烟酰胺核糖对实验性自身免疫性脑脊髓炎小鼠T细胞的免疫调节作用

Immunomodulatory effects of nicotinamide riboside on T cellin EAE mice

目的探讨烟酰胺核糖(NR)治疗实验性自身免疫性脑脊髓炎(EAE)的效果以及对T细胞的免疫调节作用。方法C57BL/6雌性小鼠通过髓鞘少突胶质细胞糖蛋白35-55(MOG_(35-55))诱导,制备EAE小鼠模型,随机分为EAE组和NR组。从免疫后第3天到免疫后第27天,EAE组灌胃给予生理盐水,NR组灌胃给予NR,连续给药25 d。从免疫当天到免疫后第28天,记录各组小鼠临床评分和体质量。免疫后第28天处死小鼠,制备脾细胞悬液和脊髓组织冰冻切片。HE染色、髓鞘染色分别检测脊髓炎性细胞浸润和髓鞘脱失,免疫荧光染色检测脊髓CD4^(+)T细胞数量,流式细胞术检测脾脏CD4^(+)T细胞亚群比例,ELISA检测脾细胞培养液中IFN-γ、IL-4、IL-10和IL-17含量。结果与EAE组相比,NR能有效推迟EAE发病时间,缓解EAE临床症状(P<0.05),减少小鼠体质量丢失,抑制外周炎性细胞浸润脊髓(P<0.01),减轻髓鞘脱失(P<0.05),减少脊髓CD4^(+)T细胞数量(P<0.05),下调脾脏CD4^(+)IFN-γ^(+)T细胞、CD4^(+)IL-17^(+)T细胞比例,上调CD4^(+)IL-10^(+)T细胞、CD4^(+)FOXP3^(+)T细胞比例(P<0.05),减少EAE小鼠脾细胞培养液中炎性因子IFN-γ、IL-17分泌,增加抗炎因子IL-4、IL-10分泌(P<0.05)。结论NR可能通过上调EAE小鼠脾脏抗炎型T细胞亚群比例,下调促炎型T细胞亚群比例;促进EAE小鼠脾脏抗炎因子分泌,抑制促炎因子分泌;调节机体免疫平衡,减轻机体炎症反应,改善EAE小鼠临床症状。

The study was designed to investigate the therapeutic effect of nicotinamide riboside(NR)on experimental autoimmune encephalomyelitis(EAE)and its immunomodulatory effect on T cells.C57BL/6 female mice were induced by myelin oligodendrocyte glycoprotein 35-55(MOG_(35-55))to construct EAE mouse model,which then divided into EAE group and NR group randomly.From the 3^(rd) day to the 27^(th) day after immunization,the mice in EAE group were given normal saline and the mice in NR group were given NR by intragastric administration for 25 consecutive days.Clinical score and body weight of mice were recorded from the day of immunization to the 28th day after immunization.The mice were sacrificed and splenic cell suspension and frozen sections of spinal cord were prepared on day 28 after immunization.HE staining and myelin staining were used respectively to detect spinal inflammatory cell infiltration and myelinoclasis;the number of CD4^(+)T cells in spinal cord was detected by immunofluorescence staining;the proportion of CD4^(+)T cell subsets in spleen was detected by flow cytometry;the contents of IFN-γ,IL-4,IL-10 and IL-17 in splenic cell culture medium were detected by ELISA.Data showed that compared with EAE group,NR effectively delayed the onset of EAE,alleviated the clinical symptoms of EAE(P<0.05),relieved the weight loss of mice,prevented the infiltration of peripheral inflammatory cells into the spinal cord(P<0.01),inhibited demyelination(P<0.05),reduced the number of CD4^(+)T cells in the spinal cord(P<0.05),down-regulated the proportion of CD4^(+)IFN-γ^(+)T cells and CD4^(+)IL-17^(+)T cells,and upregulated the proportion of CD4^(+)IL-10^(+)T cells and CD4^(+)FOXP3^(+)T cells(P<0.05).Furthermore,the secretion of IFN-γand IL-17 was decreased,and the secretion of IL-4 and IL-10 in splenic cell culture medium was increased in NR group,as compared with EAE group(P<0.05).In conclusion,NR can regulate immune balance,reduce inflammatory response and alleviate clinical symptoms of EAE mice,possibly by up-regulating the proportion of anti-inflammatory T cell subsets and down-regulating the proportion of pro-inflammatory T cell subsets,promoting the secretion of anti-inflammatory factors and inhibiting the secretion of pro-inflammatory factors in spleen of EAE mice.