The Effect Of Guben Cough Relief Recipe On The Immune Cells And Factors Of The Respiratory Mucosa Of The COPD Model Mice

Background:Chronic obstructive pulmonary disease(COPD)is an obstructive pulmonary disease characterized by incompletely reversible airflow restriction.Acute and chronic inflammation persists in the respiratory tract and the whole body,which damages the airway structure.Repeated damage and repair process causes airway remodeling and emphysema and leads to airway obstruction and gas retention,which is manifested as exertional dyspnea,bringing heavy burden to the patients'daily life and society.Curative effect of Chinese traditional medicine in the treatment of COPD is considerable and advantaged,the mechanism of which is lack in-depth research.The mucosal immunity plays an important role in a variety of diseases,but the research on the influence of traditional Chinese medicine on the respiratory mucosal immunity is in its infancy.According to the characteristics of pathogenesis of COPD,Gubenzhike recipe has the effect of strengthening the vital qi and dispelling the invading pathogenic factors.Preliminary clinical studies have shown that Gubenzhike recipe can effectively relieve cough,wheeze,sputum and other symptoms,improve lung function and regulate immune function of patients.Animal experiments showed Gubenzhike recipe reduced airway inflammation,improved lung function and pathological injury in the airway,the mechanism of which needs further research.Objective:1.To explore the influence of Gubenzhike recipe on ??T cells and their cytokines KGF in respiratory mucosal immune system and the mechanism of this recipe strengthening vital qi to eliminate pathogens.2.To research the influence of Gubenzhike recipe on the inflammatory factors in circulation and locally in the mucous membrane,and discuss the mechanism of improving the patients' symptoms and the severity of disease progression.3.To provide experimental basis for a link between mucosal immune function and "wei qi",and for traditional Chinese medicine realizing "treat before desease attack" and provide new ideas for the treatment of COPD.Methods:Astragalus,Epimedium,Rhizoma Atractylodes,Radix Stemonae,Radix Scutellariae,Radix Paeoniae Rubra and Radix Saposhnikoviae were mixed at a ratio of 4:3:2:3:3:2:1.Dry extract containing 3.6g raw herb per milliliter was made in the department of pharmaceutical preparation of China-Japan Friendship Hospital.Then we calculated the dose depending upon the per kg body mass dose conversion coefficient table between animals and adults.The high,medium and low-dose solution was administrated at 3.5g raw herb/kg,1.8g raw herb/kg,0.58g raw herb/kg bodyweight of mouse separately.A total of 125 healthy SPF grade ICR female mice were randomly divided into 5 groups:blank control group,model group,high-dose Gubenzhike recipe group,medium-dose Gubenzhike recipe group and low-dose Gubenzhike recipe group,with 25 mice in each group.Mice were exposed to successive periods of cigarette smoke(CS)for 7days/week for 12 weeks(except the days using LPS).The LPS(1.5mg/kg)in distilled water was implemented by instillation through the nasal cavity to respiratory tract on day 1,day29 and day57.Mice's weight was measured every 4 weeks and their health status was monitored.After the model establishment,an equal volume(0.2ml/day/animal)of distilled water or solution of dry extract of Gubenzhike recipe were administered separately by oral gavage to blank control group,model group,and Gubenzhike recipe groups once a day for 4 weeks.Lung function was measured by a small-animal ventilator.After the pulmonary function test,BALF,serum and intestinal mucus were collected and stored for detection of inflammatory factors IL-6 and IL-13.Morphological changes and cell infiltration were observed in lung tissue sections after HE staining,and compared with the typical pathological changes of COPD to evaluate the quality of model.Immunohistochemical staining was performed on the lung sections to detect the expression of ??TCR and ??TCR,and the secretion of KGF and KGFR in the lung tissues of mice.Based on the method of separation of mucosal epithelial lymphocyte in mice' small intestinal,we innovatively summarized the method of separation of lymphocytes in mice's lung tissue.Cells were stained with FITC anti-??TCR and PE anti-??TCR,then detected by flow cytometer.After lung tissues of mice were homogenized,KGF and KGFR proteins were detected by Western Blot,the content of KGF protein was detected by ELISA,and the mRNA level of KGF was detected by RT-PCR.Results:1.Gubenzhike recipe increased the survival rate and body weight of mice with COPD.None of the blank control group died.They had smooth fur and gentle breath in moderate frequency and uniform rhythm.Mice of the blank control group behaved normally and gradually gained weight.10 of the COPD model group died.Fur of mice in the model group was dry and dark,some of which lost.Mice moved and jumped restlessly when exposed to cigarette smoke.In the later stage,they often huddled and curled up,somewhile their whole body trembled.They breathed rapidly even with their mouths open.Their chest and abdomen undulated obviously,and they sometimes nodded irregularly.Body weight of mice with COPD was significantly lower than that of the blank control group(P<0.001).High-dose and medium-dose Gubenzhike recipe groups had 1 death,respectively,while the low-dose group had 4.Compared with the model group,mice in three Gubenzhike recipe groups had softer and shinier fur,less agitation,slower respiratory rate,and significantly heavier weight(P<0.001).2.The pathological manifestations of COPD include degeneration,necrosis,and detachment of bronchial mucosa epithelium,with the cilia being shorter and adhered.The trachea cavity becomes narrow because of hypertrophy of goblet cells and mucous cells,retention of abundant mucous,airway wall congestion,and edema.The alveoli atrophy and collapse,the alveolar septa is destroyed and ruptured,and adjacent alveoli fuse to form large alveoli.A large number of chronic