火针刺激骨关节炎模型兔犊鼻、内膝眼穴位后软骨细胞外基质及Wnt信号通路的变化
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摘要
背景:针灸能够改善骨关节炎的关节症状、降低炎症反应。火针属于针灸的一种,有学者报道称火针能够治疗骨关节炎,疗效确切,但具体机制尚不明确。目的:明确火针刺激犊鼻、内膝眼对兔骨关节炎模型作用效应及具体机制。方法:4月龄新西兰大白兔32只,购自上海斯莱克实验动物有限责任公司。实验分为4组:假手术对照组、骨关节炎组、西药治疗组、火针治疗组。除假手术对照组外,其他3组兔通过高分子固定带固定新西兰兔右膝的方法制造膝关节骨性关节炎模型,造模开始后的第7天,分别用玻璃酸钠、火针刺激犊鼻及内膝眼对西药治疗组、火针治疗组家兔进行治疗,每隔3d治疗1次,连续6次。疗程结束后采用ELISA法测定各组家兔血清中基质金属蛋白酶1、金属蛋白酶组织抑制剂1水平;番红-O染色用于骨关节炎组织学评估,Mankin评分评估骨关节炎模型;定量PCR和Western-blotting检测软骨组织中Wnt信号通路的变化。结果与结论:(1)西药治疗组、火针治疗组家兔软骨Mankin评分及组织学染色显示,软骨退化水平相较于模型组有下调趋势;(2)经治疗后,西药治疗组、火针治疗组家兔血清中基质金属蛋白酶1和金属蛋白酶组织抑制剂1表达较骨关节炎组分别出现显著的降低和显著升高(P <0.05);(3)西药治疗组、火针治疗组家兔软骨中基质金属蛋白酶1、金属蛋白酶组织抑制剂1及Wnt信号通路分子β-catenin的m RNA和蛋白表达相较于模型组分别有下调、上调及下调的趋势,差异有显著性意义;(4)结果说明,火针刺激犊鼻、内膝眼是治疗膝关节骨性关节炎的一种简便有效方法,其作用机制可能是通过降低血清中异常升高的基质金属蛋白酶1水平,调节基质金属蛋白酶1和金属蛋白酶组织抑制剂1比例关系,调节细胞外基质的降解过程及影响Wnt信号通路,从而减缓软骨退变的进程。
BACKGROUND: Acupuncture and moxibustion can improve the articular symptoms of osteoarthritis and alleviate inflammatory reaction. Fire-needle moxibustion has been reported to treat osteoarthritis, showing exact efficacy, but the underlying mechanism remains unclear. OBJECTIVE: To clarify the effect of fire-needle moxibustion at Dubi(ST 35) and Neixiyan on the rabbit models of osteoarthritis, and the underlying mechanism. METHODS: Thirty-two 4-month-old New Zealand rabbits were selected(provided by Shanghai Slac Laboratory Animals Co., Ltd.). There were four groups, including sham operation, osteoarthritis, Western medicine, and fire-needle moxibustion groups. The rabbit knees in the latter three groups were fixed using polymer fixing band to establish the model of knee osteoarthritis. Seven days later, the rabbits were treated by sodium hyaluronate, and fire-needle moxibustion at Dubi and Neixiyan, respectively, for 6 consecutive courses with 3 days in between. The levels of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in serum of rabbits in each group were determined by ELISA after treatment. Safranin-O staining was used for osteoarthritis histological evaluation. Mankin score was used to evaluate osteoarthritis model. Changes of Wnt signaling pathway in cartilage tissues were detected by qualified PCR and western blot assay. RESULTS AND CONCLUSION: Mankin score and histological staining results showed that the cartilage degradation level in the Western medicine, and fire-needle moxibustion groups was downregulated compared with the osteoarthritis group. After treatment, compared with the osteoarthritis group, the serum levels of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 were significantly decreased and increased in the Western medicine and fire-needle moxibustion groups, respectively(P < 0.05). The levels of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1, and β-catenin mRNA and protein in cartilage in the Western medicine and fire-needle moxibustion groups were significantly lower, higher and lower than those in the osteoarthritis group, respectively. These results indicate that fire-needle moxibustion at Dubi and Neixiyan is a simple and effective method for knee osteoarthritis. The mechanism may be through reducing the abnormally increased metalloproteinase-1 level, regulating ratio of metalloproteinase-1 and tissue inhibitor of metalloproteinase-1, regulating the degradation process of extracellular matrix and affecting the Wnt signaling pathway, thereby alleviating the progression of cartilage degeneration.
