不同时间针刺足三里穴对炎性大鼠脊髓、背根神经节中TRPV4蛋白表达的影响
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摘要
目的研究不同时间针刺足三里穴对炎性大鼠脊髓及背根神经节中香草酸瞬时电位受体4(TRPV4)蛋白表达的影响。方法将54只清洁级的雄性SD大鼠驯化7 d后,进行基础痛阈筛选,然后按痛阈结果随机分组为造模组36只、空白组18只,造模组在右足足垫部皮内注射完全弗氏佐剂0.1 mL,空白对照组注射0.1 mL生理盐水。造模成功的大鼠又按照基础痛阈随机分为模型组18只和针刺组18只,每组按照不同的处理时间又分为ZT8(15:00)、ZT16(23:00)2个时间亚组,每组9只。在2个时间点进行处理,针刺组在相应时间点手针针刺患侧足三里30 min,其间每5 min捻转1 min,频率为120次/min,模型组、空白组捆绑固定30 min,不做其他处理,1次/d,共治疗7 d。在第7天治理结束后检测相应时间点大鼠的痛阈,痛阈检测后取材,提取大鼠腰椎4～6的脊髓及背根神经节,采用免疫组化的方法检测大鼠背根神经节以及脊髓中TRPV4的表达。结果①在ZT8时间点:模型组大鼠与空白组相比,背根神经节中TRPV4表达量显著增加(P<0.01);脊髓组织TRPV4蛋白表达量明显上调(P<0.05)。针刺组大鼠与模型组相比,针刺组背根神经节TRPV4表达量显著下降(P<0.01),脊髓组织TRPV4蛋白表达量无明显差异(P>0.05)。②在ZT16时间点:与空白组相比,模型组大鼠背根神经节TRPV4表达量明显增加(P<0.05),脊髓组织TRPV4蛋白表达量明显上调(P<0.01)。与模型组相比,针刺组大鼠背根神经节TRPV4表达量无明显差异(P>0.05),脊髓组织TRPV4蛋白表达量降低(P<0.05)。结论炎性痛能够诱发大鼠脊髓和背根局部神经节TRPV4蛋白表达的上调;针刺足三里穴能够调节炎性痛大鼠脊髓与背根神经节TRPV4的蛋白含量。
Objective To study the effects of acupuncture at ST36(Zusanli) on the protein expression of transient vanillin receptor potential channel 4(TRPV4) in the spinal cord and dorsal root ganglia of inflammatory rat. Methods A total of 54 male SD rats of clean grade were acclimated for 7 days, and the basic pain thresholds were screened. Then, these rats were randomly divided into model(n =36) and control(n =18) groups based on the pain threshold results. 0.1 mL of complete Freund's adjuvant was injected internally, and 0.1 mL of physiological saline was injected into the blank control group. Rats with successful modeling were further divided into model group(n =18) and model+acupuncture group(n =18). Each group was needled at ZT8(15:00) or ZT16(23:00) as two different treatment time subgroup(n =9). At two time points, the needle group was needled at the corresponding time point for 30 minutes of the affected side, and the frequency was 120 times/min every 5 minutes. The model group and the control group were fixed for 30 minutes without any further treatment. After the end of the treatment on the 7 th day, the pain threshold of the rats at the corresponding time points was detected. The spinal cord and dorsal root ganglia of the lumbar vertebrae 4-6 were extracted, and the rat dorsal root ganglion was detected by immunohistochemistry. Section and expression of TRPV4 in the spinal cord. Results ①At the time of ZT8: compared with the blank group, the expression level of TRPV4 in the dorsal root ganglion increased significantly in the model group(P<0.01); the expression level of TRPV4 protein in spinal cord tissue was significantly up-regulated(P<0.05). Compared with the model group, the TRPV4 expression level in the dorsal root ganglion of the model group was significantly decreased(P<0.01). There was no significant difference in the TRPV4 protein expression level in the spinal cord tissue(P>0.05). ② At the time of ZT16: compared with the blank group, the expression level of TRPV4 in the dorsal root ganglion of the model group was significantly increased(P<0.05); the expression level of TRPV4 protein in the spinal cord was significantly up-regulated(P<0.01). Compared with the model group, there was no significant difference in the expression level of TRPV4 in the dorsal root ganglion of the model group(P>0.05); the expression level of TRPV4 protein in the spinal cord tissue decreased(P<0.05). Conclusion Inflammatory pain can induce the up-regulation of TRPV4 protein expression in the spinal cord and dorsal root ganglion of rats. Acupuncture at Zusanli can regulate the protein expression level of TRPV4 in the spinal cord and dorsal root ganglion of rats with inflammatory pain.
