Bone Marrow Mesenchymal Stem Cell Derived Exosomes Induce Alveolar Macrophage Polarization To Ameliorate The Lung Inflammation Of Smoke Inhalation Injury

ObjectiveReducing lung inflammation following injury is the key to treating smoke inhalation injuries at an early stage.this study aimed to investigate the effects of SD rats bone marrow Mesenchymal stem cell-derived exosomes on the inflammation and protection of the lungs when injured by inhalation of smoke in vivo and in vitro.,and to explore its potential mechanism,to provide new ideas for the treatment of smoke inhalation injury.Methods1.Extracted and cultured SD rat bone marrow mesenchymal stem cells identified by flow cytometry and induced osteogenic/adipogenesis differentiation.2.Collect the bone marrow supernatant mesenchymal stem cells grown in the P3-P5 generation,and extract the exosomes.The morphology of exosomes was observed under the electron microscope,the particle size distribution of exosomes was detected by Nanoparticle Tracking Analysis(NTA),and the expression of exosomes surface protein markers has been detected by Western blot.3.Use the smoke generator to force SD rats to inhale the smoke to cause injury,and the smoke inhalation injury pattern has been established.Pulmonary tissue underwent hematoxylin-eosin(H&E)staining to observe structural changes and detect mRNA expression of inflammatory factors in pulmonary tissue by quantitative reverse transcription Polymerase chain reaction(RT-qPCR);collect the bronchoalveolar lavage fluid for the BCA method to detect protein quantitation,and the enzyme-linked immunosorbent assay(ELISA)was used to determine the expression of inflammatory factors.Examine the regulation effect of exosomes derived from mesenchymal bone marrow stem cells of lung inflammation.4.Cells NR8383(rat alveolar macrophage)were damaged by smoke extract to construct a smoke inhalation injury cell model.RT-qPCR measured mRNA expression of the inflammatory factor.Immunofluorescence was used to determine the expression of the surface marker proteins of M1/M2 macrophages in each cell group to understand the polarization of macrophages.5.Western blot to detect NF-κB/STAT3 pathway-related protein expression within each NR8383 cell group.Results1.Mesenchymal bone marrow stem cells were successfully extracted and cultured,showing a specific morphology of stem cells.The results of the flow cytometry matched the characteristics of the mesenchymal stem cells of the bone marrow.After the induction of osteogenesis/adipogenesis,the successful induction was observed under an optical microscope.2.The exosomes have been successfully extracted.The results of NTA showed that the particle size distribution was 5o to 150 nm.Electron microscopy results showed small round membrane vesicles less than 100 nm in diameter.Western blot results showed that the surface markers of exosomes CD9,CD63,and CD81 are all positive.3.Following smoke inhalation injuries,unlike untreated rats,exosome-treated rats had a more complete alveolar structure;the weakest thickening of the pulmonary septum;less inflammatory infiltration and hemorrhage;lower expression of inflammatory factors TNF-α and IL-6,the expression of the anti-inflammatory factor IL-10 was higher;the protein concentration of bronchoalveolar lavage fluid decreased;the TNF-α concentration was lower;the IL-10 concentration was higher.4.After exosomes treatment of NR8383 cells damaged by smoke extract,the expression of TNF-α and IL-6 is reduced,and the expression of IL-10 is higher than that of cells damaged by simple smoke extract;Immunofluorescence showed that the fluorescence i NOS of the M1 surface marker protein decreased,and the fluorescence of the M2 surface marker protein Arg-1 increased.5.Results from Western blot showed that in comparison with the cells damaged by smoke extract,the cells after exosomes treatment had a lower expression of IKB-αand NF-κB,also the expression of STAT3 and p-STAT3 decreased.ConclusionExosomes derived from bone marrow mesenchymal stem cells can induce alveolar macrophages polarize to M2 type,and at the same time may inhibit the activation of NF-κB/STAT3 pathway.Therefore,reducing the expression of inflammatory factors and lung injury,play the role of alleviating inflammation and protecting lung tissue following a smoke inhalation injury.