| Objective: Bronchial asthma is a complicated disease involving the interaction of genes and the environment,but the etiology of asthma is still not completely understood.Airway epithelial cells offer an important protective layer between the intrapulmonary and external environment.Airway epithelium,the first airway barrier,clears pathogens by swinging the cilia,releasing antimicrobial peptides,chemokines,cytokines,and recruiting and activating,other types of immune cells.Airway epithelial cells are associated with the airway inflammation and airway remodeling of bronchial asthma by enhancing proinflammatory activity and releasing growth factors after injury of airway epithelial cells.A number of studies have proved that the level of autophagy in asthmatic airway epithelial cells is increased,and the expression of E-cadherin is down-regulated in asthmatic epithelial cells.HMGB1 is a delayed inflammatory cytokine.Recent studies have shown that HMGB1 is up-regulated in induced sputum and BALF of asthmatic patients.HMGB1 is a nuclear protein that,after stress,can be transported to the cytoplasm and released to the outside of the cell.HMGB1 is closely connected with autophagy and oxidative stress.To clarify the relationship between HMGB1-related autophagy and airway epithelial EMT transformation,relevant animal and,cell experiments were conducted in this study.Methods: First,animal experiments 1.Animal model establishment and grouping: Female BALB / C mice were divided into PBS group,OVA group,3-MA group,EP group and BUD group.Model with OVA plus aluminum hydroxide sensitization,2% OVA challenge five times a week for 4 weeks.The PBS group was substituted for PBS,while the 3-MA group was intraperitoneally injected with 15 mg / kg 3-MA 1h before the challenge.The EP group was given intraperitoneal injection of 75 mg / kg EP 1h before challenge and 2mg BUD before BUD challenge.2.Noninvasive pulmonary function test: The lung function of mice was tested within 24 hours to 48 hours after the last inhalation of nebulization,and the path value under different concentrations of Mch was measured.3.Alveolar lavage fluid cell count and ELISA detection: 2% pentobarbital sodium(50mg / ml)anesthetized mice,intravenous catheter intubation trachea,1ml sterile syringe 0.5ml sterile PBS injection along the indwelling needle recovery,repeated three times,the recovery was greater than 80%.The bronchoalveolar lavage fluid took 4 3000 rpm / min,centrifuged 10 min.Resuspend the pellet after rejection,Wright-Giemsa staining,cell counting.Supernatants were analyzed for HMGB1 levels by ELISA kit instructions.4.Pulmonary histological staining: Left lung paraformaldehyde fixed paraffin embedded lung tissue sections.Left lung sections were used to assess airway inflammation,goblet cell metaplasia and subepithelial collagen deposition with HE,AB-PAS and MASSON staining respectively.5.Immunohistochemical staining: Left lung tissue sections were routinely deparaffinized,antigen retrieval,incubated with secondary antibodies for α-SMA,HMGB1,E-cadherin,N-cadherin and Vimentin primary antibody for 2 h.6.Western blot: Extract the tissue protein,BCA protein quantification,making protein samples,12% SDS-PAGE running gel,PVDF membrane transfer,milk blocking,antibody incubation ECL luminescent liquid luminescence.Second,cell experiments 1.Cell culture: HBE cells were cultured in medium containing 10% fetal bovine serum and induced with autophagy at different times(1h,2h,4h and 6h)and different concentrations of rapamycin(0.1μM,0.2μM,0.4μM and 0.6 μM)for 24 h.The 3-MA inhibitor was pretreated with 2m M 3-MA for 24 h before induction of autophagy.EP was pretreated with media containing 10 m M ethyl pyruvate 24 h before autophagy induction.2.Plasmid transfection over expression of HMGB1 and si RNA silencing transacted HMGB1: The 12.5cm2 flasks were planted.When the degree of cell fusion reached 80%,over expression was performed on 6μg of c DNA and 18μl of lipo 2000 and silenced with 20μM si RNA and 10μl Lipo 2000.3.Immunofluorescence: 