Molecular Regulation Mechanism And Intervention Of Epithelial-mesenchymal Transition In Asthma Airway Remodelling

Objective1.To investigate the molecular pathogenesis of epithelial-mesenchymal transition(EMT)in asthmatic mice by establishing the mouse allergic asthma model,and observe the changes of airway inflammation,remodelling and reactivity.2.New theory and its evidence were provided for the better prevention of airway remodelling,by observing the effects of IKKβ inhibitor BMS-345541 and brominated domain protein inhibitor JQ1 on airway inflammation,airway remodeling and EMT in asthmatic mice.Methods1.Mice were randomly divided into ovalbuminova(OVA)asthma group and normal control group(each group,n = 8).In the OVA group,the asthma model was established by using OVA and aluminum hydroxide.The mice were sensitized three times and then stimulated by 5%OVA atomization daily for 7 days.The control group was sensitized and challenged by phosphate buffered solution(PBS)instead of OVA.Observation indicators include:(1)The behavior of mice asthma was observed after OVA challenged;(2)Lung resistance(RL)was measured by whole volume method using the animal pulmonary function instrument,and the changes of RL while increasing the concentration of acetylcholine(Ach)inhalation was observed;(3)The percentage of eosinophil and the total cell counts of bronchoalveolar lavage fluid(BALF)were calculated;(4)Enzyme-linked immunosorbent assaysl assay(ELISA)was used to determine the concentration of transforming growth factor β 1(TGFβ1)in serum and BALF;(5)Lung tissue sections were stained with hematoxylin and eosinhe(HE),periodic acid-schiff(PAS)and modified Weigert resorcinol complex red staining to observe the lung morphologys of mice;(6)Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of E-Cadherin and vimentin which are biomarkers of EMT in asthmatic mice;(7)The expression of E-Cadherin and vimentin protein was detected by western blot.2.On the basis of successful establishment of asthmatic mice model,mice were equally divided into four groups randomly: control,OVA,BMS-345541 interfered group(BMS+OVA)and dimethyl sulfoxide(DMSO)interfered control group(DMSO+OVA),each group,n = 8.In BMS+OVA group,BMS-345541 was dissolved in 1% DMSO,and the resulting solution was diluted with sterile water to a final concentration of 10 μg/μl.BMS-345541(50μg/g)was administered orally to the mice before daily OVA challenge using a feeding needle for continuous 7 days.In DMSO+OVA group,vehicle(20 μl DMSO in a total of 200μl saline)was administered orally to the mice using the same method.Observation indicators include:(1)the behavior of asthmatic mice was observed;(2)RL was measured;(3)The number of total cells and the percentage of inflammatory cells(such as eosinophils,lymphocytes,neutrophils and macrophages)of BALF were calculated;(4)ELISA was used to determine the concentration of TGFβ1 in serum and BALF;(5)HE,PAS staining and modified Weigert resorcinol complex red staining were performed to evaluate the Lung tissue histopathology;(6)The thickness of tracheal wall epithelium was measured;(7)The expression of E-Cadherin and vimentin were observed by Immunohistochemical analysis,and the mean integrated option density(IOD)was detected;(8)The expression of E-Cadherin and vimentin were observed by RT-qPCR;(9)The expression of E-Cadherin and vimentin were observed by western blot.3.Twenty-four mice were randomly divided into control group,OVA induced asthma group(OVA),and JQ1 treated asthma group(JQ1+OVA),each group,n=8.Asthma model was established successfully.OVA group mice were treated with JQ1 solution(50μg/g)via intraperitoneal injection for continuous 7 days.Observation indicators include:(1)The behavior of mice asthma was observed;(2)The total cell count and the percentage of eosinophil of BALF were calculated;(3)Lung tissue sections were stained with HE,PAS and modified Weigert resorcinol complex red staining to observe the lung morphologys of mice;(4)The expression of E-Cadherin and vimentin were observed by RT-qPCR;(5)The expression of E-Cadherin and vimentin were observed by western blot.Results 1.The results of the first part of experiment(1)Compared with the control mice,the behavior of the OVA mice showed agitation,hypokinesia,dyspnea,incontinence,and so on.