Study On The Efficacy And Molecular Regulation Mechanism Of BM-MSCs In Repairing Acute Lung Injury Through Notch3 Pathway

Acute lung injury(ALI)/ Acute respiratory distress syndrome(ARDS)is mainly caused by increased permeability of lung endothelium and epithelial cells,and is significantly characterized by acute hypoxemia and respiratory failure,and other clinical syndrome.There are approximately 3 million patients compromised of ALI /ARDS in the worldwide each year,accounting for 10% of patients admitted to Intensive Care Units(ICU),but the mortality of ARDS is very high at 30%-46%.In recent years,medication of ALI / ARDS were mainly supportive,while other treatments include high-frequency low-tidal volume ventilator-assisted ventilation,extracorporeal carbon dioxide removal(ECCO2R),prone ventilation,statins and high-frequency oscillatory ventilation(High frequency ventilation,HFOV).Effective treatments of ARDS are still unclear,and life supportive treatments such as mechanical ventilation have not significantly reduced mortality.Therefore,exploring effective treatments for ALI /ARDS has become the main objective of acute lung injury and repair in the future.Bone mesenchymal stem cells(BM-MSCs)are the earliest discovered mesenchymal stem cells(MSCs).They have low immuno-resistance potency,multi-directional differentiation,immune regulation,inflammatory intervention,and homing capacity.Its potentials of cell therapy could be applied in treatments for ALI.Previously,MSCs have the potential to reduce lung tissue damage caused by ALI /ARDS,but therapeutic mechanisms of MSCs has not been fully elucidated.In this study,we first performed primary isolation,culture,proliferation,cell viability testing,and phenotypic characterization of BM-MSCs.We found that cultured BM-MSCs were spindle-shaped and obtained osteogenic,adipogenic,and chondrogenic differentiation.The differentiation ability and phenotype biomarkers are in accordance with MSCs standards established by the International Society for Celluar Therapy.Mouse model of acute lung injury were used to explore the molecular mechanism of BM-MSCs in the treatment of ALI.After LPS-induced mice model were treated by BM-MSCs,blood,alveolar lavage fluid(BAL),and lung tissue specimens were obtained at 6 and 24 hours after model establishment.The examinations included histopathology analysis,ELISA,immunofluorescence,and Western blotting.Which were used to show changes in injury and apoptosis,inflammatory factor activity,and expression activity of signal molecules related to the Notch signaling pathway.In a LPS-treated mouse model of acute lung injury,groups treated with BM-MSCs transplantation were found that a significant reduction of inflammation by histopathological examination,decreased expressions of proinflammatory factors IL-1β,IL-6 and TNF-α in serum and alveolar lavage fluid.Results of TUNNEL test showed that lung cells had reduced apoptosis in BM-MSCs treated groups.Immunofluorescence showed that mouse lung epithelial cells co-expressed SP-C combined by GFP significantly,along with BM-MSCs embeded in the lung tissues for 24 hours.Notch signaling pathway is one of the important molecular mechanisms in the development of lung tissues.Results of Westernblot test found that Notch2,Notch3 and its ligandsJagged1 and nuclear protein Hes1 protein in the LPS group were higher than those in the sham groups at 6hrs,and significantly decreased after receiving BM-MSCs treatments(LPS+CELL groups).At 24 hours,Notch3,Jagged1,and Hes1 were significantly increased in the LPS group,while Notch3,Jagged1 and nuclear protein Hes1 were significantly higher in LPS+CELL groups than that of LPS groups.Conclusively,it was found that BM-MSCs obtained from bone marrow by adherence method,and accordance with the biological characteristics of MSCs.It was found that the Notch3-Jagged1-Hes1 signal pathway may be one of the molecular mechanisms of BM-MSCs transplantation involved in repairing acute lung injury models,which could make a further progress of research on acute lung injury and repair.