[Effect of heat-reinforcing needling on the inflammation and necrotizing apoptosis of synovial cells in synovial tissues of knee joint in rabbits with cold syndrome rheumatoid arthritis].

To observe the effect of heat-reinforcing needling (HRN) on inflammatory factors and necrotizing apoptosis of synovial cells in synovial tissues of knee joint in rabbits with cold syndrome rheumatoid arthritis (RA), so as to explore its underlying mechanisms in treating RA.By using the random number table method, 40 New Zealand rabbits were randomly divided into normal, model, antagonist(AG), twist-reinforceing needling (TRN) and HRN groups, with 8 rabbits in each group. The model of cold syndrome RA was established by ovalbumin induction combined with Freund's complete adjuvant injection and cryogenic freezing method. In the AG group, the antagonist TAK-632 (25 mg/kg) was administered intragastrically, once every 2 days, for a total of 7 times. Rabbits of TRN and HRN groups were treated with corresponding acupuncture techniques on bilateral "Zusanli" (ST36) for 30 min, once a day for 14 days. After intervention, the changes of knee skin temperature and circumference were measured. Color Doppler ultrasound was used to observe the joint cavity effusion, synovial thickness and internal blood flow signal. The histomorphological changes of synovial tissues were observed after HE staining. ELISA was used to detect the contents of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in serum. Transmission electron microscope was used to observe the ultrastructure, necrosis and apoptosis of synovial cells. Western blot was used to detect the protein expressions of receptor-interacting protein kinase1 (RIPK1), RIPK3, mixed lineage kinase domain-like protein (MLKL), and phosphorylation （p）-MLKL in synovial tissues.Compared with the normal group, the synovial was diffusely hyperplasia, joint cavity effusion and abnormal blood flow signal were obvious, inflammatory cells were clustered, arranged closely and disordered in the model group. The findings of transmission electron microscopy showed disruption of cell membrane integrity, swollen or ruptured mitochondria, obviously ruptured nucleus, condensed and pyknotic chromatin and nucleolus in the model group. Also, the skin temperature of the knee joint was significantly decreased (P<0.01), while the circumference of the knee joint, the contents of TNF-α, IL-1β and IL-6 in serum, the protein expressions of RIPK1, RIPK3, p-MLKL and MLKL in synovial tissues were significantly increased (P<0.01) in the model group. Compared with the model group, synovial tissue hyperplasia, joint cavity effusion, abnormal blood flow signals, synovial cell proliferation, inflammatory cell infiltration, disruption of cell membrane integrity, cell swelling, cell rupture, and nuclear pyknosis were reduced to different degrees in the AG, TRN and HRN groups. Additionally, the skin temperature of the knee joint was increased (P<0.01, P<0.05), while the circumference of the knee joint, the contents of TNF-α, IL-1β and IL-6 in serum, the expressions of RIPK1, RIPK3, p-MLKL and MLKL in synovial tissues were decreased (P<0.01, P<0.05) in the AG, TRN and HRN groups. The effects of HRN and AG were notably superior to that of TRN in up-regulating skin temperature of the knee joint, and down-regulating the circumference of the knee joint, the contents of TNF-α, IL-1β and IL-6 in serum, the expressions of RIPK1, RIPK3, p-MLKL and MLKL in synovial tissues (P<0.01, P<0.05).HRN can reduce synovial inflammation of knee joint in rabbits with cold syndrome RA, which may be related to its function in inhibiting the necrotizing apoptosis of synovial cells.目的：观察热补针法对类风湿关节炎(RA)寒证家兔膝关节滑膜组织炎性因子及滑膜细胞坏死性凋亡的影响，探讨热补针法治疗RA的抗炎机制。方法：新西兰家兔按随机数字表法分为正常组、模型组、抑制剂组、捻转补法组、热补针法组，每组8只。采用卵蛋白诱导联合弗氏完全佐剂混合液注射，配合低温冷冻法复制RA寒证模型。抑制剂组予TAK-632(25 mg/kg)灌胃，每2 d治疗1次，共治疗7次；捻转补法组和热补针法组取双侧“足三里”分别施以捻转补法及热补针法操作，均留针30 min，1次/d，共治疗14次。干预结束后，测量各组家兔膝关节皮温、周径；彩色多普勒超声观察各组家兔膝关节腔积液、滑膜厚度及内部血流信号；HE染色法观察各组家兔膝关节滑膜组织病理形态学变化；ELISA法检测各组家兔血清肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β及IL-6含量；透射电镜法观察各组家兔膝关节滑膜细胞超微结构及坏死情况；Western blot法检测各组家兔膝关节滑膜组织受体相互作用蛋白(RIPK)1、RIPK3、混合谱系激酶结构域样蛋白(MLKL)、磷酸化(p)-MLKL的表达。结果：与正常组比较，模型组家兔膝关节皮温明显降低(P<0.01)，周径明显增大(P<0.01)，彩色多普勒超声示滑膜弥漫性增生，关节腔积液及异常血流信号明显；HE染色示滑膜细胞明显增生，大量炎性细胞簇集，且排列紊乱；血清TNF-α、IL-1β、IL-6含量升高(P<0.01)；透射电镜示细胞膜完整性破坏，线粒体肿胀甚则破裂呈空泡样，细胞核破裂明显，染色质、核仁显著凝聚固缩；膝关节滑膜组织RIPK1、RIPK3、p-MLKL及MLKL蛋白表达显著升高(P<0.01)。与模型组比较，抑制剂组、捻转补法组、热补针法组膝关节皮温均升高，周径均缩小(P<0.01，P<0.05)；滑膜组织增生、关节腔积液及异常血流信号不同程度减轻；滑膜细胞增生、炎性细胞浸润不同程度减轻；血清TNF-α、IL-1β及IL-6含量降低(P<0.01，P<0.05)；细胞膜结构破坏及线粒体肿胀、破裂、核固缩不同程度减轻；滑膜组织RIPK1、RIPK3、p-MLKL及MLKL蛋白表达显著降低(P<0.01，P<0.05)。与捻转补法组比较，抑制剂组和热补针法组各指标变化更明显(P<0.01，P<0.05)。结论：热补针法能减轻RA寒证家兔膝关节滑膜炎性反应，其机制可能与抑制滑膜细胞坏死性凋亡有关。.