悬灸与直接灸治疗类风湿关节炎小鼠的对比研究
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摘要
目的:比较悬灸与直接灸对类风湿关节炎小鼠治疗作用的异同。方法:DBA/1J小鼠36只随机分为正常组、模型组、不治疗组、悬灸组、直接灸组和安慰治疗组。复制Ⅱ型胶原诱导的小鼠类风湿关节炎模型并予以艾灸治疗,观察悬灸与直接灸对关节肿胀、关节滑膜病理表现的影响,流式细胞术分析Th17、Treg细胞变化,ELISA检测相关细胞因子IL-1β/IL-6/IL-10/IL-17/TGF-β的含量。结果:经两次免疫后,小鼠足踝关节及脚掌、足趾关节出现红肿,CIA模型成功建立。治疗15天后,悬灸组小鼠关节肿胀有明显消退,关节评分低于模型组、不治疗组、直接灸组、安慰治疗组(P <0. 05)。病理表现上,悬灸组小鼠病变关节面粗糙较前缓解,滑膜层较治疗前变厚,与正常组病理表现较为接近;直接灸组关节面有一定恢复,但较悬灸组差;不治疗组及安慰治疗组无明显变化。ELISA检测发现CIA模型小鼠脾脏中IL-1β、IL-6、IL-17等致炎细胞因子表达增高、抑炎因子IL-10、TGF-β表达降低(均P <0. 01);悬灸和直接灸治疗后致炎因子含量与模型组比较相应降低、抑炎因子含量明显提高(均P <0. 01),且两组之间无统计学差异(均P> 0. 05)。流式细胞术显示:与正常小鼠相比,CIA模型小鼠调节性T细胞下降,而Th17细胞则上升(均P <0. 01);治疗后悬灸组Treg细胞的数量显著上升、Th17细胞的数量显著下降(均P <0. 01);而直接灸组、不治疗组、安慰治疗组Treg细胞、Th17数量与模型组比较无显著统计学差异(P> 0. 05)。结论:悬灸、直接灸均能减轻CIA小鼠关节滑膜病变、下调炎症因子和上调抗炎细胞因子含量;但与悬灸治疗比较,直接灸治疗对CIA小鼠关节肿胀度和脾Treg/Th17细胞失衡状态无显著作用。直接灸治疗CIA小鼠的疗效及作用机理还需进一步研究。
Objective: To compare the effect difference between suspended moxibustion and direct moxibustion in mice with rheumatoid arthritis( RA). Methods: 36 DBA/1 J mice were randomly divided into the normal group,the model group,the non-treatment group,the suspended moxibustion group,the direct moxibustion group,and the placebo group. RA model was induced by injecting typeⅡ collagen. Moxibustion treatment was given to RA mice,the ankle swelling was evaluated by RA scale. The pathological changes of articular cavity and synovial membrane were observed by HE analysis. Flow cytometry was applied to evaluate the changes of Treg and Th17. Expressions of IL-1β,IL-6,IL-10,IL-17 and TGF-β were analyzed by ELISA method. Results: RA model was successfully established by the injection of typeⅡ collagen. The joint swelling of RA mice partly recovered in the suspended moxibustion group,the joint score was lower than that in the other RA groups( P < 0. 05). The joint synovial layer in RA mice was rougher and thinner than that of the normal mice,the synovial layer was smoother and thicker in the suspended moxibustion group after the treatment; Similar changes were observed in the direct moxibustion group,but they were less than those in the suspended moxibustion group; no changes were seen in the non-treatment group and the placebo group. IL-1β,IL-6,and IL-17 these inflammation related cytokines were increased and anti-inflammatory related cytokines,such as IL-10 and TGF-β were decreased in RA mice( P < 0. 01). The rise of IL-1β,IL-6,IL-17 and the decline of IL-10 and TGF-β were observed in the suspended moxibustion group and the direct moxibustion group( P < 0. 01),but no difference was observed between the two groups( P > 0. 05). Treg cells were decreased and Th17 cells were increased in RA mice( P < 0. 01); with the suspended moxibustion intervention,Treg cells were increased and Th17 cells were decreased( P < 0. 01); however,no difference was observed in the other groups( P > 0. 05). Conclusion: Both suspended moxibustion and direct moxibustion can improve joint synovial pathological changes,down-regulate inflammation related cytokines and up-regulate anti-inflammatory related cytokines. But direct moxibustion has no effect on joint swelling and Treg/Th17 cell imbalance.The influence of direct moxibustion on CIA mice need further study.
