人参皂甙Rd对EAE模型小鼠Tregs/Th17细胞失衡的调节作用研究  

Regulatory effect of ginsenoside Rd on regulatory T cells/helper T cell 17 imbalance in experimental autoimmune encephalomyelitis mice models

作　　者：刘媚[1,2] 黄绮玲 马晓萌[1] 刘莹莹[1] 陈晓红[1] Liu Mei;Huang Qiling;Ma Xiaomeng;Liu Yingying;Chen Xiaohong(Department of Neurology, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China;Department of Neurology, Zhejiang Provincal People’s Hospital (People’s Hospital of Hangzhou Medical College), Hangzhou 310014, China)

机构地区：[1]中山大学附属第三医院神经病学科,广州510630 [2]浙江省人民医院(杭州医学院附属人民医院)神经内科,杭州310014

出　　处：《中华神经医学杂志》2019年第5期507-514,共8页Chinese Journal of Neuromedicine

摘　　要：目的探讨人参皂甙Rd对实验性自身免疫性脑脊髓炎(EAE)模型小鼠调节性T细胞(Tregs)/辅助性T细胞17(Th17)失衡的影响。方法将54只6~8周龄雌性C57BL/6小鼠按随机数字表法分为人参皂甙Rd组、磷酸盐缓冲液(PBS)组、空白对照组,每组18只。前2组采用经典的髓鞘少突胶质细胞糖蛋白35-55(Mog35-55)肽段免疫诱导方法制备成EAE模型,并在接种免疫后第13天起将人参皂甙Rd 40 mg/(kg·d)溶解于PBS中腹腔注射入人参皂甙Rd组(1次/d),PBS组和空白对照组予同等剂量PBS腹腔注射。各组小鼠于接种免疫后第1~20天每天进行临床症状评分,并于评分结束当天取小鼠腰髓进行组织病理学评估(炎症评分及脱髓鞘评分)。在接种免疫后第20天,取各组小鼠脾脏,行体外脾细胞培养48 h后取上清液,采用酶联免疫吸附法(ELISA)检测白介素-10(IL-10)、白介素-17(IL-17)水平;采用流式细胞仪检测脾组织中Tregs细胞、Th17细胞的比例;取各组小鼠腰髓,采用实时荧光定量PCR检测腰髓中Janus激酶1(JAK1)、Janus激酶2(JAK2)、信号转导与转录激活因子3(STAT3)、维甲酸相关孤核受体γt(ROR-γt)、叉头型基因p3(Foxp3) mRNA的表达水平。结果(1)人参皂甙Rd组的临床症状评分、炎症评分均明显低于PBS组,差异均有统计学意义(P<0.05)。PBS组腰髓中有大量炎性细胞浸润及大片髓鞘脱失,而人参皂甙Rd组的髓鞘脱失较PBS组明显减少,2组间脱髓鞘评分比较差异有统计学意义(P<0.05)。(2)与PBS组相比,人参皂甙Rd组脾细胞培养上清液中IL-10水平明显升高,IL-17水平明显降低,差异均有统计学意义(P< 0.05)。(3)与PBS组相比,人参皂甙Rd组脾组织中CD4+IL-17+细胞比例明显降低,CD4+CD25+Foxp3+细胞比例明显升高,差异均有统计学意义(P<0.05)。(4)与PBS组相比,人参皂甙Rd组腰髓中JAK1、JAK2、STAT3、ROR-γt mRNA的表达水平明显降低,Foxp3 mRNA的表达水平明显升高,差异均有统计学意义(P<0.05)�Objective To explore the effect of ginsenoside Rd on regulatory T cells (Tregs)/helper T cell 17 (Th17) imbalance in experimental autoimmune encephalomyelitis (EAE) mice models. Methods Fifty-four female C57BL/6 mice, aged 6-8 weeks, were randomly divided into ginsenoside Rd group, phosphate buffered saline (PBS) group and blank-control group (n=18). The EAE mice models in the first two groups were induced by classical Mog35-55 peptide immunoinduction method;ginsenoside Rd of 40 mg/(kg·d) was dissolved in PBS and intraperitoneally injected into the mice daily in the ginsenoside Rd group on 13th d of immunization;the mice in the PBS group and control group were intraperitoneally injected with same dose of PBS. Daily clinical symptom scale scores were obtained from the first d to the 20th d of immunization, and the lumbar spinal cords of the mice were taken for histological evaluation (inflammation scale and demyelinating scale) after the last symptom scale. On the 20th d of immunization, the spleens of each group were taken for cultivation;splenocytes were cultured in vitro for 48 h, and the culture supernatant was taken for detecting interleukin (IL)-10 and IL-17 levels by enzyme-linked immunosorbent assay (ELISA);the proportions of Tregs and Th17 in spleen tissues were detected by flow cytometry;real-time quantitative PCR was used to detect the mRNA expressions of Janus kinase 1 (JAK1), Janus kinase 2 (JAK2), signal transduction and activator of transcription 3 (STAT3), retinoic acid-related orphan nuclear receptors γt (ROR-γt) and forkhead gene p3 (Foxp3) in the lumbar spinal cords of mice in each group. Results (1) The clinical symptom scale scores and inflammation scale scores of ginsenoside Rd group were significantly lower than those of PBS group (P<0.05);there were a large number of inflammatory cells infiltration and loss of myelin in the lumbar spinal cord of PBS group, while the loss of myelin sheath in ginsenoside Rd group was obviously decreased as compared with that in PBS group;the demyelination

关 键 词：人参皂甙RD 实验性自身免疫性脑脊髓炎 调节性T细胞 辅助性T细胞17 细胞失衡 

分 类 号：R285.5[医药卫生—中药学]