热补针法对类风湿关节炎寒证家兔膝关节滑膜组织嘌呤能离子通道型受体7、Nod样受体蛋白3及半胱氨酸天冬氨酸蛋白酶-1表达的影响

Effect of heat-reinforcing needling on expression of P2X7R,NLRP3and Caspase-1in synovial tissues of knee joint in rabbits with cold syndrome rheumatoid arthritis

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嘌呤能离子通道型受体7(P2X7R)、Nod样受体蛋白3(NLRP3)、半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)表达的影响,探讨热补针法治疗RA的机制。方法:新西兰大白兔随机分为正常组、模型组、平补平泻组、热补针法组、拮抗剂组,每组6只。采用卵蛋白联合弗氏完全佐剂诱导及低温冷冻法复制RA寒证模型。热补针法组及平补平泻组分别于“足三里”施以相应的针刺手法,留针30 min,1次/d,治疗7 d;拮抗剂组腹腔注射A438079(2.5 mg/kg),1次/d,治疗7 d。运用彩色多普勒超声诊断仪观察各组家兔关节腔积液、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形态学改变;荧光定量PCR法检测各组家兔膝关节滑膜组织P2X7R、NLRP3、Caspase-1 mRNA的相对表达量;Western blot法检测各组家兔膝关节滑膜组织白细胞介素(IL)-1β、IL-6、肿瘤坏死因子α(TNF-α)的蛋白表达水平。结果:与正常组比较,模型组膝关节滑膜增厚,线状及点状血流信号增多,关节积液明显,滑膜被覆细胞肿大、滑膜基质重度增生,炎性细胞浸润明显,滑膜组织中P2X7R、NLRP3、Caspase-1 mRNA表达及IL-1β、IL-6、TNF-α蛋白表达均显著升高(P<0.01);与模型组比较,各针刺组和拮抗剂组滑膜厚度、关节积液、异常血流信号,滑膜被覆细胞、滑膜基质增生情况及炎性细胞浸润情况均改善,滑膜组织中的P2X7R、NLRP3、Caspase-1 mRNA表达及IL-1β、IL-6、TNF-α蛋白表达均明显降低(P<0.05,P<0.01);与平补平泻组比较,热补针法组、拮抗剂组P2X7R、NLRP3、Caspase-1 mRNA表达水平,IL-1β、IL-6、TNF-α蛋白表达降低更为明显(P<0.01,P<0.05)。结论:热补针法可改善RA家兔的膝关节滑膜炎性损伤,其机制可能与调节P2X7R/NLRP3/Caspase-1信号通路有关。

Objective To observe the effects of heat-reinforcing needling on the expression of purinergic ligand-gated ion channel 7 receptor(P2 X7 R),Nodlike receptor protein 3(NLRP3)and Caspase-1 in synovium tissues of knee joint of rabbits with cold syndrome rheumatoid arthritis(RA),so as to explore the anti-inflammatory mechanism of heat reinforcing needling in the treatment of RA.Methods Thirty male New Zealand white rabbits were randomly divided into normal group,model group,reinforcing-reducing needling group(RRG),heat-reinforcing needling group(HRG),and antagonist group(AG),with 6 rabbits in each group.The model of cold syndrome RA was established by ovalbumin combined with Freund’s adjuvant and cryogenic freezing.Rabbits of RRG and HRG were treated with corresponding acupuncture techniques on both sides of“Zusanli”(ST36)for 30 min,once a day for 7 days;Rabbits of the AG was intraperitoneally injected with A438079(2.5 mg/kg),once a day for 7 days.After intervention,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 by HE staining.Quantitative real-time PCR method was used to detect the mRNA expression levels of P2 X7 R,NLRP3,and Caspase-1 in synovial tissues.Western blot was used to detect the protein expression of interleukin(IL)-1β,IL-6 and tumor necrosis factor(TNF)-αin synovial tissues.Results Compared with the normal group,the synovial tissues in the model group was thickened,linear and punctate blood flow signals were increased,joint effusion was obvious,synovial coating cells were enlarged,the synovial matrix was severely hyperplasia,inflammatory cells infiltration was obvious.The mRNAs expression levels of P2 X7 R,NLRP3,Caspase-1 and IL-1β,IL-6,TNF-αproteins in synovial tissues were significantly increased(P<0.01).Compared with model group,abnormal blood flow signals,synovial thickness,joint effusion,proliferation of synovial matrix,inflammatory cell infiltration and synovial coating cells in the RRG,HRG and AG were improved.The mRNAs expression levels of P2 X7 R,NLRP3,Caspase-1 and IL-1β,IL-6,TNF-αproteins in synovial tissues were decreased in the RRG,HRG and AG(P<0.05,P<0.01).Compared with the RRG,the above indexes were lower in the HRG and AG(P<0.05,P<0.01).There was no significant difference in all indexes above between the HRG and AG(P<0.01,P<0.05).Conclusion Heat reinforcing needling can improve synovial inflammation of RA,which may be related to regulating the expressions of P2 X7 R/NLRP3/Caspase-1 signaling pathway.