芍药甘草汤对中枢性肌张力增高大鼠脑内氨基酸及其受体的影响
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摘要
探讨芍药甘草汤对中枢性肌张力增高大鼠模型的神经递质及其受体的影响。采用线拴法所致的脑卒中恢复期痉挛性肌张力增高大鼠模型,以巴氯芬为对照,实验组给予芍药甘草汤3∶1配伍比例,给药3周后测定其神经功能评分、肌张力、痛阈的变化,并采用HPLC测定大脑皮层GABA,Gly,Glu,Asp含量,免疫组化法测定大脑皮层GABA受体Aα1,B与NMDA受体亚基NR1,NR2A,NR2B蛋白表达。结果显示芍药甘草汤3∶1配伍剂量组对脑卒中后痉挛偏瘫状态有较好的改善作用,能显著改善神经症状(P<0.01),降低肌张力(P<0.01),改善痛域(P<0.05);能够显著增加脑内抑制性氨基酸GABA,Gly的含量(P<0.01),对于兴奋性氨基酸Glu,Asp有降低的趋势,但没有统计学意义;能够显著增强大脑抑制性神经递质受体GABA受体Aα1和B的表达(P<0.05),并减少大脑兴奋性神经递质受体NMDA各亚型NR1,NR2A,NR2B的表达(P<0.05)。研究表明,芍药甘草汤3∶1配伍剂量具有较好的解痉止痛之效,不仅能够增加脑卒中后痉挛状态大鼠脑内抑制性氨基酸的水平,同时还可增加其受体的表达,从而增加抑制性神经信号转导的强度;对于脑内兴奋性氨基酸水平虽然没有显著性作用,但也有降低的趋势,同时对于兴奋性氨基酸受体的表达也有抑制作用,从而减弱兴奋性神经信号转导的强度,减轻兴奋性神经毒性。结果表明,芍药甘草汤可通过调节脑内抑制性和兴奋性神经能系统的平衡,从而达到缓解脑卒中痉挛状态,起到柔筋止痉的作用。
To explore the effects of Shaoyao Gancao decoction on contents of amino acids and expressions of receptors in the brains of spastic paralysis rats,the spastic paralysis rat models of stroke convalescence were made by line tethering method. Baclofen was used as the control group,and the experiment group received Shaoyao Gancao decoction at 3∶ 1 proportions. After 3 weeks,the neurobehavioral scores,muscular tension and pain threshold were measured and compared. High performance liquid chromatography( HPLC) was used to detect the contents of GABA,Gly,Glu,Asp in cerebral cortex. The protein expressions of GABA receptors Aα1,B; NMDA receptor NR1,NR2 A and NR2 B in cerebral cortex were determined by immunohistochemistry assay. The results showed that the Shaoyao Gancao decoction at 3 ∶ 1 proportion could improve the spastic paralysis state after stroke,significantly improve neurological symptoms( P < 0. 01),decrease muscular tension( P < 0. 01) and improve pain threshold( P < 0. 05) as compared with model group.Simultaneously,the contents of inhibitory amino acids GABA and Gly were increased significantly( P < 0. 01),while with a decrease tendency in excitatory amino acids Glu and Asp( with no statistical significance). In addition,it could significantly increase the protein expressions of neurotransmitter GABA receptors Aα1,and B( P < 0. 05); reduce the expressions of neurotransmitter NMDA receptors NR1,NR2 A and NR2B( P < 0. 05). These results suggested that the Shaoyao Gancao decoction at 3: 1 proportion could effectively relieve spasm and pain. The mechanism might be associated with increasing the contents of inhibitory amino acids and increasing the expressions of their receptors in spastic paralysis rats after stroke,which would consequently enhance the signal transduction of inhibitory amino acids. Meanwhile,there was a decrease tendency in excitatory amino acids,although no significant effect was observed,and it could suppress the expressions of excitatory amino acids receptors,thus weaken the excitatory signal transduction. Thereby the neurotoxicity was relieved eventually. These findings indicated that Shaoyao Gancao decoction could regulate the balance of neurotransmitter system to relieve the spasticity,and eventually achieve tendon tonifying and spasm relieving effect.
