“三法三穴”推拿手法对坐骨神经损伤大鼠痛觉功能和脊髓背角CX3CL1/CX3CR1表达的调节

doi:10.3969/j.issn.1006-9771.2020.02.009

摘要/Abstract

摘要：

目的通过观察脊髓背角趋化因子(C-X3-C基元)配体1 (CX3CL1)/趋化因子(C-X3-C基元)受体1 (CX3CR1)蛋白和mRNA表达变化,探讨“三法三穴”推拿手法对坐骨神经损伤大鼠痛觉功能改善的作用机制。方法 74只雄性Sprague-Dawley大鼠,随机分为正常组(n = 12)、假手术组(n = 24)、模型组(n = 25)和三法三穴组(n = 13)。模型组和三法三穴组制作坐骨神经损伤模型。假手术组仅暴露坐骨神经。三法三穴组造模后第7天起用按摩推拿手法模拟仪按摩殷门、承山、阳陵泉三穴。分别于造模后7 d、干预20 d进行光热耐痛阈测定;造模后7 d、干预10 d、干预20 d进行累积疼痛评分测定;并于造模后7 d和干预20 d取材,对脊髓背角CX3CL1/CX3CR1表达进行Western blotting和RT-PCR检测,干预20 d对脊髓背角小胶质细胞进行免疫荧光观察。结果造模后7 d,与正常组比较,模型组和假手术组光热耐痛阈升高(P < 0.05);与假手术组比较,模型组和三法三穴组累积疼痛评分升高( P < 0.05);干预10 d后,三法三穴组累积疼痛评分低于模型组( P < 0.05);干预20 d后,三法三穴组光热耐痛阈和累积疼痛评分均低于模型组( P < 0.05)。造模后7 d和干预20 d后,各组CX3CL1/CX3CR1蛋白和mRNA的表达变化均无显著性差异( P > 0.05)。干预20 d后模型组小胶质细胞呈部分活化或完全活化状态,三法三穴组呈未活化或部分活化状态。结论 “三法三穴”推拿手法对坐骨神经损伤大鼠痛觉功能有改善,可能是通过CX3CL1/CX3CR1以外的途径调节小胶质细胞实现的。

关键词: 坐骨神经损伤, 痛觉, 推拿, 趋化因子(C-X3-C基元)配体1/趋化因子(C-X3-C基元)受体1, 大鼠

Abstract:

Objective To investigate the mechanism of Three Handing-Three Points on pain function in sciatic nerve injury rats by observing the changes of chemokine (C-X3-C motif) ligand 1, CX3CL1)/chemokine (C-X3-C motif) receptor 1 (CX3CR1) protein and mRNA expression in spinal dorsal horn.Methods A total of 74 male Sprague-Dawley rats were randomly divided into normal group (n= 12), sham group (n = 24), model group (n = 25), and Three Handing-Three Points group (Tuina group, n = 13). The model group and Tuina group prepared the sciatic nerve injury model. The sham group exposed sciatic nerve only. Tuina group received Tuina on Yinmen (BL37), Chengshan (BL57) and Yanglingquan (GB34) with Tuina manipulation emulator. The photothermal pain threshold was measured seven days after modeling and after 20 days of intervention; cumulative pain score was measured seven days after modeling, and after ten days and 20 days of intervention. The spinal dorsal horn tissues were extracted to detect the protein and mRNA expression of CX3CL1/CX3CR1 with Western blotting and RT-PCR seven days after modeling and after 20 days of intervention. The microglia morphology in spinal dorsal horn was observed with immunofluorescence after 20 days of intervention.Results Seven days after modeling, compared with the normal group, the photothermal pain tolerance threshold increased in the model group and the sham group (P < 0.05); compared with the sham group, the cumulative pain score increased in the model group and Tuina group ( P < 0.05). After ten days of intervention, the cumulative pain score was lower in Tuina group than in the model group ( P < 0.05). After 20 days of intervention, both the photothermal pain tolerance threshold and cumulative pain score were lower in Tuina group than in the model group ( P < 0.05). There was no significant difference in the expression of CX3CL1/CX3CR1 protein and mRNA on the seven days after modeling and after 20 days of intervention ( P > 0.05). The microglia in the model group were partially activated or completely activated, while those in Tuina group were unactivated or partially activated after 20 days of intervention. Conclusion Three Handing-Three Points can improve the pain function of sciatic nerve injured rats, which may associate with regulating microglia through the pathway other than CX3CL1/CX3CR1.

