The Role Of Bone Marrow-derived Mesenchymal Stem Cells In The Regulation Of Inflammatory Response And The Repair Of Alveolar Epithelial Cells In Acute Lung Injury Of The Juvenile SD Rats

Objective:To establish an acute lung injury(ALI)model in juvenile SD rats by inhalation of lipopolysaccharide(LPS).To investigate the changes in expression levels of inflammatory factors such as Toll-like receptor(TLR)-4,nuclear factor(NF)-?B,tumor necrosis factor(TNF)-?,interleukin(IL)-17a and vascular factors such as vascular endothelial growth factor(VEGF),von Willebrand factor(vWF)in lung tissue of ALI animal model.To observe the degree of repair of lung tissue injury after transplantation of exogenous bone marrow-derived mesenchymal stem cells(BMSCs)in ALI animal model.To establish a model of LPS attacking alveolar type II epithelial cells(AEC?),simulate endotoxin-induced lung injury,and to explore whether BMSCs could attenuate the damage of LPS to AEC?and promote its repairing by using indirect co-culture method.Methods:1.Animal experiment:144 juvenile male SD rats were randomly divided into three groups:LPS group(model group),control group,LPS+BMSCs group(treatment group),and each group was divided into 3h,6h,12 h,1d,2d,3d,7d,14d,8subgroups(6 rats in each group).The rats in the model group and the treatment group were aerosol inhaled with LPS(5mg/ml,2ml,20min)to establish the juvenile rats model of ALI.The rats in the control group were inhaled with the equal amounts of saline.After 1h,the rats in the treatment group were injected the BMSCs into the tail vein(1×10~7/kg,1×10~6/ml),the model group and the control group by tail vein injection of equal amounts of dulbecco's modified eagle medium(DMEM).HE staining was used to observe the pathological changes of lung tissue,and,to evaluate pulmonary edema by using W/D ratio and BALF cell count and cell smear.The expression of VEGF,NF-?B and IL-17a were detected by immunohistochemistry and VEGF,vWF,TLR-4,NF-?B,TNF-?,IL-17 were detected by western blotting.2.Cell experiment:four groups:A:DMEM+BMSCs group,B:DMEM group,C:LPS group,D:LPS+BMSCs group.The AEC?in C and D group were stimulated with 10?g/ml LPS.The same amount of DMEM was added to Group A and B.After 4 h,BMSCs were seeded in small aperture 0.4?m transwell inserts in A and D groups,B and C group only add a transwell insert,without BMSCs.After culturing the cells for 24 h,the activity of each group of AEC?was detected by cell proliferation-toxicity detection reagent.Results:1.Animal experiment:Histopathological examination showed typically visible inflammation of lung tissue in model group,including the disappearance of the normal structure of alveolar,alveolar wall thickening,alveolar capillary congestion,alveolar hemorrhage and inflammatory cell infiltration,pulmonary interstitial edema,hyaline membrane formation and lung injury lesions;the degree of lung injury in the treatment group was significantly lower than that in the model group.At early phase,compared with the control group,the model group of juvenile rat lung tissue W/D ratio,BALF cell count and smear and the expression of VEGF,vWF,TLR-4,TNF-?,NF-?B,IL-17(a)protein were increased significantly(P<0.05).Compared with the model group,these were significantly decreased in the treatment group(P<0.05).2.Cell experiment:The absorbance(the OD value)of each group was detected by CCK-8.There was no significant difference between the OD values of group A and group B,indicating that BMSCs had no effect on normal AEC?.The OD value of group C was significantly lower than that of group B(P<0.05),indicating that the AEC?injury by using LPS was successful.The OD value of group D was significantly higher than that of group C(P<0.05),indicating that BMSCs could attenuate the AEC?injury and promote the repair of AEC?.Conclusion:The pathogenesis of ALI induced by LPS may be related to the expression of VEGF,vWF,TLR-4,NF-?B,TNF-?and IL-17(a).Compared with the model group,the damage of lung injury in treatment group was mitigated with application of BMSCs,and the expression of VEGF,vWF,TLR-4,NF-?B,TNF-?and IL-17(a)protein in the lung tissue of the treatment group were significantly decreased.Those indicate that BMSCs can inhibit the inflammatory response of ALI lung tissue and promote the repair of lung injury.BMSCs have no effect on normal AEC?,but can reduce LPS damage to AEC?and promote its repair.