Azithromycin Influences Airway Remodeling In Asthma Via The PI3K/Akt/mTOR/HIF-1α/VEGF Pathway

BackgroundAsthma is affecting about 235 million of people all over the word.The prevalence rate of asthma in children under 14 years of age in Chinese cities was significantly increased,it increased by 43.4% in the past 10 years,and was up to 147.9% in the past 20 years.The data indicate that the incidence of asthma didn’t reduce along with the improvement of economic conditions.Therefore,the professionals of respiration are consistent in focus on the asthma,and a lot of manpower and financial resources have been put into the study of asthma.Macrolide drugs,such as azithromycin,roxithromycin,clarithromycin,not only have strong antibacterial effect,but also can inhibit the release of inflammatory mediators,reduce airway responsiveness,and regulate immune by affecting cytokine secretion of leukocyte,macrophage and T lymphocyte,or directly acting on a variety of cytokines.Because azithromycin is better than other macrolides at gastrointestinal tolerance,tissue permeability and half-life,and its efficacy and patient compliance are high,it was chose by us to perform the present study.Azithromycin has broad-spectrum antibacterial activity and shows activity against both gram-positive and gram-negative pathogens via inhibiting bacterial protein synthesis by interfering 23 S ribosomal RNA and specific ribosomal proteins.Moreover,it exerts a certain regulating action on virulence factors of bacteria and production of toxins.This thesis consists of three parts to study the intervention of azithromycin on asthma.The first part is clinical study.The control group was treated with aerosol inhalation of budesonide,and treatment group was treated with oral administration of azithromycin combined with aerosol inhalation of budesonide.Through comparing the therapeutic effect,and detecting the expression of NF-kappa B and STAT1 in peripheral blood mononuclear cells,this part aimed to find the possible mechanism of azithromycin on treating asthma and confirm its clinical efficacy.In the second part,the acute phase model of mouse asthma was used to expose the therapeutic effect of azithromycin on the acute phase of asthma by promoting the repair of airway epithelial barrier via Wnt/β-catenin pathway,the expression of MUC5 AC protein,and the improvement of histopathology.In the third part,after the airway remodeling model of asthmatic mice was established,the change of the PI3K/Akt/ mTOR /HIF-1 Alpha/VEGF pathway in this model was completely verified,and the intervention of azithromycin on this signal pathway was explored.In the present study,the intervention of azithromycin was researched from three levels of pediatric asthma clinical,acute asthmatic mice model and the airway remodeling model in asthma,its aim is to excavate the immunotherapy effect of azithromycin,find a more effective way to control asthma,so as to improve the control rate of asthma in children.Part I The effect of azithromycin combined with budesonide on NF-κB and STAT1 in the peripheral blood mononuclear cells of the children with asthmaObjective: To investigate the differences of NF-κB and STAT1 in the peripheral blood mononuclear cells,and changes of pulmonary function indexes of FEV1,FEV1/FVC and PEF% in the children with asthma treated by azithromycin or azithromycin combined with glucocorticoid budesonide.Methods: Sixty of children with acute asthma attack were divided into a control group(n=23)and treat group(n=38).The control group was administrated by aerosol inhalation of budesonide.The treat group was combined with azithromycin based on aerosol inhalation of budesonide,sequential therapy of azithromycin dry suspension was given by oral administration and the regimen was as followed: 1 time per day,4 days per course,total 3 courses and an interval of 3 days between each course.After two groups were continuously treated for 15 days,5 mL of fasting venous blood was collected and density gradient centrifugation with Ficoll was used to isolate peripheral blood mononuclear cells(PBMC).And,RT-qPCR and Western blot methods were adopted to determine mRNA expression of NF-κB and STAT1,and protein expression NF-κB and STAT1,respectively.Furthermore,the pulmonary function was detected.Results: There was significantly difference between two groups in the mRNA and protein levels of NF-kB and STAT1.And,the pulmonary function indexes of FEV1,FEV1/FVC and PEF% were significantly higher in the treat group than in the control group.These results indicated that the budesonide combined with azithromycin can improve the therapeutic effect of children with asthma.Conclusions:(1)The expression levels of mRNA and protein of NF-kB and STAT1 in the control group were significantly increased.(2)The expression levels of mRNA and protein of NF-kB and STAT1 in treat group were significantly decreased.(3)The combination treatment of azithromycin and budesonide is superior to single therapy of budesonide,and can obviously improve the lung function and clinical effect in children with asthma.Part II Azithromycin promotes the repair of airway epithelial barrier via Wnt/β-catenin pathway in acute asthma model of mouseObjective: To compare the changes of airway structure and the expression of Wnt/ beta-catenin and MUC5 AC after azithromycin was combined to interven the asthmatic mice,and further explore the repair effects of the combined treatment on airway epithelial barrier.Methods: Male BALB/c mice of 4~6 weeks and weight 18~22g were randomly divided into three groups with ten animals per group.Group 1 was the asthma model,mice were sensitized by intraperitoneal injecting the mixed solution of ovalbulmin(OVA)and aluminum hydroxide at 1st,8th and 15 th day,and were stimulated to establish the asthma model by OVA aerosol inhalation every day during 15~28d;group 2 was model + azithromycin,mice were administrated 0.09 mg/g azithromycin by gavage at 2 h before inhalation of OVA;group 3 was the control,mice were given the same treatments except administrated normal saline instead of drug.Samples were collected at 28 th day.HE staining,the measurement of the thickness of bronchial vessel wall and airway smooth muscle were performed to observe the histomorphology.And,OVA-sIgE was assessed by ELISA method.Moreover,western blot method was adopted to determine the protein expression levels of Wnt7 b and β-catenin in lung tissue.In addition,the mRNA of Wnt7 b,β-catenin and MUC5 AC were detected by RT-qPCR.Results:(1)The asthma model group showed the obvious injury of airway,and is characterized by the disorder and shedding of epithelial cell,the thickening and stenosis of bronchial wall,hyperemia and edema of mucosa,the thickening and fracture of the basement membrane,the infiltration of a large number of inflammatory cells.While,the azithromycin treatment alleviated the hyperemia and edema of mucosa in the bronchial wall,reduced the thickness of the basement and decreased the infiltration degree of inflammatory cells.