Role Of IL-17A Regulating Autophagy Of Bronchial Fibroblasts In COPD Airway Inflammation And Airway Remodeling

Objective:1.Establishing COPD airway remodeling model by single airway injection adenovirus with high expression of IL-1β.To observe the expression of IL-17 A,CCL20,autophagy-related proteins and related signal pathways,and collagen in mice lung tissue.2.Isolating and culturing COPD primary mice bronchial fibroblasts(MBFs).To investigate whether IL-17 A affects the production of collagen and CCL20 inflammatory cytokines by interfering with autophagy in MBFs,participating in airway inflammation and airway remodeling.Methods:1.Twenty C57BL/6J mice aged 6-8 weeks were randomly divided into two groups after adaptive feeding for one week,Ad-Lac Z group and Ad-IL-1β group.The COPD airway remodeling model was established by airway injection of adenovirus with high expression of IL-1β.Pulmonary function,HE staining and Masson staining were used to evaluate the establishment of COPD airway remodeling model.The expression of IL-17 A,CCL20 and other inflammatory factors in lung tissue of mice was detected by ELISA.The autophagy related proteins LC3II/LC3 I,Beclin-1,P62,PI3K/AKT/m TOR signal pathway related proteins,Collagen I and Collagen III were detected by WB.2.COPD primary MBFs were cultured by enzyme combined digestion and tissue block adherent methods.The marker of COPD MBFs,vimentin was identified by immunofluorescence.CCK-8 was used to explore the optimal concentration and time of IL-17 A and rapamycin on MBFs.The experiment was divided into four groups : control group,rapamycin(Rapa)group,IL-17 A group,and IL-17A+rapamycin group(IL-17A+Rapa).The expressions of autophagy-related proteins LC3II/LC3 I,Beclin-1,P62,Collagen I and Collagen III in the four groups were detected by WB,and the expressions of inflammatory cytokines CCL20,IL-10 and IL-6 in the cell supernatant were detected by ELISA.Results:1.COPD airway remodeling model: Compared with Ad-Lac Z group,the pulmonary function in Ad-IL-1β group,FEV0.1/FVC and Cdyn were decreased(p<0.05).HE and Masson staining of lung tissue showed,compared with the Ad-Lac Z group,the Ad-IL-1βgroup had a large number of inflammatory cells near the small airway,airway wall and smooth muscle thickening and Collagen fibrils were increased.2.Expression of inflammatory cytokines in lung tissue.ELISA results showed that compared with Ad-Lac Z group,the expressions of IL-17 A,CCL20 and IL-6 in Ad-IL-1βgroup were increased(P<0.05),and the expression of IL-10 was decreased(P<0.05).3.The expression of autophagy-associated proteins and PI3K/AKT/m TOR signal pathway-associated proteins in lung tissue.WB results showed that compared with Ad-Lac Z group,LC3II/LC3 I and Beclin-1 in Ad-IL-1β group were decreased(P<0.05),P62 protein was increased(P<0.05),P-PI3K/total PI3 K,p-AKT/total AKT,p-m TOR/total m TOR were increased in Ad-IL-1β group(p<0.05).4.The expression of Collagen I and Collagen III in lung tissue.Compared with Ad-Lac Z group,the expression of Collagen I and Collagen III in Ad-IL-1β group were increased(p<0.01).5.Culturing and identifying COPD primary MBFs.MBFs were isolated and cultured by enzyme combined digestion and tissue block adherent methods.MBFs were fusiform and polygonal.Immunofluorescence showed that vimentin,the surface marker of MBFs,was strongly positive.The growth curve of MBFs measured by CCK-8 was approximately s-type.6.Intervention concentration and intervention time of Rapa and IL-17 A.CCK-8 results showed that the optimal intervention concentrations of Rapa and IL-17 A on MBFs were 2μmol /L and 10 ng/m L respectively,and the optimal intervention time was 24 h.7.IL-17 A inhibited MBFs autophagy.WB results showed that compared with the Control group MBFs,IL-17 A group MBFs LC3II/LC3 I,Beclin-1 were decreased(P<0.05),P62 was increased(P<0.05),Rapa group MBFs LC3II/LC3 I was increased(P<0.05),P62 was decreased(P<0.05),the expression of Beclin-1was no significant difference.Compared with the Rapa group,the expressions of LC3II/LC3 I and Beclin-1 in the IL-17A+ Rapa group were decreased(P<0.05),and the expression of P62 was increased(P<0.05).8.IL-17 A inhibited autophagy of MBFs and promoted collagen production.WB results showed that compared with the Control group,Collagen I and Collagen III in IL-17 A group were increased(P<0.01),but Collagen I and Collagen III were decreased in Rapa group(P<0.01).Compared with the Rapa group,Collagen I and Collagen III were increased in IL-17 A + Rapa group(P<0.01).9.The effect of IL-17 A on inflammatory factors after inhibiting autophagy of MBFs.ELISA results showed that compared with the Control group,CCL20 was increased in the IL-17 A group(P<0.01),and IL-10 was decreased(P<0.01).There was no significant difference for CCL20 in Rapa group,and IL-10 was decreased(P<0.01).Compared with Rapa group,CCL20 was increased in IL-17 A + Rapa group(P<0.01)and IL-10 was decreased(P<0.01).The expression of IL-6 in the four groups was not statistically significant.Conclusion:1.IL-17 A and CCL20 was increased in the lung tissue of COPD mice.2.The level of autophagy in the lung tissue of COPD mice was decreased.Activation of PI3K/AKT/m TOR signaling pathway may be the mechanism of autophagy decreasing in COPD mice.3.The expression of Collagen I and Collagen III were increased in lung tissue of mice with COPD.4.IL-17 A inhibited COPD primary MBFs autophagy.5.IL-17 A promoted the expression of Collagen I and Collagen III by modulating COPD primary MBFs autophagy.6.IL-17 A promoted the secretion of CCL20 by COPD primary MBFs and decreased the secretion of IL-10.