| Objectives:1.To observe the intervening effect of Kangzhi syrup on airway remodeli-ng of cough variant asthma by experiments.2.To investigate the mechanism of Kangzhi syrup on airway remodeling in cough variant asthma.Methods:The airway remodeling model of guinea pig cough variant asthma was established by the method for sensitization and stimulation with ovalbumin,the guinea pigs were randomly divided into blank control group,model group,Kangzhi syrup group,dexamethasone group,CAPE(caffeic acid phenethyl ester)group.The serum,bronchoalveolar lavage fluid(BALF)and lung tissue of guinea pigs were collected.Hematoxylin eosin staining(HE)and Masson trichrome staining(MASSON)were used to observe the pulmonary inflammation,mucus secretion and collagen deposition in guinea pigs.ELISA technique was used to detect the content of hydroxyproline in the trachea,the serum levels of MMP-9 and TIMP-1 and the level of TGF-β1 in BALF.Immunohistochemistry was used to detect the expression of TGF-1 in lung tissue of guinea pigs.Western Blot method was used to detect the protein expression level of NF-κB and IκB in lung tissue,and the Real-time PCR method was used to detect the gene expression level of NF-κB and IκB in lung tissue.Results:1.Cough frequency:Compared with the blank group,the number of coughs in the model group increased significantly(P<0.01);Compared with the model group,the number of cough in the Kangzhi syrup group,dexamethasone group and CAPE group were significantly reduced(P<0.01),there was no significant difference between the three groups(P<0.05).2.Classification and counting of cells in BALF:Compared with the blank control group,the number of white blood cells in BALF of model group increased significantly(P<0.01),at the same time,the absolute counting of EOS increased obviously(P<0.01);MAC ratio decreased,but absolute counting increased(P<0.05);The ratio of EOS and LYM in BALF increased significantly(P<0.05),especially in EOS(p<0.01).Compared with the model group,the total number of white blood cells,EOS absolute counting and EOS classification in BALF were significantly lower in Kangzhi syrup group,dexamethasone group and CAPE group(P<0.01),there was no significant difference between the three groups(P<0.05).3.HE staining results of lung tissue under microscope:In the model group,there was a structure disorder of connective tissue around the bronchus and blood vessel,the necrosis and abscission of airway epithelial cell;There were a large number of inflammatory cells infiltrated in the trachea,and eosinophils,lymphocytes and neutrophils were more common;Submucosa and smooth muscle thickened,goblet cells and mucus glands increased,the wall was thickened and the lumen was narrowed,the mucus plug could be observed,alveolar wall thickened,airway mucosal edema,folds increased,a large amount of collagen like material was deposited in the submucosa stroma.Compared with the model group,there was a small amount of inflammatory cell infiltration in the bronchial epithelium,airway smooth muscle was slight thickening,there was no obvious thickening of bronchial wall,mucosal was smooth,submucosal collagen decreased,lumens were regular in Kangzhi syrup group,dexamethasone group and CAPE group.4.Morphological changes of lung tissue under microscope:Compared with the blank control group,Wam/Pbm and Wai/Pbm were significantly increased in the model group,the difference was statistically significant(P<0.01);Compared with the model group,Wam/Pbm and Wai/Pbm were significantly decreased in Kangzhi syrup group,dexamethasone group and CAPE group(P<0.01);the difference was statistically significant(P<0.01);Wam/Pbm and Wai/Pbm ratio between the three groups were not statistically significant(P>0.05).5.Masson staining results under microscope:There was a thin layer of collagen fibers around the trachea in the blank control group,while the airway subepithelial collagen deposition was significantly increased to form a thick dense layer,which was statistically significant compared with blank control group(P<0.01);In Kangzhi syrup group,dexamethasone group and CAPE group,compared with the model group,the collagen deposition around trachea decreased significantly,and the difference was statistically significant(P<0.01);there was no significant difference between the three groups(P<0.05).6.Comparison of hydroxyproline content in trachea:Compared with the blank control group,the hydroxyproline content in the model group was significantly increased,the difference was statistically significant(P<0.01);Compared with the model group,the hydroxyproline content was significantly decreased in Kangzhi syrup group,dexamethasone group and CAPE group,the difference was statistically significant(P<0.01);There was no significant difference of the hydroxyproline content between the three groups in the dexamethasone group,the Kangzhi syrup group and the CAPE group.7.Detection of MMP-9 and TIMP-1 content and results of MMP-9/TIMP-1 in serum:Compared with the blank control