Therapeutic Effect Of Bone Marrow Stromal Stem Cell Supernatant In Experimental Autoimmune Encephalomyelitis

Background and PurposeMultiple sclerosis(MS)is a common nervous system autoimmune disease.The central nervous system has its own unique structural and functional characteristics.Studies at this stage show that the disease has not been effectively treated due to the lack of effective therapeutic targets and high-precision regulatory elements.Recently,the immunoregulatory and neuroprotective capabilities of bone marrow stromal stem cells(BMSCs)have been reflected in multiple sclerosis.At present,the clinical researches on stem cell treatment diseases focus on stem cell transplantation treatment,tissue injection and other directions.At the same time,bone marrow stromal stem cells,as ideal cells with multi-directional differentiation potential,can also exert some therapeutic effects through paracrine production of a number of factors and exosomes.Therefore,the subject is based on experimental animal model of multiple sclerosis,experimental autoimmune encephalomyelitis.Intervention treatment of EAE disease by administering non-invasive treatment via nasal mucosal administration to mice with BMSC secreted factors.The treatment effect is analyzed.The method belongs to noninvasive treatment,which has high safety,good clinical prognosis,simple and convenient operation,and has far-reaching clinical application prospects.MethodsIn vivo experiments: We established B cell-mediated r MOG-induced mouse EAE models.On the third day after the model immunization,nasal administration was divided into groups and divided into BMSC group and ES(specialized stem cell medium)group,and their body weight and clinical scores were monitored.At the peak of disease development,Luxol fast blue assay and immunohistochemistry were used to analyze the degree of central nervous system lesions in mice.Enzyme-linked immunosorbent assay was used to detect antibody titer and inflammatory factor secretion in the serum of mice at the peak of disease.Based on the B-cell mediated mouse EAE model,administration was performed according to different administration methods,including intraperitoneal administration,intravenous injection administration and nasal administration,and the mouse body weight and clinical score were monitored.In vitro experiments: Spleen cells and draining lymph node cells and sorted B cells and T cells of C57BL/6 mice in the BMSC group and ES group at the peak period after immunization were collected and induced in vitro for 96 hours with BMSC supernatant or ES medium.Flow cytometry was used to detect the changes in the proportion of Bcell subsets,and the expression levels of GM-CSF,IFN-γ,TNF-α,and IL-1βproinflammatory cytokines,and the proportion of T-cell subsets.ResultsIn vivo experiments: Monitoring the weight and clinical scores of r MOG-induced EAE mice shows that nasal administration of BMSC supernatant can significantly delay the development of r MOG-induced EAE and significantly reduce the severity of EAE.At the same time,on the 19 th day,the BMSC group and ES group of mice were sampled.The results of LFB staining showed that the treatment of BMSC supernatant could reduce the demyelination of the central nervous system in EAE mice.The results of immunohistochemical experiments showed that BMSC supernatant treatment can reduce the accumulation of inflammatory cells in the central nervous system of mice.In addition,after the mice were collected at the peak period,the serum collected were subjected to ELISA experiments.The results showed that BMSC supernatant treatment can reduce the antibody titer and inflammatory cytokine secretion in the serum of EAE mice,including GM-CSF,IFN-γ,IL-1β,TNF-α.Furthermore,we also used control experiments to analyze the differences in the therapeutic effects of different administration routes on EAE disease.According to weight and clinical scores,there was no statistical difference between the therapeutic effects of different administration methods.In vitro experiments: Flow cytometry was performed on 96 hours culture of spleen B cells from peak-period EAE mice,and the results showed that the percentage of B cells that produced GM-CSF was significantly reduced and the proportion of Breg cells that secreted IL-10 was significantly increased(P <0.001).At the same time,B cell secreted antibodies and pro-inflammatory cytokines were significantly reduced after cultured with BMSC supernatant.It is worth noting that the BMSC supernatant did not have a significant effect on the proportion of T cell subpopulations when culturing individual T cells in vitro,and when BMSC supernatant was used to culture mixed cells in vitro,the T cell subsets Th1,Th17 and Treg cells were affected(P <0.05,P <0.05,P<0.001).In addition,we used flow cytometry to test whether the phenotype of BMSC used in the experiment was stable,and detected BMSC surface markers,CD29,CD34,CD44,Sca-1,CD90.2,CD31,and CD117.Flow cytometry results showed that the BMSC cells we used were stable in phenotype.ConclusionsBMSC supernatant nasal treatment for EAE mice can reduce the clinical symptoms of EAE disease and improve the pathological changes of central nervous system in EAE mice.And the treatment can affect the function of T lymphocytes through B lymphocytes and thus intervene in the progression of EAE disease.The nasal mucosal administration of BMSC secreted factors has no obvious difference in the treatment effect on EAE mice compared with other common administration routes,but this method has higher safety,simpler operation.