Objective: To observe the effect of electroacupuncture (EA) of "Zusanli"(ST36) and "Guanyuan"(CV4) on the apoptosis rate of synoviocytes and protein expression of Fas, FasL and Caspase-3 in synovial tissue of adjuvant-induced arthritis (AIA) rats, so as to explore its mechanisms underlying improvement of rheumatoid arthritis （RA）.
Methods: A total of 24 rats were randomly divided into normal, model, medication and EA groups, with 6 rats in each group. The AIA model was established by injection of complete Freund's adjuvant (CFA, 0.1 mL) into the left hindlimb paw. The rats in the medication group received intraperitoneal injection of 0.35 mg/kg of methotrexate, twice a week for 4 weeks. The rats in the EA group received EA stimulation of ST36 and CV4 (20 Hz/50 Hz, 1 mA) for 20 min, 6 times a week for 4 weeks. The left hind paw volume was measured using a paw volume meter, and histopathological changes of synovial tissue were observed by light microscope after H.E. staining. The serum contents of tumor necrosis factor-α（TNF-α） and interleukin-1 （IL-1） were measured by ELISA. The apoptosis of synoviocytes was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL), and the expression of apoptosis-associated proteins Fas, FasL and Caspase-3 in synovium were detected by Western blot.
Results: Compared with the normal group, the left hind paw volume from day 3 to 24 after administration of CFA, serum IL-1 and TNF-α contents were significantly increased (P<0.01), while the expressions of Fas, FasL and Caspase-3 proteins and apoptotic rate of synoviocytes were significantly decreased in the model group (P<0.01). In comparison with the model group, the paw volume from day 17 to 24 after modeling, the serum IL-1 and TNF-α contents were significantly reduced (P<0.01), while the apoptotic rate of synoviocytes, expressions of Fas protein in both medication and EA groups, Caspase-3 protein in the acupuncture group and FasL protein in the medication group were increased (P<0.01, P<0.05). Compared with the medication group, the expression of FasL protein was decreased in EA group (P<0.05). H.E. stain showed obvious hyperplasia of the synovial lining layer, and disordered arrangement of synovial cells, with edema and enlargement in some cells in the model group, which was relatively milder in both medication and EA groups.
Conclusion: EA of ST36 and CV4 can promote the apoptosis of synoviocytes and the expressions of Fas and FasL proteins in AIA rats, which may contribute to its role in relieving synovitis through activating Fas/FasL signaling.
目的：观察电针“足三里”“关元”对佐剂性关节炎(AIA)大鼠滑膜组织细胞凋亡率及其他凋亡蛋白含量的影响, 探讨电针通过死亡受体（Fas）凋亡途径调节滑膜细胞凋亡和抗炎治疗类风湿关节炎（RA）的机制。方法：SD大鼠按照随机数字表法分为正常组、模型组、甲氨蝶呤组、电针组, 每组6只。选用弗氏完全佐剂注射法制备AIA大鼠模型。造模后第3天开始干预, 甲氨蝶呤组按照0.35 mg/kg的剂量进行灌胃, 每周2次；电针组予以电针“足三里”“关元”, 每次20 min, 每周6次；两组均干预4周。用足趾容积测量仪测量各组大鼠左后肢足趾容积, HE染色观察踝关节滑膜组织形态变化, ELISA法检测大鼠血清肿瘤坏死因子-α(TNF-α)、白细胞介素-1(IL-1)含量, TUNEL染色观察踝关节滑膜细胞凋亡率, Western blot法检测踝关节滑膜组织中凋亡相关基因Fas、死亡受体配体(FasL)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的蛋白表达变化。结果：与正常组比较, 模型组大鼠同时点左足趾容积增加（P<0.01）, 血清IL-1、TNF-α含量升高(P<0.01), 滑膜细胞凋亡率及滑膜组织Fas、FasL、Caspase-3蛋白表达降低(P<0.01)。与模型组比较, 甲氨蝶呤组、电针组大鼠第17、24天足趾容积减小（P<0.01）, 血清IL-1、TNF-α含量降低（P<0.01）, 滑膜细胞凋亡率增加(P<0.01), 甲氨蝶呤组滑膜组织Fas、FasL蛋白表达升高(P<0.01, P<0.05), 电针组滑膜组织Fas、Caspase-3蛋白表达升高(P<0.05)。与甲氨蝶呤组比较, 电针组滑膜组织FasL蛋白表达降低(P<0.05)。HE染色显示模型组大鼠滑膜组织滑膜衬里层增生明显, 滑膜细胞排列紊乱, 部分细胞出现水肿, 有纤维组织增生, 炎性细胞浸润；甲氨蝶呤组大鼠滑膜部分增厚, 炎性细胞浸润较模型组减少；电针组大鼠滑膜形态较模型组显著改善, 滑膜增生情况较模型组减轻, 炎性细胞浸润减少。结论：电针“足三里”“关元”能增加AIA大鼠滑膜细胞凋亡率, 上调滑膜组织Fas、Caspase-3蛋白表达, 表明电针治疗RA可能通过Fas凋亡途径促进滑膜细胞凋亡, 发挥缓解滑膜炎性反应的作用。.
Keywords: Caspase-3; Electroacupuncture; Fas; FasL; Rheumatoid arthritis; Synoviocyte apoptosis.