电针对颈椎病大鼠椎间盘软骨细胞及MCP-1/CCR2信号通路的影响
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摘要
目的:观察电针对颈椎病大鼠椎间盘软骨细胞及MCP-1/CCR2信号通路的影响。方法:将45只雄性SD大鼠,按照随机数字表法分为3组,假手术组、模型组和电针组,每组15只。除假手术组外均建立动静力失衡性颈椎病模型,并于造模后3个月开始电针干预双侧颈夹脊穴,30 min/次,1次/d,14 d为1个疗程。研究中通过电子透射电镜观察颈椎间盘软骨细胞的形态判定颈椎病造模是否成功;而后采用免疫组化检测颈椎间盘软骨细胞中肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)的表达情况;接着采用实时聚合酶链式反应(Real time PCR)检测MCP-1/CCR2信号通路,观察电针对MCP-1/CCR2信号通路的影响。结果:1)假手术组的颈椎间盘软骨细胞表面较光滑,可见微绒毛样凸起,细胞整体形态上类似椭圆形,细胞核形态类似一个肾脏,且细胞核完整无破损,染色质均匀;而细胞外的基质中含有丰富且排列有序的胶原纤维;模型组中的颈椎间盘软骨细胞出现退行性改变,其细胞表面的微绒毛样凸起明显减少,细胞核破损,染色质明显固缩,细胞外基质中的胶原纤维明显减少,排列无序。提示颈椎病造模成功。2)与假手术组比较,模型组和电针组大鼠TNF-α和IL-6表达均较密集,阳性表达增多(P <0. 05),其中是电针组TNF-α和IL-6表达明显较模型组降低(P <0. 05),提示电针能够抑制颈椎病大鼠椎间盘软骨细胞的TNF-α和IL-6表达。3)与假手术组比较,模型组和电针组颈椎间盘中MCP-1/GAPDH和CCR2/GAPDH均增高,差异有统计学意义(P <0. 05);电针组与模型组比较,颈椎间盘中MCP-1/GAPDH和CCR2/GAPDH低于模型组,差异有统计学意义(P <0. 05)。结论:电针可以抑制颈椎病大鼠椎间盘软骨细胞中TNF-α和IL-6表达,可能与抑制MCP-1/CCR2信号通路的激活有关。
Objective: To observe the effect of electroacupuncture on chondrocyte and MCP-1/CCR2 signaling pathway of intervertebral disc in rats with cervical spondylosis. Methods: A total of 45 male SD rats were divided into three groups according to random number table,a sham operation group,a model group and an electro-acupuncture group,with 15 rats in each group. Except for the sham operation group,the models of dynamic and static imbalance cervical spondylosis were established,and electro-acupuncture intervention on bilateral cervical Jiaji( EX-B2) acupoints was started 3 months after modeling,30 minutes a day for 14 days. In this study,the morphology of cervical intervertebral disc chondrocyte was observed by transmission electron microscopy to determine the success of cervical spondylosis modeling. Then the expression of tumor necrosis factor-alpha( TNF-alpha) and interleukin-6( IL-6) in cervical intervertebral disc chondrocyte was detected by immunohistochemistry. Then real-time polymerase chain reaction( Real-time PCR) was used to detect MCP-1/CCR2 signaling pathway and observe the effect of electroacupuncture on MCP-1/CCR2 signaling pathway. Results: 1) The surface of cervical intervertebral disc chondrocyte in sham operation group was smooth,with microvilli-like protuberance,oval shape of cell as a whole,and the shape of nucleus was similar to that of a kidney,and the nucleus was intact without damage and the chromatin was uniform; the extracellular matrix contained abundant and orderly collagen fibers;the degeneration of cervical intervertebral disc chondrocyte appeared in the model group. The microvilli-like protrusions on the cell surface were significantly reduced,the nucleus was damaged,the chromatin was obviously condensed,and the collagen fibers in the extracellular matrix were significantly reduced and arranged in disorder. It is suggested that cervical spondylosis was successfully modeled. 2) Compared with sham operation group,the expression of TNF-alpha and IL-6 in model group and electro-acupuncture group were more intensive and positive( P < 0. 05). Among them,the expression of TNF-alpha and IL-6 in electro-acupuncture group was significantly decreased than that in the model group( P < 0. 05),suggesting that electro-acupuncture could inhibit the expression of TNF-alpha and IL-6 in intervertebral disc chondrocytes of rats with cervical spondylosis. 3) Compared with sham operation group,MCP-1/GAPDH and CCR2/GAPDH in cervical intervertebral disc of the model group and the electro-acupuncture group increased,with statistical significance( P < 0. 05); compared with model group,MCP-1/GAPDH and CCR2/GAPDH in cervical intervertebral disc of electro-acupuncture group were lower than model group,with statistical significance( P < 0. 05). Conclusion: Electroacupuncture can inhibit the expression of TNF-alpha and IL-6 in the chondrocyte of intervertebral disc of rats with cervical spondylosis,which may be related to the inhibition of the activation of MCP-1/CCR2 signaling pathway.
