| Objective:The purpose of this study is to explore the mechanism of Pingchuan granules in inhibiting the transformation of airway epithelial stroma in asthma by means of network pharmacology,and to verify the predictive mechanism by animal experimentsMethods:1.To analyze the potential mechanism of Pingchuan granules in inhibiting airway epithelial stromal transformation in asthma by network pharmacology.The specific steps are as follows:through the intersection of asthma,airway remodeling and epithelial stromal transformation related targets obtained in Drug Bank,OMIM and genecards,through TCMSP,Chinese Academy of Sciences compound reference database was used to obtain the compounds and target genes of Pingchuan granules,and the intersection was used to obtain the target of Pingchuan granules acting on the disease.The intersection target is imported into string database,and PPI network of Pingchuan granules disease target is constructed.Cluster analysis of Pingchuan granules disease target is carried out by MCODE plug-in.By using autodock Vina software,13 inflammatory targets of cluster 1 cluster of Pingchuan granules disease target and 8 main active components of Pingchuan granules were butted respectively.It was verified that the active components of Pingchuan granules were the material basis of improving the effect of Pingchuan granules by inhibiting the inflammatory reaction and the process of epithelial stromal transformation.The 13 core targets of cluster 1 were enriched by go and KEGG using David database,and the results were imported into the bio information analysis platform for visualization analysis of go enrichment plot to obtain the signal pathway involved in the treatment of asthma by Pingchuan granules.Finally,the analysis results are analyzed2.Based on the results of network pharmacology analysis,animal experiments were designed to further verify the partial mechanism of Pingchuan granules inhibiting the epithelial stromal transformation of asthma Experiment:40 BALB/c mice were randomly divided into 4 groups,10 in each group,which were blank group(NC group),model group(HDM group),Pingchuan granules group(PCG group),budesonide group(BUD group).In addition to NC group,the remaining three groups of mice were used from the first day to duplicate the asthma model.10 μl of HDM induction solution was given to the mice 5 days a week,resting for 2 days and inducing for 5 weeks The PCG group was treated with Pingchuan granules concentrate(3 1.2g/kg/D),while the NC group,HDM group and BUD group were treated with normal saline of equal volume.BUD group(0.9%normal saline 2 ml+BUD group aerosol suspension 1 mg)was given toBUD group mice one hour before HDM induction for 20 minutes each time for 5 weeks,once a day.AHR was detected on the 34th day(24h after the last HDM induction)after the establishment of the model.The mice were anesthetized by intraperitoneal injection of 3%Pentobarbital Sodium Solution(30mg/kg).Observation indexes:(1)detect the AHR of asthmatic mice in each group;(2)HE staining,PAS staining and Masson staining were used to observe the pathological changes of lung tissue in asthmatic mice in each group;(3)ELISA was used to detect the concentration of IgE and IL-6 in BALF and serum;(4)immunohistochemistry,RT-PCR and Western The expression levels of IL-6,α-SMA,E-cadherin,N-cadherin and their mRNA in lung tissues were detected by blot;(5)the expression levels of p-JAK2,p-STAT3,JAK2 and STAT3 in lung tissues were detected by immunohistochemistry and Western blot.To explore the mechanism of Pingchuan granules in inhibiting airway epithelial stromal transformation in asthmaResults:1.A total of 115 Pingchuan granules with OB≥ 30%and DL≥ 0.18 and 257 target proteins of traditional Chinese medicine were obtained.958 common targets of airway remodeling and epithelial stromal transformation of asthma were obtained.120 targets of Pingchuan granules for treatment of diseases(asthma airway remodeling epithelial stromal transformation)were obtained,and the visualization network of Pingchuan granules disease target was constructed.The top 20 targets were obtained respectively They are Jun,AKT1,rela,mapkl,mapk3,TNF,hsp90aa1,IL-6,Mapk14,ESR1,Rb1,FOS,EGFR,Rb1,mapk8,NR3C1,CXCL8,VEGFA,CDKN1A and CCND1.These targets play an important role in the pathological process of airway remodeling and epithelial stromal transformation in asthma.By using autodock Vina software,13inflammatory targets of cluster 1 of Pingchuan granules disease target and 8 main active components of Pingchuan granules screened in 2.4 were butted and analyzed.It was found that 8 main active components of Pingchuan granules had certain binding activity with 13 inflammatory targets,and the stability of 8 main active components of Pingchuan granules and IL-6 was the best.It is suggested that Pingchuan granules may inhibit the epithelial stromal transformation by inhibiting the inflammatory reaction.According to the bioinformatics analysis of 13 core targets of cluster 1 in David database,it can be seen that the effect of Pingchuan granules in the treatment of airway epithelial stromal transformation