Objective: To compare the effect difference between suspended moxibustion and direct moxibustion in mice with rheumatoid arthritis( RA). Methods: 36 DBA/1 J mice were randomly divided into the normal group,the model group,the non-treatment group,the suspended moxibustion group,the direct moxibustion group,and the placebo group. RA model was induced by injecting typeⅡ collagen. Moxibustion treatment was given to RA mice,the ankle swelling was evaluated by RA scale. The pathological changes of articular cavity and synovial membrane were observed by HE analysis. Flow cytometry was applied to evaluate the changes of Treg and Th17. Expressions of IL-1β,IL-6,IL-10,IL-17 and TGF-β were analyzed by ELISA method. Results: RA model was successfully established by the injection of typeⅡ collagen. The joint swelling of RA mice partly recovered in the suspended moxibustion group,the joint score was lower than that in the other RA groups( P < 0. 05). The joint synovial layer in RA mice was rougher and thinner than that of the normal mice,the synovial layer was smoother and thicker in the suspended moxibustion group after the treatment; Similar changes were observed in the direct moxibustion group,but they were less than those in the suspended moxibustion group; no changes were seen in the non-treatment group and the placebo group. IL-1β,IL-6,and IL-17 these inflammation related cytokines were increased and anti-inflammatory related cytokines,such as IL-10 and TGF-β were decreased in RA mice( P < 0. 01). The rise of IL-1β,IL-6,IL-17 and the decline of IL-10 and TGF-β were observed in the suspended moxibustion group and the direct moxibustion group( P < 0. 01),but no difference was observed between the two groups( P > 0. 05). Treg cells were decreased and Th17 cells were increased in RA mice( P < 0. 01); with the suspended moxibustion intervention,Treg cells were increased and Th17 cells were decreased( P < 0. 01); however,no difference was observed in the other groups( P > 0. 05). Conclusion: Both suspended moxibustion and direct moxibustion can improve joint synovial pathological changes,down-regulate inflammation related cytokines and up-regulate anti-inflammatory related cytokines. But direct moxibustion has no effect on joint swelling and Treg/Th17 cell imbalance.The influence of direct moxibustion on CIA mice need further study.
引文
[1]叶伟胜,张铁良.类风湿关节炎流行病学进展[J].国际骨科学杂志,2009,30(3):144-147.
[2]胡晓敏,宗英,余珊珊,等.类风湿关节炎治疗药物的研发进展及趋势[J].中国新药杂志,2017,26(1):36-43.
[3]胡玲,郝峰,钟峰,等.艾灸治疗类风湿关节炎的多中心随机对照临床研究[J].环球中医药,2011,6(5):401-405.
[4]郝锋,胡玲,吴子建,等.隔姜灸治疗活动期类风湿关节炎量效关系的临床研究[J].时珍国医国药,2013,24(3):677-680.
[5]陈开慧,李仁保,李凯,等.艾灸治疗类风湿关节炎的临床效果及其对血IL-6、CRP水平的研究[J].中药药理与临床,2015,31(1):303-304.
[6]俞红五,朱艳,潘喻珍,等.艾灸辅助治疗类风湿关节炎患者临床疗效观察及机制探讨[J].中国针灸,2016,36(1):17-20.
[7]何悦硕,梁维超.麦粒灸治疗类风湿性关节炎疗效观察[J].上海针灸杂志,2010,29(7):449-450.