Objective: To observe the effect of electroacupuncture on chondrocyte and MCP-1/CCR2 signaling pathway of intervertebral disc in rats with cervical spondylosis. Methods: A total of 45 male SD rats were divided into three groups according to random number table,a sham operation group,a model group and an electro-acupuncture group,with 15 rats in each group. Except for the sham operation group,the models of dynamic and static imbalance cervical spondylosis were established,and electro-acupuncture intervention on bilateral cervical Jiaji( EX-B2) acupoints was started 3 months after modeling,30 minutes a day for 14 days. In this study,the morphology of cervical intervertebral disc chondrocyte was observed by transmission electron microscopy to determine the success of cervical spondylosis modeling. Then the expression of tumor necrosis factor-alpha( TNF-alpha) and interleukin-6( IL-6) in cervical intervertebral disc chondrocyte was detected by immunohistochemistry. Then real-time polymerase chain reaction( Real-time PCR) was used to detect MCP-1/CCR2 signaling pathway and observe the effect of electroacupuncture on MCP-1/CCR2 signaling pathway. Results: 1) The surface of cervical intervertebral disc chondrocyte in sham operation group was smooth,with microvilli-like protuberance,oval shape of cell as a whole,and the shape of nucleus was similar to that of a kidney,and the nucleus was intact without damage and the chromatin was uniform; the extracellular matrix contained abundant and orderly collagen fibers;the degeneration of cervical intervertebral disc chondrocyte appeared in the model group. The microvilli-like protrusions on the cell surface were significantly reduced,the nucleus was damaged,the chromatin was obviously condensed,and the collagen fibers in the extracellular matrix were significantly reduced and arranged in disorder. It is suggested that cervical spondylosis was successfully modeled. 2) Compared with sham operation group,the expression of TNF-alpha and IL-6 in model group and electro-acupuncture group were more intensive and positive( P < 0. 05). Among them,the expression of TNF-alpha and IL-6 in electro-acupuncture group was significantly decreased than that in the model group( P < 0. 05),suggesting that electro-acupuncture could inhibit the expression of TNF-alpha and IL-6 in intervertebral disc chondrocytes of rats with cervical spondylosis. 3) Compared with sham operation group,MCP-1/GAPDH and CCR2/GAPDH in cervical intervertebral disc of the model group and the electro-acupuncture group increased,with statistical significance( P < 0. 05); compared with model group,MCP-1/GAPDH and CCR2/GAPDH in cervical intervertebral disc of electro-acupuncture group were lower than model group,with statistical significance( P < 0. 05). Conclusion: Electroacupuncture can inhibit the expression of TNF-alpha and IL-6 in the chondrocyte of intervertebral disc of rats with cervical spondylosis,which may be related to the inhibition of the activation of MCP-1/CCR2 signaling pathway.
引文
[1]徐丽莺,邵斌.电针夹脊穴联合补肾通络方治疗脊髓型颈椎病30例临床观察[J].中国民族民间医药,2018,27(22):109-111.
[2]Wang XY,He W,Shi H,et al. Effects of Electroacupuncture at PC6and ST36 on HeartRate Variability in Anesthetized Mice[J]. World J Tradit Chin Med,2015,1(3):67-70.
[3]施金杉.电针疗法“颈四针”穴与“颈夹脊”穴治疗神经根型颈椎病的疗效对照观察[J].中医临床研究,2018,10(31):30-32.
[4]Li W,Nyhan MM,Wilker EH,et al. Recent exposure to particle radioactivity and biomarkers of oxidative stress and inflammation:The Framingham Heart Study[J]. Environ Int,2018,121(Pt 2):1210-1216.
[5]刘改霞,孙雨瑶,陈子璐,等.炎性疼痛对小鼠外周组织炎性反应及TNF-α和MCP-1的表达水平的影响[J].西安交通大学学报:医学版,2019,40(1):1-6.
[6]王先海,李珍斌,李小妹,等.通经定晕丸阻断颈椎病大鼠NF-κB激活MCP-1/CCR2通路及TNF-α及IL-1β、IL-6表达的研究[J].中国临床药理学与治疗学,2018,23(4):370-376.
[7]赵崇清,薛原,薛兴平.颈椎病与中医辨证关系的探讨(附100例分析)[J].中国中西医结合影像学杂志,2006,4(3):199-200,203.
[8]Karateev AE,Erdes ShF. Use of nonsteroidal anti-in-flammatory drugs in case of ankylosing spondylitis:long and persistently[J]. Ter Arkh,2014,86(11):123-127.
[9]Puvanesarajah V,Jain A,Shimer AL,et al. Complications and mortality following one to two-level anterior cervical fusion for cervical spondylosis in patients above 80 years of age[J]. Spine(Phila Pa 1976),2017,42(9):E509-E514.