BACKGROUND: Acupuncture and moxibustion can improve the articular symptoms of osteoarthritis and alleviate inflammatory reaction. Fire-needle moxibustion has been reported to treat osteoarthritis, showing exact efficacy, but the underlying mechanism remains unclear. OBJECTIVE: To clarify the effect of fire-needle moxibustion at Dubi(ST 35) and Neixiyan on the rabbit models of osteoarthritis, and the underlying mechanism. METHODS: Thirty-two 4-month-old New Zealand rabbits were selected(provided by Shanghai Slac Laboratory Animals Co., Ltd.). There were four groups, including sham operation, osteoarthritis, Western medicine, and fire-needle moxibustion groups. The rabbit knees in the latter three groups were fixed using polymer fixing band to establish the model of knee osteoarthritis. Seven days later, the rabbits were treated by sodium hyaluronate, and fire-needle moxibustion at Dubi and Neixiyan, respectively, for 6 consecutive courses with 3 days in between. The levels of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in serum of rabbits in each group were determined by ELISA after treatment. Safranin-O staining was used for osteoarthritis histological evaluation. Mankin score was used to evaluate osteoarthritis model. Changes of Wnt signaling pathway in cartilage tissues were detected by qualified PCR and western blot assay. RESULTS AND CONCLUSION: Mankin score and histological staining results showed that the cartilage degradation level in the Western medicine, and fire-needle moxibustion groups was downregulated compared with the osteoarthritis group. After treatment, compared with the osteoarthritis group, the serum levels of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 were significantly decreased and increased in the Western medicine and fire-needle moxibustion groups, respectively(P < 0.05). The levels of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1, and β-catenin mRNA and protein in cartilage in the Western medicine and fire-needle moxibustion groups were significantly lower, higher and lower than those in the osteoarthritis group, respectively. These results indicate that fire-needle moxibustion at Dubi and Neixiyan is a simple and effective method for knee osteoarthritis. The mechanism may be through reducing the abnormally increased metalloproteinase-1 level, regulating ratio of metalloproteinase-1 and tissue inhibitor of metalloproteinase-1, regulating the degradation process of extracellular matrix and affecting the Wnt signaling pathway, thereby alleviating the progression of cartilage degeneration.
引文
[1]Martel-Pelletier J,Barr AJ,Cicuttini FM,et al.Osteoarthritis.Nat Rev Dis Primers.2016;2:16072
[2]吕苏梅,张瑞丽.中老年膝骨关节炎的流行病学研究进展[J].中国老年学,2016,36(16):4133-4135.
[3]Litwic A,Edwards MH,Dennison EM,et al.Epidemiology and burden of osteoarthritis.Br Med Bull.2013;105:185-199.
[4]卢宏伟,罗飞,侯天勇,等.硫酸氨基葡萄糖联合非甾体抗炎药在轻中度膝骨关节炎中的作用[J].中国医药导报,2016,13(17):137-139.
[5]张晨,马骏,党晓谦,等.非甾体抗炎药治疗骨关节炎的研究进展[J].中华关节外科杂志(电子版),2017,11(2):66-69.
[6]Wen Y,Li J,Wang L,et al.Udp-glucose dehydrogenase modulates proteoglycan synthesis in articular chondrocytes:Its possible involvement and regulation in osteoarthritis.Arthritis Res Ther.2014;16(6):484.
[7]马妮.针灸治疗膝骨关节炎近十年研究进展及贺氏火针治疗的初步疗效观察[J].北京中医药大学学报,2014,17(22):485-487.
[8]刘骏达,吴明霞.从血液流变学探讨瘀血阻滞型膝骨性关节炎的针灸治疗[J].亚太传统医药,2017,13(8):59-62.
[9]张婧怡,陈卫东,刘玉蕊,等.火针疗法的源流及现代应用[J].亚太传统医药,2016,12(3):42-44.
[10]黄昌锦,黄应杰,陈楚云.火针疗法的发展源流[J].中国针灸,2013,33(5):455-458.
[11]曾红文,聂斌,史琳琳.刺血合火针点刺治疗膝关节骨性关节炎疗效观察[J].中国针灸,2008,28(7):493-495.