[8]谢丽琼,谢祖艺,陈丽,等.改良温和悬灸疗法在类风湿关节炎痹症中的应用及护理[J].齐鲁护理杂志,2014,20(11):22-24.
[9]丁放.单纯灸法治疗类风湿关节炎[J].针灸临床杂志,2007,23(10):40-41.
[10]张小芹,窦杰.雷火灸治疗痰瘀痹阻型类风湿关节炎临床观察[J].泰州职业技术学院学报,2015,15(2):64-65.
[11]陈晓莉,黄迪君,宋开源,等.麦粒灸对佐剂性关节炎大鼠细胞免疫功能的影响[J].上海针灸杂志,2003,22(1):14-16.
[12]杨馨,杨慎峭,周海燕,等.艾灸对实验性RA家兔滑膜细胞MAPK信号通路影响的研究[J].中华中医药学刊,2007,25(3):470-474.
[13]高骏,周海燕,刘旭光,等.艾灸对实验性RA大鼠血浆Th1-Th2细胞因子平衡影响的实验研究[J].成都中医药大学学报,2008,31(1):21-23.
[14]魏铮,杨露晨,杨馨,等.艾灸对类风湿性关节炎家兔滑膜细胞CyclinD1及CDK4蛋白表达的影响[J].中华中医药学刊,2012,30(7):1503-1505.
[15]姚剑,胡玲,宋小鸽,等.艾灸“肾俞”“足三里”穴对类风湿关节炎大鼠滑膜组织中Ras-MAPK信号通路的影响[J].世界针灸杂志:英文版,2013,23(2):29-33.
[16]江星,孙志岭,周丹萍,等.艾灸对类风湿关节炎模型大鼠滑膜病变的影响[J].中国老年学杂志,2015,35(19):5386-5389.
[17]袁娟,胡玲,宋小鸽,等.艾灸对类风湿性关节炎大鼠关节滑膜组织Toll样受体4骨髓样分化因子88核转录因子κB信号通路的影响[J].针刺研究,2015,40(3):199-204.
[18]张传英,邵芙蓉,蔡荣林,等.艾灸对类风湿性关节炎大鼠关节滑膜组织转录信号转导因子1、细胞因子信号负调控因子基因表达的影响[J].针刺研究,2015,40(3):205-209.
[19]Hu ZM,Cao JP,He L,et al.Effects of different moxibustion times on TRPV3 ion channel protein and synovial cell apoptosis in rats with rheumatoid arthritis[J].J Acupunct Tuina Sci,2016,14(2):67-72.
[20]殷媛,陈天帷,张瑞,等.艾灸对胶原诱导性关节炎大鼠血清白介素-17和肿瘤坏死因子α含量的影响[J].针刺研究,2017,42(2):149-152.
[21]Zhang H,Ma XP,Wu HG,et al.Effect of moxibustion on tumor necrosis factor-αand nuclear transcription factor kappa B in ankle joints of rats with rheumatoid arthritis[J].J Acupunct Tuina Sci,2017,15(3):171-176.
[22]彭传玉,胡玲.艾灸“肾俞”、“足三里”对类风湿关节炎模型大鼠基质金属酶-9的影响[J].浙江中医药大学学报,2015,39(1):6-8.
[23]黄迪君,韩哲林,罗荣.麦粒灸对实验性RA大鼠足跖肿胀度及滑膜组织Caspase-3表达的影响[J].成都中医药大学学报,2003(2):29-30,64.
[24]郭江燕,姜姝姝,高宇晨,等.麦粒灸对佐剂型关节炎大鼠屈关节疼痛实验(缩腿嘶鸣)评分及足容积肿胀度的影响[J].中医药导报,2015,21(11):7-9.
[25]范薇,杨剑,夏丽娜,等.从“少阳主骨”看阳陵泉穴对艾灸治疗佐剂性关节炎大鼠的抗炎增效作用[J].辽宁中医杂志,2017,44(4):852-854.
