Objectives: To observe the effect of heat-reinforcing needling on the level of tumor necrosis factor receptor associated factor 6 (TRAF6)-K63 ubiquitination and the nuclear factor-κB (NF-κB) pathway in the synovial tissue of the knee joints of a rabbit model with rheumatoid arthritis (RA) of cold syndrome, so as to explore the mechanism by which the heat-reinforcing needling inhibits inflammatory response in RA synovitis.
Methods: Eight out of 32 rabbits were randomly selected and assigned to the normal group. The remaining rabbits were induced to establish the RA cold syndrome model using ovalbumin combined with complete Freund's adjuvant and low-temperature cryogenic methods. After modeling evaluation, the rabbits were randomly divided into model group, inhibitor group, and heat-reinforcing needling group, with 6 rabbits in each group. Rabbits in the inhibitor group received intraperitoneal injection of C25-140 solution (2.5 mg/kg), twice a day for 14 days. Rabbits in the heat-reinforcing needling group received acupuncture at "Zusanli"(ST36) according to the operation standard of the heat-reinforcing needling and the needle was retained for 30 min, once a day, for 14 days. After the intervention, knee joint circumference and pain threshold of the rabbits were measured. Color Doppler ultrasound was used to observe changes in knee joint imaging. HE staining was performed to observe morphological changes in the synovial tissue. Immunohistochemical method was used to observe the expression of inflammatory factors tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the synovial tissue. Western blot was used to detect the protein expression of ubiquitin-conjugating enzyme E2 13 (Ubc13) and NF-κB p65 in the synovial tissue, and co-immunoprecipitation was used to detect the level of TRAF6-K63 ubiquitination in the synovial tissue.
Results: Compared with the normal group, the model group showed a decreased pain threshold (P<0.05), increased knee joint circumference (P<0.05), and increased average optical density of TNF-α, IL-1β, and IL-6 in the synovial tissue(P<0.05), as well as elevated protein expression of NF-κB p65 and TRAF6-K63 ubiquitination levels (P<0.05), while Ubc13 protein expression was decreased (P<0.05). Compared with the model group, the heat-reinforcing needling group and the inhibitor group had increased pain thresholds and reduced knee joint circumferences (P<0.05), and significantly reduced average optical density of TNF-α, IL-1β, and IL-6 in the synovial tissue (P<0.05), as well as decreased protein expression of NF-κB p65, TRAF6-K63 ubiquitination levels (P<0.05), while Ubc13 protein expression was increased (P<0.05). Compared to the heat-reinforcing needling group, the inhibitor group showed reduced pain threshold (P<0.05), and lower average optical density values of TNF-α, IL-1β, IL-6 in the synovial tissue (P<0.05), as well as decreased expression of NF-κB p65 protein, TRAF6-K63 ubiquitination levels (P<0.05), while Ubc13 protein expression was increased (P<0.05). The imaging changes of the knee joints and the pathological morphological changes of the synovial tissues in the two treatment groups are both less severe than those in the model group.
Conclusions: The heat-reinforcing needling may inhibit inflammatory response in synovitis by suppressing TRAF6-K63 ubiquitination levels, down-regulating the activity of the NF-κB pathway, and reducing the secretion of downstream inflammatory factors.
目的: 观察热补针法对类风湿关节炎（RA）寒证家兔模型膝关节滑膜组织中肿瘤坏死因子受体相关因子6（TRAF6）-K63泛素化水平及核因子-κB（NF-κB）通路的影响，探讨热补针法抑制RA滑膜炎性反应的机制。方法: 32只家兔中随机抽取8只为正常组。剩余家兔采用卵蛋白联合完全弗氏佐剂诱导及低温冷冻法建立RA寒证模型，评估后随机分为模型组、抑制剂组、热补针法组，每组各6只。抑制剂组给予C25-140溶液（2.5 mg/kg）腹腔注射，2次/d，共14 d；热补针法组采用热补针法的操作标准针刺“足三里”，留针30 min，1次/d，共14 d。干预完成后测量家兔膝关节周径和痛阈值，彩色多普勒超声观察膝关节影像学变化，HE染色观察膝关节滑膜组织形态学变化，免疫组织化学法检测滑膜组织中肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1β、IL-6的表达，Western blot法检测滑膜组织中泛素结合酶E2 13（Ubc13）、NF-κB p65的蛋白表达，免疫共沉淀法检测滑膜组织中TRAF6-K63泛素化水平。结果: 与正常组比较，模型组痛阈值降低（P<0.05），膝关节周径增加（P<0.05），滑膜组织中TNF-α、IL-1β、IL-6的平均吸光度值升高（P<0.05），NF-κB p65蛋白表达、TRAF6蛋白K63泛素化水平升高（P<0.05），Ubc13蛋白表达降低（P<0.05），多普勒超声见膝关节滑膜广泛性增生、肥厚，关节积液明显，HE染色示滑膜细胞肿胀变大明显，形态破坏，广泛的炎性细胞及淋巴细胞的浸润；与模型组比较，热补针法组及抑制剂组的痛阈值升高（P<0.05），膝关节周径减小（P<0.05），滑膜组织中TNF-α、IL-1β、IL-6的平均吸光度值降低（P<0.05），NF-κB p65蛋白表达、TRAF6蛋白K63泛素化水平明显降低（P<0.05），Ubc13蛋白表达升高（P<0.05），多普勒超声见滑膜增生、肥厚程度有所改善，血流信号减少，关节积液明显减少，HE染色示滑膜细胞增生、肿大及炎性浸润较模型组均有所改善；与热补针法组比较，抑制剂组家兔痛阈值降低（P<0.05），膝关节滑膜组织TNF-α、IL-1β、IL-6的平均吸光度值降低（P<0.05），NF-κB p65蛋白表达、TRAF6蛋白K63泛素化水平降低（P<0.05），Ubc13蛋白表达升高（P<0.05）。结论: 热补针法可通过抑制TRAF6-K63泛素化水平，下调NF-κB通路活性，减少下游炎性因子的分泌，从而起到抑制滑膜炎性反应的作用。.
Keywords: Heat-reinforcing needling; Inflammatory response; Nuclear factor-κB pathway; Rheumatoid arthritis; TRAF6 ubiquitination.