[10]Nagashima H,Morio Y,Yamane K,et al. Tumor necrosis factor-alpha,interleukin-1beta,and interleukin-6 in the cerebrospinal fluid of patients with cervical myelopathy and lumbar radiculopathy[J]. Eur Spine J,2009,18(12):1946-1950.
[11]史哲.夹脊穴临床功效分析[J].辽宁中医药大学学报,2016,18(1):153.
[12]林秋吉,沈晓明.颈型、神经根型颈椎病针灸治疗近况[J].中医药信息,2005,22(4):40-42.
[13]刘鹏宇,郑健刚.针刺夹脊穴治疗颈、腰椎病临床研究进展[J].长春中医药大学学报,2012,28(1):178-180.
[14]谭奇纹.论华佗夹脊穴与经络脏腑的联系[J].中医药信息,2004,21(2):6-7.
[15]刘迪,陈璐璐,耿楠,等.“动光明”刺灸法治疗郁病的临床应用[J].环球中医药,2018,11(11):1706-1708.
[16]刘道龙,褚雪菲,萨仁.电针委阳穴、承山穴结合毫火针治疗老年性不安腿[J].长春中医药大学学报,2018,34(4):730-732.
[17]周丽,刘晓君,毛玮,等.针刺治疗功能性消化不良的选穴规律[J].湖北中医药大学学报,2018,20(2):124-126.
[18]李建宇,宋雪艾,蔡昱,等.仰韶文化人面鱼纹寓意与足太阳经功能相关理论渊源初探[J].中医学报,2018,33(3):405-410.
[2]Wang XY,He W,Shi H,et al. Effects of Electroacupuncture at PC6and ST36 on HeartRate Variability in Anesthetized Mice[J]. World J Tradit Chin Med,2015,1(3):67-70.
[3]施金杉.电针疗法“颈四针”穴与“颈夹脊”穴治疗神经根型颈椎病的疗效对照观察[J].中医临床研究,2018,10(31):30-32.
[4]Li W,Nyhan MM,Wilker EH,et al. Recent exposure to particle radioactivity and biomarkers of oxidative stress and inflammation:The Framingham Heart Study[J]. Environ Int,2018,121(Pt 2):1210-1216.
[5]刘改霞,孙雨瑶,陈子璐,等.炎性疼痛对小鼠外周组织炎性反应及TNF-α和MCP-1的表达水平的影响[J].西安交通大学学报:医学版,2019,40(1):1-6.
[6]王先海,李珍斌,李小妹,等.通经定晕丸阻断颈椎病大鼠NF-κB激活MCP-1/CCR2通路及TNF-α及IL-1β、IL-6表达的研究[J].中国临床药理学与治疗学,2018,23(4):370-376.
[7]赵崇清,薛原,薛兴平.颈椎病与中医辨证关系的探讨(附100例分析)[J].中国中西医结合影像学杂志,2006,4(3):199-200,203.
[8]Karateev AE,Erdes ShF. Use of nonsteroidal anti-in-flammatory drugs in case of ankylosing spondylitis:long and persistently[J]. Ter Arkh,2014,86(11):123-127.
[9]Puvanesarajah V,Jain A,Shimer AL,et al. Complications and mortality following one to two-level anterior cervical fusion for cervical spondylosis in patients above 80 years of age[J]. Spine(Phila Pa 1976),2017,42(9):E509-E514.
[10]Nagashima H,Morio Y,Yamane K,et al. Tumor necrosis factor-alpha,interleukin-1beta,and interleukin-6 in the cerebrospinal fluid of patients with cervical myelopathy and lumbar radiculopathy[J]. Eur Spine J,2009,18(12):1946-1950.
[11]史哲.夹脊穴临床功效分析[J].辽宁中医药大学学报,2016,18(1):153.
[12]林秋吉,沈晓明.颈型、神经根型颈椎病针灸治疗近况[J].中医药信息,2005,22(4):40-42.
[13]刘鹏宇,郑健刚.针刺夹脊穴治疗颈、腰椎病临床研究进展[J].长春中医药大学学报,2012,28(1):178-180.
[14]谭奇纹.论华佗夹脊穴与经络脏腑的联系[J].中医药信息,2004,21(2):6-7.
[15]刘迪,陈璐璐,耿楠,等.“动光明”刺灸法治疗郁病的临床应用[J].环球中医药,2018,11(11):1706-1708.
[16]刘道龙,褚雪菲,萨仁.电针委阳穴、承山穴结合毫火针治疗老年性不安腿[J].长春中医药大学学报,2018,34(4):730-732.
[17]周丽,刘晓君,毛玮,等.针刺治疗功能性消化不良的选穴规律[J].湖北中医药大学学报,2018,20(2):124-126.
[18]李建宇,宋雪艾,蔡昱,等.仰韶文化人面鱼纹寓意与足太阳经功能相关理论渊源初探[J].中医学报,2018,33(3):405-410.