| Background: Multiple sclerosis(MS) is a chronic demyelinating neurodegenerative disease of the central nervous system(CNS); however, neither the etiology nor the mechanism of disease is fully understood. Currrently, it is thought that the disease is mediated by pathogenic T cell responses across the blood-brain barrier(BBB), leading to axonal demyelination of neurons. It has been reported that chondroitin disaccharide is the degradation product of chondroitin sulfate proteoglycan and it promotes repair in the central nervous system.MicroRNAs(miRNAs) are a class of evolutionally highly-conservated small noncoding single-stranded RNAs, with approximately 22 nucleotides in length, wchich have recently been discovered to be regulatory modulators of gene expression post-transcriptionally, either by targeting mRNA degradation or by inhibition of protein translation. miRNAs directly modulate the expression of regulatory proteins that are required for normal development and function of the immune system. Emerging evidence underlines an involvement of miRNAs in the pathogenesis of Multiple Sc1 erosis. A number of mi RNAs have been found to be dysregulated in blood cells, brain lesions as well as biological fluids such as serum and plasma from MS patients. When poor1 y regu1ated, microRNAs are clitically involved in a range of human diseases and potentially serve as good diagnostic markers.Objective: In this study we investigated the effects of chondroitin disaccharide and its induced miR-29 b and mi R-199 b on CD44 expression in mouse lymphocytes, as well as the effects of miR-29 b and miR-199 b on the development and progression of experimental autoimmune encephalomyelitis(EAE) in mice, an animal model of MS.Methods:1. Effects of chondroitin sulfate disaccharide on the changes of cellular immunology such as lymphocytes, infiltrating cells and cytokine interferon-γ as well as expression of genes including CD44, miR-29 b,miR-199 b,IL-17, and FoxP3 were investigated in EAE mice induced by MOG and PTX.2. Silico studies were used to study the interactions between miR-29 b or miR-199 b and the 3’-untranslation region of CD44 mRNA.3.Real-time PCR and flow cytometry were used to investigate the effects of the antagonists and analogues of miR-29 b and miR-199 b on CD44 expression in mouse spleen lymphocytes after electroporation.4. The antagonists and analogues of miR-29 b and miR-199 b were tranduced into mouse splenocytes. After culture for 2 days, the splenocytes were adoptively transferd into mice intravenonsly. After 14 days of adoptive transfer, the changes of immune cells and cytokines were analyzed by flow cytometry and ELISA.Results: Chondroitin disaccharide up-regulated several specific miRNAs such as miR-29 b and miR-199 b, and induced significant inhibition of experimental autoimmune encephalomyelitis(EAE) in mice.In vitro studies discovered that miR-29 b and mi R-199 b could up-regulated the expression of CD44 in splenocytes. At the same time,it can restrain the proliferation of lymphocytes.Stduies by adoptive transfer experiments showed that mi R-29 b and miR-199 b could regulate the expression of IL-17.Conclusion: Chondroitin sulfate disaccharide could up-regulate the expression of miR-29 b and miR-199 b, and inhibit the development and progression of experimental autoimmune encephalomyelitis in mice. Computer simulation discovered that miR-29 b and miR-199 b could bind to the 3-’noncoding sequence of CD44 mRNA. Our studies revealed that miR-29 b and miR-199 b could inhibit the expression of CD44 and thus suppress lymphocyte proliferation and migration, leading to the significant inhibition of EAE development. miR-29 b and mi R-199 b could also modulate the expression of IL-4 and IL-17 in the lymphocytes from EAE mice, which may be involved in the control of EAE development. |
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