weichangan on the subcutaneous transplantation of human gastric cancer in nude mice.417 of them were up-regulated,more than 1.2 times,340 were down-regulated,less than 0.83 times.Through GO analysis of bioinformatics gene ontology,the results showed that there were 542 GO terms significantly related to compound gastrointestinal security at the level of molecular function(MF).There are 1940 GO terms that are significantly related to gastrointestinal security at the level of biological process(BP).There are 1 251 GO terms that are significantly related to gastrointestinal security at the level of cellular component(CC).KEGG signaling pathway enrichment analysis showed that there were 45 signaling pathways significantly related to compound gastrointestinal antagonist,involving multiple cancer-related pathways.In the constructed protein network,20 core protein clusters were selected.3.The core targets of network pharmacology,active compounds and differential proteins screened by proteomics were analyzed.The results showed that Compound Weichangan was enriched in eight signaling pathways in network pharmacology and proteomics,namely TNF Signaling Pathway,IL-17 Signaling Pathway,PI3K-Akt Signaling Pathway,Renin-otensin System,Focal adhesion,TNF Signa Pathway.Ling Pathway,NF-kappaa B Signaling Pathway,Glycolysis/Glucone Genesis.The network pharmacology and proteomics network of Fufang Weichangan were analyzed jointly.In the overlap analysis of 8 core targets and 20 core protein clusters in the network pharmacology network,ABCC1,PTGS2,Cycs and Bax were the four overlapping proteins.The results of Q-PCR and Western Blot validation showed that,compared with the control group,Fufang Weichangan could down-regulate the expression of ABCC1 and PTGS2 proteins and up-regulate the expression of Bax and Cycs proteins in the subcutaneous transplantation model of human gastric cancer in nude mice.4.The experimental results showed that luteolin,an important active compound of Weichangan,inhibited the proliferation of gastric cancer cells in a time-dose-dependent manner.At the same time,it blocked the cell cycle at S stage,induced the apoptosis of gastric cancer cells,decreased the mitochondrial membrane potential,increased the expression of Bax,Caspase 3 and Cycs,decreased the expression of Bcl-2 and downregulated the expression of p-Akt.After adding PI3K-Akt pathway inhibitor LY294002,the expression of Bax,Caspase 3 and Cycs protein increased,the expression of Bcl-2 protein decreased and the expression of p-Akt protein decreased compared with luteolin group.Conclusion: 1.Compound Weichangan has the effect of anti-gastric cancer through multiple targets and pathways.Its mechanism may involve eight signaling pathways and four core targets,including ABC1,PTGS2,Cycs and Bax.2.The anti-gastric cancer mechanism of Compound Weichangan involves up-regulating the expression of Cycs and Bax and down-regulating the expression of ABCC1 and PTGS2.3.Luteolin,an important active compound in Fufang Weichangan,can inhibit the proliferation of gastric cancer cells and block the cell cycle in S phase;induce apoptosis of gastric cancer cells,which may be related to up-regulation of Bax protein expression and down-regulation of Bcl-2 protein expression,and may involve in PI3K-Akt signaling pathway. |
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