| Background:Combining the holistic idea of traditional Chinese medicine(TCM)and reductionism of western medicine conforms to the trend of the development of the modern medicine.Based on the viewpoint of systems biology,systems pharmacology pointed out the direction for the mechanism elucidation of TCM on the molecular level by fusing the achievement of systems biology and modern medicine.For the elucidation of TCM mechanism,we should not only research the biological pathways which affected by TCM in cell at the micro-level(molecule,biological network),but also learn the diffusion of TCM effect among the cell at the macro-level(tissue,organ,whole body).That is,elucidate the mechanism of body changes which affected by TCM on the multi-scale(time-space)level.At present,the comprehensive regulation of multi-components,multi-targets and multi-pathways has already become the basic consensus of researchers in the field of TCM.However,some limitations are still existing in network pharmacology and systems pharmacology which developed by these consensus when they were used for elucidation of TCM.For example,the molecular type was single in network,they were unable to realize batch processing,they did not realize the cell specificity and could not effectively combine with the experimental data,etc.Therefore,how to elucidate the specific pathway and comprehensive regulative process of TCM efficiently and systematacially is still a challengeable work in TCM.Objective:In order to form a comprehensive understanding of TCM mechanism,not only include the effect of TCM on the expression of genes,proteins and the topological structure of biological network,but also the diffusion of TCM effect in networks,this study will establish the formal expression method and inference system between them,and the technology system of elucidation of TCM mechanism based on cell type-specific network.Methods:(1)Integrating the gene regulatory network,small molecular metabolic network,human biology reaction and signaling pathways,the background network was constructed;on the basis of background network,taking the specific expression data of the gene and protein as constraint condition,this study constructed the cell type-specific network by Entity Grammar System(EGS);according to the locating information of cell signaling molecules and their receptors,the network model of tissue,organ and system which contained multiple cells by connected cell type-specific networks were constructed.(2)The network topology parameters and the proportion of the shared edges to all edges were calculated;the simulating capability of networks was evaluated by clustering analysis,enrichment analysis and the mechanism elucidation of nimodipine which mechanism was definite.(3)Taking the gene expression data of HepG2 which affected by component A and MCF-7 which affected by SiWu-Tang(SWT)as examples,and with EGS as the key technology,the method of TCM mechanism elucidation was established based on network comparison and qualitative simulation.(4)Take the anti-breast cancer mechanism elucidation of SWT as an example,this study specifically illustrate the mechanism elucidation method of TCM based on cell typespecific networks:the new pharmacological effects of SWT was discovered by TCM repositioning;the mechanism of new pharmacological effects and the diffusion of effect were elucidated based on cell type-specific networks and the multiple cells communication network;identify the active ingredients cluster of SWT by molecular docking and initial transcription process-based identification(ITPI)which put forward in this study;validate the anti-breast cancer effect of verbascoside by CCK8,flow cytometry and western blot.That is to say,verified the reliability of the identification method of TCM effective components cluster which based on the cell type-specific network.Results:(1)This study has constructed the specific networks of 83 normal cells and 48 tissues/organs.(2)Clustering analysis showed that the accuracy of 83 normal cells and 48 tissues/organs are 97.59%and 64.6%respectively;the enrichment analysis showed that the mainly biological pathways which the proteins in myocardial cell type-specific network participate were muscle formation,contraction and filament sliding.These pathways were all closely related to the electrophysiological and mechanical physical properties of myocardial cell;the mechanism elucidation based on the multiple cell network of smooth muscle cell-nerve cell-vascular endothelial cell showed that nimodipine could inhibit the smooth muscle contraction by the theory of Ca2+ overload and excitatory amino acids toxicity,inhibit apoptosis and promote cell proliferation,thus played the role of protective effect to nerve cell and vascular endothelial cell.The mechanism of nimodipine that we got in this study conformed to the known mechanism.(3)The data which were used to construct cell/tissue/organ type-specific networks were divided into two types,omics and traditional data.This study focused on the TCM mechanism elucidation which combined with omics data.Considering that the biomolecule amount and expression quantity in human body were all likely to change after drug action,this study divided the method of cell/tissue/organ type-specific network construction and mechanism elucidation into two cases:the biomolecule amount were different(such as HepG2 affected by component A)and same(such as MCF-7 affected by SWT),the TCM mechanism elucidation method were network comparison and qualitative simulation respectively.Finally,this study established the corresponding methods of cell type-specific network construction and mechanism elucidation according to the four cases,which included omics data,traditional data,different and same biomolecule amount.(4)Similarity scores of gene expression profile between SWT and 1294 small molecule drugs in Cmap were obtained by the method of TCM repositioning;according to the top 10 small molecule drugs which have definite pharmacological actions and records in DrugBank,this study found SWT has the effect of anti-breast cancer;the mechanism elucidation based on MCF-7 specific network indicated the anti-breast cancer mechanism of SWT were regulation of cell apoptosis and proliferation,response to hormone stimulus,influence gland development,regulation of cell migration and influence the drug resistance of MCF-7;this study construct the communication network of MCF-7-normal mammary glandular cell-macrophage-myoepithelial cell.Through communication network analysis,this study found SWT could not only regulate the MCF-7 but also influence the immunologic function of normal mammary glandular cell,macrophage and myoepithelial cell,and influence the MCF-7 proliferation and migration by cellular signalling transduction;46 active ingredients were identified by molecular docking based on the result of SWT repositioning;61 active ingredients were identified by ITPI,the transcription factors and differentially expressed genes which affected by active ingredients were also provided;the combination of ITPI and molecular docking could identify the active ingredient cluster of SWT more comprehensively from the aspects of gene and protein;the results of CCK8 showed that Verbascoside had the effect of antiproliferative activities.The IC50 of Verbascoside were respectively 8.85,4.405 and 4.565?M in the treattime of 24h,48h and 72h.Verbascoside had the dual-directional regulation effects which phytoestrogen possessed:under the concentration of 5?40?M,Verbascoside exhibited higher inhibition of MCF-7 cell proliferation;while,under the concentration of 5?40?M,it showed a estrogenic effect;under the concentration of 2?32?M,the results of cell apoptosis showed that Verbascoside had the anti-apoptosis effect and presented a well linear relationship after 48h treatment.With the increase of concentration,the antiapoptotic effect also increased and the highest apoptotic rate was 73%;by the western blot,the expression of AP1 increased,Cdk6 and MYC decreased under the concentration of 1?32?M,and the effect was best under the concentration of 32?M;in conclusion,the results of verify experiment agreed with the results which based on the MCF-7 specific network:Verbascoside could enhance the phosphorylation of SMAD3,promote the expression of AP-1 and suppress the expression of MYC and Cdk6;by suppressing proliferation and promoting the apoptosis of MCF-7,Verbascoside played the effect of anti-breast cancer.Conclusions:The cell type-specific network which constructed in this study has a well simulating capability.Well simulating capability lays a foundation for mechanism elucidation of TCM.The method of mechanism elucidation which based on the cell type-specific network could illuminate the influence and regulatory mechanism of TCM on cell biological process in detail,and discover the diffusion of TCM effects among the various levels of the models,such as molecule,cell and multiple cells,etc.This study not only elucidated the anti-breast cancer mechanism of SWT but also identified its active ingredients cluster.All these will provide a basis for the quality control and combination drug design of SWT.The TCM mechanism elucidation based on cell type-specific network will provide an efficient and objective technology system for the interpretation of some TCM theories,such as processing,property and combination,etc. |
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