| Objective: The main pathway and target of Xiaoning oral liquid in the treatment of children's bronchial asthma were predicted by constructing "drug-target-disease" network.Combined with the results of network pharmacology analysis and verified by animal experimental studies,the mechanism of Xiaoning oral liquid on the immune regulation of asthma mice was preliminarily revealed,which provided reference and ideas for further in-depth interpretation of the scientific connotation of the overall regulatory effect of Traditional Chinese medicine compound and the improvement of clinical efficacy.Methods:1.Component screening and target prediction: According to the pharmacokinetic parameters OB and DL in TCMSP,the potential active ingredients of Xiaoning oral liquid with OB > 30% and DL > 0.18 were screened out.TCMSP,Pub Chem,Chemical Book,Chem Spider and other databases and related literature were consulted to obtain the Chemical structure of relevant drug components.Swiss Target Prediction database predicted the potential targets of Xiaoning oral liquid.Integration of OMIM(http://www.omim.org/),CTD(http://ctdba-se.org/),Gene Cards three database(https://www.genecards.org/)screeningand predict asthma disease targets.Cytoscape3.7.2 software and String(https://string-db.org/)database constructs a single drug active ingredient action target PPI network and drug actiontarget-asthma disease target PPI network.Network Analyzer Plugin was used to analyze Network topology parameters and obtain the key nodes of Xiaoning oral liquid and asthma disease.DAVID database was used for cluster enrichment analysis of core targets and KEGG signal pathway analysis to predict the main mechanism of Xiaoning oral liquid in the treatment of asthma.2.Animal experiment verification: Based on the results of network pharmacology analysis,animal experiment was designed to study the immune regulation mechanism of Xiaoning oral liquid on asthmatic mice.Experiments: An OVA asthmatic mouse model was constructed.Sixty female BALB/ C mice were randomly divided into 4groups(blank group,model group,Budesonide group,and Xiaoning group)with 15 mice in each group.The general state of mice was observed.Blood,bronchoalveolar lavage fluid(BALF),lung tissue and intestinal mucous membrane were collected after fasting for 24 h after the last intervention.HE staining was used to observe the pathology of lung tissue.The total number and classification count of white blood cells were observed under light microscope.The percentage of CD3+CD8+IL-17+cells(Th17 cells)and CD4+CD25+T cells(Treg cells)were detected by flow cytometry.Ig E,IL-2,IL-6,IL-10,TNF-?,BALF and intestinal mucous membrane SIgA were detected by ELISA.Results:1.Results of network pharmacologyA total of 105 compounds with oral availability(OB)> 30% and drug similarity(DL)> 0.18 were obtained.257 potential targets of Drug action were analyzed by Swiss Target Prediction database,and 471 targets related to asthma were obtained by combining OMIM,Gene Cards,Drug Bank and other databases.Through the topological parameter analysis of PPI network of single drug's active component-action target,130 core targets were obtained,and these targets may be the main nodes of Xiaoning oral liquid.Through the construction of PPI network of drug target-asthma disease target of Xiaoning oral liquid,55 key nodes were obtained,which were mainly inflammatory and immune-related factors.DAVID database carries out functional annotation analysis of 130 core targets.Hint Xiaoning oral liquid with immune response in the treatment of asthma,inflammation,regulation and control of RNA polymerase,lipopolysaccharide-mediated,and chemokines-mediated signaling pathways,ERK1,ERK2 cascade control,transcriptional regulation,DNA template,protein kinase B signaling,glucose metabolism,regulation of T cell proliferation,cell response to lipopolysaccharide,angiogenesis 13 biological function related.36 asthma-related pathways were obtained through KEGG pathway enrichment analysis.It is speculated that the process of Xiaoning oral liquid in treating asthma mainly interacts with TNF signaling pathway,Cytokine-Cytokine receptor interaction and cancer Pathways Cancer,Influenza A,Toll-like receptor signaling Pathway and T Cell receptor signaling pathway Pathway,JAK-Stat signaling Pathway,Tuberculosis,PI3K-Akt signaling pathway Pathway,Chemokine signaling Pathway and other signaling pathways.2.Results of animal experiments(1)Cell