| Objective: Gastric cancer is one of the most common malignant tumors with the highest morbidity and mortality.At present,the treatment methods of gastric cancer still have various limitations,and the search for more effective therapeutic drugs may bring a new turnaround for gastric cancer patients.The traditional Chinese medicine Salvia miltiorrhiza is a traditional blood-activating drug with potential clinical value in the treatment of gastric cancer,but its main active ingredients as well as molecular mechanisms for the treatment of gastric cancer still need to be further investigated.Based on the network pharmacology research platform,this study identifies the important multi-target active ingredients of Salvia miltiorrhiza,and verifies the mechanism of important action of multi-target active ingredients in the treatment of gastric cancer by combining molecular docking and in vitro experiments,to provide a scientific basis for the treatment of gastric cancer with Salvia miltiorrhiza.Methods: The active ingredients and targets of Salvia miltiorrhiza were looked up using the Traditional Chinese Medicine Systems Pharmacology(TCMSP)database and analytical platform.Using “Gastric cancer” or “GC” as the search term,the gastric cancer disease targets in multiple databases were collected and integrated.Cytoscape＿v3.8.0 software generates “active ingredient-target-disease” networks to screen potentially important multi-target active ingredients and their candidate targets.Based on this,the STRING database was utilized to build the protein-protein interaction networks(PPI),and the Cyto NCA program was employed to topologically screen the core targets.Then R package "Cluster Profiler" was used to conduct functional analysis of the Gene Ontology(GO)and pathway enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes(KEGG)to identify important functions and pathways.Core targets and important multi-target active ingredients were molecularly docked using Auto Dock-Vina software,and the results were visualized with Py MOL and BIOVIA Discovery Studio Visualizer.The findings of network pharmacology and molecular docking were then in vitro validated.CCK8 assay was used to detect the cell viability or drug cytotoxicity of gastric mucosal epithelial cells and gastric cancer cells.Cell clone formation assay was used to analyze the proliferation ability of gastric cancer cells.Cell scratch assay and Transwell migration assay were used to analyze the migration ability of gastric cancer cells.Western blot was used to detect the expression changes of related proteins.Results: A total of 65 Salvia miltiorrhiza active ingredients and 117 drug targets were tested,as well as 12,337 gastric cancer disease targets.Among them,there were106 intersecting targets of Salvia miltiorrhiza and gastric cancer.In the constructed“active ingredient-target-disease” network,the second multi-target active ingredient was Tanshinone IIA.In the constructed "active ingredient-target-disease" network,Tanshinone IIA is of great research value.According to the number of targets corresponding to the active ingredient,the important multi-target active ingredient Tanshinone IIA was sorted out.The core targets of Salvia miltiorrhiza and Tanshinone IIA were calculated independently by Cytoscape＿v3.8.0 software,and the intersection of the two yielded seven common core targets,namely MMP9,PTGS2,TP53,FOS,JUN,MYC and CASP3.The GO functional annotation of Tanshinone IIA candidate targets was collated to obtain 948 biological processes(BP),43 cellular components(CC)and 125 molecular functions(MF).Among them,BP was related to drug response and G protein-coupled receptor signaling pathway,CC was related to synaptic membrane and membrane microstructure,and MF was related to DNA-binding transcription factors.A total of 113 pathways were collated by KEGG pathway enrichment analysis,mainly including Apoptosis,c AMP,IL-17 and TNF.TP53 and BCL-2 are the intersecting targets of two important pathways in Apoptosis as well as Gastric cancer.Molecular docking results showed that Tanshinone IIA had a high potential binding ability with core targets MMP9,PTGS2,TP53 and FOS,and a relatively weak binding ability with JUN,MYC and CASP3.The results of CCK8 assay and colony formation assay showed that TanshinoneⅡA and Cisplatin could inhibit the cell viability and colony formation of gastric cancer cells(AGS and HGC27)in a concentration-dependent manner.Cell scratch assay and Transwell migration assay showed that TanshinoneⅡA significantly inhibited the wound healing rate and reduced the number of migration of gastric cancer cells.Tanshinone IIA significantly increased the percentage of early and late apoptosis of gastric cancer cells as detected by flow cytometry.Western blot results showed that with the increase of Tanshinone IIA concentration,the expression of TP53 transcriptional independent apoptosis pathway-related proteins was changed.Among them,there was no significant change in the expression of total Caspase-3 and BAX proteins,the expression of PARP1 and BCL-2 proteins was significantly down-regulated,the expression of cleaved-PARP1 and TP53 proteins was significantly up-regulated,while the expression of cleaved-Caspase3 protein was significantly up-regulated only in HGC27 cells.Conclusion: Tanshinone IIA is an important multi-target active ingredient of the traditional drug Salvia miltiorrhiza for the treatment of gastric cancer,which can regulate the cell proliferation,apoptosis,and migration process of gastric cancer by acting with seven core targets(PTGS2,MMP9,CASP3,MYC,FOS,TP53 and JUN).Among them,the transcriptional non-dependent apoptotic pathway of TP53 may be the key mechanism of Tanshinone IIA against gastric cancer.This study systematically explored the multi-target active ingredients of Salvia miltiorrhiza in the treatment of gastric cancer and their mechanism of action,and the research and development of multi-target active ingredients is expected to provide new strategies for the treatment of gastric cancer. |
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