| Objective:In this study,we established the hormone intervention model of asthmatic rats and used the clinically effective Jiajian Wumei Pill granule as therapeutic drugs to evaluate the effect of the drug on the model rat TGF-?1/Smad signaling pathway and the resistance to airway remodeling.Inhibition effect,clarify the target and mechanism of Jiajian Wumei Pill granule for inhibiting airway remodeling in asthmatic rats with hormone intervention model.Method:Healthy male SD rats were randomly divided into the normal group,the asthma group,the dexamethasone group,the Pulmicort group and the Chinese medicine group,and fed for 1 week.Except for the normal group,all other groups were sensitized by intraperitoneal injection of ovalbumin(OVA)and aluminum hydroxide adjuvant mixture on day 1 and day 8 of the experiment.Starting on the 15th day,1%OVA was used to spray the next day.Except for the normal group and the asthma group,the remaining groups were given intraperitoneal injection of dexamethasone sodium phosphate injection 0.5 mg/kg prior to nebulization challenge,and the dose of dexamethasone was withdrawn at a rate of 0.1 mg/kg per week.The Pulmicort group received 0.413 mg/kg atomized inhalation daily.The Chinese medicine group was given intragastric administration of 1.86g/kg Jiajian Wumei Pill granule.The medication was used for 7 weeks,observe the behavioral changes of rats in each group.Extract the specimen 24 hours after the last dose.(1)Comparison of lung ventilation function and airway remodeling in each group:Test the lung ventilation function of rats in each group after anesthesia,record forced vital capacity(FVC),forced expiratory volume in 0.2 seconds(FEV0.2),average flow rate in 0.2 seconds(FEV0.2/FVC%),forced maximum expiratory flow rate(PEF),mid-expiratory forced expiratory flow rate(FEF25-75%).After the rats were sacrificed,a part of the left lung tissue was fixed and embedded in paraffin.Using HE and Masson staining to observe the pathological and morphological changes of lung tissue in each group of rats.Using medical image analysis software to determine the airway wall area(Wat),airway smooth muscle area(Wam)and the percentage of the total airway area.(2)Compare the changes of Collagen ?,Collagen ? protein and TGF-?1/Smad signaling pathway indicators in each group:Alveolar lavage of rat right lung,the content of TGF-?1,Smad2,Smad3,Smad6 and Smad7 in alveolar lavage fluid of each group was detected by ELISA.A part of the left lung tissue of the rat was taken and the average optical density values of TGF-?1,Smad2,Smad3,Smad6,Smad7,Collagen ?,and Collagen ? were determined by immunohistochemistry.The upper lobe and middle lobe of the right lung of the rats were taken for Western Blotting and RT-qPCR,and the relative expression levels of TGF-?1,Smad2,Smad3,Smad6,Smad7,Collagen ? and Collagen ? protein and mRNA in the lung tissue of each group were compared.Result:(1)Compared with the normal group,rats in the asthma group were observed to have shortness of breath,scratching the nose and mouth,irritability,abdominal muscles twitching,and sometimes sneezing after being stimulated with egg protein.The dexamethasone group also has the above performance,but the weight is significantly reduced,the hair color is more dull,the degree of irritability,irritability is more obvious,and it is more aggressive.All the above symptoms in the Chinese medicine group were improved,and the wheezing sound was reduced.Pulmonary ventilation function indexes FVC,FEV0.2,FEV0.2/FVC%,PEF,FEF25-75%in asthma group were significantly lower than those in normal group(P<0.05),and persistent airflow obstruction occurred.There was no significant difference between the dexamethasone group and the asthma group(P>0.05).All indexes of the Chinese medicine group were significantly improved(P<0.05),and the symptoms of airflow obstruction were alleviated.The results of HE staining and Masson staining showed that the airway epithelial cells in the asthma group were degenerated and shed.A large number of inflammatory cells infiltrated around the airway.The airway lumen was significantly narrowed.The thickness of the airway wall,smooth muscle layer and basement membrane layer increased significantly Interstitial fiber hyperplasia,collagen deposition increased,Wat%,Wam%were significantly higher than the normal group(P<0.05),airway remodeling was obvious.Compared with the asthma group,the airway changes in the dexamethasone group were reduced,and the Wat%was significantly reduced(P<0.05).The airway changes in the Chinese medicine group were all reduced compared with the two groups.Airway epithelial cells were slightly degenerated,occasionally shed,a small number of inflammatory cells infiltrated around the airway,the thickness of the airway wall,smooth muscle layer and basement membrane layer decreased,collagen deposition decreased,and Wat%significantly decreased(P<0.05).(2)Compared with the normal group,the contents of TGF-?1,Smad2,and Smad3 in the alveolar lavage fluid of the asthma group were significantly increased(P<0.05),and the contents of Smad6 and Smad7 were significantly reduced(P<0.05).The relative expression levels of TGF-?1,Smad2,Smad3,Collagen I,Collagen ? protein and mRNA in lung tissue were significantly increased(P<0.05),and the relative expression levels of Smad6,Smad7 protein and mRNA were significantly decreased(P<0.05).Compared with the asthma group,the TGF-?1 content in the alveolar lavage fluid of the dexamethasone group was significantly reduced(P<0.05).The relative expression levels of TGF-?1,Smad2,Smad3,Collagen ?,Collagen ?protein and mRNA in lung tissue were significantly reduced(P<0.05),and the relative expression levels of Smad6,Smad7 protein and mRNA were significantly increased(P<0.05).Compared with the dexamethasone group,the contents of TGF-?1,Smad2,and Smad3 in the alveolar lavage fluid of the Chinese medicine group were significantly reduced(P<0.05),and the contents of Smad6 and Smad7 were significantly increased(P<0.05).The relative expression levels of TGF-?1,Smad2,Smad3,Collagen I,Collagen ? protein and mRNA in lung tissue were significantly reduced(P<0.05),and the relative expression levels of Smad6,Smad7 protein and mRNA were significantly increased(P<0.05)Conclusion:Jiajian Wumei Pill granule restore the balance of Smad2/3 and Smad6/7 by regulating TGF-?1/Smad signaling pathway,inhibit the transduction of TGF-?1 signal in cells,and thereby reduce airway smooth muscle hyperplasia and Collagen I,Collagen III protein deposition,inhibits airway wall thickening,delays the development of airway remodeling,and improves lung function in rats. |
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