count and classification under light microscope: Compared with blank group,the total number of white blood cells and the percentage of eosinophils in BALF in model group were significantly increased(P<0.01),while the percentage of neutrophils and lymphocytes were significantly increased(P<0.05).Compared with model group,the total number of white blood cells in BALF in Budesonide group and Xiaoning group was significantly decreased(P<0.01),and the percentage of eosinophil,neutrophil and lymphocyte in Budesonide group were decreased(P<0.05),among which the percentage of eosinophil in Xiaoning group was significantly decreased(P<0.01),neutrophil and lymphocyte were decreased(P<0.05).(2)HE staining showed that the morphology and structure of lung tissues in the blank group were basically normal.The model group showed the pathological characteristics of typical allergic bronchial asthma,including inflammatory cellinfiltration,significantly reduced alveolar number,changes of smooth muscle cells around bronchi or vascular lumen,and increased intraluminal secretions.Compared with the model group,Budesonide group and Xiaoning group showed relatively mild asthma lesions,and significantly reduced inflammatory cell infiltration,epithelial cell shedding and airway mucus secretion.(3)ELISA related results.(1)The results of serum Ig E showed that the level of serum Ig E in model group was significantly higher than that in blank group(P<0.01).Compared with model group,serum Ig E level in Budesonide group and Xiao ning group was significantly decreased(P<0.01).(2)The results of immune factors IL-6,TNF-?,IL-10 and IL-2 showed that compared with blank group,IL-6 and TNF-? in model group were increased(P<0.05),while IL-10 and IL-2 were decreased(P<0.05).Compared with model group,IL-6 and TNF-? were decreased in Budesonide group and Xiaoning group(P<0.05),IL-10 and IL-2 were increased in Budesonide group(P< 0.05),IL-10 was increased in Xiaoning group(P<0.05)and IL-2 was significantly increased(P<0.01).(3)The results of BALF and intestinal mucosa SIgA showed that compared with the blank group,the content of BALF and intestinal mucosa SIgA in model group was lower than that in blank group.Compared with model group,intestinal SIgA and BALF SIgA content in Xiaoning group was higher than that in model group(P<0.05).BALF SIgA content in Budesonide group was higher than that in model group(P< 0.05),but there was no statistical significance in intestinal mucosa SIgA content(P> 0.05).(4)Flow cytometry results showed that compared with blank group,Th17 cells in peripheral blood of model group were increased(P<0.05),while Treg cells were decreased(P<0.05).Compared with model group,Th17 cells in peripheral blood of all treatment groups were decreased(P<0.05)and Treg cells were increased(P<0.05).Conclusion:1.Through network pharmacology analysis,it is shown that the connotation of“multi-component-multi-target-multi-pathway-multi-pathway” of Traditional Chinese medicine compound Xiaoning oral liquid mainly involves:105 kinds of active ingredients of 10 kinds of drugs,10 kinds of the same compounds,257 potential targets,469 asthma related targets information,to establish “drug-targets – disease”resulting from the network core targets 130,targets-55 asthma disease targets key nodes,13 biological process information,36 asthma related pathways.It is speculated that Xiaoning oral liquid can regulate the whole body to treat asthma by participating in immunity,inflammatory reaction,gene expression and transcription.2.TNF Signaling Pathway,Toll-like receptor signaling Pathway and T Cell receptor signaling pathway and JAK-Stat signaling Pathway may be the main mechanism of Xiaoning oral liquid in asthma intervention.Its signaling pathway is closely related to asthma immunity.3.Xiaoning oral liquid can improve the general condition of asthma mice,reduce airway inflammatory infiltration,inhibit airway inflammation and remodeling.At the same time,BALF and intestinal mucosa SIgA of asthma mice were improved,and lung and intestinal mucosa were repaired.4.Xiaoning oral liquid can play a therapeutic role in the treatment of children's bronchial asthma through the regulation of asthma immunity.The mechanism of action may be mainly through the regulation of Th17 cells,Treg cells and related immune factors,and the correction of Th17/Treg immune imbalance to treat asthma. |
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