of asthma is mainly related to secretion regulation,positive regulation of cytokine production,acute inflammatory response,acute phase response,positive regulation of MHC class Ⅱ biosynthesis process and other molecular functions.Through the analysis of KEGG enrichment,it is concluded that the process of Pingchuan granules in the treatment of asthma is mainly related to asthma,Fc epsilon RI signal pathway,JAK-STAT signal pathway,MAPK signal pathway,T cell receptor signal pathway,toll like receptor signal pathway and nod like receptor signal pathway2.Compared with NC group,the airway hyperresponsiveness of model group was increased,and the airway hyperresponsiveness of PCG group and BUD group was improved,and the curative effect was similar(P>0.05)3.HE staining:compared with NC group,the airway lumen of the asthmatic mice in HDM group was narrowed,the mucous membrane was obviously edematous,the gland was hypertrophic,the wall and mucous membrane were obviously thickened,and a large number of inflammatory cells were infiltrated around the airway.Compared with the HDM group,the above pathological characteristics of PCG group and BUD group decreased There was no significant difference between PCG group and BUD group(P>0.05).4.PAS staining:compared with NC group,airway mucus and goblet cells in HDM group were significantly increased;compared with HDM group,mucus and goblet cells in PCG group and BUD group were significantly reduced.There was no significant difference between PCG group and BUD group(P>0.05)5.Masson staining:there were only a few collagen fibers around the airway of NC group mice.Compared with NC group,there was a large amount of collagen deposition in the lung tissue of HDM group,which was widely distributed around the airway.After Masson staining,collagen deposition in lung tissue of PCG group and BUD group was significantly reduced than that of HDM group(P<0.05),while the pathological changes in PCG group were more significant than that in BUD group(P<0.05)6.The results of ELISA showed that the levels of IgE and IL-6 in BALF and serum of model group were significantly higher than those of NC group(P<0.05);the levels of IgE and IL-6 in BALF and serum of PCG group and BUD group were significantly lower than those of HDM group(P<0.05);the level of IL-6 in BALF and serum of PCG group was significantly lower than that of BUD group(P<0.05)7.Immunohistochemistry,RT-PCR and Western blot were used to detect the expression level of IL-6,E-cadherin,N-cadherin and α-SMA in lung tissue.Compared with NC group,the expression level of IL-6,α-SMA,N-cadherin protein and mRNA in HDM group was significantly higher,while the expression level of E-cadherin protein and mRNA was significantly lower(P<0.01).Compared with the HDM group,the expression level of IL-6 protein and mRNA in PCG group and BUD group was significantly lower(P<0.01),the expression of α-SMA and N-cadherin protein and mRNA in PCG group and BUD group was significantly lower,the expression of E-cadherin protein and mRNA was significantly increased,and the difference was statistically significant(PCG vs.HDM P<0.01 and bud vs.HDM P<0.05).The difference between PCG group and BUD group was statistically significant(P<0.05)8.Immunohistochemistry and Western blot were used to detect the expression level of key proteins in JAK2/STAT3 signaling pathway in lung tissue:compared with NC group,the expression level of p-JAK2 and p-STAT3 protein in HDM group increased significantly(P<0.01),but there was no significant change in JAK2 and STAT3 protein in HDM group(P>0.05)Compared with HDM,the protein expression of p-JAK2 and p-STAT3 in PCG group and BUD group was significantly lower,the difference was statistically significant(PCG vs.HDM P<0.01 and BUD vs.HDM P<0.05).The protein expression of p-JAK2 and p-STAT3 in PCG group was significantly lower than that in BUD group(P<0.05)Conclusions:1.In this study,the network pharmacology method was used to analyze and obtain the conclusion that Pingchuan granules interfere with the epithelial mesenchymal transformation of asthma through multiple pathways and targets,and its pharmacological mechanism may be to inhibit the inflammatory response by acting on the IL-6-mediated JAK2/STAT3 signaling pathway,thereby inhibiting the epithelial mesenchymal transformation of asthma and slowing down the process of airway remodeling2.Pingchuan granules can reduce the pathological changes of airway hyperresponsiveness,airway inflammatory cell infiltration,goblet cell proliferation,airway collagen deposition and tube wall thickness in asthmatic mice,and inhibit the formation of airway remodeling3.Pingchuan granules can reduce IgE and IL-6 levels in serum and BALF of asthmatic mice,reduce expression of IL-6,α-SMA,N-cadherin protein and mRNA in lung tissue,increase expression of E-cadherin protein and mRNA,and decrease expression of p-JAK2 and p-STAT3 protein4.Pingchuan granules inhibit the activation of the IL-6-mediated JAK2/STAT3 signaling pathway,inhibit the inflammatory response,and thereby inhibit the transformation of the epithelial-mesenchymal in asthma,and reduce airway remodeling. |
