Effect And Mechanism Of Human Umbilical Cord Mesenchymal Stem Cell-derived Exosomes In The Treatment Of Severe Steroid-resistant Asthma

Background:Severe steroid-resistant asthma(SSRA)is a serious problem in clinical asthma management.The affected patients have severe clinical symptoms,deteriorated quality of life,and are ineffective in the first-line treatment of asthma with steroids.People urgently need effective and effective treatment programs.It will be helpful to find an effective treatment plan through exploring and understanding of the pathogenesis of SSRA in depth.Airway inflammation is one of the important pathological manifestations of SSRA,and it is also a key factor that mediates the occurrence and development of the disease.Therefore,reducing airway inflammation is the key to controlling asthma symptoms.Immune cells and related cytokines play an important role in regulating airway inflammation and airway remodeling.Among them,macrophages are the most abundant immune cells in lung tissue,being involved in the process of airway inflammation.Studies have shown that under different conditions,macrophages can be polarized into two types,the M1 type that promotes inflammation,and the M2 type,which is mainly involved in tissue damage repair.Macrophages with different polarization states will affect the progression and outcome of airway inflammation.Therefore,regulating the polarization phenotype of macrophages can be a new treatment option for SSRA.As a feasible and effective biological therapy,mesenchymal stem cell therapy has a significant role in immune regulation and tissue damage repair.It has broad application prospects in the treatment of immune-related diseases.Current studies believe that the immunomodulatory effect of mesenchymal stem cells is mainly due to its paracrine mechanism.Mesenchymal stem cell exosomes(MSC-Exo),as an important paracrine factor,have immunomodulatory functions similar to their secreted cells.Moreover,MSC-Exo is more suitable for clinical application than mesenchymal stem cells,because nanoscale exosomes do not have problems such as pulmonary vascular bed obstruction and immune rejection.MSC-Exo has become the most attractive candidate drug for the treatment of pulmonary inflammatory diseases due to its powerful immunomodulatory effect.However,it is still unclear how MSC-Exo plays a role in regulating pulmonary inflammatory response and whether it can play a therapeutic role on SSRA by regulating macrophage polarization.Therefore,it is of great significance to elucidate the inherent immunopathological mechanism of SSRA and provide new medical strategies for its clinical treatment.Objective : This study intends to investigate whether MSC-Exo can reduce the pulmonary inflammatory response of SSRA by regulating the transformation of macrophage polarization phenotypes,and to explore possible molecular mechanisms,so as to provide theoretical support and experimental basis for the application of its transformation in clinical treatment.Method:(1)Exosomes were isolated from medium supernatant of human umbilical cord mesenchymal stem cell(h UCMSC)using Qiagen Exoeasy Maxikit.Exosomes were identified by transmission electron microscopy,particle tracking analysis and flow cytometry.(2)An OVA/ CFA-induced SSRA mouse model was established for the treatment of MSC-Exo by in-situ bronchial injection.The therapeutic effect of MSCExo on SSRA was evaluated by detecting the airway lung function,lung histopathology and the levels of macrophage polarization related cytokines in SSRA mice.(3)The macrophages in mice were systematically depleted by intraperitoneal injection of clodronate liposomes.The depletion effect was identified by flow cytometry.And further investigated whether the therapeutic effect of MSC-Exo was weakened after the clearance of macrophages.(4)In vitro inflammatory model of LPS-stimulated RAW264.7 cells was established and was co-cultured with MSC-Exo.The macrophage polarization markers of M1 and M2 type macrophages were detected by q RT-PCR,ELISA,Western blot,immunofluorescence and flow cytometry.The regulation effect of MSC-Exo on macrophage polarization and its related mechanism were further discussed.(5)Quantitative proteomics sequencing using Tandem Mass Tags(TMT)tagging was used to detect the changes in protein expression profile during MSC-Exoregulated macrophage polarization.(6)The key proteins screened by proteomics sequencing were verified by Western blot,and the role of the key protein in MSC-Exo regulating macrophage polarization was further verified by si RNA silencing and overexpressed plasmid transfection techniques.Result:(1)Disk-like vesicles with circular lipid twin layer structure and typical exosome characteristics were successfully isolated from h UCMSC,with particle diameters between 50 and 150 nm and high expression of specific marker proteins CD63 and CD81 on the surface.(2)Tracheal administration of MSC-Exo can effectively improve the lung function of SSRA mice,reduce the infiltration of inflammatory cells in lung tissue,inhibit the hypersecretion of airway mucus and reduce the level of inflammatory factors in lung tissue.(3)Systemic depletion of macrophages weakened the therapeutic effect of MSC-Exo,suggesting that macrophages may be the target of the immunomodulatory effect of MSC-Exo.(4)In an in vitro inflammatory model of LPS-stimulated RAW 264.7 cells,it was found that MSC-Exo inhibited M1 macrophage polarization and promoted M2 macrophage polarization.(5)Tumor necrosis factor receptor-associated factor 1(TRAF1)was found to be the key protein in the regulation effect of MSC-Exo on macrophage polarization by TMT labeled quantitative proteomics sequencing analysis.The expression level of TRAF1 was verified both in vivo and in vitro and the experimental results were consistent with the results of proteomics sequencing.(6)In an in vitro inflammatory model of LPSstimulated RAW 264.7 cells,RAW264.7 cells were transfected with TRAF1 si RNA and overexpressed plasmid respectively,and the related protein pathways were verified.It was found the influence of MSC-Exo on the NF-?B and PI3K/AKT signaling pathways that regulate the macrophage polarization was TRAF1 dependent.Conclusion:1.MSC-Exo can reduce the pulmonary inflammation in the SSRA mouse model induced by OVA/CFA.2.MSC-Exo exerts an inhibitory effect on the pulmonary inflammation in SSRA mice induced by OVA/CFA through macrophages.3.MSC-Exo may regulate NF-?B and PI3K/ AKT signaling pathways by inhibiting TRAF1 to inhibit M1 polarization and promote M2 polarization of macrophages.Innovation:1.This study found for the first time that the therapeutic effect of MSC-Exo on OVA/CFA-induced SSRA mouse models,which supplemented the development value of MSC-Exo as a candidate therapeutic drug for SSRA,and provided a theoretical basis for formulating new and effective SSRA treatment strategies.2.This study is the first to clarify the important role of macrophages in the treatment of OVA/CFA-induced SSRA mouse model by MSC-Exo.3.This study for the first time clarified the molecular mechanism of MSC-Exo in the treatment of SSRA,which is regulating macrophage polarization via inhibiting TRAF1.