Key words: sciatic nerve injury, pain function, Tuina, chemokine (C-X3-C motif) ligand 1/chemokine (C-X3-C motif) receptor 1, rats

中图分类号:

R745.4

莫岩君,张羽墨,于天源,邵帅,沈熠,罗宇婷,吕桃桃. “三法三穴”推拿手法对坐骨神经损伤大鼠痛觉功能和脊髓背角CX3CL1/CX3CR1表达的调节[J]. 《中国康复理论与实践》, 2020, 26(2): 189-196.

MO Yan-jun,ZHANG Yu-mo,YU Tian-yuan,SHAO Shuai,SHEN Yi,LUO Yu-ting,LÜ Tao-tao. Effect of Three Handing-Three Points on Pain Function and Expression of CX3CL1/CX3CR1 in Spinal Cord Dorsal Horn of Rats with Sciatic Nerve Injury[J]. 《Chinese Journal of Rehabilitation Theory and Practice》, 2020, 26(2): 189-196.

图/表 10

表1

表2

表3

表4

图1

表5

图2

表6

表7

图3

参考文献 29

[1]	李鸿浩. 止痛药会损伤听力吗?[J]. 心血管病防治知识, 2013, 78(6):44.
[2]	刘志林, 陈晓容, 彭丽雅. 药代动力学和药效动力学结合模型对急慢性镇痛药物的评估作用[J]. 保健医学研究与实践, 2016, 13(6):88-91.
[3]	周玉颖, 肖适崎, 孙健. 防治麻醉性镇痛药毒副作用的护理体会[J]. 辽宁药物与临床, 2003, 6(3):145.
[4]	李同军, 刘旭东, 于志国, 等. 五段拔伸推拿法治疗腰椎间盘突出症胫腓神经损伤的临床研究[J]. 针灸临床杂志, 2018, 34(9):63-66.
[5]	吴文刚, 杨清滨, 吴振坤, 等. 推拿治疗周围神经损伤1245例疗效观察[J]. 中国中医药信息杂志, 1999, 6(4):66-67.
[6]	马建强, 李凤岩, 吕忠礼. 推拿手法治疗分娩性臂丛神经损伤的临床观察[J]. 中国中医骨伤科杂志, 2018, 26(3):46-49.
[7]	冼思彤, 于天源, 潘璠, 等. 推拿对坐骨神经损伤大鼠背根神经节降钙素基因相关肽表达的影响[J]. 中医学报, 2015, 30(9):1311-1314.
[8]	鲁梦倩, 于天源, 姚斌彬, 等. 推拿对坐骨神经损伤模型大鼠神经超微结构的影响[J]. 南京中医药大学学报, 2015, 31(4):349-352.
[9]	张林峰, 于天源, 潘璠, 等. 推拿对坐骨神经损伤大鼠髓鞘碱性蛋白表达的影响[J]. 中华中医药杂志, 2016, 31(8):3045-3048.
[10]	高玉峰, 姚斌彬, 吴剑聪, 等. 推拿对大鼠坐骨神经损伤后MAP-2和NF-M表达的影响[J]. 中医药导报, 2014, 20(1):11-13.
[11]	李易真, 杨超, 于天源, 等. “三法三穴”推拿手法对坐骨神经损伤大鼠运动功能和脊髓NogoA及其受体表达的影响[J]. 中国康复理论与实践, 2018, 24(12):1403-1407.
[12]	吴帅, 于天源, 杨超, 等. “三法三穴”对坐骨神经损伤模型大鼠冷感觉超敏反应的行为学研究[J]. 中医药导报, 2017, 23(22):8-11, 22.
[13]	Charo I F, Ransohoff R M. The many roles of chemokines and chemokine receptors in inflammation[J]. N Engl J Med, 2006, 354(6):610. doi: 10.1056/NEJMra052723
[14]	Conroy M J, Maher S G, Melo A M, et al. Identifying a novel role for fractalkine (CX3CL1) in memory CD8(+)T cell accumulation in the omentum of obesity-associated cancer patients[J]. Front Immonul, 2018, 9(3):1867.
[15]	Sheridan G K, Murphy K J. Neuron-glia crosstalk in health and disease: fractalkine and CX3CR1 take centre stage[J]. Open Biol, 2013, 3(12):130181-130182. doi: 10.1098/rsob.130181 pmid: 24352739
[16]	Zhu W, Acosta C, Macneil B, et al. Elevated expression of fractalkine (CX3CL1) and fractalkine receptor (CX3CR1) in the dorsal root ganglia and spinal cord in experimental autoimmune encephalomyelitis: Implications in multiple sclerosis-induced neuropathic pain[J]. BioMed Res Int, 2013, 1(14):874-880.
[17]	Verge G M, Milligan E D, Maier S F, et al. Fractalkine (CX3CL1) and fractalkine receptor (CX3CR1) distribution in spinal cord and dorsal root ganglia under basal and neuropathic pain conditions[J]. Eur J Neurosci, 2004, 20(5):119-123.
[18]	Sun J L, Xiao C, Lu B, et al. CX3CL1/CX3CR1 regulates nerve injury-induced pain hypersensitivity through the ERK5 signaling pathway[J]. J Neurosci Res, 2013, 91(4):545-553. doi: 10.1002/jnr.v91.4
[19]	Zhou Q Q, Chen S S, Zhang Q Q, et al. Cerebrospinal fluid-contacting nucleus mediates nociception via release of fractalkine[J]. Braz J Med Biol Res, 2017, 50(9):e6275. doi: 10.1590/1414-431x20176275
[20]	Old E A, Nadkarni S, Grist J, et al. Monocytes expressing CX3CR1 orchestrate the development of vincristine-induced pain[J]. J Clin Invest, 2014, 124(5):2023-2036. doi: 10.1172/JCI71389
[21]	Clark A K, Malcangio M. Microglial signalling mechanisms: Cathepsin S and Fractalkine[J]. Exp Neurol, 2012, 234(2):283-292. doi: 10.1016/j.expneurol.2011.09.012 pmid: 21946268
[22]	Aldskogius H, Kozlova E. Microglia and neuropathic pain[J]. Glia, 2013, 13(4):2792-2799.
[23]	Watkins L R, Milligan E D, Maier S F. Glial activation: a driving force for pathological pain[J]. Trends Neurosci, 2001, 24(8):450-455. pmid: 11476884
[24]	Nair S, Sobotka K S, Joshi P, et al. Lipopolysaccharide-induced alteration of mitochondrial morphology induces a metabolic shift in microglia modulating the inflammatory response in vitro and in vivo[J]. Glia, 2019, 12(5):468-472.
[25]	Joana M D, Rita G, Liliana C, et al. Region: pecific control of microglia by adenosine A2A receptors: uncoupling anxiety and associated cognitive deficits in female rats[J]. Glia, 2018, 67(3):278-281.
[26]	Owolabi S A, Saab C Y. Fractalkine and minocycline alter neuronal activity in the spinal cord dorsal horn[J]. FEBS Lett, 2006, 580(18):4290-4310.
[27]	Begum F, Zhu W, Cortes C, et al. Elevation of tumor necrosis factor alpha in dorsal root ganglia and spinal cord is associated with neuroimmune modulation of pain in an animal model of multiple sclerosis[J]. J Neuroimmune Pharm, 2013, 8(3):677-690. doi: 10.1007/s11481-013-9449-5 pmid: 23483352
[28]	Scholz J, Woolf C J. The neuropathic pain triad: neurons, immune cells and glia[J]. Nat Neurosci, 2007, 10(11):1361-1368. doi: 10.1038/nn1992
[29]	Abdo H, Calvo-Enrique L, Lopez J M, et al. Specialized cutaneous Schwann cells initiate pain sensation[J]. Science, 2019, 365(6454):695-699. doi: 10.1126/science.aax6452

