法舒地尔修饰的巨噬细胞治疗实验性变态反应性脑脊髓炎的分子机制

Molecular mechanism of fasudil-modified macrophages in the treatment of experimental allergic encephalomyelitis

目的探讨法舒地尔修饰的巨噬细胞治疗实验性变态反应性脑脊髓炎(EAE)的分子机制。方法采用髓鞘少突胶质细胞糖蛋白(MOG)诱导C57BL/6小鼠建立主动免疫EAE模型,免疫9 d后制备腹腔巨噬细胞,用PBS或法舒地尔处理72 h(对照组和实验组)。将对照组和实验组巨噬细胞经尾静脉回输至EAE模型小鼠中,每只5×10^(7)个细胞。治疗10 d后分析对照组和实验组小鼠临床行为和体质量变化;经腹腔分离2组小鼠巨噬细胞,采用流式细胞术分析2组M1和M2型巨噬细胞比例;采用荧光定量PCR分析M1和M2巨噬细胞标志物的表达情况;采用ELISA分析白细胞介素(IL)-10和IL-12的水平。结果实验组小鼠临床症状评分较对照组下调,差异有统计学意义(F=6.135,P<0.001)。与对照组比较,实验组小鼠体质量下降较少,差异有统计学意义(F=3.105,P=0.011)。与对照组细胞比较,实验组细胞M2型标志物精氨酸酶(Arg-1)、YM-1和FIZZ mRNA与IL-10细胞因子水平升高,M1型标志物一氧化氮合酶(iNOS)mRNA和IL-12细胞因子水平下降,差异均有统计学意义(t=7.091,P<0.001;t=11.094,P<0.001;t=6.182,P<0.001;t=3.942,P<0.001;t=4.132,P<0.001;t=3.198,P<0.001)。与对照组相比较,实验组的F4/80^(+)CD206^(+)巨噬细胞比例增加,而F4/80^(+)CD16/32^(+)巨噬细胞比例下调,差异有统计学意义(t=10.618,P<0.001;t=12.105,P<0.001)。结论法舒地尔修饰的巨噬细胞可改善EAE小鼠的临床症状,主要是通过改善机体巨噬细胞极性实现的。

Objective To explore the molecular mechanism of fasudil-modified macrophages in the treatment of experimental allergic encephalomyelitis(EAE). Methods C57 BL/6 mice were induced by myelin oligodendrocyte glycoprotein(MOG) to establish an active immune EAE model. Peritoneal macrophages were prepared after 9 days of immunization and the mice were either unable to be treated with or without fasuldil for 72 h(control group and experimental group). Macrophages from the control group and the experimental group were transfused into EAE mice models, with each mouse containing 5×10^(7) cells. After treatment for 10 days,the changes of clinical behavior and body weight of mice in the control group and experimental group were analyzed. Macrophages in the two groups were isolated from the abdominal cavity and the ratio of M1 and M2 macrophages in the two groups was analyzed by flow cytometry. The expression of macrophage markers in M1 and M2 was analyzed by fluorescence quantitative PCR. The levels of interleukin-10(IL-10) and interleukin-12(IL-12) were analyzed by enzyme-linked immunosorbent assay(ELISA). Results The clinical symptom score of the experimental group was significantly lower than that of the control group(F=6.135, P<0.001). Compared with the control group, the weight loss of mice in the experimental group was less, and the difference was statistically significant(F=3.105, P=0.011). Compared with the control group, the M2-labeled arginase(Arg-1), YM-1, FIZZ and IL-10 cytokine levels in the observation group increased significantly, while the M1-labeled nitric oxide synthase(iNOS) and IL-12 cytokine levels decreased significantly(t=7.091,P<0.001;t=11.094,P<0.001;t=6.182, P<0.001;t=3.942,P<0.001;t=4.132,P<0.001;t=3.198, P<0.001). Compared with the control group, the proportion of macrophages in F4/80-CD206 increased significantly, while the proportion of macrophages in F4/80-CD16/32 decreased significantly(t=10.618, P<0.001;t=12.105,P<0.001). Conclusion Fasudil-modified macrophages can significantly improve the clinical symptoms of EAE mice, and its mechanism is mainly through improving the polarity of macrophages.