24-well plate cells to grow,given different processing conditions,the final cell fusion degree of about 30-40%,immunofluorescence experiments,inverted microscopy.Results:First,Role of HMGB1-related autophagy in epithelial-mensenchymal transformation of airway epithelial cells in bronchial asthmatic mice 1.3-MA inhibition of autophagy significantly inhibited airway inflammation and airway hyperresponsiveness EBSS starvation induced autophagy and RAP-induced autophagy can lead to decreasing E-cadherin,N-cadherin and Vimentin in airway epithelial cells.EBSS and RAP induced EMT levels decreased after 3-MA suppression of autophagy.2.3-MA inhibition of autophagy can reduce the level of EMT in asthmatic airway epithelial cells The results of immunohistochemistry and western blot in lung tissue sections of 3-MA inhibitor group showed that E-cadherin,N-cadherin and Vimentin were extended in 3-MA group mouse epithelial cells.3.EP inhibited HMGB1 metastasis and inhibited airway inflammation and airway hyperresponsiveness EP group showed that EP could inhibit the expression of HMGB1 in airway epithelial cells,meanwhile,the total number of cells in alveolar lavage fluid of mice decreased obviously and eosinophils decreased.HE staining showed that infiltration of inflammatory cells in the lungs improved,MASSON staining showed reduced deposition of collagen in the subcutaneous and AB-PAS showed decreased goblet cell metaplasia.Pulmonary function of small animals showed that airway responsiveness in the EP group was significantly lower than that in the OVA group.4.Inhibition of autophagy in airway epithelial cells by EP inhibited the translocation of HMGB1 Electron microscopy showed that autophagosomes in airway epithelium of 3-MA group and EP group were markedly decreased.Protein electrophoresis showed that autophagy level decreased in 3-MA group and EP group.5.EP inhibited the migration of HMGB1 and decreased the EMT level of asthmatic airway epithelial cells Compare to the asthma group,the EP inhibition group increased the expression of E-cadherin and decreased the expression of N-cadherin and Vimentin in airway epithelial cells.Second,study on HMGB1 autophagy induced mesenchymal transition of airway epithelial cells 1.Starvation and RAP induced autophagy can lead to EMT in airway epithelial cells EBSS starvation induced autophagy and RAP-induced autophagy can lead to decreasing E-cadherin,N-cadherin and Vimentin in airway epithelial cells.EBSS and RAP induced EMT levels decreased after 3-MA suppression of autophagy.2.HMGB1 protein and over expression of HMGB1 can lead to airway epithelial cells in EMT The effect of HMGB1 protein in HBE cells showed that EMT level increased with the time and concentration of HMGB1.HMGB1 over expression showed high expression of HMGB1 in the cytoplasm of HBE cells.E-cadherin also decreased in HBE cells over expressing and N-cadherin and Vimentin increased.3.Inhibition of autophagy can reduce HMGB1-induced EMT HMGB1 and over-expression of HMGB1 can lead to elevated autophagy in HBE cells,3-MA inhibition of autophagy decreased EMT levels of HBE cells.4.EP and silencing HMGB1 can inhibit EBSS-induced EMT After pretreatment with HBE cells,EBSS was required to induce autophagy.The EMT level in HBE cells was much lower than that in untreated cells.EBSS-induced autophagy in HBE cells silenced with HMGB1 also showed that the level of EMT reduced.Conclution: 3-MA inhibits autophagy and EP inhibits HMGB1 translocation can significantly reduce autophagy in airway epithelial cells of OVA-induced asthma mice,reduce airway inflammation,reduce airway hyperresponsiveness,and reduce the level of epithelial mesenchymal transition.Cell experiments further prove that EBSS or RAP-induced autophagy can lead to the occurrence of EMT HBE cells,HMGB1 protein and over-expression of HMGB1 also lead to autophagy and EMT in HBE cells.3-MA inhibited autophagy,EP pretreatment of HBE cells and silencing of HMGB1 inhibited HBE cells in EMT levels. |
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