group,the MMP-9 and TIMP-1 content and MMP-9/TIMP-1 ratio in serum of model group increased significantly,the difference was statistically significant(P<0.01);Compared with the model group,the MMP-9 and TIMP-1 content decreased significantly in Kangzhi syrup group,dexamethasone group and CAPE group,the difference was statistically significant(P<0.01);Compared with the model group,MMP-9/TIMP-1 ratio decreased significantly in dexamethasone group,the difference was statistically significant(P<0.01),MMP-9/TIMP-1 ratio decreased significantly in Kangzhi syrup group and CAPE group,the difference was statistically significant(P<0.01);There was no significant difference of MMP-9,TIMP-1 and MMP-9/TIMP-1 ratio in serum between the dexamethasone group and CAPE group(P>0.05).8.Detection results of TGF-β1 in guinea pigs:8.1 Detection results of TGF-β1 in BALF by ELISA:Compared with the blank control group,the content of TGF-β1 in BALF was significantly increased in model group(P<0.05);Compared with the model group,the content of TGF-β1 in BALF was significantly decreased in Kangzhi syrup group and CAPE group(P<0.05);There was no significant difference of the content of TGF-β1 in BALF between the three groups in the dexamethasone group,the Kangzhi syrup group and the CAPE group(P>0.05).8.2 Detection results of TGF-β1 in BALF by immunohistochemical staining:TGF-β1 positive staining was mainly found in trachea and bronchial epithelial cells,submucosa,trachea and vascular smooth muscle layer,which was brown or dark yellow.Compared with the blank control group,the expression of TGF-β1 was significantly increased in the model group,the difference was statistically significant(P<0.01);Compared with the model group,the expression of TGF-β1 was significantly decreased in Kangzhi syrup group and dexamethasone group,the difference was statistically significant(P<0.01);There was no significant difference of the expression of TGF-β1 between the three groups in the dexamethasone group,the Kangzhi syrup group and the CAPE group(P>0.05).9.Detection results of NF-κB and IκB in lung tissue of guinea pigs9.1 Results of NF-κB protein expression in lung tissue by Western Blot method:Compared with the blank control group,the expression of NF-κB protein in lung tissue of model group was significantly increased,the difference was statistically significant(P<0.01);Compared with the model group,the expression of NF-κB protein was significantly decreased,the difference was statistically significant(P<0.01);There was no significant difference between the dexamethasone group,the Kangzhi syrup group and the CAPE group(P>0.O5).9.2 Results of IκB protein expression in lung tissue by Western Blot method:Compared with the blank control group,the expression of NF-κB protein in lung tissue of model group was significantly decreased,the difference was statistically significant(P<0.01);Compared with the model group,the expression of NF-κB protein was significantly increased,the difference was statistically significant(P<0.01);There was no significant difference between the dexamethasone group,the Kangzhi syrup group and the CAPE group(P>0.05).9.3 Results of NF-κB RNA expression in lung tissue by Real-time PCR:Compared with the blank control group,the expression of NF-κB RNA in lung tissue of model group was significantly increased,the difference was statistically significant(P<0.01);Compared with the model group,the expression of NF-κB RNA was significantly decreased,the difference was statistically significant(P<0.01);There was no significant difference between the dexamethasone group,the Kangzhi syrup group and the CAPE group(P>0.05).9.4 Results of IκB RNA expression in lung tissue by Real-time PCR:Compared with the blank control group,the expression of IκB RNA in lung tissue of model group was significantly decreased,the difference was statistically significant(P<0.01);Compared with the model group,the expression of IκB RNA was significantly increased,the difference was statistically significant(P<0.01);There was no significant difference between the dexamethasone group,the Kangzhi syrup group and the CAPE group(P>0.05).Conclusions:1.Kangzhi syrup could inhibit airway remodeling in cough variant asthma.2.Kangzhi syrup could regulate the balance of MMP-9/TIMP-1 and reduce extracellular matrix deposition though lowered the MMP-9 levels in guinea pigs,to inhibit airway remodeling in cough variant asthma.3.Kangzhi syrup could reduce extracellular matrix deposition though lowered the TGF-β1levels in guinea pigs,to inhibit airway remodeling in cough variant asthma.4.Kangzhi syrup could lower the NF-κB levels and inhibit NF-κB signaling path though raised the IκB levels in guinea pigs,to inhibit airway remodeling in cough variant asthma.5.Kangzhi syrup might lower downstream factors level of MMP-9 and TGF-β1 and reduce extracellular matrix deposition though inhibited NF-κB signaling path,to inhibit airway remodeling in cough variant asthma. |
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