Objective: To explore the mechanism of moxibustion on the treatment of rheumatoid arthritis (RA) in the perspective of programmed cell death-1 and its ligand 1 (PD-1/PD-L1).
Methods: A total of 30 Japanese big ear white rabbits were randomly divided into a control group, a model group and a moxibustion group, 10 rabbits in each one. In the model group and the moxibustion group, RA model was prepared by the injection of Freund's complete adjuvant (FCA) into the hind knee joint cavities of each rabbit. In the control group, 0.9% sodium chloride solution of the same dose was injected. On the 8th day of experiment, in the moxibustion group, moxibustion was applied to "Shenshu" (BL 23) and "Zusanli" (ST 36), 5 cones at each acupoint, on the bilateral sides alternatively, once a day, 6 treatments as one course, with an interval of 1 days between the treatment courses. Totally, 3 courses of treatment were required. On the 1st, 7th, 14th, 21st and 28th days of experiment, successively, the circumference of the bilateral knee joints was measured with the tape. On the 28th day of experiment, H.E. staining was adopted to observe the histopathological morphology and to evaluate the score of knee synovial tissue. ELISA was used to determined the concentrations of soluble PD-1 (sPD-1) and its ligand 1 (sPD-L1), the interleukin 2 (IL-2) and IL-17 in knee synovial fluid and the concentrations of sPD-1 and sPD-L1 in serum. The histochemistry method was used to determine the expressions of membrane PD-1 (mPD-1) and its ligand 1 (mPD-L1) in spleen tissue.
Results: On the 14th, 21st and 28th days of experiment, the circumference of both knee joints was increased in each of the rabbits in the model group as compared with the control group (P<0.01), and it was reduced significantly in the moxibustion group as compared with the model group (P<0.01). Compared with the control group, the hyperplasia of synovial tissue and fibrous tissue, as well as inflammatory cell infiltration were increased obviously in the model group (P<0.01), and they were reduced significantly in the moxibustion group as compared with the model group (P<0.01, P<0.05). Compared with the control group, the concentrations of IL-2 and IL-17 in knee synovial fluid were increased in the rabbits of the model group (P<0.01). Compared with the model group, after the intervention with moxibustion, the concentrations of IL-2 and IL-17 in knee synovial fluid were reduced in the rabbits of the moxibustion group (P<0.05). Compared with the control group, the concentrations of sPD-1 and sPD-L1 in knee synovial fluid and serum in the rabbits of the model group were all increased (P<0.05). Compared with the model group, the concentration of sPD-1 in the knee synovial fluid and serum were reduced in the rabbits of moxibustion group (P<0.05). Compared with the control group, the expressions of mPD-1 and mPD-L1 in spleen tissue were increased obviously in the rabbits of the model group (P<0.05). Compared with the model group, the expression of mPD-L1 in spleen tissue was up-regulated in the rabbits of the moxibustion group (P<0.05).
Conclusion: Moxibustion could inhibit the over-activation of T cells by enhancing the negative regulation of PD-1/PD-L1 signaling pathway so as to play its effect in treatment of RA.
目的：从程序性死亡因子1及其配体1（PD-1/PD-L1）入手，探讨艾灸治疗类风湿性关节炎（RA）的机制。方法：将30只日本大耳白兔随机分为对照组、模型组和艾灸组，每组10只。模型组和艾灸组家兔予双后肢膝关节腔内注射弗氏完全佐剂（FCA）制备RA模型；对照组注射等量0.9%氯化钠溶液。艾灸组于实验第8天予艾灸“肾俞”“足三里”，每穴5壮，双侧交替，每日1次，6 次为一疗程，疗程之间休息1 d，共治疗3个疗程。实验第1、7、14、21、28天采用软皮尺测量家兔双侧膝关节周长；实验第28天，HE染色观察膝关节滑膜组织病理形态并评分，ELISA法检测膝关节滑膜液可溶性型程序性死亡因子1及其配体1（sPD-1、sPD-L1）、白介素-2（IL-2）、白介素-17（IL-17）及血清sPD-1、sPD-L1的含量，免疫组化法检测脾脏组织细胞膜型程序性死亡因子1及其配体1（mPD-1、mPD-L1）的表达。结果：实验第14、21、28天，模型组家兔双侧膝关节周长较对照组增加（P<0.01），艾灸组较模型组显著降低（P<0.01）。与对照组比较，模型组家兔膝关节滑膜组织、纤维组织增生，炎性细胞浸润明显增加（P<0.01），艾灸组较模型组均明显减少（P<0.01，P<0.05）。与对照组比较，模型组家兔膝关节滑膜液IL-2、IL-17含量升高（P<0.01）；与模型组比较，艾灸组家兔干预后膝关节滑膜液IL-2、IL-17含量降低（P<0.05）。与对照组比较，模型组家兔sPD-1、sPD-L1在膝关节滑膜液和血清中含量均升高（P<0.05）；与模型组比较，艾灸组家兔sPD-1在膝关节滑膜液和血清中含量降低（P<0.05）。与对照组比较，模型组家兔脾脏组织mPD-1、mPD-L1的表达明显升高（P<0.05）；与模型组比较，艾灸组家兔脾脏组织mPD-L1的表达上调（P<0.05）。结论：艾灸可能通过增强PD-1/PD-L1信号通路的负性调控，抑制T细胞的过度活化，从而发挥治疗RA的作用。.
Keywords: anti-inflammatory effect; moxibustion; programmed cell death-1 and its ligand 1 (PD-1/PD-L1); rheumatoid arthritis (RA).