To explore the effects of Shaoyao Gancao decoction on contents of amino acids and expressions of receptors in the brains of spastic paralysis rats,the spastic paralysis rat models of stroke convalescence were made by line tethering method. Baclofen was used as the control group,and the experiment group received Shaoyao Gancao decoction at 3∶ 1 proportions. After 3 weeks,the neurobehavioral scores,muscular tension and pain threshold were measured and compared. High performance liquid chromatography( HPLC) was used to detect the contents of GABA,Gly,Glu,Asp in cerebral cortex. The protein expressions of GABA receptors Aα1,B; NMDA receptor NR1,NR2 A and NR2 B in cerebral cortex were determined by immunohistochemistry assay. The results showed that the Shaoyao Gancao decoction at 3 ∶ 1 proportion could improve the spastic paralysis state after stroke,significantly improve neurological symptoms( P < 0. 01),decrease muscular tension( P < 0. 01) and improve pain threshold( P < 0. 05) as compared with model group.Simultaneously,the contents of inhibitory amino acids GABA and Gly were increased significantly( P < 0. 01),while with a decrease tendency in excitatory amino acids Glu and Asp( with no statistical significance). In addition,it could significantly increase the protein expressions of neurotransmitter GABA receptors Aα1,and B( P < 0. 05); reduce the expressions of neurotransmitter NMDA receptors NR1,NR2 A and NR2B( P < 0. 05). These results suggested that the Shaoyao Gancao decoction at 3: 1 proportion could effectively relieve spasm and pain. The mechanism might be associated with increasing the contents of inhibitory amino acids and increasing the expressions of their receptors in spastic paralysis rats after stroke,which would consequently enhance the signal transduction of inhibitory amino acids. Meanwhile,there was a decrease tendency in excitatory amino acids,although no significant effect was observed,and it could suppress the expressions of excitatory amino acids receptors,thus weaken the excitatory signal transduction. Thereby the neurotoxicity was relieved eventually. These findings indicated that Shaoyao Gancao decoction could regulate the balance of neurotransmitter system to relieve the spasticity,and eventually achieve tendon tonifying and spasm relieving effect.
引文
[1]张秋香.芍药甘草汤合血府逐瘀汤加减治疗中风后痉挛性瘫痪35例临床观察[J].河北中医,2012,34(5):690.
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[3]刘超.芍药甘草汤治疗中风后肢体痉挛症[J].中国医药导刊,2011,13(8):1352.
[4]杨旭,王景霞,张建军,等.芍药甘草汤对中枢性肌张力增高大鼠解痉止痛最佳配比的筛选研究[J].北京中医药大学学报,2015,38(1):33.
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[7]杨慎峭,朱天民,金荣疆,等.康复训练结合电针对脑卒中肢体痉挛大鼠γ-氨基丁酸能中间神经元表达的影响[J].中医康复医学杂志,2013,28(3):198.
[8]陈党红.舒筋颗粒对脑卒中后肌张力增高大鼠神经行为学及痛阈的影响[J].安徽中医学院学报,2009,28(1):44.
[9]胡学英.浅谈偏瘫肢体肌肉痉挛的运动疗法治疗[J].继续医学教育,2011,25(5):68.
[10]岳增辉,袁建菱,姜京明.经筋论治脑卒中后痉挛状态及对脑脊液Glu、GABA的影响[J].中国针灸,2004,24(8):565.
[11]倪克锋,葛芳,包烨华.脑卒中后肌张力增高的机制研究[C].嘉兴:2008年浙江省物理医学与康复学术年会,2008.
[12]公维军,张通,孙新亭.脑卒中后痉挛性偏瘫的研究现状[J].中国康复理论与实践,2008,14(3):212.
[13]Yezierski Robert P.Spinal cord injury pain:spinal and supraspinal mechanisms[J].J Rehabil Res Dev,2009,46(1):95.
