艾灸对实验性类风湿性关节炎家兔滑膜组织NLRP3炎性小体激活因素Cathepsin-B、ROS水平的影响

Effects of Moxibustion on Levels of Cathepsin-B and ROS of NLRP3 Inflammasome in Synovial Tissue of Experimental RA Rabbits

目的观察艾灸对类风湿性关节炎(RA)的抗炎消肿作用,研究艾灸对实验性RA家兔滑膜NLRP3 mRNA、组织蛋白酶B(Cathepsin-B)mRNA的表达和活性氧(ROS)含量的影响,探索艾灸对RA中NLRP3炎性小体激活状态的影响。方法日本大耳白兔24只,按体质量、性别分层,随机分为空白对照组(简称空白组)、RA模型组(简称模型组)、艾灸治疗组(简称艾灸组),每组8只。将弗氏完全佐剂(FCA)按0.5 mL/kg体质量剂量注入模型组、艾灸组动物双膝关节腔内。同法将等量的无菌生理盐水注入空白组动物作为对照。艾灸治疗艾灸组动物双侧“肾俞”“足三里”,每穴各5壮,每日1次。6 d为1个疗程,共治疗3个疗程,疗程之间休息1 d。治疗后观测各家兔左、右膝关节周长,治疗后第21天进行膝关节滑膜组织取材,采用RT-PCR法检测各组家兔膝关节滑膜组织中NLRP3 mRNA、Cathepsin-B mRNA的表达;采用ROS检测试剂盒检测各组家兔膝关节滑膜组织中ROS的含量。结果造模后白兔双膝关节周长较空白组明显增高(P<0.01);艾灸治疗后白兔双膝关节周长较模型组显著下降(P<0.05)。与空白组比较,模型组的NLRP3 mRNA、Cathepsin-B mRNA的表达和ROS荧光强度均显著升高(P<0.01);而与模型组比较,艾灸组的NLRP3 mRNA、Cathepsin-B mRNA的表达和ROS荧光强度均降低明显(P<0.05)。结论艾灸对实验性RA家兔膝关节滑膜炎症过程具有明显的抑制作用;艾灸“肾俞”“足三里”穴通过下调Cathepsin-B的表达和抑制ROS的生成从而抑制NLRP3的激活可能是艾灸治疗RA改善其病理过程的机制之一。

Objective To observe the anti-inflammatory and detumescence effect of moxibustion on rheumatoid arthritis(RA),study the effect of moxibustion on the expressions of NOD-like receptor protein 3(NLRP3)mRNA and cathepsin-B mRNA and reactive oxygen species(ROS)content in synovium of experimental RA rabbits and explore the effect of moxibustion on the activation state of NLRP3 inflammasome in RA.Methods Twenty-four Japanese Big-ear white rabbits were randomly divided into the blank control group(the blank group),the RA model group(the model group)and the moxibustion treatment group(the moxibustion group)with 8 rabbits in each group.Freund’s complete adjuvant(FCA)(0.5 mL/kg)was injected into the knee joint cavities of the model group and the moxibustion group.In the same way,the same dose of sterile normal saline was injected into the blank group as the control.In the moxibustion group,moxibustion was used at“Shenshu(BL23)”and“Zusanli(ST36)”with 5 Zhuang for each acupoint,once a day.Continuous 6 days was one course of treatment,there were a total of 3 courses of treatment and a day off between treatment courses.After treatment,the perimeters of the left and right knee joints of the rabbits were observed.On the 21 st day after treatment,the synovium tissue of knees was sampled for analyzing the expressions of NLRP3 mRNA and Cathepsin-B mRNA and the content of ROS in each group by RT-PCR and ROS Assay Kit.Results Compared with those of the blank group,the perimeters of bilateral knee joints in the model group were significantly increased(P<0.01).Compared with those of the model group,the perimeters of bilateral knee joints in the moxibustion group were significantly decreased(P<0.05).Compared with those of the blank group,the expressions of NLRP3 mRNA and Cathepsin-B mRNA and ROS fluorescence intensity in the model group were significantly increased(P<0.01).Compared with those of the model group,the expressions of NLRP3 mRNA and Cathepsin-B mRNA and ROS fluorescence intensity in the moxibustion group were significantly decreased(P<0.05).Conclusion Moxibustion can obviously inhibit the synovitis process of experimental RA rabbits.Moxibustion at“Shenshu(BL23)”and“Zusanli(ST36)”can inhibit the activation of NLRP3 by down-regulating the expression of Cathepsin-B and inhibiting the production of ROS,which may be one of the mechanism of moxibustion to improve the pathological process of RA.