Effect And Mechanism Of Exosomes Derived From Bone Marrow Mesenchymal Stem Cells On EMT In COPD Rats

ObjectiveThe aim of this study was to investigate the inhibitory effect of exosomes derived from bone marrow mesenchymal stem cells（BMSC-Exos）on epithelial-mesenchymal transformation（EMT）induced by lipopolysaccharide and cigarette in chronic obstructive pulmonary disease（COPD）rats,and to explore the mechanism of this inhibitory effect.Methods1.Isolation and identification of bone marrow mesenchymal stem cells and extraction of exosomesThe BMSCs were cultured by whole bone marrow adherence method and detected by flow cytometry.The exosomes from the third generation BMSCs were isolated and purified by exosomal concentration kit and purification kit,and identified by transmission electron microscopy（TEM）,Nanoparticle Tracking Analysis（NTA）and Western Blotting.2.Establishment of COPD rat modelThe COPD rat model was established by LPS and fumigation.On the 1st and 14 th day,LPS was injected into the trachea of each rat except the control group,200μg/200μl.Passive smoking（tar 25mg;CO 13mg;nicotine 1.1mg）shall be put in the smoke box in the morning and afternoon for 2 13 and 15 28 days,and the interval between two fumigations shall be 0.5h each time or equal to 6h.Rats in control group were injected with the same amount of saline.3.Experimental groupsForty-two 4-week-old SD rats were randomly divided into 7 groups,6 in each group.Control group（Control）: intratracheal infusion and atomization equivalent amount of PBS;Model group（Model）: LPS infusion combined with fumigation model,atomization equivalent amount of PBS;Low dose BMSC-Exos atomization group（Low-MSC-Exos（IN））: model rats,atomization inhalation 0.5×108 particles/kg of BMSC-Exos suspension 1ml;Medium dose BMSC-Exos atomization group（Mid-MSC-Exos（IN））: model rats,atomization inhalation 1.0×108 particles/kg of BMSC-Exos suspension 1ml;High dose BMSC-Exos atomization group（High-MSC-Exos（IN））: model rats,atomization inhalation 1.5×108particles/kg of BMSC-Exos suspension 1ml;BMSC-Exos tail vein injection group（MSC-Exos（IV））:model rats were injected with 1.5×108 particles/kg BMSC-Exos suspension in tail vein;Hormone nebulization group（Hormone（IN））: model rats,atomization inhalation pulmicort（1mg/rat）.Each treatment group was treated continuously for 5 days for 30 minutes.4.Effects of atomized BMSC-Ex OS on systemic and local inflammation and EMT-related mechanisms in COPD ratsOn the 28 th day after the intervention treatment,the pulmonary function was measured,the rats were dissected,lung tissue samples,serum and BALF were collected.The pulmonary function of each group was compared and COPD model was established successfully.The levels of pro-fibrosis factor-β1（TGF-β1）,interleukin-1β（IL-1β）and tumor necrosis factor-α（TNF-α）in BALF and serum were determined by ELISA.Pulmonary tissue changes were assessed using hematoxylin and eosin（HE）and masson staining.Western blotting detects the expression of markers associated with the EMT process,including E-cadherin（E-cad）,cytokeratin-19（CK19）andα-smooth muscle actin（α-SMA）,and immunohistochemical staining（IHS）detects the expression of markers associated with the Wnt/β-catenin signaling pathway,including P-GSK-3β,GSK-3β,β-catenin and cyclin D1.Results1.Characterization of BMSCs and BMSC-ExosIn the third generation,rat BMSCs showed long spindle shape and aggregated growth.Flow cytometry analysis showed that BMSCs highly expressed of CD73,CD90 and CD105,and lowly expressed of CD14,CD19,CD34,CD45 and HLA-DR.Exosomes were separated and purified by molecular exclusion chromatography（SEC）and affinity chromatography concentration.The results of TEM showed that the exosomes were typical cup-shaped.NTA results indicate that the peak diameter of BMSC-Exos is 91.7nm,accounting for 97.3% of all particles,and the particle concentration was 3.3×106/ml.The results of Western blotting showed that exosome surface proteins CD9 and CD63 were highly expressed.2.Lung function of rats in each groupCompared with the control group,the lung function of the model group was severely damaged,but the lung function of COPD rats was significantly improved after BMSC-Exos atomization.Compared with the model group,the levels of 0.2 second forced expiratory volume（FEV0.2）,0.3 second forced expiratory volume（FEV0.3）,0.2sec forced expiratory volume/Forced lung capacity（FEV0.2/FVC）,0.3sec forced expiratory volume/Forced lung activity（FEV0.3/FVC）,Expiratory flow rate at25%FVC（FEF25%）,Expiratory flow rate at 50%FVC（FEF50%）were significantly increased in all of the BMSC-Exos atomization groups（P < 0.05）。