[26]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:327-329.
[27]季辉,王玲玲,周攀,等.不同灸温对急性佐剂性关节炎大鼠血清IL-1β、IL-2及TNF-α含量的影响[J].上海针灸杂志,2015,34(7):597-599.
[28]马文彬,刘旭光,周海燕.择时艾灸对类风湿性关节炎大鼠下丘脑-垂体-上腺轴昼夜节律的影响[J].针刺研究,2016,41(2):100-107.
[29]Hu ZM,Cao JP,He L,et al.Effects of different moxibustion times on TRPV3 ion channel protein and synovial cell apoptosis in rats with rheumatoid arthritis[J].J Acupunct Tuina Sci,2016,14(2):67-72.
[30]Yim YK,Lee H,Hong KE,et al.Electro-acupuncture at acupoint ST36 reduces inflammation and regulates immune activity in CollagenInduced Arthritic Mice[J].Evid Based Complement Alternat Med,2007,4(1):51-57.
[31]高秀花,王兴丽,刘旭光,等.不同灸法对类风湿关节炎家兔HPAA功能的影响[J].中国中医基础医学杂志,2015,21(7):1140-1142.
[32]刘鳐,张付平,王蕾,等.不同灸法艾灸血清对类风湿关节炎滑膜成纤维细胞的影响[J].西部医学,2015,27(10):1445-1448.
[2]胡晓敏,宗英,余珊珊,等.类风湿关节炎治疗药物的研发进展及趋势[J].中国新药杂志,2017,26(1):36-43.
[3]胡玲,郝峰,钟峰,等.艾灸治疗类风湿关节炎的多中心随机对照临床研究[J].环球中医药,2011,6(5):401-405.
[4]郝锋,胡玲,吴子建,等.隔姜灸治疗活动期类风湿关节炎量效关系的临床研究[J].时珍国医国药,2013,24(3):677-680.
[5]陈开慧,李仁保,李凯,等.艾灸治疗类风湿关节炎的临床效果及其对血IL-6、CRP水平的研究[J].中药药理与临床,2015,31(1):303-304.
[6]俞红五,朱艳,潘喻珍,等.艾灸辅助治疗类风湿关节炎患者临床疗效观察及机制探讨[J].中国针灸,2016,36(1):17-20.
[7]何悦硕,梁维超.麦粒灸治疗类风湿性关节炎疗效观察[J].上海针灸杂志,2010,29(7):449-450.
[8]谢丽琼,谢祖艺,陈丽,等.改良温和悬灸疗法在类风湿关节炎痹症中的应用及护理[J].齐鲁护理杂志,2014,20(11):22-24.
[9]丁放.单纯灸法治疗类风湿关节炎[J].针灸临床杂志,2007,23(10):40-41.
[10]张小芹,窦杰.雷火灸治疗痰瘀痹阻型类风湿关节炎临床观察[J].泰州职业技术学院学报,2015,15(2):64-65.
[11]陈晓莉,黄迪君,宋开源,等.麦粒灸对佐剂性关节炎大鼠细胞免疫功能的影响[J].上海针灸杂志,2003,22(1):14-16.
[12]杨馨,杨慎峭,周海燕,等.艾灸对实验性RA家兔滑膜细胞MAPK信号通路影响的研究[J].中华中医药学刊,2007,25(3):470-474.
[13]高骏,周海燕,刘旭光,等.艾灸对实验性RA大鼠血浆Th1-Th2细胞因子平衡影响的实验研究[J].成都中医药大学学报,2008,31(1):21-23.
[14]魏铮,杨露晨,杨馨,等.艾灸对类风湿性关节炎家兔滑膜细胞CyclinD1及CDK4蛋白表达的影响[J].中华中医药学刊,2012,30(7):1503-1505.