[12]王立兴.火针结合电针治疗膝关节骨性关节炎的临床研究[D].广州中医药大学,2016.
[13]毛雪文,王世广,王桂玲,等.贺氏火针刺骨法治疗膝骨性关节炎80例疗效观察[J].中医临床研究,2016,8(35):20-23.
[14]林国华,李万瑶,苏国龙.火针对实验性小鼠膝骨关节炎关节软骨病理改变的影响[J].广州中医药大学学报,2005,22(5):373-376.
[15]Lu W,Wang L,Wo C,et al.Ketamine attenuates osteoarthritis of the knee via modulation of inflammatory responses in a rabbit model.Mol Med Rep.2016;13(6):5013-5020.
[16]Ogura N,Tobe M,Sakamaki H,et al.Interleukin-1 beta induces interleukin-6 mrna expression and protein production in synovial cells from human temporomandibular joint.J Oral Pathol Med.2002;31(6):353-360.
[17]Krajewska-Wlodarczyk M,Owczarczyk-Saczonek A,Placek W,et al.Articular cartilage aging-potential regenerative capacities of cell manipulation and stem cell therapy.Int J Mol Sci.2018;19(2).pii:E623.
[18]Yu D,Xu J,Liu F,et al.Subchondral bone changes and the impacts on joint pain and articular cartilage degeneration in osteoarthritis.Clin Exp Rheumatol.2016;34(5):929-934.
[19]雷鸣,于斐,肖德明.骨关节炎临床表现特点及沉默信息调节因子1在其发病中的作用[J].中国组织工程研究,2017,21(12):1933-1939.
[20]王昌盛,杨海涛,邓茗中,等.膝关节骨关节炎软骨下水肿与MRI及临床表现的相关性研究[J].实用放射学杂志,2017,33(8):1236-1240.
[21]Huang H,Zheng J,Shen N,et al.Identification of pathways and genes associated with synovitis in osteoarthritis using bioinformatics analyses.Sci Rep.2018;8(1):10050.
[22]Mathiessen A,Conaghan PG.Synovitis in osteoarthritis:Current understanding with therapeutic implications.Arthritis Res Ther.2017;19(1):18.
[23]Ma CH,Lv Q,Cao Y,et al.Genes relevant with osteoarthritis by comparison gene expression profiles of synovial membrane of osteoarthritis patients at different stages.Eur Rev Med Pharmacol Sci.2014;18(3):431-439.
[24]葛伟韬,高云,刘珍珠,等.膝骨关节炎中医病名辨识[J].中医杂志,2016,57(23):1989-1992.
[25]吴玖斌,李蒙,谢雁鸣,等.膝关节骨性关节炎当代中医学术团队研究[J].北京中医药大学学报,2016,39(6):516-519.
[26]刘源,郭艳幸,郭珈宜,等.膝骨关节炎的中医诊疗进展[J].亚太传统医药,2017,13(1):56-59.
[27]刘保延.火针[M].北京:中医古籍出版社,1994.
[28]李彬,谢新才,王麟鹏.火针治疗膝骨关节炎临床观察[J].北京中医药,2011,30(12):923-925.
[29]Blazek AD,Nam J,Gupta R,et al.Exercise-driven metabolic pathways in healthy cartilage.Osteoarthritis Cartilage.2016;24(7):1210-1222.
[30]Huang G,Chubinskaya S,Liao W,et al.Wnt5a induces catabolic signaling and matrix metalloproteinase production in human articular chondrocytes.Osteoarthritis Cartilage.2017;25(9):1505-1515.
[31]Sun MM,Beier F,Pest MA.Recent developments in emerging therapeutic targets of osteoarthritis.Curr Opin Rheumatol.2017;29(1):96-102.
[32]高宁阳,曹月龙,刘婷,等.Wnt信号通路与骨关节炎[J].中国骨伤,2010,23(4):320-323.
[33]徐伟丽,牛玲玲,王文侠,等.经典Wnt信号通路对骨代谢的调节作用[J].中国骨质疏松杂志,2016,22(3):376-380.
[34]Bejsovec A.Wingless signaling:A genetic journey from morphogenesis to metastasis.Genetics.2018,208(4):1311-1336.
[35]Hui T,Zhou Y,Wang T,et al.Activation of beta-catenin signaling in aggrecan-expressing cells in temporomandibular joint causes osteoarthritis-like defects.Int J Oral Sci.2018;10(2):13.