3.Levels of TNF-α,IL-6 and TGF-β1 in serum and BALF of rats in each groupAtomized exosomes can reduce the inflammatory response and fibrosis process of COPD,and the effect of low dose atomization is the most significant.Compared with model group,the contents of TNF-α,IL-6 and TGF-β1 in serum and BALF were decreased of BMSC-Exos atomization group,BMSC-Exos tail vein group and hormone group（P < 0.05）.Among them,the inflammatory factors and fibrosis factors in the low-dose BMSC-Exos atomization group were decreased more significantly than those in the other treatment groups（P < 0.05）.4.Pathological changes of lung tissues in each groupAfter treatment with BMSC-Exos,there was a significant reduction in airway inflammation and alveolar destruction in COPD.But the effect of caudal vein injection of BMSC-Exos and aerosol hormone was not significant.HE staining and Masson staining showed that the alveolar structure of the control group was normal,no obvious infiltration of inflammatory cells was observed,and collagen deposition was less under the trachea.Compared with the control group,the alveolar structure of model group was damaged,and a large number of inflammatory cells infiltrated around blood vessels and bronchi,with more blue collagen fiber positive areas.However,after BMSC-Exos atomization treatment,alveolar fusion and inflammatory cell infiltration decreased,the area of blue collagen fibers decreased,and the inflammatory score decreased significantly（P < 0.05）.5.Expression of EMT-related proteins in different treatment groupsLPS in combination with cigarette smog-induced COPD promoted EMT processes in airway epithelial cells,whereas low and medium dose BMSC-Exos in atomized form inhibited this process.Western blotting results showed that,compared with control group,The expression levels of CK19 and E-cad in the epithelial cells of the model group were significantly reduced,while α-SMA protein expression level was significantly increased（P < 0.05）.The levels of CK19 and E-cad increased and α-SMA decreased after low and medium doses of BMSC-Exos and hormone nebulization treatment（P < 0.05）.The level of α-SMA in high dose BMSC-Exos atomization group and tail vein group was significantly higher than that in model group（P < 0.05）.6.Expression of Wnt/β-catenin signaling pathway related proteins in different treatment groupsAfter BMSC-Exos atomization treatment,the Wnt/β-catenin signaling pathway showed high expression of negative regulatory genes and low expression of downstream genes.The results of Immunohistochemical showed that the expression of Cyclin D1,β-catenin and P-GSK-3β were increased and GSK-3β expression was decreased in the model group compared with the control group（P < 0.05）.The expression of Cyclin D1,β-catenin and P-GSK-3β decreased and GSK-3β increased after low dose of BMSC-Exos atomization（P < 0.05）.In the high dose BMSC-Exos atomization group,tail vein group and hormone group,there was no significant difference in the expression of β-catenin and P-GSK-3β compared with the model group（P < 0.05）.Conclusion1.Atomized BMSC-Exos can improve lung function,inflammation and fibrosis,reduce epithelial cell transformation and reverse the process of EMT in COPD model rats.2.In COPD model rats,the Wnt/β-catenin signaling pathway,which is closely related to EMT,is abnormally activated,and this signal is attenuated after atomization of BMSC-Exos,which may be one of the mechanisms of its inhibition of EMT process.