Liu Zhidan, Li Xiaoyan, Zhao Chuang, Chen Chunlan, Li Miao, Tan Qian, Zhang Ling, Liang Wei
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.
Zhongguo Zhen Jiu. 2017 Oct 12;37(10):1083-91. doi: 10.13703/j.0255-2930.2017.10.015.
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.
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 mRNA and protein expression of Foxp3, Galectin-9, RORγt, CARMA1, NF-κB were analyzed by Real-time PCR and Western Blotting method.
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 (<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 cytometry test indicated the cell count of Treg in the model group was reduced but that of Th17 was increased than the normal group (both <0.01); the moxibustion could increase significantly the cell count of Treg (<0.05), but no effect was observed on Th17 (>0.05). The results of ELISA test indicated the differences of increasing of IL-1β, IL-6, IL-17, IL-23, TGF-βas well as the reducing of IL-10 were not significant between the sham moxibustion group and the moxibustion group (all >0.05); moxibustion treatment could increase the content of Galectin-9 which was reduced in RA mice (<0.05). The results of RT-PCR and Western blotting test indicated the mRNA and protein expression of Foxp3, Galectin-9 were reduced in the model group (all <0.01), which could be up-regulated by moxibustion treatment (<0.05, <0.01); the mRNA and protein expression of RORγt, CARMA1, NF-κB was increased (all <0.01), which could be down-regulated by moxibustion treatment (<0.05, <0.01).
Moxibustion could improve the swelling of joint and inflammatory reaction of joint synovial in RA mice; the mechanism may be related to the regulation of Treg cells number in spleen and the expression of Foxp3, Galectin-9, RORγt, CARMA1, NF-κB, mRNA and protein expression.
观察艾灸对类风湿关节炎（RA）小鼠Treg/Th17失衡及相关信号通路的影响，以探讨艾灸治疗RA的作用机制。
将24只DBA/1J雄性小鼠随机分为正常组、模型组、假艾灸组和艾灸组，每组6只。除正常组外，其余小鼠采用皮下注射Ⅱ型胶原和佐剂的方法诱导RA模型。艾灸组小鼠取“足三里”（ST 36）和“肾俞”（BL 23）进行艾灸，每穴1壮，每壮1mg，每次每穴6壮。连续治疗6天为1个疗程，共治疗2个疗程，疗程间隔2天。假艾灸组小鼠处理同艾灸组，但不进行艾灸。采用胶原诱导性关节炎（CIA）的RA评分评估艾灸对关节肿胀的影响；通过HE染色观察关节炎症的病理变化；采用流式细胞术分析脾脏中Th17和Treg细胞计数；采用ELISA法分析细胞因子IL-1β、IL-6、IL-10、IL-17、IL-23、TGF-β和Galectin-9的含量；采用实时荧光定量PCR和蛋白质免疫印迹法分析Foxp3、Galectin-9、RORγt、CARMA1、NF-κB的mRNA和蛋白表达。
二次免疫后10～12天，观察到踝关节、足和趾关节红肿，表明RA模型建立成功。艾灸治疗15天后，艾灸组和假艾灸组关节肿胀均有所改善，艾灸组改善更明显（P<0.05）。病理变化方面，与正常组相比，模型组关节面粗糙，滑膜层变薄，假艾灸组有一定程度恢复；艾灸组关节面光滑，滑膜层增厚，接近正常组。流式细胞术检测结果显示，模型组Treg细胞计数低于正常组，Th17细胞计数高于正常组（均P<0.01）；艾灸可显著增加Treg细胞计数（P<0.05），对Th17细胞计数无影响（P>0.05）。ELISA检测结果显示，假艾灸组和艾灸组IL-1β、IL-6、IL-17、IL-23、TGF-β升高及IL-10降低的差异均无统计学意义（均P>0.05）；艾灸治疗可增加RA小鼠中降低的Galectin-9含量（P<0.05）。实时荧光定量PCR和蛋白质免疫印迹检测结果显示，模型组Foxp3、Galectin-9的mRNA和蛋白表达降低（均P<0.01），艾灸治疗可上调其表达（P<0.05，P<0.01）；RORγt、CARMA1、NF-κB的mRNA和蛋白表达升高（均P<0.01），艾灸治疗可下调其表达（P<0.05，P<0.01）。
艾灸可改善RA小鼠关节肿胀及关节滑膜炎症反应；其机制可能与调节脾脏中Treg细胞数量及Foxp3、Galectin-9、RORγt、CARMA1、NF-κB的mRNA和蛋白表达有关。
