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目的探讨Rho激酶抑制剂Fasudil衍生物FSD-C11治疗实验性自身免疫性脑脊髓炎(EAE)的免疫调节机制。方法采用MOG35-55多肽建立C57BL/6小鼠EAE模型,随机分为FSD-C11组和Saline组,于免疫后第3天起腹腔注射FSD-C11化合物和Saline。免疫后28 d处死小鼠,FACS法检测脾组织CD4+T细胞亚群,ELISA法检测外周免疫系统中细胞因子的分泌情况,Western blot法测定脊髓ROCKⅡ、i NOS、Arg-1、TLR-2和TLR-4的蛋白表达。结果 FSD-C11干预EAE能够抑制脊髓中ROCKⅡ表达,减少外周CD4+IFN-γ+T细胞,增加CD4+IL-10+和CD4+CD25+T细胞(P<0.05),减少外周免疫系统炎性细胞因子IFN-γ、IL-17、IL-6、IL-1β和TNF-α的含量(P<0.05),而增加IL-10的含量(P<0.05),抑制脊髓组织中巨噬细胞标志蛋白i NOS表达、增加Arg-1的表达(P<0.05)。抑制脊髓组织中TLR-2和TLR-4蛋白表达(P<0.05)。结论 FSD-C11可调节外周免疫细胞活化和增殖,抑制外周免疫系统分泌炎性因子,增加保护性的细胞因子,改善炎性微环境,促进M1型巨噬细胞向M2型转化,控制CNS的炎性细胞侵润,从而达到减轻或改善EAE的临床症状。
Objective To investigate the immunoregulatory mechanism of the Rho kinase inhibitor Fasudil derivative FSD-C11 in the treatment of experimental autoimmune encephalomyelitis (EAE). Methods The C57BL / 6 mice model of EAE was established by MOG35-55 peptide and divided into FSD-C11 group and Saline group. FSD-C11 compound and Saline were injected intraperitoneally on the third day after immunization. Mice were sacrificed 28 days after immunization. The subsets of CD4 + T cells were detected by FACS and the secretion of cytokines in the peripheral immune system by ELISA. The expressions of ROCKⅡ, iNOS, Arg-1, TLR-2 And TLR-4 protein expression. Results The intervention of FSD-C11 in EAE could inhibit ROCKⅡ expression in the spinal cord, decrease peripheral CD4 + IFN-γ + T cells and increase CD4 + IL-10 + and CD4 + CD25 + T cells (P <0.05) (P <0.05), but increased the content of IL-10 (P <0.05), and inhibited the expression of macrophage markers in spinal cord Protein i NOS expression increased Arg-1 expression (P <0.05). Inhibition of TLR-2 and TLR-4 protein expression in spinal cord tissue (P <0.05). Conclusion FSD-C11 can regulate the activation and proliferation of peripheral immune cells, inhibit the secretion of inflammatory cytokines by peripheral immune system, increase the protective cytokines, improve the inflammatory microenvironment, promote the transformation of M1 macrophages into M2 and control the inflammation of CNS Sexual invasion, so as to reduce or improve the clinical symptoms of EAE.