| Objective:(1)The potential targets and mechanisms of Shengxian Wumei Decoction(SXWMD)in the treatment of asthma were revealed by means of network pharmacological analysis,which provided theoretical basis for experimental studies.(2)Based on wnt7/β-catenin signaling pathway,the effect targets and possible mechanisms of SXWMD in inhibiting airway remodeling in glucocorticoids intervention model of asthmatic rats were explored.Methods:(1)Network pharmacological analysis:The data platforms such as Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and a Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine(BATMAN-TCM)were searched to screen the main active components and targets of SXWMD,and the disease target databases such as the human gene database(Genecards),Therapeutic Target Database(TTD)and Comparative Toxicogenomics Database(CTD)were used to search the related targets of asthma disease.The common targets between active ingredient targets of SXWMD and disease targets of asthma were obtained by Wayne analysis.And then the protein interactions of the common targets were analyzed by String database and the key targets were screened out by functional modularization analysis of MCODE in Metascape,a gene function annotation tool.The database for Annotation,Visualization and Integrated Discovery(David)was used to analyze the Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of the active component-disease common targets,and the representative results were visualized as a bubble map.(2)Experimental study:Totally 50 healthy male SD rats were randomly divided into normal group,asthma group,glucocorticoids intervened asthma group,Budesonide group and SXWMD group,10 in each group.Except the normal group,the animal models were induced by sensitization and stimulation with ovalbumin(OVA)And except for rats in normal and asthma groups,rats in the other groups were intraperitoneally injected with glucocorticoids.Based on the glucocorticoids intervention.rats in Budesonide group were given atomization inhalation with Budesonide[2 mL/(20 min·day)]and rats in SXWMD group were treated with intragastric administration[0.81g/(kg· d)]for 7 weeks.Lung function indices,including forced vital capacity(FVC),forced expiratory volume in 0.3 second(FEV0.3),FEV0.3/FVC%,minute ventilation volume(MVV)and peak expiratory flow(PEF)were recorded by pulmonary ventilation function test.Airway hyperresponsiveness indices,such as inspiratory resistance(Ri),expiratory resistance(Re)and respiratory dynamic compliance(Cdyn)were recorded and calculated by bronchial provocation test.Pathological changes of rat lung tissues were observed by HE and Masson staining.Nucleus count of airway wall smooth muscle cells(NCASMc),area enclosed by internal perimeter(Ai),Wall area of tube(Wat)and percentage(%TAt),Wall area of smooth muscle(WAm)and percentage%WAm were assessed and calculated with Image-Pro Plus 6.0.The concentrations of transforming growth factor-β 1(TGF-β1),interleukin-4(IL-4)and interferon-y(INF-y)in bronchoalveolar lavage fluid(BALF)were detected by ELISA,and the ratio of IL-4/INF-γ was calculated.Immunohistochemistry,Western blot and RT-qPCR techniques were used to detect the protein and mRNA expression of Wnt7,β-catenin,Collagen Ⅰ and Collagen Ⅲ in lung tissue of rats in each group.Results:(1)Network pharmacological analysis:165 main active components,163 potential targets and 5974 asthma targets were screened.After that,127 intersection targets were obtained by Wayne analysis,and then the most significant functional modules and 40 key target genes such as APP,MAPK8 and GSK3B were obtained by cluster analysis of PPI network with MCOBE in Metascape.GO and KEGG enrichment analysis showed that these targets involved in cell signal transduction,negative regulation of smooth muscle cell contraction and proliferation,cell proliferation regulation and other biological processes,as well as PI3K-Akt,NF-κB and other signal pathways.(2)Experimental study:①Compared with the normal group,the histopathological indexes of airway remodeling such as NCASMc,WAt/Pi,WAm/Pi,%WAm and%TAt in the asthma group and glucocorticoids intervened asthma group were significantly increased(P<0.05),while the Ai/Pi in the asthma group was significantly decreased(P<0.05).The pulmonary ventilation function indexes such as FEV0.3,FVC,PEF and FEV0.3/FVC in the asthma group were significantly decreased(P<0.05)and FEV0.3 and FEV0.3/FVC in the glucocorticoids intervened asthma group were significantly decreased(P<0.05).Ri_R(3),Ri_R(4),Re_R(2),Re_R(3)and Re_R(4)in the asthma group were significantly increased(P<0.05),and Ri_R(4)and Re_R(4)in the glucocorticoids intervened asthma group were significantly increased(P<0.05).In both asthma group and glucocorticoids intervened asthma group Cdyn_R was significantly decreased(P<0.05),TGF-β 1,IL-4 and IL-4/INF-γ were significantly increased(P<0.05),INF-γ was significantly decreased(P<0.05)and the expression of Wnt7,β-catenin,ECM,Collagen Ⅰ,Collagen Ⅲ and mRNA was significantly increased.②Compared with the asthma group,the levels of histopathological indexes of airway remodeling,such as NCASMc,%WAm and%TAt,the levels of airway responsiveness indexes such as Ri_R(3),Ri_R(4),Re_R(2),Re_R(3)and Re_R(4),and airway inflammation indexes such as TGF-β1,IL-4 and IL-4/INF-γ were decreased in all treatment groups(P<0.05).The AOD value and relative expression level of Wnt7,β-catenin,Collagen Ⅰ and Collagen Ⅲ,and the relative expression level of mRNA of Wnt7,β-catenin and Collagen Ⅲ were decreased in all treatment groups(P<0.05).While,the levels of ventilation function indexes FEV0.3 and FEV0.3/FVC,airway responsiveness index Cdyn_R and airway inflammation index INF-γwere increased in each treatment group(P<0.05).In addition,in SXWMD group,the levels of airway remodeling indexes such as WAt/Pi and WAm/Pi,and the relative expression level of mRNA of Collagen Ⅰ were decreased(P<0.05),and the level of Ai/Pbm were increased(P<0.05).③Compared with glucocorticoids intervened asthma group,SXWMD could further reduce the levels of NCASMc,%WAm and%TAt(P<0.05),increase the levels of FEV0.3/FVC and FEV0.3(P<0.05),decrease the level of RE(4)(P<0.05),and increase the levels of Cdyn_R(3)and Cdyn_R(4)(P<0.05).Besides,it could Further decrease TGF-β1,IL-4 and IL-4/INF-γ(P<0.05),increase INF-γ(P<0.05),decrease the AOD value and relative expression level of Wnt7,β-catenin,Collagen Ⅰ and Collagen Ⅲ protein(P<0.05),and decrease the mRNA relative expression level of Wnt7,β-catenin and Collagen Ⅲ(P<0.05).Conclusion:(1)SXWMD contains a variety of active components,and its mechanisms are complex.It could comprehensively interfere with asthma by regulating immune inflammation,proliferation and differentiation of smooth muscle cells.(2)SXWMD could inhibit the progress of airway remodeling in the glucocorticoids intervention models of asthmatic rats,delay the decline of pulmonary ventilation function and the increase of airway responsiveness in rats,and its mechanisms may be related to the regulation of abnormal activation of Wnt7/β-catenin signaling pathway,so as to inhibit the proliferation of ASMC and intervene airway inflammation. |
PDF Full Text Request