Fasudil对实验性自身免疫性脑脊髓炎小鼠致脑炎性T细胞的免疫调节作用

目的：探讨法舒地尔(Fasudil)修饰的脾单个核细胞(mononuclear cells，MNCs)治疗实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis，EAE)的疗效及可能的分子机制。方法：雌性C57BL/6小鼠采用髓鞘少突胶质细胞糖蛋白35–55诱导，建立主动免疫EAE模型，免疫后第9天制备脾MNCs，和/无Fasudil培养。作用72 h后收集上述细胞，检测T细胞亚群的变化、Rho激酶(Rho kinase，ROCK)活性和细胞因子水平；以5×107个细胞/只小鼠腹腔注射诱导，建立被动转移EAE模型，并将小鼠分为PBS-MNCs组和Fasudil-MNCs组，每组8只。观察各组小鼠临床评分和体质量变化。结果：免疫第9天EAE小鼠脾致脑炎性MNCs可通过被动转移实验诱导EAE模型的建立，而体外Fasudil修饰的MNCs不能诱导EAE的发生；与PBS-MNCs组相比，Fasudil-MNCs组小鼠体质量减轻较少(P<0.05)。体外实验显示：Fasudil处理的MNCs ROCK活性降低(P<0.01)；Fasudil可抑制IFN-γ和IL-17的CD4+T细胞比例(IFN-γ：P<0.01；IL-17：P<0.05)，并增强TGF-β和IL-10的CD4+T细胞分泌(均P<0.001)；此外，Fasudil可降低细胞因子IL-17水平(P<0.001)和增强细胞因子IL-10分泌(P<0.05)。结论：Fasudil介导的细胞治疗通过抑制辅助性T细胞1(helper T cell 1，Th1)和Th17炎性反应，促进Th2免疫调节作用，影响EAE的发生和发展。

Immunoregulative effect of Fasudil on encephalomyelitic T cells in experimental autoimmune encephalomyelitis mice

Objective: To explore the therapeutic effect of Fasudil-modified splenic mononuclear cells (MNCs) in experimental autoimmune encephalomyelitis (EAE) and the possible mechanisms. Methods: C57BL/6 female mice were immunized with myelin oligodendrocyte glycoprotein peptide 35–55 to establish active immunity EAE model. Splenic MNCs were isolated on the 9th day after immunization and treated with or without Fasudil for 72h in vitro. These cells were collected for analysis of the variance of T cell subtypes, the level of cytokines and the activity of Rho kinase (ROCK). MNCs (5×107 cells) were resuspended in 500 µL of phosphate buffer solution (PBS) and transferred into EAE model (intraperitoneal injection), which was divided into a PBS-MNCs group and a Fasudil-MNCs group. Changes of body weight and clinical symptom scores were observed. Results: Splenic encephalitogenic MNCs from EAE mice on the 9th day after immunization could establish passive transfer EAE model. But Fasudil-treated MNCs did not trigger EAE development. Compared with the PBS-MNCs group, the loss of body weight was less in the Fasudil-MNCs group. The in vitro experiment indicated that Fasudil could suppress the activity of ROCK on MNCs (P<0.01), decrease the percentage of CD4+ T cells with the expression of interferon-γ (IFN-γ) and interleukin-17 (IL-17) (IFN-γ: P<0.01; IL-17: P<0.05), while increase the secretion of CD4+ T cells with the expression of transforming growth factor-β (TGF-β) and IL-10 (all P<0.001) . Furthermore, Fasudil could inhibit the release of IL-17 (P<0.001) and enhance the level of IL-10 (P<0.05). Conclusion: Fasudil-modified cell therapy affects the occurrence and development of EAE by inhibiting the inflammatory reaction of helper T cell 1 (Th1) and Th17 while enhancing the immunoregulative effect of Th2.