艾灸对类风湿关节炎小鼠Treg/Th17细胞及其信号通路的影响  

Effects of moxibustion on Treg/Th17 cell and its signal pathway in mice with rheumatoid arthritis

作　　者：刘志丹 李晓燕[1,2] 赵创 陈春兰[1,2] 李淼 谭倩[4] 张玲 梁薇[1,2] LIU ZhiNan LI Xiaouan ZHAO Chuang CHEN Chunlan LI Miao TAN Qian ZHANG Ling LIANG Wi(Department of Acupuncture and Moxibustion, Shanghai Baoshan Hospital of Integrated Traditional Chinese Medicine and Western Medicine, Shanghai 201999, China Baoshan Branch of Shuguang Hospital, Shanghai University of TCM Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of TCM College of Basic Medical Science, Nanjing University of CM)

机构地区：[1]上海市宝山区中西医结合医院针灸科,上海201999 [2]上海中医药大学附属曙光医院宝山分院 [3]上海中医药大学附属岳阳中西医结合医院 [4]南京中医药大学基础医学院

出　　处：《中国针灸》2017年第10期1083-1092,共10页Chinese Acupuncture & Moxibustion

基　　金：国家自然科学基金青年科学基金项目:81403465;上海市"杏林新星"计划项目:ZY3-RCPY-2-2041;上海市宝山区卫生青年医学人才培养项目:bswsyq-2014-A 05

摘　　要：目的:观察艾灸对类风湿关节炎(RA)小鼠调节性T细胞(Treg)/辅助性T细胞(Th17)失衡及相关信号通路的影响,探讨艾灸治疗类风湿关节炎的作用机制。方法:24只雄性DBA/1J小鼠随机分为正常组、模型组、假治疗组和艾灸组,每组6只。将正常组之外的小鼠用Ⅱ型胶原加佐剂于尾部皮下注射,建立类风湿关节炎模型。艾灸组小鼠在"足三里""肾俞"穴予以1 mg/壮的艾灸治疗,每只小鼠每穴灸6壮,连续治疗6 d为一疗程,共治疗2个疗程,疗程间休息2 d。假治疗组仅进行与艾灸组一样的抓取。采用胶原诱导的关节炎模型(collagen induced arthritis,CIA)多发性关节炎指数评分标准观察艾灸对关节肿胀的影响,病变关节矢状面切片苏木素伊红(HE)染色观察关节炎症病理表现,流式细胞术分析小鼠脾脏Th17、Treg细胞数量,酶联免疫分析(ELISA)检测相关细胞因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、白细胞介素-17(IL-17)、白细胞介素-23(IL-23)、转化生长因子-β(TGF-β)及半乳糖凝集素9(Galectin-9)的含量,实时定量荧光PCR(Real-time PCR)及Western blotting方法分析基因Foxp3、Galectin-9、RORγt、CARMA1、核转录因子(NF-κB)mRNA及蛋白表达。结果:二次免疫后10~12d,小鼠1~2个后足踝关节及脚掌、足趾关节出现红肿,类风湿关节炎模型成功建立。艾灸治疗开始后第15天,艾灸组和假治疗组小鼠关节肿胀均有所好转,艾灸组疗效优于假治疗组(P<0.05)。病理形态上,RA模型小鼠关节面较正常小鼠粗糙,滑膜层变薄,假治疗组小鼠上述变化有一定程度的自行恢复,而艾灸组小鼠关节面恢复平整,滑膜层较治疗前变厚,与正常组表现较为接近。流式细胞术检测显示,RA模型小鼠脾脏中Treg细胞数量较正常组下降、Th17数量细胞上升(均P<0.01);艾灸治疗能使Treg细胞数量显著上升(P<0.05),而对Th17细胞数无显著影响(P>0.05)。ELISA检测Objective To observe the effects of moxibustion on Treg/Th17 imbalance and related signal pathway in mice with rheumatoid arthritis（RA）,so as to explore the action mechanism of moxibustion on RA.Methods Twenty-four DBA/1J male mice were randomly divided into a normal group,a model group,a sham moxibustion group and a moxibustion group,6 mice in each one.RA model was induced by subcutaneous injection of typeⅡcollagen and adjuvant at tail in mice other than the normal group.The mice in the moxibustion group were treated with moxibustion at＂Zusanli＂（ST 36） and ＂Shenshu＂（BL 23）,1 mg per cone,6 cones per acupoint.The consecutive 6-day treatment was taken as one course,and totally 2 courses were given with an interval of 2 d between courses.The mice in the sham moxibustion group were treated with immobilization as the moxibustion group.The effects of moxibustion on joint swelling was evaluated by RA scale of collagen induced arthritis（CIA）;the pathological changes of joint inflammation were observed by HE staining;the cell count of Th17 and Treg in spleen was analyzed by flow cytometry;the content of cytokine IL-1β,IL-6,IL-10,IL-17,IL-23,TGF-β and Galectin-9 were analyzed by ELISA;the m RNA and protein expression of Foxp3,Galectin-9,RORγt,CARMA1,NF-κB were analyzed by Real-time PCR and Western Blotting method.Results Ten to 12 d after the secondary immune,red and swelling of ankle joint,feet and toe joint were observed,indicating successful establishment of RA model.15 d into moxibustion treatment,the joint swelling was improved in the moxibustion group and the sham moxibustion group,which was superior in the moxibustion group（P〈0.05）.As for pathological changes,compare with the normal group,the articular surface was rougher and synovial layer thinner in the model group,which was recovered to a certain extent in the sham moxibustion group;the articular surface was smooth and synovial layer was thicker in the moxibustion group,which was similar to the normal group.The results of flow

关 键 词：类风湿关节炎 灸法 痹症 调节性T细胞 免疫失衡 

分 类 号：R245[医药卫生—针灸推拿学]