The Effects And Mechanisms Of Exosomes Derived From Alveolar Macrophages Of COPD On The Proliferation Of Airway Epithelial Cells

Background and ObjectivesThe occurrence and development of chronic obstructive pulmonary disease are usually related to mechanisms such as chronic inflammation,oxidative stress and protease-antiprotease imbalance.The airway epithelial metaplasia and the excessive mucus secretion caused by goblet cell hyperplasia are the characteristic pathological changes of COPD,the specific mechanism of which is not very clear.Previous studies have shown that alveolar macrophages play an important role in the pathogenesis of COPD,the studies about their effects on airway epithelial cells are limited.The role of exosomes and their secreted mi RNAs in intercellular communication has intrigued researchers recently.It has not been reported whether alveolar macrophages,one of the important source of exosomes,play a role in airway epithelial cells and relevant mechanism as we know.Epidemiological studies show that the annual morbidity of lung cancer in COPD patients is 4-5 times higher than that in the normal population,suggesting that COPD is closely related to lung cancer.Both diseases involve changes in the biological function and structure of airway epithelial cells.In this study we extracted the alveolar macrophages from animal model of COPD rats and cocultured with human airway epithelial cells line(16HBE)in vitro.CCK8 detection,clone formation and transwell invasion were used to detect the proliferation and migration of airway epithelial cells.The expression of related mucins and inflammatory mediators was assessed by western blot and ELISA.Bioinformatics methods were used to find differentially expressed mi RNA.Exosomes were isolated and identified from macrophages.The effects of exosomal mi RNAs in intercellular communication with airway epithelial cells were explored to expand understanding of the pathogenesis in COPD.It may provide a theoretical basis for further clarifying the correlation between COPD and lung cancer and finding possible therapeutic targets to reduce the occurrence of COPD with lung cancer in the future.Materials and Methods1.Effects of COPD-derived alveolar macrophages on airway epithelial cellsCigarette smoke and lipopolysaccharide-induced methods were used to establish an animal model of COPD rats.HE staining was used to observe the pathological changes of lung tissue.Macrophages were isolated from the alveolar lavage fluid.The macrophages were co-cultured with airway epithelial cells(16HBE).The proliferative activity and migration ability of airway epithelial cells were detected by CCK8 method,clone formation test and transwell invasion test.Western Blot and Elisa methods were used to determine the expression of mucin and the proinflammatory mediators such as IL-6 and TNF-?.2.Effects of exosomes derived from alveolar macrophages of COPD on the function of airway epithelial cellsExosomes were extracted from alveolar macrophages of COPD rats and healthy control rats by kit method.The morphological characteristic of exosomes was observed by electron microscope.The differentially expressed mi RNAs in peripheral blood of COPD patients and normal people were analyzed and compared from the GEO database.The differentially expressed mi RNAs of macrophage exosomes were detected by corresponding mi RNA array analysis.The mi RNA was overexpressed in airway epithelial cells.The effects of overexpressed mi RNA on the biological function of airway epithelial cells were observed by CCK8,transwell invasion experiments and western blot.3.The mechanism of the effect of exosomes derived from alveolar macrophages of COPD on the function of airway epithelial cellsUsing bioinformatics methods to predict target gene of exosomal mi RNA.CCK8was used to analyze the influence of blocking the target gene on the proliferation of airway epithelial cells.ResultsI.Effects of COPD-derived alveolar macrophages on airway epithelial cells1.The proliferative activity and migration ability of airway epithelial cells was significantly enhanced after co-culture with alveolar macrophages from COPD than from healthy controls(P<0.05).2.Alveolar macrophages from COPD promoted the production of mucins MUC5AC,MUC5B and MUC2 in airway epithelial cells and inhibited the expression of CFTR.3.The levels of tumor necrosis factor(TNF-?)and interleukin-6(IL-6)secreted by airway epithelial cells co-cultured with alveolar macrophages from COPD weresignificantly higher than those from the control group(P<0.001).II.Effects of exosomes derived from alveolar macrophages of COPD on the function of airway epithelial cells1.The exosomes could be successfully extracted by SBI kit.The specific marker protein on the surface of exosomes was detected by Western Blot.The structure of exosome was clear and recognizable under the electron microscope,and the vesicle-like structure was visible.The median diameter of exosomes derived from macrophages in the COPD group was 139.6nm,with a concentration of 2.8×10~7/ml,and the median diameter of exosomes derived from macrophages in the control group was 139.8nm,with a concentration of 2.1×10~7/ml by Nanoparticle Tracking Analysis.There was no significant difference in the size and concentration of exosomes between the two groups.2.GEO database analysis and mi RNA array analysis showed that mi R-380 was the significantly up-regulated mi RNA in exosomes derived from alveolar macrophages.Exosomal mi R-380 enhanced the proliferative activity and migration ability of airway epithelial cells(P<0.05),promoted the expression of mucins such as MUC5AC,MUC5B and MUC2 and inhibited the expression of CFTR in airway epithelial cells(16HBE).III.The mechanism of the effect of exosomes derived from alveolar macrophages of COPD on the function of airway epithelial cells1.The target gene of exosomal mi R-380 is cystic fibrosis transmembrane conductance regulator(CFTR).2.The construction of CFTR-si RNA in airway epithelial cells led to the decrease of CFTR.The proliferation activity and migration ability of airway epithelial cells 16HBE was enhanced after the target gene CFTR was blocked(P<0.05).ConclusionsAlveolar macrophages from COPD can enhance the proliferation and migration of airway epithelial cells,and promote the expression of mucin and proinflammatory mediators such as IL-6 and TNF-?in 16HBE.The content of mi R-380 in exosomes derived from alveolar macrophages of COPD was significantly increased.The increased proliferative activity and migration ability of airway epithelial cells and the increased expression of mucin may be related to the exosome delivering mi R-380 to airway epithelial cells as well as the negative regulation of CFTR gene leads to the down-regulation of CFTR protein.