热补针法对类风湿关节炎寒证模型家兔血清炎性因子及膝关节滑膜组织自噬的影响

Effect of heat-reinforcing needling on expression of serum inflammatory factors and autophagy of knee joint synovial tissue in rheumatoid arthritis rabbits with cold syndrome

目的:观察热补针法对类风湿性关节炎(RA)寒证模型家兔血清炎性因子及膝关节滑膜组织自噬的影响,探讨热补针法治疗RA的抗炎机制。方法:清洁级新西兰家兔随机分为正常组、模型组、热补针法组、抑制剂组和激动剂组,每组10只。采用弗氏佐剂、卵蛋白混合溶液注射联合低温冷冻+风寒湿法建立RA寒证模型。热补针法组予“足三里”施热补针法干预,留针30 min, 1次/d,干预14 d;抑制剂组和激动剂组分别腹腔注射自噬抑制剂3-甲基腺嘌呤(1.9 mg/kg)和自噬激动剂雷帕霉素(2.5 mg/kg),隔日1次,共7次。测量各组家兔膝关节周径、皮肤温度;用彩色多普勒超声检查家兔膝关节滑膜厚度、关节腔积液及内部血流信号;ELISA法检测血清肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、IL-6及C反应蛋白(CRP)含量;透射电镜观察滑膜细胞超微形态结构及自噬小体;Western blot法检测滑膜组织自噬相关蛋白Atg5、Unc-51样自噬激活激酶1(ULK1)、微管相关蛋白轻链3B(LC3B)及Beclin-1的表达水平;荧光定量PCR法检测滑膜组织NOD样受体蛋白3(NLRP3)、核因子-κB(NF-κB)mRNA的表达水平。结果:与正常组比较,模型组家兔膝关节周径显著增大(P<0.01),皮肤温度明显降低(P<0.01),血清TNF-α、IL-1β、IL-6及CRP含量升高(P<0.01),滑膜组织Atg5、ULK1、Beclin-1蛋白表达水平及LC3BⅡ/LC3BⅠ显著降低(P<0.01),NLRP3、 NF-κB mRNA表达水平显著升高(P<0.01)。与模型组、抑制剂组比较,热补针法组及激动剂组膝关节周径缩小(P<0.01),皮肤温度升高(P<0.01),血清TNF-α、IL-1β、IL-6及CRP含量降低(P<0.01),滑膜组织Atg5、ULK1、Beclin-1表达水平及LC3BⅡ/LC3BⅠ升高(P<0.01),NLRP3、NF-κB mRNA表达水平显著降低(P<0.01)。正常组家兔关节滑膜光滑平整,无关节腔积液及异常血流信号,滑膜细胞核染色致密均匀,核膜光滑完整;模型组滑膜增厚、血流信号丰富,关节囊积液明显,滑膜细胞核染色质固缩,核膜模糊残缺,偶见自噬小体;热补针法组及激动剂组滑膜变薄、血流信号减弱,滑膜细胞核染色固缩不明显,细胞核膜较清晰,可见吞噬泡及自噬小体;抑制剂组上述改善均不明显。结论:热补针法能显著缓解RA寒证模型家兔膝关节滑膜炎性反应,其机制可能与增强滑膜细胞自噬活性,抑制炎性因子TNF-α、IL-1β、IL-6及CRP的合成与释放有关。

Objective To observe the effect of heat-reinforcing needling on the expression of serum inflammatory factors and autophagy of knee synovial tissue in rheumatoid arthritis(RA) rabbits with cold syndrome, so as to explore its mechanism of anti-inflammatory in the treatment of RA. Methods Fifty rabbits were randomly divided into normal, model, heat-reinforcing needling, inhibitor and agonist groups(n=10 rabbits in each group). The model of RA with cold syndrome was established by Freund’s adjuvant and ovalbumin mixed solution injection combined with freezing and wind-cold dampness method. Heat-reinforcing needling was applied at “Zusanli”(ST36) for 30 min, once a day for 14 days. Rabbits of the inhibitor and agonist groups were given intraperitoneally injected with autophagy inhibitor 3-methyladenine(3-MA) or autophagy agonist rapamycin, once every 2 days for 7 days. The knee circumference and skin temperature of the rabbits in each group were measured. Color doppler ultrasonography was applied to examine the synovial membrane, joint effusion and blood flow signals in the knee joints of the rabbits in each group. Serum tumor necrosis factor(TNF)-α, interleukin(IL)-1β, IL-6 and C-creactive protein(CRP) were detected by ELISA. Transmission electron microscopy was applied to observe the ultrastructure and autophagosomes of synovial cells. The protein expressions of autophagy-related protein Atg5, serine/threonine protein kinase-dysregulated 51-like kinase 1(ULK1), microtubule-associated protein light chain 3 B(LC3 B), and Beclin-1 were detected by Western blot. Fluorescence quantitative PCR was used to detect the mRNA expressions of NOD-like receptor 3(NLRP3) and nuclear factor-κB(NF-κB). Results Compared with the normal group, the circumference of the knee joint was increased(P<0.01), the skin temperature was decreased(P<0.01), the knee joint synovium was thickened and the blood flow signal was abundant, the contents of serum TNF-α, IL-1β, IL-6, and CRP were increased(P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3 BⅡ/LC3 BⅠof synovial tissue were significantly decreased(P<0.01), the mRNA expressions of NLRP3 and NF-κB were increased(P<0.01) in the model group. In comparison with the model and inhibitor groups, the circumference of the knee joint was decreased(P<0.01), whlie the skin temperature was increased(P<0.01), the synovial membrane became thinner and the blood flow signal was wea-kened, the contents of TNF-α, IL-1β, IL-6 and CRP were decreased(P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3 B Ⅱ/LC3 B Ⅰ were increased(P<0.01), and the mRNA expressions of NLRP3 and NF-κB were decreased(P<0.01) in the heat-reinforcing needling and agonist groups. Conclusion Heat-reinforcing needling can alleviate the inflammatory response of the knee joint synovium in RA rabbits with cold syndrome, which may be related to its function in enhancing the autophagy activity of synovial cells and inhibiting the synthesis and release of inflammatory factors TNF-α, IL-1β, IL-6 and CRP.