| Research background and purpose:Cough variant asthma is a special type of chronic respiratory disease,also known as atypical asthma.Most patients have cough without obvious sputum as the main or only clinical manifestation.Although patients with this disease do not have obvious symptoms such as chest tightness and shortness of breath,the patient’s condition is prone to repeated attacks and protracted recovery.After high-intensity exercise and at night,the patient’s cough is prone to appear and even worsen.Repeated chronic coughing affects patients’ rest and work,causing patients to seek medical treatment repeatedly,and some patients may even suffer from mental illnesses such as depression and anxiety.If there is no effective treatment,it will turn into a typical asthma,which will increase the family’s economic and social burdens.Cough variant asthma has the characteristics of chronic airway inflammation,airway remodeling,and airway hyperresponsiveness caused by a variety of inflammatory cells and inflammatory transmitters,and its treatment principles are similar to those of typical asthma.The treatment plan of western medicine is based on inhaled glucocorticoids plus β2-receptor agonists,supplemented with theophylline drugs,anticholinergic drugs,leukotriene modulators,etc.according to the patient’s specific condition.The course of treatment is generally more than 2 months.Western medicine has good short-term effects and can quickly control symptoms.It is the main clinical treatment drug for acute attacks.However,due to the long course of treatment of western medicine,poor drug tolerance,poor treatment compliance of patients,and high recurrence rate after stopping the drug,it is difficult for western medicine to substantially settle the problem.Traditional Chinese medicine has a long history of treating cough variant asthma.It has the advantages of improving the lung and spleen function,strengthening the vitality of the body,regulating the physical fitness of patients,preventing the recurrence of the disease,and having no obvious side effects.It has attracted increasing attention from researchers.Therefore,we should give full play to the therapeutic advantages of Western medicine and traditional Chinese medicine,and adopt a combination of Chinese and Western medicine to treat cough variant asthma.It is better than pure Western medicine in improving patients’ clinical symptoms,lung function and quality of life.In this study,Elisa,Western Blot,RT-PCR,proteomics analysis and other techniques will be used to study cough variant asthma rats model.Through in vivo and in vitro experiments,the improvement of Huanglong Zhike granlues combined with dexamethasone tablets will be preliminarily explored.To further study the possible mechanism and targets of Huanglong Zhike granlues in the treatment of cough variant asthma,and provide a theoretical basis for the development of new drugs for the treatment of cough variant asthma.Research methods:1.Randomly divided 50 SD rats into normal group,model group,traditional Chinese medicine group(Huanglong Zhike granule),Western medicine group(dexamethasone tablets),and combination group(Huanglong Zhike granules combined with dexamethasone tablets).That means fifty rats were equally divided into five groups.Except for the normal group,the rats in the other groups used the OVA method to prepare cough variant asthma rat models;the non-invasive ventilation lung function instrument was used to detect the airway reactivity of the rats in each group.2.Wright-Giemsa staining method was used to to detect the inflammatory cells in the BALF of each group of rats;The lung tissues of rats in each group were stained with HE to observe the pathological changes.3.The contents of IL-4,IL-5 and IL-13 in BALF of each group of animal models in this experiment were measured by ELISA method,and ELISA was used to detect the content of Ig E in the serum of animal model in each group.In order to observe m RNA changes of IL-4,IL-5 and IL-13 in the lung tissue of animal model,RT-PCR was used in this experiment;the expression of β-catenin and GSK-3β protein in lung tissue of animal model in this experiment were measured by Western blot.4.TMT labeling,high performance liquid chromatography grading technology and mass spectrometry-based quantitative proteomics technology was used to to detect the difference in protein expression between the combined group and the normal group and the model group.Software/methods such as Max Quant,KEGGMapper,KAAS,Blast,R package network D3 was used to perform function classification,function enrichment and cluster analysis based on function enrichment of differentially expressed proteins.After comparing with the STRING(v.11.0)protein