[14]杨远滨,金爽.痉挛状态与疼痛[J].中国康复医学杂志,2011,26(3):292.
[15]Rietman J S,Geertzen J H.Efficacy of intrathecal baclofen delivery in the management of severe spasticity in upper motor neuron syndrome[J].Acta Neurochir suppl,2007,97(1):205.
[16]羊继平,张锡庆,王晓东,等.痉挛型脑瘫患儿脑脊液兴奋性、抑制性氨基酸的研究[J].中华小儿外科杂志,1998,19(5):282.
[17]周鸿铭,雷娜,鲁亚平.甘氨酸神经递质研究进展[J].生物学杂志,2011,28(1):79.
[18]陈小飞,王永安.氨基酸及其受体在脑缺血中的作用及相关药物应用[J].中国药物应用与监测,2010,7(2):116.
[19]Zhou Cui,Li Chong,Yu Hong-min,et al.Neuroprotection of gamma-aminobutyric acid receptor agonists via enhancing neuronal nitric oxide synthase(Ser847)phosphorylation through increased neuronal nitric oxide synthase and PSD95 interaction and inhibited protein phosphatase activity in cerebral ischemia[J].J Neurosci Res,2008,86(13):2973.
[20]Gladding Clare M,Raymond Lynn A.Mechanisms underlying NMDA receptor synaptic/extrasynaptic distribution and function[J].Mol Cell Neurosci,2011,48(4):308.
[21]Mittmann T,QüM,Zilles K,et al.Long term cellular dysfunction after focal cerebral isehemic:in vitro analyses[J].Neuroscience,1998,85(1):15.
[22]Fukui M,Song J H,Choi J,et al.Mechanism of glutamate induced neurotoxicity in HT22 mouse hippocampal cells[J].Eur J Pharmacol,2009,617(1/3):1.
[23]陈虎,蔡定芳,沈思钰,等.电针对局灶性脑缺血大鼠海马兴奋性氨基酸受体的影响[J].上海针灸杂志,2003,22(5):13.
[24]Zepeda A,Sengpiel F,Guagnelli M A,et al.Functional reorganization of visual cortex maps after ischemic lesions is accompanied by changes in expression of cytoskeletal proteins and NMDA and GABAA receptor subunits[J].J Neurosci,2004,24(8):1812.
[25]Friedman C J,Lyden P D,Nanez S,et al.High dose baclofen is neuroprotective but also causes intracerebral hemorrhage:a quantal bioassay study using the intraluminal suture occulsion method[J].Exp Neurol,1997,147:346.
[26]李冬梅,车薇,李霞,等.芍药苷对脑缺血再灌注沙土鼠ATP酶和兴奋性氨基酸的影响[J].中国药物警戒,2014,11(12):717.
[27]王景霞,张建军,屈胜胜,等.芍药苷对血虚肝郁证候模型大鼠海马CA3区组织形态及一氧化氮通路的影响[J].北京中医药大学学报,2015,38(2):85.
[2]张颖,杨坚,高宁沁.芍药甘草汤结合运动疗法治疗脑卒中后肘关节屈曲痉挛的临床研究[J].上海中医药杂志,2012,46(11):47.
[3]刘超.芍药甘草汤治疗中风后肢体痉挛症[J].中国医药导刊,2011,13(8):1352.
[4]杨旭,王景霞,张建军,等.芍药甘草汤对中枢性肌张力增高大鼠解痉止痛最佳配比的筛选研究[J].北京中医药大学学报,2015,38(1):33.
[5]陈乐乐,岳增辉,朱小姗.大鼠脑卒中痉挛性瘫痪模型制备方法研究[J].上海中医药大学学报,2012,26(2):75.
[6]Bohannon R W,Smith M B.Interrater reliability of a modified ashworth scale of muscle spasticity[J].Phys Ther,1987,67:206.
[7]杨慎峭,朱天民,金荣疆,等.康复训练结合电针对脑卒中肢体痉挛大鼠γ-氨基丁酸能中间神经元表达的影响[J].中医康复医学杂志,2013,28(3):198.