[36]Kremer R,Gilsanz V.Fat and bone:An odd couple.Front Endocrinol(Lausanne).2016;6:190.
[37]Zhou X,Li W,Jiang L,et al.Tetrandrine inhibits the wnt/beta-catenin signalling pathway and alleviates osteoarthritis:An in vitro and in vivo study.Evid Based Complement Alternat Med.2013;2013:809579..
[38]史纪元,易智,刘宗智,等.不同程度骨关节炎中Wnt/β-catenin信号通路、OPN和MMP-13的相关性的研究[J].现代生物医学进展,2017,17(24):4639-4644.
[39]Nguyen LT,Sharma AR,Chakraborty C,et al.Review of prospects of biological fluid biomarkers in osteoarthritis.Int J Mol Sci.2017;18(3).pii:E601.
[40]Rose BJ,Kooyman DL.A tale of two joints:The role of matrix metal oproteases in cartilage biology.Dis Markers.2016;2016:4895050.
[41]Murakami K,Maeda S,Yonezawa T,et al.Synovial fluid matrix metalloproteinase-2 and-9 activities in dogs suffering from joint disorders.J Vet Med Sci.2016;78(6):1051-1054.
[2]吕苏梅,张瑞丽.中老年膝骨关节炎的流行病学研究进展[J].中国老年学,2016,36(16):4133-4135.
[3]Litwic A,Edwards MH,Dennison EM,et al.Epidemiology and burden of osteoarthritis.Br Med Bull.2013;105:185-199.
[4]卢宏伟,罗飞,侯天勇,等.硫酸氨基葡萄糖联合非甾体抗炎药在轻中度膝骨关节炎中的作用[J].中国医药导报,2016,13(17):137-139.
[5]张晨,马骏,党晓谦,等.非甾体抗炎药治疗骨关节炎的研究进展[J].中华关节外科杂志(电子版),2017,11(2):66-69.
[6]Wen Y,Li J,Wang L,et al.Udp-glucose dehydrogenase modulates proteoglycan synthesis in articular chondrocytes:Its possible involvement and regulation in osteoarthritis.Arthritis Res Ther.2014;16(6):484.
[7]马妮.针灸治疗膝骨关节炎近十年研究进展及贺氏火针治疗的初步疗效观察[J].北京中医药大学学报,2014,17(22):485-487.
[8]刘骏达,吴明霞.从血液流变学探讨瘀血阻滞型膝骨性关节炎的针灸治疗[J].亚太传统医药,2017,13(8):59-62.
[9]张婧怡,陈卫东,刘玉蕊,等.火针疗法的源流及现代应用[J].亚太传统医药,2016,12(3):42-44.
[10]黄昌锦,黄应杰,陈楚云.火针疗法的发展源流[J].中国针灸,2013,33(5):455-458.
[11]曾红文,聂斌,史琳琳.刺血合火针点刺治疗膝关节骨性关节炎疗效观察[J].中国针灸,2008,28(7):493-495.
[12]王立兴.火针结合电针治疗膝关节骨性关节炎的临床研究[D].广州中医药大学,2016.
[13]毛雪文,王世广,王桂玲,等.贺氏火针刺骨法治疗膝骨性关节炎80例疗效观察[J].中医临床研究,2016,8(35):20-23.
[14]林国华,李万瑶,苏国龙.火针对实验性小鼠膝骨关节炎关节软骨病理改变的影响[J].广州中医药大学学报,2005,22(5):373-376.
[15]Lu W,Wang L,Wo C,et al.Ketamine attenuates osteoarthritis of the knee via modulation of inflammatory responses in a rabbit model.Mol Med Rep.2016;13(6):5013-5020.
[16]Ogura N,Tobe M,Sakamaki H,et al.Interleukin-1 beta induces interleukin-6 mrna expression and protein production in synovial cells from human temporomandibular joint.J Oral Pathol Med.2002;31(6):353-360.
[17]Krajewska-Wlodarczyk M,Owczarczyk-Saczonek A,Placek W,et al.Articular cartilage aging-potential regenerative capacities of cell manipulation and stem cell therapy.Int J Mol Sci.2018;19(2).pii:E623.