(2)The result of ELISA showed that the serum level of OVA sIgE was significantly higher in the asthma group than that in the control group(P < 0.05),and although it was still higher in the azithromycin than the control group,was significantly lowered compared to asthma model group(P<0.05),suggesting the modeling is successful.(3)The results of western blotting showed that the protein level of Wnt7 b in lung tissue was significantly increased in the asthma model group compared to azithromycin group(P < 0.05);and β-catenin protein level in lung tissue was higher in the asthma group than that in control group,and was decreased in azithromycin group but was still higher than the control group.(4)The results of RT-qPCR showed Wnt7 b,β-catenin and MUC5 AC mRNA in lung tissue were significantly higher in the model mice than those in the control mice(P < 0.05).While,these mRNA levels was significantly decreased in the azithromycin group compared to the model group(P < 0.05),but was still higher than the control group.(5)The results of WAt /PBM,WAm /PBM and N /PBM showed that the WAt/PBM and WAm /PBM were lower in azithromycin group than those in the asthma group,and the difference was statistically significant.Conclusions:(1)In the OVA sensitized mouse asthma model,the expressions of Wnt7 b,β-catenin and MUC5 AC were significantly higher,which is associated with asthma;(2)The pretreatment with azithromycin can attenuate the expressions of Wnt7 b,β-catenin and MUC5 AC in lung tissue of OVA sensitized mice,repair the disorders in the airway epithelial cells of asthmatic mice,and alleviate the symptoms of asthma.(3)The mechanism of azithromycin on asthma might be due to suppression of Wnt7b/ β-catenin signaling pathway and inhibition of expression of MUC5 AC,prevention of further diffusion of inflammation,and repair of the airway epithelial cells of asthmatic mice.Part Ⅲ The influence of Azithromycin on airway remodeling of asthma through PI3K/Akt/mTOR/HIF-1 Alpha/VEGF PathwayObjective: To completely verify the changes of PI3K/Akt/mTOR/HIF-1 Alpha/ VEGF pathway in the airway remodeling based on the asthma model,and to explore the influence of azithromycin on airway remodeling in asthma mice through PI3K/Akt/ mTOR/HIF-1 Alpha/VEGF Pathway.We expected that azithromycin can reduce the airway remodeling degree in asthma through its dual action both anti-inflammatory and immunoregulation,so as to add the treatment method of asthma and increase the control rate of asthma.Methods: Sixty of male BALB/c mice were randomly divided into five groups with 12 animals per group.Group 1 was normal control(non-sensitized animals);group2 was asthma model,mice were sensitized by the mixed solution of ovalbumin and aluminum hydroxide,and subsequently challenged by OVA;group 3 was model + azithromycin treatment of short-course;group 4 was model + azithromycin treatment of long-course;and group 5 was positive control of budesonide.The mice were intervened by short-term or long-term administration of azithromycin,or budesonide according to the experimental design.The apparatus for the measurement of lung function in laboratory animals was used to determine the airway responsiveness of mice,and the fresh lung tissue was taken for HE staining.And,the serum level of IgE was determined using the blood collected via the caudal vein.The irrigating solution of PBS was recycled for ELISA.Moreover,HIF-1α mRNA and VEGF mRNA in lung tissue were assessed RT-qPCR method.Furthermore,the VEGF protein in lung tissue was measured by ELISA,and western blot method was adopted to determine the protein expression of PI3 K,Akt,mTOR and HIF-1 α in lung tissue.Results:(1)The asthma mice model was successfully established,indicated by more frequent activities,shortness of breath,twitches of abdominal muscle,urine or fecal incontinence and hair rising in the process of modeling,and lags in response and sluggish action in some model mice.The airway responsiveness was significantly higher in the model mice than that in the control mice.Microscopic examination showed that there was the infiltration of a large number of inflammatory cell around the airway in the model group,while the normal control group had more complete alveolar wall structure,no thickening in airway epithelium,and no obvious infiltration of inflammatory cells around the airway.(2)In the lung tissue of mice,the expression of PI3 K,Akt,mTOR and HIF-1 Alpha proteins was significantly higher in the model group than that in the control group.(3)Compared with the asthma model group,all of drug treatment groups,including short-course azithromycin,long-course azithromycin and budesonide,abated the infiltration of inflammatory cells which was observed under microscope.And,compared with the short-course azithromycin and the budesonide groups,the infiltration of inflammatory cells was more obviously decreased in the long-course azithromycin.(4)All of drug treatment groups,i.e.short-course azithromycin,long-course azithromycin and budesonide,can reduce the levels of PI3 K,Akt,mTOR,HIF-1 α and VEGF,among them,the long-course azithromycin was the best,and the statistically significant differences were observed between long-course and short-course azithromycin,as well as between long-course azithromycin and budesonide.Conclusions:(1)PI3K,Akt,mTOR,HIF-1 Alpha and VEGF were highly expressed in the lungs of the asthmatic mice.(2)Azithromycin can inhibit the immune response of asthmatic mice by regulating PI3K/Akt/mTOR/HIF-1 Alpha/VEGF signaling pathway,thereby alleviate airway remodeling in asthma.