The Research Of Protective Effects Of BMSC-derived Exosomes On Smoke Inhalation Lung Injury

ObjectiveSmoke inhalation lung injury is a serious acute respiratory disease in clinic.Due to the limited effectiveness of current therapies,patients with smoke inhalation lung injury have high disability rate,mortality and poor prognosis.The study explored the therapeutic effect of rat bone marrow mesenchymal stem cells derived exosomes on smoke inhalation lung injury in vivo and in vitro.MethodsThe first step: Bone marrow mesenchymal stem cells isolated from the femur and tibia of SD rats were cultured by adherent culture method.The second step: The morphology of bone marrow mesenchymal stem cells was observed under a microscope.And the expression of bone marrow mesenchymal stem cells surface marker proteins CD29,CD44,CD90,CD105,hematopoietic stem cell surface marker proteins CD34 and CD45 were detected by flow cytometry.The third step: Exosome extraction kit was used to extract the rat bone marrow mesenchymal stem cells derived exosomes.Nano particle tracking analysis(NTA)was used to mearsue size distribution of exosomes.Transmission electron microscopy was used to scan exosome structure.Western blot was used to detect the expressions of surface markers(CD9,CD63,CD81).The fourth step: A selfmade smoke generator was used to construct smoke inhalation lung injury models of SD rats,which subsequently treated with the exosomes.H&E staining was used to detect the pathological change of rat lung tissue.The apoptosis of lung tissue was measured by TUNEL staining.The pulmonary vascular permeability was detected by lung wet-dry ratio,and the concentration of protein in bronchoalveolar lavage fluid was detected via BCA method.The releases of TNF-α,IL-1 β and IL-6 in bronchoalveolar lavage fluid(BALF)were detected by enzyme linked immunosorbent assay(ELISA),and the expressions of inflammatory and apoptosis related proteins in lung tissue were detected by qRT-PCR and western blot.The fifth step: BEAS-2B and A549 cells were treated with smoke medium made by self-made smoke generator to establish the cell model of smoke inhalation injury.Then the cells were treated with exosomes.CCK-8 assay was used to detect the changes of cell proliferation,LDH assay was used to detect the cytotoxicity,ELISA assay was used to detect the releases of TNF-α,IL-1 β and IL-6 in the supernatant of cell culture medium.qRT-PCR and westernblot were used to detect the expressions of inflammatory and apoptosis-related proteins.The sixth step: After the treatment of smoke medium,BEAS-2B and A549 cells were treated with exosomes and HMGB1 overexpression plasmid respectively.CCK-8 assay was used to detect the cell proliferation,LDH assay was used to detect the cytotoxicity,ELISA assay was used to detect the releases of TNF-α,IL-1 β and IL-6 in the supernatant of cell culture medium.qRT-PCR and westernblot were used to detect the expression of inflammatory,apoptosis-related proteins and NF-κB signal pathway.Results1.Bone marrow mesenchymal stem cells were successfully isolated and cultured by adherent culture method,and the cell morphology showed a unique fusiform shape of bone marrow mesenchymal stem cells.The flow cytometry results showed that the positive rates of surface markers of hematopoietic stem cells such as CD34,CD45 were less than 3%.The positive rates of surface markers CD44,CD90,CD105 and CD29 of bone marrow mesenchymal stem cells were all more than 90%.2.The results of NTA showed that the diameter of exosome was mainly distributed between 30-150 nm,and the results of transmission electron microscope showed vesicles with clear membrane structure.Western blot results showed that the surface markers CD9,CD63 and CD81 were expressed.Combined with these results,it confirmed that the vesicles extracted from bone marrow mesenchymal stem cells are exosomes.3.After the exosomes treatment for 48 h,the results showed that compared with rats with inhalation injury,the injury of lung tissue was significantly alleviated,the texture and alveolar structure of lung tissue were clearer,the thickening of pulmonary septum and intrapulmonary hemorrhage were significantly improved.The dry-wet ratio results of lung suggested that the pulmonary vascular permeability was significantly improved,the concentration of protein in bronchoalveolar lavage fluid was significantly decreased.TUNEL staining results indicated that the apoptosis of lung tissue was significantly reduced.ELISA results showed that the releases of TNF-α,IL-1 β and IL-6 in bronchoalveolar lavage fluid were decreased significantly.And the results of qRT-PCR and western blot showed that the expression of inflammatory and apoptosis related proteins in lung tissue decreased significantly.4.Compared with BEAS-2B and A549 cells treated with smoke medium alone,the proliferation of smoke-injured cells treated with smoke/exosomes was significantly enhanced and the cytotoxicity was decreased.ELISA results suggested that the releases of inflammatory factors decreased.qRT-PCR results suggested that the m RNA expression level of inflammatory factors decreased,and western blot results shown that the expression levels of inflammatory and apoptosis-related proteins were decreased.5.On the basis of smoke medium treatment of BEAS-2B and A549 cells,exosome could inhibit the decrease of cell proliferation,increase of cytotoxicity,production and release of inflammatory and apoptotic factors caused by smoke medium.Overexpression of HMGB1 could aggravate the effect of smoke medium.However,exosomes could effectively inhibit the pro-inflammatory and apoptotic effects of HMGB1.Conclusion1.Bone marrow mesenchymal stem cells were successfully isolated and cultured by adherent culture method,and the exosomes derived from bone marrow mesenchymal stem cells were successfully extracted and confirmed.2.The self-made smoke generator successfully established the model of smoke inhalation lung injury in SD rats.3.The exosomes derived from bone marrow mesenchymal stem cells could effectively inhibit smoke inhalation lung injury in SD rats.4.Exosomes derived from bone marrow mesenchymal stem cells could effectively suppress smoke injury in BEAS-2B and A549 cells.5.Exosomes derived from bone marrow mesenchymal stem cells could play the protective role in cells treated with smoke injury by inhibiting HMGB1/NF-κB signal pathway.