[8]陈党红.舒筋颗粒对脑卒中后肌张力增高大鼠神经行为学及痛阈的影响[J].安徽中医学院学报,2009,28(1):44.
[9]胡学英.浅谈偏瘫肢体肌肉痉挛的运动疗法治疗[J].继续医学教育,2011,25(5):68.
[10]岳增辉,袁建菱,姜京明.经筋论治脑卒中后痉挛状态及对脑脊液Glu、GABA的影响[J].中国针灸,2004,24(8):565.
[11]倪克锋,葛芳,包烨华.脑卒中后肌张力增高的机制研究[C].嘉兴:2008年浙江省物理医学与康复学术年会,2008.
[12]公维军,张通,孙新亭.脑卒中后痉挛性偏瘫的研究现状[J].中国康复理论与实践,2008,14(3):212.
[13]Yezierski Robert P.Spinal cord injury pain:spinal and supraspinal mechanisms[J].J Rehabil Res Dev,2009,46(1):95.
[14]杨远滨,金爽.痉挛状态与疼痛[J].中国康复医学杂志,2011,26(3):292.
[15]Rietman J S,Geertzen J H.Efficacy of intrathecal baclofen delivery in the management of severe spasticity in upper motor neuron syndrome[J].Acta Neurochir suppl,2007,97(1):205.
[16]羊继平,张锡庆,王晓东,等.痉挛型脑瘫患儿脑脊液兴奋性、抑制性氨基酸的研究[J].中华小儿外科杂志,1998,19(5):282.
[17]周鸿铭,雷娜,鲁亚平.甘氨酸神经递质研究进展[J].生物学杂志,2011,28(1):79.
[18]陈小飞,王永安.氨基酸及其受体在脑缺血中的作用及相关药物应用[J].中国药物应用与监测,2010,7(2):116.
[19]Zhou Cui,Li Chong,Yu Hong-min,et al.Neuroprotection of gamma-aminobutyric acid receptor agonists via enhancing neuronal nitric oxide synthase(Ser847)phosphorylation through increased neuronal nitric oxide synthase and PSD95 interaction and inhibited protein phosphatase activity in cerebral ischemia[J].J Neurosci Res,2008,86(13):2973.
[20]Gladding Clare M,Raymond Lynn A.Mechanisms underlying NMDA receptor synaptic/extrasynaptic distribution and function[J].Mol Cell Neurosci,2011,48(4):308.
[21]Mittmann T,QüM,Zilles K,et al.Long term cellular dysfunction after focal cerebral isehemic:in vitro analyses[J].Neuroscience,1998,85(1):15.
[22]Fukui M,Song J H,Choi J,et al.Mechanism of glutamate induced neurotoxicity in HT22 mouse hippocampal cells[J].Eur J Pharmacol,2009,617(1/3):1.
[23]陈虎,蔡定芳,沈思钰,等.电针对局灶性脑缺血大鼠海马兴奋性氨基酸受体的影响[J].上海针灸杂志,2003,22(5):13.
[24]Zepeda A,Sengpiel F,Guagnelli M A,et al.Functional reorganization of visual cortex maps after ischemic lesions is accompanied by changes in expression of cytoskeletal proteins and NMDA and GABAA receptor subunits[J].J Neurosci,2004,24(8):1812.
[25]Friedman C J,Lyden P D,Nanez S,et al.High dose baclofen is neuroprotective but also causes intracerebral hemorrhage:a quantal bioassay study using the intraluminal suture occulsion method[J].Exp Neurol,1997,147:346.
[26]李冬梅,车薇,李霞,等.芍药苷对脑缺血再灌注沙土鼠ATP酶和兴奋性氨基酸的影响[J].中国药物警戒,2014,11(12):717.
[27]王景霞,张建军,屈胜胜,等.芍药苷对血虚肝郁证候模型大鼠海马CA3区组织形态及一氧化氮通路的影响[J].北京中医药大学学报,2015,38(2):85.