[18]Yu D,Xu J,Liu F,et al.Subchondral bone changes and the impacts on joint pain and articular cartilage degeneration in osteoarthritis.Clin Exp Rheumatol.2016;34(5):929-934.
[19]雷鸣,于斐,肖德明.骨关节炎临床表现特点及沉默信息调节因子1在其发病中的作用[J].中国组织工程研究,2017,21(12):1933-1939.
[20]王昌盛,杨海涛,邓茗中,等.膝关节骨关节炎软骨下水肿与MRI及临床表现的相关性研究[J].实用放射学杂志,2017,33(8):1236-1240.
[21]Huang H,Zheng J,Shen N,et al.Identification of pathways and genes associated with synovitis in osteoarthritis using bioinformatics analyses.Sci Rep.2018;8(1):10050.
[22]Mathiessen A,Conaghan PG.Synovitis in osteoarthritis:Current understanding with therapeutic implications.Arthritis Res Ther.2017;19(1):18.
[23]Ma CH,Lv Q,Cao Y,et al.Genes relevant with osteoarthritis by comparison gene expression profiles of synovial membrane of osteoarthritis patients at different stages.Eur Rev Med Pharmacol Sci.2014;18(3):431-439.
[24]葛伟韬,高云,刘珍珠,等.膝骨关节炎中医病名辨识[J].中医杂志,2016,57(23):1989-1992.
[25]吴玖斌,李蒙,谢雁鸣,等.膝关节骨性关节炎当代中医学术团队研究[J].北京中医药大学学报,2016,39(6):516-519.
[26]刘源,郭艳幸,郭珈宜,等.膝骨关节炎的中医诊疗进展[J].亚太传统医药,2017,13(1):56-59.
[27]刘保延.火针[M].北京:中医古籍出版社,1994.
[28]李彬,谢新才,王麟鹏.火针治疗膝骨关节炎临床观察[J].北京中医药,2011,30(12):923-925.
[29]Blazek AD,Nam J,Gupta R,et al.Exercise-driven metabolic pathways in healthy cartilage.Osteoarthritis Cartilage.2016;24(7):1210-1222.
[30]Huang G,Chubinskaya S,Liao W,et al.Wnt5a induces catabolic signaling and matrix metalloproteinase production in human articular chondrocytes.Osteoarthritis Cartilage.2017;25(9):1505-1515.
[31]Sun MM,Beier F,Pest MA.Recent developments in emerging therapeutic targets of osteoarthritis.Curr Opin Rheumatol.2017;29(1):96-102.
[32]高宁阳,曹月龙,刘婷,等.Wnt信号通路与骨关节炎[J].中国骨伤,2010,23(4):320-323.
[33]徐伟丽,牛玲玲,王文侠,等.经典Wnt信号通路对骨代谢的调节作用[J].中国骨质疏松杂志,2016,22(3):376-380.
[34]Bejsovec A.Wingless signaling:A genetic journey from morphogenesis to metastasis.Genetics.2018,208(4):1311-1336.
[35]Hui T,Zhou Y,Wang T,et al.Activation of beta-catenin signaling in aggrecan-expressing cells in temporomandibular joint causes osteoarthritis-like defects.Int J Oral Sci.2018;10(2):13.
[36]Kremer R,Gilsanz V.Fat and bone:An odd couple.Front Endocrinol(Lausanne).2016;6:190.
[37]Zhou X,Li W,Jiang L,et al.Tetrandrine inhibits the wnt/beta-catenin signalling pathway and alleviates osteoarthritis:An in vitro and in vivo study.Evid Based Complement Alternat Med.2013;2013:809579..
[38]史纪元,易智,刘宗智,等.不同程度骨关节炎中Wnt/β-catenin信号通路、OPN和MMP-13的相关性的研究[J].现代生物医学进展,2017,17(24):4639-4644.
[39]Nguyen LT,Sharma AR,Chakraborty C,et al.Review of prospects of biological fluid biomarkers in osteoarthritis.Int J Mol Sci.2017;18(3).pii:E601.
[40]Rose BJ,Kooyman DL.A tale of two joints:The role of matrix metal oproteases in cartilage biology.Dis Markers.2016;2016:4895050.
[41]Murakami K,Maeda S,Yonezawa T,et al.Synovial fluid matrix metalloproteinase-2 and-9 activities in dogs suffering from joint disorders.J Vet Med Sci.2016;78(6):1051-1054.