(2)Lung tissue histopathology: OVA induced mice showed the influx of inflammatory cells around the bronchi and bronchioles and along with submucosal edema;Lung tissue from OVA mice displayed obvious airway remodelling,including enlargement of airway smooth muscle,basement membrane thickening,goblet cell hyperplasia,mucus hypersecretion,epithelium damage,subepithelial fibrosis.(3)Determination of RL: administration of Ach at doses increasing progressively from 3.125 to 25 mg/ml leads to increased dramatically RL in the OVA group compared with the control group(P﹤0.01).(4)Total cells and percentage of eosinophil in BALF: the total cell counts and the percentage of eosinophil in the OVA group were significantly higher than the control group(P﹤0.01).(5)Determination of TGF β 1 in serum and BALF: compared with the control group,the TGFβ1 levels of the serum and BALF were markedly elevated in the OVA group(P﹤0.01).(6)The E-cadherin and vimentin expression of mRNA and protein: Mice challenged by OVA had decreased expression of the E-Cadherin and increased expression of vimentin.They were well consistent with the results of RT-qPCR and western blotting(P﹤0.05).2.The results of BMS-345541 intervention in OVA asthmatic mice(1)The symptoms of asthmatic mice were relieved after being treated with BMS-345541.(2)The BMS-345541 treatment group exhibited less inflammatory cell infiltration and subepithelial fibrosis.(3)The treatment group with BMS-345541 resulted in a significant decrease in RL(P﹤0.01).(4)After BMS-345541 treatment,the increased cell counts and eosinophil percentage were markedly decreased(P﹤0.05).(5)The epithelial thickness was markedly alleviated by the administration of BMS-345541(P﹤0.001).(6)After administrating BMS-345541,the concentrarion of TGFβ1 of serum and BALF were 42.04±28.38 pg/ml,25.45±14.15 pg/ml.The OVA group were 84.41±38.18 pg/ml,44.36±10.98 pg/ml.The BMS+OVA group had significantly decreased than the OVA group(P﹤0.05).(7)The value of IOD by immunohistochemical staining represented the expression of each protein.The E-Cadherin IOD in BMS+ OVA group was 0.22±0.07,the vimentin IOD was 0.17±0.04.After BMS-345541 daily administration,compared with the OVA group,the expression of E-Cadherin was increased,while the expression of vimentin was decreased(P﹤0.05).(8)After BMS-345541 daily administration,in mRNA and protein level,the expression of E-Cadherin was increased,while the expression of vimentin was decreased(P﹤0.05).(9)There was no significant difference between the BMS+OVA and the control group(P﹥0.05).3.The results of JQ1 intervention in OVA asthmatic mice(1)The symptoms of asthmatic mice were relieved after treated with JQ1.(2)Compared with OVA group,the airway inflammatory cell infiltration,airway wall damage,airway spasm,mucus exudation,goblet cell proliferation and subepithelial fibrosis were decreased in JQ1+OVA group.(3)Compared with OVA group,the cell counts and eosinophil percentage were markedly decreased after JQ1 treatment(P﹤0.01).(4)After JQ1 daily administration,in mRNA and protein level,the expression of E-Cadherin was increased,while the expression of vimentin was decreased(P﹤0.01).Both mRNA and protein levels showed the same results.(5)There was no significant difference between the JQ1+OVA and the control group(P﹥0.05).Conclusion1.Allergic asthmatic mouse model was established successfully by OVA sensitized/challenged.2.The OVA asthmatic mouse showed the typical airway inflammation,airway hyperresponsiveness and airway remodelling.3.The increasing secretion of TGFβ1 was observed in OVA asthmatic mouse.TGFβ1 maybe related to asthma EMT.4.EMT evidence was observed in airway remodelling.The decreased expression of E-Cadherin and the increased expression of vimentin could result in the occurrence of EMT.5.Both the IKKβ inhibitor BMS-345541 and the bromodomain protein inhibitor JQ1 may block the airway inflammation,reduce the airway hyperactivity and airway remodelling,and inhibit the EMT in asthmatic mice.6.The EMT was related to the molecular regulations of TGFβ1,NF-κB and BRD4.The regulation of NF-κB/RelA-BRD4 signaling pathway may provide a new target for the future treatment of asthma.