| Objective:To establish the asthma remission mice model,and explore the mechanism of Gu-Ben-Fang-Xiao Decoction(GBFXD)in improving airway remodeling of asthma remission model mice by network pharmacology,proteomics,metabonomics,and molecular biology.Methods:(1)The asthma remission mice were induced by OVA-RSV.The HE staining was used to evaluate the chronic inflammation of the lung.Meantime,the Sirius red staining,coupled with the immunohistochemistry and ELISA detecting the expression levels of E-cadherin,Vimentin,?-SMA and HYP in the lung tissue of mice,was applied to assess the degree of airway remodeling in mice with asthma remission,and the effect of GBFXD on airway remodeling.(2)Through the network pharmacology,the possible target points of GBFXD were selected.Then,the GO annotation and KEGG analysis were carried out on the related target points,so as to predict the potential regulatory network of GBFXD.(3)The iTRAQ quantitative proteomics method was used to find the differentially expressed proteins among control,model and GBFXD group.GO annotation and KEGG analysis were performed on these differentially expressed proteins.Combined with the results of network pharmacology,the mechanism of GBFXD on improving airway remodeling was further explored.(4)According to the results of proteomic,the ECM related proteins which were also differential proteins such as collagen(Col1a1,Col1a2,Col4a1,Col4a2),laminin(LAMA3,LAMA5,LAMB2,LAMB3)and fibronectin(Fnl)were detected by RT-PCR.In addition,the expression levels of Col1a2,Col4a1,RTN4B and the regulation of PI3K/AKT pathway by GBFXD were detected by Western Blotting.(5)Liquid chromatography-mass spectrometry(LC-MS)was used to analyze the metabolic profile of mice in the asthma remission,and to verify the differential lipid metabolites and the realted pathways to explore the mechanism of GBFXD on airway remodeling.Results:(1)The results of lung tissue pathology and airway remodeling related marker protein detection showed that compared with the control group,there were obvious inflammatory cell infiltration and collagen deposition in the airway and around the blood vessels in the asthma remission model group,and GBFXD intervention could effectively reduce the chronic airway inflammation and remodeling in mice.In addition,the change of airway remodeling markers such as reduced E-cadherin and elevated Vimentin,?-SMA and HYP in the lung tissue was shown in the model group compared with the control group.While GBFXD could significantly reverse the expression of the above proteins,indicating that airway remodeling existed in the lung tissue of the model group,and GBFXD could alleviate airway remodeling to a certain extent.(2)The results of network pharmacology showed that the potential targets of GBFXD were 1830,corresponding disease targets were 1717.Meantime,a total of 525 common targets were obtained,after the intersection of the two targets and standardized annotations by the Uniprot database.Through bioinformatics analysis of the 525 common targets,the results showed that common targets in BP mainly involved in regulating cell adhesion,epithelial cell proliferation,angiogenesis and PI3K pathway,CC mainly involved in extracellular matrix,collagen containing extracellular matrix,protein extracellular matrix,and PI3K complex,while MF mainly related to PI3K activity and phosphatidylinositol kinase activity.KEGG pathway is mainly enriched in pathways related with airway remodeling,such as PI3K/AKT signaling pathway,VEGF signaling pathway,TGF-? signaling pathway and sphingolipid signaling pathway.(3)The results of proteomics showed that a total of 79 differentially expressed proteins were identified in the model/control group,among which BP was mainly involved in muscle contraction,ECM composition and cell adhesion,CC was mainly involved in smooth muscle fiber,ECM composition and collagen trimer,MF was mainly involved in receptor binding,ECM structural composition and cell adhesion molecule binding.A total of 24 differentially expressed proteins were identified in GBFXD/model group,among which BP was mainly involved in cell adhesion,ECM composition and extracellular structure,CC was mainly involved in ECM,ECM composition and basement membrane,MF was mainly involved in receptor binding,cell adhesion molecule binding and structural component activity.In addition,there were 18 overlapping differential proteins,most of which were ECM proteins,which were involved in development of airway remodling.Meantime,KEGG analysis showed that the main regulatory pathways of GBFXD were ECM receptor interaction,focal adhesion and PI3K/AKT signaling pathway.(4)The expression levels of collagen(Col1a1,Col1a2,Col4a1,Col4a2),laminin(LAMA3,LAMA5,LAMB2,LAMB3)and fibronectin(Fn1)in lung tissue of mice in model group were significantly increased,while GBFXD could significantly reduce their expression.Additionally,PI3K/AKT pathway was activated in the lung tissue of the model group mice,and GBFXD could inhibit its activation.(5)A total of 42 significantly different metabolites were identified,especially the levels of fatty acids,acylcarnitine,phosphatidylcholine,phosphatidylethanolamine,phosphatidylinositol,triglycerides and diacylglycerols have changed.Targeted metabolomics verified that the expression level of fatty acids(FA 16:1)which is basically consistent with the results of non-targeted metabolomics.In addition,GBFXD can activate AMP-activated protein kinase(AMP-activated protein kinase,AMPK)pathway.Conclusion:(1)Mice in remission of asthma still have airway remodeling,while GBFXD could reduce collagen deposition in airway and perivascular,improve airway remodeling and protect lung function.(2)GBFXD could reduce the expression levels of ECM protein and RTN4B,which may be related to inhibiting the activation of PI3K/AKT pathway mediated by RTN4B.(3)GBFXD may affect fatty acid metabolism by regulating AMPK,thereby improving airway remodeling. |
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