Studies On The Effects Of Bone Marrow Mesenchymal Stem Cells Against Sulfur Mustard-induced Injuries

Sulfur mustard?SM,C₄H₈Cl₂S,bis?2-chloroethyl?sulfide,CAS: 505-60-2?,a bifunctional alkylating agent,is a typical representative of blister agents and a major chemical threat since its introduction on the battlefield.It has been used in a series of conflicts including World War?40?,Japan invasion of China,and Iran-Iraq war,etc.In addition,SM is also one of the major chemical weapons abandoned by Japan in China,which has resulted in severe damage to our society and has been imposing a enduring threat to the environment and the public health.What is more,SM is regarded as one of the most wanted chemical agents for its easy availablity and synthesis.ISIL has been reported to succeed in applying chemical agents including SM to attack civilians recently and caused numerous casualties in Syria and Iraq.Because of the complex mechanism of sulfur mustard,currently there are no specific antidotes and the state-of-the-art treatment of SM poisoning is primarily supportive.Therefore,mechanism investigation,antidotes searching and development of effective therapeutic measures are hot topics in this field,which are very important to public security and public health.Lung is one of the primary targets in SM-induced injuries.Acute pulmonary injuries with secondary infection is the main reason of earlier death in SM poisoning.Various studies have proved that the acute pulmonary injuries induced by SM have close relationship with inflammation,immunological responses and oxidative stress.Recently,the bone marrow mesenchymal stem cells?BMSCs?therapy has become a hot topic in stem cell therapy.BMSCs have been reported to be able to repair many damaged tissues,especially in treating pulmonary injuries,which has been well established in several animal models.On the one hand,BMSCs may provide anti-inflammartory,immunomudulatory and antioxidative activities through secreting soluble cytokines,active molecules and inhibitory factors et al.,on the other hand,it could also exert the effects through inhibiting T cell proliferation and down-regulating the activities of the immunocytes,such as macrophages and dendritic cells.As lung is one of the primary targets in SM-induced injuries,it promotes us to explore the possiblity of applying BMSCs to treat SM-induced lung injuries.In this study,we used BMSCs to treat SM poisoning for the first time.The survival rate of mice exposed to SM was investigated,and the efficacy of BMSCs in ameliorating SM-induced pulmanory pathophysiological changes was evaluated.Furthermore,The possible mechanism was also explored and discussed.A simple and efficient protocol for isolation,cultivation,purification of ICR mice BMSCs was applied to get the BMSCs.The biological characteristics of the isolated cells were detected by flow cytometry,revealing that they don't express hematopoietic cell surface biomarkers,e.g.,CD34 and CD45,but express stromal cell surface biomarkers including CD29 and CD44.They could differentiate into adipocytes and osteoblast.Then we evaluated the efficacy of BMSCs in improving the survival rate of mice exposed to SM subcutaneously?s.c.?40 mg/kg.It turned out that,after injection of BMSCs to the mice exposed to SM through tail vein with a dose of 1?107 /cells,the survival rate of mice treated with BMSCs was remarkbly improved?P<0.05?compared with the SM group.In addition,the symptoms,such as the decrease of the body weight,anorexia,diarrhea and even aganoblepharon,which are typical in the SM-exposed mice,have all been significantly ameliorated.Therefore,BMSCs therapy showed promising treatment efficacy against SM exposure in mice.Using the same mice model as described above,after the injection of the ready BMSCs,and the conditions of histopathological changes,inflammatory cells infiltrate,the total proteinin?TP?in bronchoalveolar lavage fluid?BALF?and the wet weight/dry weight ratio?W/D?of the lung were examined comparatively.The hematoxylin and eosin?HE?staining showed that BMSCs therapy could remarkably alleviate SM-induced lung injury in histopathological analysis compared with the SM group?P<0.05?.While immunohistochemistry analysis indicated that in the BMSCs group,the counts of MPO+ neutrophils,CD3⁺ T lymphocytes and CD68⁺ macrophages were obiviously lower than those of the SM group?P<0.01?.It implies that BMSCs markedly reduced inflammatory cell infiltration in the lung tissues.Furthermore,in comparision with SM group,BMSCs treatment could greatly decrease the ratio of wet weight/dry weight?W/D?of the lung?P<0.05?and the total protein in BALF?P<0.01?.It could be inferred from the above results that BMSCs could significantly improve the SM-induced lung injures.BMSCs' mechanism of improving SM-induced lung injuries were investigated accordingly.We examined the expression of TNF-a,IL-1b and IL-10 in lung tissues.Elisa and quantitative real-time PCR were applied for the analysis.The results indicated that BMSCs therapy remarkably decreased the expression levels of TNF-a and IL-1b,and significantly increased the expression level of IL-10?P<0.01?.In addition,immunohistochemistry were applied to detect the expression level of NF-kB,and we found BMSCs therapy also remarkably down-regulated the expression of NF-kB.The results indicated that BMSCs may provide the anti-inflammatory activities through down regulation of TNF-a and IL-1b,and through up-regulation of IL-10,together with the suppression of NF-kB pathway.In addition,in the systemic analysis using the technique of Luminex Performance Assays,BMSCs markedly decreased the expression leves of LIX/CXCL5,P-10/CXCL10,MCP-1/CCL2,MIP-1b/CCL4,RANTES/CCL5,M-CSF?P<0.05?,while the expression of KC/CXCL1,MIP-2/CXCL2,MIG/CXCL9,and MIP-1a/CCL3 have not been significantly affected.It implies that the effects of BMSCs therapy may related to their immunomodulatory activities.BMSCs therapy also markedly decreased the leves of MDA?P<0.01?,and significantly increased the leves of SOD and GSH?P<0.01?.Moreover,Western Blot analysis revealed that BMSCs significantly up-regulated the level of nuclear factor erythroid 2-related factor 2?Nrf-2?and heme oxygenase-1?HO-1??P<0.05?.These results suggest that the effects of BMSCs therapy may also related to their anti-oxidative activities,partly through Nrf-2/HO-1 signaling pathway.