Objective To study the effects of acupuncture at ST36(Zusanli) on the protein expression of transient vanillin receptor potential channel 4(TRPV4) in the spinal cord and dorsal root ganglia of inflammatory rat. Methods A total of 54 male SD rats of clean grade were acclimated for 7 days, and the basic pain thresholds were screened. Then, these rats were randomly divided into model(n =36) and control(n =18) groups based on the pain threshold results. 0.1 mL of complete Freund's adjuvant was injected internally, and 0.1 mL of physiological saline was injected into the blank control group. Rats with successful modeling were further divided into model group(n =18) and model+acupuncture group(n =18). Each group was needled at ZT8(15:00) or ZT16(23:00) as two different treatment time subgroup(n =9). At two time points, the needle group was needled at the corresponding time point for 30 minutes of the affected side, and the frequency was 120 times/min every 5 minutes. The model group and the control group were fixed for 30 minutes without any further treatment. After the end of the treatment on the 7 th day, the pain threshold of the rats at the corresponding time points was detected. The spinal cord and dorsal root ganglia of the lumbar vertebrae 4-6 were extracted, and the rat dorsal root ganglion was detected by immunohistochemistry. Section and expression of TRPV4 in the spinal cord. Results ①At the time of ZT8: compared with the blank group, the expression level of TRPV4 in the dorsal root ganglion increased significantly in the model group(P<0.01); the expression level of TRPV4 protein in spinal cord tissue was significantly up-regulated(P<0.05). Compared with the model group, the TRPV4 expression level in the dorsal root ganglion of the model group was significantly decreased(P<0.01). There was no significant difference in the TRPV4 protein expression level in the spinal cord tissue(P>0.05). ② At the time of ZT16: compared with the blank group, the expression level of TRPV4 in the dorsal root ganglion of the model group was significantly increased(P<0.05); the expression level of TRPV4 protein in the spinal cord was significantly up-regulated(P<0.01). Compared with the model group, there was no significant difference in the expression level of TRPV4 in the dorsal root ganglion of the model group(P>0.05); the expression level of TRPV4 protein in the spinal cord tissue decreased(P<0.05). Conclusion Inflammatory pain can induce the up-regulation of TRPV4 protein expression in the spinal cord and dorsal root ganglion of rats. Acupuncture at Zusanli can regulate the protein expression level of TRPV4 in the spinal cord and dorsal root ganglion of rats with inflammatory pain.
引文
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[8] Wang K,Xiang XH,Qiao N,et al.Genomewide analysis of rat periaqueductal gray-dorsal horn reveals time-,region- and frequency-specific mRNA expression changes in response to electroacupuncture stimulation[J].Scien tific Reports,2014,4(4):6713-6720.
[9] 张杨,王永慧,丁欣利,等.TRPV4在介导大鼠背根神经节持续受压后机械和热痛敏中的作用[J].中国康复医学杂志,2010,25(12):1123-1130.Zhang Y,Wang YH,Ding XL,et al.The role of transient receptor potential vanilloid 4 in mediating mechanical and thermal allodynia following chronic compression of dorsal root ganglion in rats[J].Chinese Journal of Rehabilita tion Medicine,2010,25(12):1123-1130.
[10] 曲玉娟,张晓,魏慧,等.大鼠背根神经节持续受压后脊髓背角TRPV4蛋白表达及分布的变化[J].中国康复医学杂志,2017,32(2):151-155.Qu YJ,Zhang X,Wei H,et al.Alternation of TRPV4 protein expression and distribution in spinal dorsal horn in chronic compression of the dorsal root ganglion model of rats[J].Chinese Journal of Rehabilitation Medicine,2017,32(2):151-155.
[2] 余曙光,郭义.实验针灸学[M].上海:上海科学技术出版社,2009.Yu SG,Guo Y.Experimental Acupuncture and Moxibustion[M].Shanghai:Shanghai Science and Technical Publisher,2009.
[3] Bellamy N,Sothern RB,Campbell J.Aspects of diurnal rhythmicity in pain,stiffness,and fatigue in patients with fibromyalgia [J].J Rheumatol,2004,31(2):379-389.
[4] 姚利钱.胆病疼痛昼夜节律探析[J].皖南医学院学报,2010,29(1):22-23.Yao LQ.Analysis of circadian rhythm of biliary pain[J].Journal of Wannan Medical University,2010,29(1):22 -23.
[5] 余早勤,张程亮,王友群,等.疼痛的昼夜节律与镇痛药物治疗[J].医药导报,2014,33(6):771-774.Yu ZQ,Zhang CL,Wang YQ,et al.Circadian rhythm of pain and analgesic medication[J].Herald of Medicine,2014,33(6):771-774.
[6] 邹明新,李思颉,邵国,等.电针刺激足三里穴对甲醛溶液所致内脏炎症痛大鼠脊髓蛋白激酶C膜转位水平的影响[J].中国疼痛医学杂志,2016,22(3):174-177.Zhou MX,Li SJ,Shao G,et al.Effects of electroacu puncture at Zusanli on spinal protein kinase C membrane translocation in formalin-induced visceral inflammatory pain rats[J].Chinese Journal of Pain Medicine,2016,22(3):174-177.
[7] 贺渊哲,邵先舫,熊辉,等.福尔马林足底炎性疼痛大鼠模型的建立及其背根神经节辣椒素受体表达水平[J].湖南中医药大学学报,2014,34(1):10-13.He YZ,Shao XF,Xiong H,et al.Establishment of rat model with inflammatory pain induced by formalin and the expression level of capsaicin receptor in dorsal root ganglion[J].Journal of Hunan University of CM,2014,34(1):10-13.
[8] Wang K,Xiang XH,Qiao N,et al.Genomewide analysis of rat periaqueductal gray-dorsal horn reveals time-,region- and frequency-specific mRNA expression changes in response to electroacupuncture stimulation[J].Scien tific Reports,2014,4(4):6713-6720.
[9] 张杨,王永慧,丁欣利,等.TRPV4在介导大鼠背根神经节持续受压后机械和热痛敏中的作用[J].中国康复医学杂志,2010,25(12):1123-1130.Zhang Y,Wang YH,Ding XL,et al.The role of transient receptor potential vanilloid 4 in mediating mechanical and thermal allodynia following chronic compression of dorsal root ganglion in rats[J].Chinese Journal of Rehabilita tion Medicine,2010,25(12):1123-1130.
[10] 曲玉娟,张晓,魏慧,等.大鼠背根神经节持续受压后脊髓背角TRPV4蛋白表达及分布的变化[J].中国康复医学杂志,2017,32(2):151-155.Qu YJ,Zhang X,Wei H,et al.Alternation of TRPV4 protein expression and distribution in spinal dorsal horn in chronic compression of the dorsal root ganglion model of rats[J].Chinese Journal of Rehabilitation Medicine,2017,32(2):151-155.