Objective: To observe the effects of heat-reinforcing needling on synovial inflammation and microRNA-155 (miR-155)/Toll-like receptor 4 (TLR4)/nuclear transcription factor-κB (NF-κB) signaling axis, so as to investigate its anti-inflammatory mechanism in rabbits with cold syndrome of rheumatoid arthritis (RA).
Methods: A total of 36 rabbits were randomly divided into normal, model, agonist, inhibitor, heat-reinforcing needling (HRN) and agonist+heat-reinforcing needling (A+HRN) groups, with 6 rabbits in each group. The RA with cold syndrome model was induced by injecting ovalbumin dry powder and Freund's complete adjuvant combined with cold freezing. Rabbits in agonist group were intraperitoneally injected with miR-155 agomir 4.5 OD; rabbits in the inhibitor group were intraperitoneally injected with miR-155 antagomir 6.1 OD; rabbits in HRN group received heat-reinforcing needling at bilateral "Zusanli" (ST36) for 30 min；rabbits in A+HRN group received the same treatment as agonist group, and 30 min later, received the same treatment as the HRN group; rabbits in the normal and model groups were grasped and fixed in the same way, all groups received continuous treatment once a day for 7 d. After modeling, the knee joints of rabbits were examined by ultrasound, the pain threshold and the circumference were determined. After the interventions, the pain threshold and knee circumference were measured; the pathological morphology of synovial tissue of the knee joints were observed by HE staining; the mRNA levels of miR-155 and suppressor of cytokine signaling protein 1 (SOCS1), the expression levels of SOCS1, TLR4, NF-κB p65, interleukin (IL)-1β and IL-17A proteins in synovial tissue of knee joints were detected by real-time PCR and Western blot respectively.
Results: Compared with the normal group, the pain threshold was significantly decreased (P<0.05), and the knee circumference was significantly increased (P<0.05); the synovial tissue of knee joints showed significant hyperplasia, abundant blood flow signal, joint cavity effusion and obvious inflammatory invasion, the pathological score was significantly increased (P<0.05), the expressions of miR-155 mRNA and IL-1β, IL-17A, TLR4, NF-κB p65 proteins were significantly increased (P<0.05), the expressions of SOCS1 mRNA and protein were significantly decreased (P<0.05) in the model group. Compared with model group, the pain threshold was significantly increased (P<0.05), the circumference of knee joint was significantly decreased (P<0.05); in synovial tissue, the pathological score was decreased (P<0.05), the expression levels of miR-155 mRNA and IL-1β, IL-17A, TLR4, NF-κB p65 proteins were significantly decreased (P<0.05), and the expressions of SOCS1 mRNA and protein were significantly increased (P<0.05) in inhibitor group and HRN group, while the above changes in agonist group were reversed (P<0.05). Compared with the agonist group, the pain threshold was significantly increased (P<0.05), the knee circumference was significantly decreased (P<0.05), the synovial pathological score was significantly decreased (P<0.05), the expressions of miR-155 mRNA and IL-1β, IL-17A, TLR4, NF-κB proteins in synovial tissue were significantly decreased (P<0.05), and the expression levels of SOCS1 mRNA and protein were significantly increased (P<0.05) in A+HRN group.
Conclusion: The heat-reinforcing needling can increase the pain threshold, reduce the knee circumference and inhibit the inflammatory response in rabbits with RA cold syndrome. The possible mechanism is related to the regulation of miR-155/TLR4/NF-κB signaling axis.
目的：观察热补针法对类风湿关节炎(RA)寒证家兔滑膜炎性反应，以及对小核糖核酸-155(miR-155)/Toll样受体4(TLR4)/核转录因子-κB(NF-κB)信号轴的影响，探讨热补针法治疗RA的抑炎机制。方法：将36只家兔随机分为正常组、模型组、激动剂组、抑制剂组、热补针法组(热补组)、激动剂+热补组，每组6只。采用卵蛋白混合弗氏完全佐剂联合低温冷冻法诱导RA寒证家兔模型。激动剂组予腹腔注射miR-155 agomir 4.5 OD；抑制剂组予腹腔注射miR-155 antagomir 6.1 OD；热补组予热补针法针刺双侧“足三里”治疗，留针30 min；激动剂+热补组予腹腔注射miR-155 agomir 4.5 OD，30 min后予热补针法针刺双侧“足三里”治疗，留针30 min。各组干预均1次/d，连续7 d。造模完成后对家兔膝关节行超声检测，测定痛阈和膝关节周径。干预结束后测定家兔痛阈、膝关节周径；采用HE染色法观察家兔膝关节滑膜组织病理形态结构；实时荧光PCR法及Western blot法检测家兔膝关节滑膜组织miR-155、细胞因子信号传导抑制蛋白1(SOCS1)mRNA水平及白细胞介素(IL)-1β、IL-17A、TLR4、NF-κB p65、SOCS1蛋白表达水平。结果：与正常组家兔比较，模型组家兔痛阈显著降低(P<0.05)，膝关节周径显著增加(P<0.05)，膝关节滑膜组织增生，血流信号丰富，关节腔积液，炎性浸润明显，病理评分显著升高(P<0.05)，滑膜组织中miR-155 mRNA水平及IL-1β、IL-17A、TLR4、NF-κB p65蛋白表达水平显著升高(P<0.05)，SOCS1 mRNA及蛋白表达水平显著降低(P<0.05)；与模型组比较，抑制剂组及热补组家兔痛阈明显升高(P<0.05)，膝关节周径显著减小(P<0.05)，滑膜组织病理评分降低(P<0.05)，滑膜组织中miR-155 mRNA表达水平及IL-1β、IL-17A、TLR4、NF-κB p65表达明显降低(P<0.05)，SOCS1 mRNA及蛋白表达显著增高(P<0.05)，而激动剂组以上变化则相反(P<0.05)；与激动剂组比较，激动剂+热补组痛阈显著升高(P<0.05)，膝关节周径显著减小(P<0.05)，滑膜组织病理评分降低(P<0.05)，滑膜组织中miR-155 mRNA表达水平及IL-1β、IL-17A、TLR4、NF-κB p65蛋白表达水平显著降低(P<0.05)，SOCS1 mRNA及蛋白表达水平显著升高(P<0.05)。结论：热补针法干预RA寒证家兔可提高痛阈，减小膝关节周径，抑制炎性反应，其作用机制与调控miR-155/TLR4/NF-κB信号轴活性相关。.
Keywords: Inflammation; Micro RNA-155; Rheumatoid arthritis; Suppressor of cytokine signaling protein 1; heat-reinforcing needling.