MiR-186-5p From M2 Macrophage-derived Exosomes Alleviates Allergic Asthma Via TRPP2 In Airway Smooth Muscle Cells In Mice

Background:Bronchial asthma is one of the most common chronic diseases in the world,with nearly 2 million deaths each year due to severe exacerbations.Allergic asthma is a common type of bronchial asthma,mostly triggered by exposure to pollen,dust and other exogenous allergens,and seriously affecting the quality of patients life.Macrophages are one of the important aspects of intrinsic immunity and play an significant role in inflammation as well as host defense.The functions performed in macrophages are determined by their different polarization states.M2 macrophages have anti-inflammatory effects and the function of exosomes depends on their secretory cells.Therefore,this study uses M2 macrophage-derived exosomes as an entry point to investigate the effects of M2 macrophage-derived exosomes on allergic asthma and their specific mechanisms.Methods: We used C57BL/6J male mice to construct an allergic asthma model to explore the effects of different macrophage-derived exosomes on respiratory rate,tracheal tone,alveolar lavage fluid inflammatory factors,serum Ig E and lung inflammatory infiltration in mice with allergic asthma;the isolated trachea of C57BL/6J male mice was selected for primary airway smooth muscle cell culture,and LPS was selected to induce primary airway smooth muscle cells.MiR-186-5p-NC,miR-186-5p-inhibitor and miR-186-5p-mimics were transfected to knockdown and overexpress miR-186-5p levels;Detection of target protein expression levels in M2 macrophages and airway smooth muscle cells by Western blot.Results:（1）M2macrophage-derived exosomes reduced respiratory rate,tracheal tone,inflammatory factor IL-5 and leukocyte count in alveolar lavage fluid and serum Ig E expression of mice.（2）CCK-8 assay detects the effect of cell viability in LPS-induced airway smooth muscle cells and shows that M2 macrophage-derived exosome reduce PCNA expression in LPS-induced airway smooth muscle cells.（3）The differentially expressed miRNAs in M2 macrophage-derived exosome and targeting pkd2 were analyzed by the Biosign database library,and then only miR-186-5p could be detected in peripheral blood by the EVmiRNA online database.Validation of miR-186-5p high expression in both M2 macrophages and M2 macrophage-derived exosome by q-PCR.（4）MiR-186-5p-inhibitor was transfected at the cellular level and verified using q-PCR that both M2 macrophages and M2 macrophage-derived exosomes were lowly expressed after knockdown.（5）After collecting knockdown miR-186-5p exosomes treated mice with asthma model show that the respiratory rate,tracheal tone,IL-5,IL-13,TNF-α and leukocyte count in alveolar lavage fluid and Ig E expression level in serum were increased.Also the TRPP2 expression were increased in airway smooth muscle cells.（6）Collection of knockdown miR-186-5p exosomes treated with LPS-induced airway smooth muscle cells showed elevated levels of PCNA and TRPP2 expression in cells.Conclusion: MiR-186-5p carried by M2 macrophage-derived exosomes alleviates allergic asthma inflammatory response and airway hyperresponsiveness by reducing TRPP2 expression in airway smooth muscle in mice.