[15]姚剑,胡玲,宋小鸽,等.艾灸“肾俞”“足三里”穴对类风湿关节炎大鼠滑膜组织中Ras-MAPK信号通路的影响[J].世界针灸杂志:英文版,2013,23(2):29-33.
[16]江星,孙志岭,周丹萍,等.艾灸对类风湿关节炎模型大鼠滑膜病变的影响[J].中国老年学杂志,2015,35(19):5386-5389.
[17]袁娟,胡玲,宋小鸽,等.艾灸对类风湿性关节炎大鼠关节滑膜组织Toll样受体4骨髓样分化因子88核转录因子κB信号通路的影响[J].针刺研究,2015,40(3):199-204.
[18]张传英,邵芙蓉,蔡荣林,等.艾灸对类风湿性关节炎大鼠关节滑膜组织转录信号转导因子1、细胞因子信号负调控因子基因表达的影响[J].针刺研究,2015,40(3):205-209.
[19]Hu ZM,Cao JP,He L,et al.Effects of different moxibustion times on TRPV3 ion channel protein and synovial cell apoptosis in rats with rheumatoid arthritis[J].J Acupunct Tuina Sci,2016,14(2):67-72.
[20]殷媛,陈天帷,张瑞,等.艾灸对胶原诱导性关节炎大鼠血清白介素-17和肿瘤坏死因子α含量的影响[J].针刺研究,2017,42(2):149-152.
[21]Zhang H,Ma XP,Wu HG,et al.Effect of moxibustion on tumor necrosis factor-αand nuclear transcription factor kappa B in ankle joints of rats with rheumatoid arthritis[J].J Acupunct Tuina Sci,2017,15(3):171-176.
[22]彭传玉,胡玲.艾灸“肾俞”、“足三里”对类风湿关节炎模型大鼠基质金属酶-9的影响[J].浙江中医药大学学报,2015,39(1):6-8.
[23]黄迪君,韩哲林,罗荣.麦粒灸对实验性RA大鼠足跖肿胀度及滑膜组织Caspase-3表达的影响[J].成都中医药大学学报,2003(2):29-30,64.
[24]郭江燕,姜姝姝,高宇晨,等.麦粒灸对佐剂型关节炎大鼠屈关节疼痛实验(缩腿嘶鸣)评分及足容积肿胀度的影响[J].中医药导报,2015,21(11):7-9.
[25]范薇,杨剑,夏丽娜,等.从“少阳主骨”看阳陵泉穴对艾灸治疗佐剂性关节炎大鼠的抗炎增效作用[J].辽宁中医杂志,2017,44(4):852-854.
[26]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:327-329.
[27]季辉,王玲玲,周攀,等.不同灸温对急性佐剂性关节炎大鼠血清IL-1β、IL-2及TNF-α含量的影响[J].上海针灸杂志,2015,34(7):597-599.
[28]马文彬,刘旭光,周海燕.择时艾灸对类风湿性关节炎大鼠下丘脑-垂体-上腺轴昼夜节律的影响[J].针刺研究,2016,41(2):100-107.
[29]Hu ZM,Cao JP,He L,et al.Effects of different moxibustion times on TRPV3 ion channel protein and synovial cell apoptosis in rats with rheumatoid arthritis[J].J Acupunct Tuina Sci,2016,14(2):67-72.
[30]Yim YK,Lee H,Hong KE,et al.Electro-acupuncture at acupoint ST36 reduces inflammation and regulates immune activity in CollagenInduced Arthritic Mice[J].Evid Based Complement Alternat Med,2007,4(1):51-57.
[31]高秀花,王兴丽,刘旭光,等.不同灸法对类风湿关节炎家兔HPAA功能的影响[J].中国中医基础医学杂志,2015,21(7):1140-1142.
[32]刘鳐,张付平,王蕾,等.不同灸法艾灸血清对类风湿关节炎滑膜成纤维细胞的影响[J].西部医学,2015,27(10):1445-1448.