| Objective: Multiple sclerosis is a chronic demyelinating disorder caused by inflammation of the central nervous system and autoimmune dysfunction,often resulting in severe lifelong disability.Currently,there is no cure for MS,and the treatment mainly relies on disease-modifying drugs and symptomatic treatment drugs to slow down the progress of the disease to reduce the recurrence and severity of the disease.Stem cell therapy has been shown in clinical studies and in many disease models to be effective in alleviating nervous system damage and slowing down disease progression.Recent studies have shown that the paracrine role of exosomes may be the main pathway for stem cells to exert their effects,and the exosomes of bone marrow mesenchymal stem cells play a therapeutic role in many autoimmune diseases and tissue damage.Microglia cells,as immune monitoring cells of CNS,are involved in the occurrence and development of MS and affect the progress of the disease due to the unbalanced proportion of the mutual transformation of M1/M2 phenotypes.In this study,we used bone marrow mesenchymal stem cell exosome to treat MS animal model---experimental autoimmune encephalomyelitis model,to explore the effect of exosome treatment on behavioral score and CNS pathological changes in rats with disease.By detecting the changes in the proportions of the M1/M2 phenotypes of microglia in the spinal cord before and after treatment,it was determined whether exosomes affected the polarization of microglia and thereby alleviated the severity of MS.Our study will explore the role and molecular mechanism of microglia polarized exosomes in the treatment of EAE model,providing a promising method to improve the treatment of autoimmune diseases and inflammatory diseases.Methods: In this study,the primary rat bone marrow mesenchymal stem cells were isolated and extracted by whole bone marrow adherent method,and the stem cell properties were identified.Exosomes were extracted from the culture supernatant of bone marrow mesenchymal stem cells by ultracentrifugation and identified for subsequent functional experiments.On this basis,it is expected to evaluate the in-vivo therapeutic effect of exosomes in the EAE rat disease model by using tail vein injection of exosomes.Subsequently,stem cells and exosomes were co-cultured with microglia using in vitro cell experiments.Immunofluorescence and real-time quantitative PCR were used to detect the expression of proteins and m RNA related to phenotypic transformation of microglia cells,determining whether stem cell exosomes regulate the immune inflammatory response of microglia cells and participate in the pathogenesis of MS.Results: 1.The rat bone marrow mesenchymal stem cells were successfully isolated and extracted,and their morphological and surface marker expressions were consistent with the characteristics of mesenchymal stem cells,with the ability of multidirectional differentiation.On this basis,exosomes of bone marrow mesenchymal stem cells with diameters between 30 and 100 nm were successfully extracted,and specific molecular markers CD9,CD63 and Alix were expressed on their surfaces.2.Efficacy evaluation of bone marrow mesenchymal stem cell exosomes on EAE rats.Compared with EAE group,exosome treatment significantly reduced neurobehavioral score and weight loss.The results of HE staining suggested that the infiltration of inflammatory cells in brain tissue was reduced in the exosome treatment group,while LFB staining suggested that the demyelination of spinal cord was reduced in the exosome treatment group.After treatment,the expression of anti-inflammatory cytokines was increased,and the expression of pro-inflammatory cytokines was decreased.Compared with the EAE group,protein expression level and m RNA level of M2 phenotype markers were significantly increased in the exosome treatment group,while protein level and m RNA level of M1 related markers were decreased.These results suggest that stem cell exosomes can not only reduce CNS inflammation and demyelination,but also regulate the central and peripheral immune environment.3.The results of in vitro experiments showed that the m RNA expression level of M2-related markers in microglia treated with stem cell exosomes was increased,the m RNA expression level of M1-related markers was decreased,the secretion of pro-inflammatory cytokines was decreased,and the secretion of anti-inflammatory cytokines was increased.After treatment with stem cell exosomes,microglia tended to transform to the M2 phenotype.4.After labelfree proteomic detection,there were 7 differentially expressed proteins in LPSactivated microglia treated with exosomes compared with the untreated group(no null value in any group,difference multiple >1.5,P<0.05).PRM method was used to verify the result of label-free proteomic detection.The results confirmed that there were 5 target differentially expressed proteins(Acadv1,Aarsd1,Acad9,RT1.a1(f),and Ehd1),which were indeed expressed differently in microglia activated by LPS before and after exosome treatment.Conclusion: 1.In this study,rat bone marrow mesenchymal stem cells were successfully extracted and exosomes in the cultured supernatant were isolated.Meanwhile,protein quantification of exosomes was conducted.2.Exosomes derived from bone marrow mesenchymal stem cells can alleviate the progress of disease in EAE rats,improve the immune environment in rats,and alleviate inflammatory cell infiltration in brain tissue and myelin loss in the spinal cord.3.Bone marrow mesenchymal stem cell exosomes can induce microglial cells to polarization toward the M2 phenotype in vivo and in vitro,playing an immunomodulatory role.4.The exosomes of bone marrow mesenchymal stem cells can induce microglia to transform into M2 phenotype by affecting the expression of 5 differential proteins and improve the severity of EAE in rats. |
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