| As a representative of erosive agent,sulfur mustard(SM,dichlorodiethyl sulfide)was first discovered and artificially synthesized in the 19 th century.SM was used in World War I for the first time and later used in the Japanese invasion of China,the Iran-Iraq War,and the Syrian Civil War,which caused a large number of casualties and property damage.Because of its simple structure,easy production and stable properties,it is also known as the king of poisons.SM has a long-lasting effect,various of poisoning pathways,strong penetration,no specific prevention and treatment,so it is still one of the major chemical warfare threats faced by our military.In addition,SM is also one of the most important chemical poisons abandoned by Japanese invasion of China,posing a serious threat to the health of people and the safety of the ecological environment.Therefore,exploring the poisoning mechanism of SM and finding effective drugs have important strategic significance for maintaining the combat effectiveness of our army,safeguarding citizens' health,and protecting the ecological environment.The SM poisoning pathways consist of lung,skin,cornea,digestive,nerve and other multi-system damage,in which lung is the most important target organ.A large number of studies have found that SM poisoning can result in inflammation of the respiratory tract and destruction of alveolar structures,causing acute lung injury and finally leading to death.Bone marrow mesenchymal stem cells(BMSCs)are a kind of adult stem cells with self-renewal and multi-differentiation ability.Studies have shown that BMSCs can act on the regulation of immune system and the repair of damaged tissues by secreting anti-inflammatory cytokines,regulating immune cell subpopulation,secreting growth factors,and differentiation.As a result,it could reduce the inflammatory reaction in lung,accelerate the repair of damaged lung tissue,and relieve acute lung injury.This study found that the BMSCs are mainly located in the lung after injection to poisoning mice.BMSCs are benefit to the repair of lung injury by promoting the differentiation of type I and type II epithelial cells,secreting growth factors such as EGF,FGF,PDGF,and strengthening cell attachment,thus reducing pulmonary edema and improving the lung function of SM poisoning mice.In addition,BMSCs can significantly reduce the expression of proinflammatory cytokines and elevate the expression of proinflammatory cytokines by affecting the differentiation of macrophages and T lymphocytes,and regulate the expression of TLR4 signaling pathway.1.The identification of BMSCs and its location in SM poisoned miceICR mouse femurs and tibias were used for primary isolation and culture of BMSCs.The surface marker antigens and stem cell differentiation were detected to identify the obtained BMSCs: the results showed that the obtained cells were highly expressed surface antigens with mesenchymal cell characteristics such as CD73,CD90,and CD105,and did not express or lowly express hematopoietic cell surface antigens such as CD11 b and CD19.The results of induction differentiation test showed that the obtained BMSCs were successfully differentiated into osteoblasts,adipocytes and chondroblasts.In this study,animal model was established by subcutaneous injection of SM in ICR mice.The cultured and expanded BMSCs were injected through the tail vein to conduct further experiments.Firstly,we identified the localization of BMSCs in mouse by in vivo imaging.We found that BMSCs mainly accumulated in bilateral lungs.The fluorescence intensity at 1 h and 2 h increased significantly,and there was still some fluorescence at 24 h.It clarified that,after mustard gas injury,BMSCs can act on the main target organ-lungs.2.The tissue repairing effect and mechanism of BMSCs in SM-induced lung injury in miceOur previous study found that BMSCs could significantly reduce the wet/dry weight ratio of lung and total protein concentration in alveolar lavage fluid in SM poisoning mice.In order to further observe the repair role of BMSCs in the treatment,we first detected the expression changes of growth factors in lung tissue.The results showed that BMSCs could significantly increase the expression of EGF,FGF,PDGF.Ki-67 staining showed that the positive cells in lungs of BMSCs-treated mice were significantly higher than that in the SM group.In addition,we also found that after SM poisoning,the surface markers of type I,type II alveolar epithelial cells named AQP5,SP-C,and the cell connexins markers VE-cadherin,Occludin,Claudin-5,ZO-1 and Caveolin-1 were significantly reduced,while BMSCs treatment could obviously elevate the expression.The above results suggested that BMSCs could promote the repair of damaged lung epithelium by secreting growth factors,promoting the proliferation and differentiation of alveolar epithelial cells,strengthening cell junction,thereby reduced pulmonary edema and improved pulmonary function.In the previous study,we found that BMSCs could significantly improve the survival rate of SM poisoning mice.In this study,we further examined the effect of BMSCs on pulmonary function.The results showed that,after SM poisoning,the peak expiratory flow rate,peak inspiratory flow rate,minute ventilation,and 50% of lung volume expiratory flow rate were significantly reduced.The above indicators were significantly increased after BMSCs treatment,which suggested that BMSCs could improve lung function in SM-poisoning mice.3.The immunomodulatory effect and mechanism of BMSCs in SM-induced lung injury in miceOur previous study found that BMSCs can significantly reduce the infiltration of macrophages,T lymphocytes,and other inflammatory cells in lungs by reducing the production of chemokines.Therefore,this study further examined the subpopulation changes of macrophages and T lymphocytes.The results showed that compared with the SM group,BMSCs significantly reduced the expression of pro-inflammatory M1 macrophages and Th17 cells,increased the expression of anti-inflammatory M2 macrophages and Treg cells.It suggested that BMSCs can reduce SM-induced lung injury by regulating the subpopulation change of immune cells.To further examine the immunomodulation effect of BMSCs,we examined the major pro-inflammatory cytokines TNF-?,IL-1?,IL-6,IL-12,IL-17 and anti-inflammatory cytokines IL-10,TGF-? expression in mouse lung tissue and cell models.The results showed that compared with the SM group,BMSCs can significantly reduce the expression of pro-inflammatory cytokines TNF-?,IL-1?,IL-6,IL-12,IL-17 and increase anti-inflammatory cytokine IL-10,TGF-? level in lung tissue.In a SM-purified mouse macrophage(Raw 264.7)and spleen lymphocytes model,the co-culture of BMSCs can also significantly reduce the expression of TNF-?,IL-1?,IL-12,IL-17 and increase the expression of IL-10 and TGF-? in culture supernatant,which suggests that BMSCs can reduce inflammation in vivo by reducing pro-inflammatory cytokines and raising anti-inflammatory cytokines.We further explored signaling pathways that may play a role in reduction of SM injury by BMSCs at the animal and cellular levels.The results showed that compared with the control group,the expression of TLR4 and its downstream NF-?B p65,p50 in lung tissue was significantly increased,and treatment with BMSCs can significantly reduce it.In Raw 264.7 model,the expression levels of TLR4,NF-?B p65,p50 were also increased,and BMSCs transwell co-culture,knocked out TLR4 receptors in macrophages,or given TLR4 inhibitor TAK242,can significantly reduce the expression of TLR4,NF-?B p65,p50.It suggested that TLR4 may play an important role in the regulation of SM lung injury by BMSCs. |
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