network interaction database,the differential protein interaction relationship was extracted.5.Image analysis software was used to measure the thickness of the bronchial wall and smooth muscle;PAS staining was used to detect goblet cell metaplasia and Masson staining was used to detect subepithelial fibrosis.6.ASMC cells was extracted from rat lung tissue and identified by Cellular immunohistochemistry;ASMC cells was divided into normal group,traditional Chinese medicine group(Huanglong Zhike granules),western medicine group(dexamethasone tablets),and combination group(Huanglong Zhike granlues combined with dexamethasone tablets).Each drug intervention group was cultured with Huanglong Zhike granules,dexamethasone tablets,Huanglong Zhike granlues and dexamethasone tablets respectively.7.The protein expressions of GSK-3β,β-catenin,Cyclin D1 and c-Myc in ASMCs of animal model in each experimental group were detected by Western Blot method;The m RNA expression levels of GSK-3β,β-catenin,Cyclin D1 and c-Myc proteins in ASMCs of animal model in each experimental group were detected by RT-PCR.8.Methyl thiazolyl tetrazolium method was used to detect the changes of ASMC proliferation activity in each group,flow cytometry was used to detect the DNA content of ASMC in each group;Transwell and cell scratch test were used to detect ASMC migration.Research results:1.Compared with the model group,each intervention group reduced the airway responsiveness of rats to varying degrees;The total amount of eosinophils and leukocytes in BALF of animal model in each intervention group,and the degree of bronchial lumen stenosis were all improved.2.The count of IL-4,IL-5,IL-13 and Ig E in BALF and the m RNA expression of IL-4,IL-5,IL-13 of asthma model increased significantly;the contents of IL-4,IL-5,IL-13,Ig E and the m RNA levels of IL-4,IL-5,IL-13 in lung tissue of intervention group were significantly reduced(P<0.05).Western blot results showed that,the protein expression of β-catenin was significantly reduced and the protein expression of GSK-3β in was significantly increased each intervention group(P<0.05).3.Proteomics analysis results showed: 1).Protein differential expression analysis(P<0.05): Compared with the normal group,the combination group has 275 up-regulated proteins and 235 down-regulated proteins;compared with the model group,the combined group has 164up-regulated proteins and 118 down-regulated proteins;compared with the model group,β-catenin,c-Myc and Cyclin D1 protein were down-regulated in the combination group,and GSK-3β protein was up-regulated;compared with the normal group,β-catenin,c-Myc and Cyclin D1 protein were up-regulated in the combination group,and GSK-3β protein was down-regulated.2).Protein function and interaction analysis: β-catenin,c-Myc,GSK-3β and Cyclin D1 were located in the protein interaction network of the most closely interacting differential proteins in different groups.4.Compared with the model group,the thickness of bronchial wall,smooth muscle thickness,the number of metaplasia airway goblet cell and subepithelial fibrosis were reduced to varying degrees in each intervention group.5.Compared with the normal group,the protein expression and m RNA expression of β-catenin,c-Myc and Cyclin D1 in ASMC in each drug intervention group were significantly decreased(P<0.05),At the same time,the protein expression and m RNA expression of GSK-3β in each drug intervention group were increased(P<0.05).6.Compared with the normal group,the proliferation ability of ASMC in each drug group was weakened to varying degrees,the G0/G1 cell cycle in each drug group was prolonged to different degrees,and the S phase and G2/M phase in each drug group were shortened to different degrees;compared with the normal group,the scratch repair and migration ability of ASMC in each drug group were weakened to varying degrees.Conclusion:1.Huanglong Zhike granlues combined with dexamethasone tablets could reduce airway inflammation of the cough variant asthma model by regulating the count of IL-4,IL-5,IL-13,Ig E and the m RNA expression of IL-4,IL-5,and IL-13.2.Huanglong Zhike granlues combined with dexamethasone tablets could regulate the protein expression and m RNA expression of β-catenin,c-Myc,GSK-3β and Cyclin D1 in ASMC;Huanglong Zhike granlues combined with dexamethasone tablets could inhibit the proliferation and migration of ASMC to reduce airway remodeling in the cough variant asthma model.3.The mechanism of Huanglong Zhike granlues combined with dexamethasone tablets reduced airway inflammation and airway remodeling in cough variant asthma models was related to the regulation of Wnt/β-catenin signaling pathway. |
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