The Protective Effect And Mechanism Of Umbilical Cord Mesenchymal Stem Cells On Lipopolysaccharide-induced Acute Lung Injury In Mice

BackgroundAcute lung injury（ALI）refers to the gradual increase in breathing difficulties after the body is attacked by various non-heart disease factors,leading to the formation of diffuse lung injury,pulmonary edema,and continuous lung inflammation,and finally developing into acute respiratory distress syndrome（ARDS）.The basic pathological changes of ALI mainly include the increased permeability of pulmonary capillary endothelial cells and alveolar epithelial cells,aggregation of inflammatory cells,release of a large number of inflammatory factors,a considerable accumulation of fibrin,formation of hyaline membrane,and fibrosis.The clinical manifestations are severe breathing difficulties.Currently,the therapeutic strategies for ALI mainly include the primary disease treatment,respiratory support treatment,fluid management,and glucocorticoids treatment.However,the above-mentioned treatment methods cannot effectively improve the prognosis of ALI,which has become a common cause of morbidity and death in clinical critical care medicine with a mortality rate as high as 35%to 40%.Therefore,a novel effective therapy for ALI is urgently needed.Recently,cell-based therapies,especially using mesenchymal stem cells（MSCs）,have been developed as an attractive strategy for tissue injury and inflammation owing to their unique immunomodulatory properties.MSCs refer to pluripotent stem cells derived from bone marrow,fat,cartilage,placenta,umbilical cord,and other tissues.They have multi-directional differentiation potential,immunomodulatory properties,self-renewal capacity,and anti-inflammatory effects.In this study,we chose human umbilical cord mesenchymal stem cells（UC-MSCs）for research due to the superiorities including painless collection procedure,easy amplification in vitro,low immunogenicity,and strong ability to secrete cytokines（such as IL-10,TSG-6,and PGE₂）and other characteristics.Therefore,the purpose of this study is to explore the therapeutic effect of UC-MSCs on lipopolysaccharide（LPS）-induced ALI in mice and its underlying mechanism.Methods1.To study the effect of UC-MSCs on LPS-induced ALI in mice and its underlying mechanism.In vivo,24 male BALB/c mice were randomly divided into 4 groups（n=6）:1)Control group:mice were inhaled 0.9%saline by aerosol for 20 min.4 h later,the tail vein was injected with 100μL of PBS;2)UC-MSCs group:mice were inhaled 0.9%saline by aerosol for 20 min.4 h later,the tail vein was injected with UC-MSCs（5×10⁵,100μL）;3)LPS group:mice were inhaled LPS by aerosol for20 min.4 h later,the tail vein was injected with 100μL of PBS;4)LPS+UC-MSCs group:mice were inhaled LPS by aerosol for 20 min.4 h later,the tail vein was injected with UC-MSCs（5×10⁵,100μL）.Mice were sacrificed 24 hours later.The pathological changes of lung tissue were observed under the light microscope.The total cell number and total protein concentration of the bronchoalveolar lavage fluid（BALF）were measured.The m RNA and protein expression of inflammatory factors in lung tissues were measured by q RT-PCR and immunohistochemistry.The level of apoptosis in lung tissue was detected by TUNEL assay.Immunofluorescence staining was used to detect the infiltration of macrophages and the expression of PD-L1 in lung tissue.2.To further explore the effect of UC-MSCs on the secretion of cytokines in mouse macrophages induced by LPS,and the mechanism of UC-MSCs on the interaction between macrophages and T cells,we designed the following in vitro experiments.（1）The effect of UC-MSCs on the secretion of PGE₂ after receiving LPS stimulation.LPS（100ng/m L）was used to stimulate UC-MSCs,and unstimulated UC-MSCs were used as controls.After 12 hours,cell supernatants and cellular proteins were collected.ELISA was used to detect PGE₂ secretion,and Western Blot was used to detect COX-2 expression.（2）The effect of UC-MSCs on the secretion of cytokines from mouse RAW264.7macrophages was analyzed.RAW264.7 cells were divided into 5 groups:1)Control group;2)UC-MSCs-condition medium（CM）group;3)LPS group（100ng/m L）;4)LPS+UC-MSCs-CM group;5)LPS+UC-MSCs-CM+PGE₂ inhibitor（Pyranoprofen）group.The above groups were treated for 12 hours.The m RNA expression of cytokines in RAW264.7 cells was detected by q RT-PCR.（3）Analysis of UC-MSCs affecting T cell activity through macrophages.Jurkat T cells were divided into 3 groups（n=3）:1)Control group;2)Jurkat+RAW264.7group;3)Jurkat+UC-MSCs-CM-treated Raw264.7 group.The above groups were treated by LPS（100 ng/m L）for 24 hours.The m RNA expression of IL-2,IFN-γand PD-1 in Jurkat T cells was detected by q RT-PCR.Results1.The total number of cells and protein concentration in BALF were significantly reduced by UC-MSCs,including reducing the damage of alveolar epithelial cells and alveolar endothelial cells and reducing permeability..2.UC-MSCs could improve the pathological damage of lung tissue,including thinning of alveolar wall,reduction of congestion and bleeding and so on,while reducing the degree of inflammatory cell infiltration and apoptosis.3.The secretion of pro-inflammatory factors（IL-1β,MCP-1 and TNF-α）in mice lung tissue was significantly reduced by UC-MSCs,and the secretion of anti-inflammatory factors（IL-10）was promoted.4.The inflammatory infiltration of macrophages in mice lung tissue was significantly reduced by UC-MSCs,and the expression of PD-L1 in lung tissue was enhanced.5.UC-MSCs could reduce the expression of pro-inflammatory factors（IL-1β,MCP-1 and IL-6）in RAW264.7 cells by secreting PGE₂,and promote the expression of anti-inflammatory factors（IL-10）and PD-L1.6.UC-MSCs could up-regulate the expression of PD-1 on the surface of T lymphocytes and reduce the expression of IL-2 and IFN-γthrough macrophages,thereby reducing inflammation.ConclusionOur research confirmed that UC-MSCs could improve acute lung injury in mice.In terms of mechanism,UC-MSCs could inhibit the secretion of pro-inflammatory factors in macrophages by secreting PGE₂ to reduce lung inflammation caused by LPS.UC-MSCs could also promote the expression of PD-L1 on the surface of macrophages by secreting PGE₂,and inhibit the immune activity of T lymphocytes through macrophages,indirectly exerts a protective effect on ALI.