上游引物	CX3CL1	FGA AAA TAG GCC CCT GCT AGT
	CX3CR1	GTG AAC AGG TGG AGA GCG TC
下游引物	CX3CL1	ATT CTC CTC CCT GCA CAC CT
	CX3CR1	TTC CAG TCA TCT TAG AAA GC

组别	n	造模后7 d	干预20 d
正常组	6	7.817±0.979	7.922±1.623
假手术组	6	11.111±0.839^a	8.206±1.490
模型组	6	17.539±1.149^a,b	15.211±2.549^a,b
三法三穴组	6		10.039±0.981^a,c
F/χ²值		147.50	15.020
P值		< 0.001	0.002

组别	n	造模后7 d	干预10 d	干预20 d
假手术组	6	1.111±0.502	0.611±0.136	0.333±0.298
模型组	6	17.056±1.290^a	18.444±1.293^a	19.222±1.682^a
三法三穴组	6	14.611±1.272^a	16.444±0.660^a,b	8.000±1.011^a,b
F/χ²值		14.5123	14.760	412.240
P值		< 0.001	< 0.001	< 0.001

组别	n	造模后7 d	干预20 d
正常组	6	0.545±0.178	0.748±0.314
假手术组	6	0.554±0.211	0.647±0.347
模型组	6	0.502±0.136	1.162±0.464
三法三穴组	6		0.803±0.226
F值		0.140	2.490
P值		0.867	0.090

组别	n	造模后7 d	干预20 d
正常组	6	0.412±0.248	0.429±0.159
假手术组	6	0.377±0.209	0.280±0.129
模型组	6	0.322±0.155	0.410±0.190
三法三穴组	6		0.447±0.110
F值		0.290	1.540
P值		0.755	0.235