inflammatory cells infiltrate around the trachea and alveoli.There are bronchi remodeling,increased collagen content,and scar formation.Gubenzhike recipe significantly improved the morphology situation of COPD mice lungs.It increased the structural integrity of the bronchi,alveoli,pulmonary mesenchyme,and the orderliness of cilia.Gubenzhike recipe also decreased the stenosis degree of airway,the number of lung bullae,and the number of inflammatory cells infiltrated around airway and pulmonary interstitium,3.Lung function parameters:Compared with that of the blank control group,the Rrs and Ers of the model control group were significantly higher(P<0.05),and the Crs was significantly lower(P<0.05).Compared with that in the model control group,the Rrs of three Gubenzhike recipe groups was significantly lower(P<0.05),the Ers of the medium-dose Gubenzhike recipe group was significantly lower(P<0.05),and the Crs was significantly higher(P<0.05).Compared with G of the model group,that of the blank control group was significantly lower(P<0.05),and that of the high-dose and low-dose Gubenzhike recipe group was significantly lower(P<0.05).Compared with that of the model group,the Est was significantly higher and the Cst was significantly lower in the high-dose Gubenzhike recipe group(P<0.05).4.Immunohistochemical staining:In the blank control group,there were a few??T cells and ??T cells in the lung tissue.As for the model group,scattering distribution of ??T cells in the alveolar tissue and hardly any ??T cells were observed.There were significantly more ??T cells around bronchi and rarefied ??T cells in the three Gubenzhike recipe groups.Positive brown cells were counted in three random fields at 400x magnification in each section with immunohistochemical staining of ??TCR and ??TCR.The ratio of ??T cell/??T cell reduced in lung tissue of COPD model mice and increased by Gubenzhike recipe.5.The lymphocytes in lung tissues were isolated through density gradient centrifugation method and then analysed by flow cytometry.The percentage of ??T lymphocytes in the low-dose Gubenzhike recipe group was significantly higher than that in the model group(P<0.05).Compared with the blank control group,the ratio of ??T/??T lymphocytes in all other groups increased.That ratio decreased in high-dose and low-dose Gubenzhike recipe groups in contrast to that of the model group.6.Immunohistochemical staining of KGF and KGFR was made,respectively,on the paraffin lung sections.In the blank control group and model group,there were a few brown particles representing little expression of KGF.Large area of brown particles was visible in three Gubenzhike recipe groups,in which KGF expression was significantly higher.The amount of KGFR was more in the model group and three Gubenzhike recipe groups than the blank control group.7.Compared to that of the model group,KGF was significantly higher in the blank control group(P<0.05)and medium-dose Gubenzhike recipe group(P<0.05),and KGFR was significantly higher in the blank control group(P<0.05),high-dose Gubenzhike recipe group(P<0.05),and low-dose Gubenzhike recipe group(P<0.05)in the western blot detection.8.ELISA results showed that KGF of the high-dose Gubenzhike recipe group was significantly higher than that of the model group(P<0.05).9.As for the RT-PCR assays,mRNA level of KGF was significantly higher in the blank control group(P<0.05),medium-dose Gubenzhike recipe group(P<0.05),and low-dose Gubenzhike recipe group(P<0.05)than in the model group.10.The levels of IL-6 and IL-13 in serum,BALF,and intestinal mucus of the mice were determined by ELISA according to the manufacturer's instructions.The IL-6 level in serum of the blank control group was significantly lower than that of the other four groups(P<0.05),while the content of IL-6 in the high-dose Gubenzhike recipe group was significantly lower than that in the model group(P<0.05).The IL-6 level in BALF of the model control group was significantly higher than that of the blank control group(P<0.05)and the medium-dose and low-dose Gubenzhike recipe groups(P<0.05).There was no significant statistical difference in IL-6 level of intestinal mucus and IL-13 level of serum between the groups.The IL-13 level in BALF of the model group was significantly higher than that of the blank control group(P<0.05)and the high-dose and medium-dose Gubenzhike recipe groups(P<0.05).The IL-13 level in intestinal mucus of the model group was significantly higher than that of the blank control group(P<0.05)and the high-dose,medium-dose,and the low-dose Gubenzhike recipe groups(P<0.05).Conclusion:This study suggested that after successive periods of cigarette smoke exposure and instillation LPS solution through the nasal cavity to respiratory tract,pathological changes of lung tissue and lung function of model mice were in conformity with the characteristic of COPD.Gubenzhike recipe could improve life quality and weight of COPD model mice,improve pathological changes of lung tissue morphology of COPD model mice,reduce the degree of airway obstruction and improve lung tissue compliance.Gubenzhike recipe could reduce IL-6 content in serum and BALF and IL-13 content in BALF and intestinal mucous of COPD model mice,effectively control the airway and systemic inflammatory.Gubenzhike recipe could adjust portion of gamma delta T cells in COPD model mice,promote the secretion of KGF and KGFR,accelerate pulmonary tissue reparation,improve respiratory tract epithelial integrity and enhance the respiratory mucosal immune function,which provided experimental evidence for its effects of reinforcing the"wei qi" by means of strengthening vital qi to eliminate pathogens.By regulating the mucosal immune function,traditional Chinese medicine can achieve the function of"nourishing the healthy and dispelling evil",thus delaying the disease progression and reducing the acute attack,providing a new idea for the treatment of COPD.The experimental method of extraction and isolation of lung lymphocytes will also provide the experimental basis for the study of respiratory mucosal immunity.