口服法舒地尔治疗EAE对巨噬细胞、T细胞及TLR/NF-B通路的作用研究

目的探讨口服盐酸法舒地尔(Fasudil)对实验性自身免疫性脑脊髓炎(experimental allergic encephalomyelitis,EAE)小鼠巨噬细胞及TLR/NF-B通路的作用。方法采用小鼠髓鞘少突胶质细胞糖蛋白35-55肽(myelin oligodendrocyte glycoprotein 35-55peptide,MOG35-55)诱导C57BL/6小鼠建立EAE模型,将EAE小鼠随机分为EAE模型组和Fasudil治疗组,免疫后第3天给予Fasudil治疗组小鼠Fasudil灌胃干预,直到免疫后第27天,EAE模型组同样处理给予等量生理盐水。光镜观察脊髓组织CD4+T细胞/CD68巨噬细胞表达的变化,Western blot法测定脊髓诱导型一氧化氮合酶(iNOS)、精氨酸酶1(Arg-1)、Toll样受体2(TLR-2)、TLR-4和磷酸化核因子B(p-NF-B)蛋白的表达,ELISA法测定培养72h脾细胞分泌细胞因子的含量。结果与EAE模型组比较,Fasudil组CD4+T细胞数和CD68巨噬细胞数明显减少(P0.01),巨噬细胞M1表型iNOS表达减少(P0.05),M2表型Arg-1表达增加(P0.01),炎性通路蛋白p-NF-B、TLR-2和TLR-4表达减少(P0.05),外周细胞炎性因子白细胞介素-6(IL-6)、IL-1β、肿瘤坏死因子α(TNF-α)、干扰素γ(IFN-γ)和IL-17分泌减少(P0.05),而IL-10分泌增加(P0.05)。结论口服Fasudil可抑制CD4+T细胞/CD68巨噬细胞的激活,促进巨噬细胞表型M1向M2转化,抑制脊髓组织中p-NF-B、TLR-2和TLR-4的表达,抑制外周免疫细胞炎性因子分泌,而增加IL-10的分泌。

Research on macrophages,Tcells and TLR/NF- B pathway in EAE mice treated with oral fasudil

Objective To explore the effect and mechanism of oral fasudil on macrophages and TLR/NF‐B pathway in mice with experimental autoimmune encephalomyelitis (EAE) .Methods EAE models were induced by myelin oligodendrocyte glycoprotein 35‐55 peptide (MOG35‐55) in C57BL/6 mice. The models were randomly divided into the EAE group and the fasudil group.Mice in the fasudil group were intragastrically infused with fasudil one time every day from the 3rd day after immunization to the 27th day.Mice in the EAE group were treated with the same dosage of saline.CD4+ T cells and CD68 macrophages expressions in the spinal cord tissue were observed by light microscope. The expression of iNOS , Arg‐1 , TLR‐2 , TLR‐4 and p‐NF‐ B in spinal cord were detected by western blot. The cytokine of splenocyte cells cultured for 72 h were analyzed by ELISA. Results The number of CD4+ T cells and CD68 macrophages decreased in the fasudil group (P<0.01) .iNOS phenotype of macrophages M1 was inhibited (P<0.05) ,and the expression of M2 phenotypic Arg‐1 increased (P<0.01) . The expression of inflammatory signaling pathway proteins including p‐NF‐ B , TLR‐2 and TLR‐4 reduced (P< 0.05) .Macrophage inflammatory cytokines including IL‐6 ,IL‐1β and TNF‐α decreased (P<0.05) ,and secretion of IL‐10 increased (P<0.05) , T cells inflammatory cytokine IL‐17 and IFN‐γ decreased (P< 0.05) .Conclusions Oral treatment with fasudil inhibits CD4+ T cells/CD68 macrophages activation , improves the conversion from macrophage phenotype M1 to M2 ,and inhibits the expression of p‐NF‐ B ,TLR‐2 and TLR‐4 in spinal cord.Inflammatory cytokines secreted by macrophages and T cells in spleen are inhibited , and the secretion of IL‐10 increases.