天穹止痛胶囊防治偏头痛的药效学研究与机理探讨
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摘要
偏头痛(migraine)是常见的神经内科疾病,反复发作,迁延难愈。WHO将重度偏头痛列为严重影响患者生活质量的慢性疾病之一。随着偏头痛的发病率呈逐年升高之势,世界各国因此而造成的医疗耗费和工作损失也相当可观。尽管现代医学治疗偏头痛的药物不断更新,但是这些药物或由于远期疗效不肯定,或由于存在不同程度的毒副作用,或由于价格比较昂贵等因素,严重影响了在我国的临床推广使用。因此,寻求安全有效的防治偏头痛药物,是当前急待解决的问题,中药整体调节作用持久缓和,相对安全,这正是中药防治偏头痛的主要切入点。本课题旨在通过文献整理和实验研究,探讨中药防治偏头痛的作用机制,为临床合理、安全、有效地用药提供指导。这对提高患者的生活质量,减轻社会负担均具有重要意义。
1.文献研究
查阅从秦汉时期到清代的古代中医学家对偏头痛的文献记载,检索2000-2007年发表在医学期刊上治疗偏头痛的最新临床报道,既系统全面地分析总结历代医家对偏头痛的名称、病因病机、证候分型、治则治法、处方用药的传统特色,又反映治疗偏头痛的时代用药特点,为进一步开展中医药防治偏头痛研究奠定文献基础,为寻找有效的防治偏头痛提供思路与方法。
文献研究分析发现,偏头痛主要是由于风、瘀、痰、虚杂合而致,风邪侵袭、肝阳上扰、瘀血阻滞是基本病机特点。治疗方法常是数法联用,以祛风止痛、平肝熄风、搜风通络法、活血祛瘀为主。疏散外风药、平熄内风药、活血化瘀药等是治疗偏头痛的常用药物。川芎“上行头目,下行血海,行血中之气,祛血中之风,走而不守”成为治疗偏头痛的第一要药。
本课题以上述研究结果为基础,结合导师临床用药经验,针对风邪外袭、肝阳上扰、瘀血阻络所致的常见型偏头痛,以平肝息风、活血通络、祛风止痛为治则,挖掘地方特色药物资源,选取传统药物与专治神经性头痛的汉桃叶相结合,拟订治疗偏头痛的中药复方——天穹止痛胶囊。
2.实验研究
在全面检索国内外最新的有关偏头痛研究进展以及实验性偏头痛动物模型研究概况的基础上,进行以下实验研究内容:
2.1基础药效研究
2.1.1镇痛实验:采用小鼠热板实验和醋酸扭体实验,通过行为学反应观察天穹止痛胶囊的镇痛作用。实验结果显示天穹止痛胶囊能提高小鼠热板实验的痛阈值,延长小鼠腹腔注射醋酸所致扭体反应潜伏期,减少扭体次数,提示天穹止痛胶囊具有一定的镇痛作用。
2.1.2镇静实验:观察天穹止痛胶囊对正常小鼠自主活动的影响,探讨其镇静作用。实验结果显示天穹止痛胶囊能够减少正常小鼠的自主活动数,提示其对中枢神经系统具有一定的抑制作用,有利于改善偏头痛患者发病时的急躁情绪。
2.2作用机制研究
2.2.1对神经递质的影响:采用皮下注射硝酸甘油偏头痛大鼠模型,以高效液相法检测给药后脑组织中5-HT、NE、DA的含量。实验结果显示,天穹止痛胶囊可以升高脑组织中5-HT水平,但对NE、DA无明显影响,提示天穹止痛胶囊可能通过促进5-HT对血管收缩和痛觉调节作用,抑制偏头痛的发作。
2.2.2对血管活性物质的影响:采用皮下注射硝酸甘油偏头痛大鼠模型,以放免法检测给药后血浆中CGRP、ET的含量。实验结果显示,天穹止痛胶囊可以降低血浆中CGRP、ET水平,提示天穹止痛胶囊可以通过抑制CGRP介导的血管扩张及CGRP对ET收缩血管的抑制作用,有利于抑制神经源性炎症,对偏头痛起到防治作用。
2.2.3对痛觉调节物质的影响:采用电刺激上矢状窦(SSS)硬脑膜偏头痛大鼠模型,以放免法检测给药后血浆中CGRP、SP、β-EP的含量。实验结果显示,天穹止痛胶囊可以降低血浆中CGRP、SP含量,升高β-EP含量,提示天穹止痛胶囊可以通过抑制伤害性刺激信息向中枢的传递过程,抑制偏头痛的发作。
2.2.4对CGRP及其受体mRNA表达的影响:采用电刺激上矢状窦(SSS)硬脑膜偏头痛大鼠模型,以RT-PCR方法检测中脑导水管周围灰质(PAG)区CGRP及其受体复合物CRLR/RAMP1 mRNA的表达。实验结果显示,天穹止痛胶囊可以抑制PAG中CGRP mRNA、CRLRm RNA、RAMP1 mRNA的过多表达,并且CGPR表达与RAMP1表达有一定的相关性,提示天穹止痛胶囊不仅可以抑制CGRP mRNA的过度表达,而且可以抑制其受体复合物CRLR/RAMP1 mRNA的过度表达,影响功能性CGRP的表达,发挥对疼痛信息传导的调节作用,抑制三叉神经节传入冲动的发放。
基于上述研究结果,我们推测天穹止痛胶囊防治偏头痛的作用途径可能是:①抑制神经源性炎症反应产生的疼痛刺激。②调节致痛物质与镇痛物质的释放平衡,调节CGRP及其受体复合物mRNA的表达,抑制伤害性信息向中枢的传递过程。
3.课题的创新性天穹止痛胶囊的组方是采用辨证与辨病相结合,传统药物与地方药物相结合而成。处方中的天麻、川芎等是中医辨证治疗偏头痛的传统用药;汉桃叶为广西常用药物,是治疗偏头痛的地方优势药物。这种组方思路有利于挖掘地方优势药材资源,丰富临床用药品种。
电刺激SSS硬脑膜作为一种可行的、先进的偏头痛动物模型,已在偏头痛的实验研究中逐渐推广使用。但是该模型目前在国内外主要是用于探讨偏头痛产生的病理机制以及西药对偏头痛的治疗作用,尚未应用于探讨中药复方治疗偏头痛的作用途径,本课题首次将该模型引入中药复方防治偏头痛的实验研究中,进行了有意义的开创研究,并取得了相应结果。
CGRP作为一种参与偏头痛病理过程的重要因子,在神经源性炎症反应和伤害性信息传递中发挥重要作用。其生物学效应不是通过结合单一受体实现,而是由受体复合物所介导,即CRLR与RAMP1组成的受体复合物与CGRP有高度亲和力,二者共同构建功能性的CGRP受体,目前防治偏头痛的药物尚未涉及相关内容的研究工作。本实验不仅探讨了天芎止痛胶囊对偏头痛动物模型CGRP的基因表达水平,还进一步探讨其受体复合物CRLR/RAMP1的基因表达水平,以及三者的相关性,进行了有意义的探索研究,并取得了相应结果。
Migraine is a common and complex neurological disorder that often attacks recurrently and is difficult to cure completely. An investigation made by WHO reveals that migraine is one of the chronic disease which seriously influence the quality of the patient’s life.It affects a significant proportion of the adult population worldwide.It represents an enormous socio-economic burden to the individual as well as to the socity. Although a great variety western medicine have been prepared,it is short of drugs which have no side effects and have positive prostecdtive efficacy.The effects of Chinese materia medica is mildly and have little side effects.This research aims at applying the theory of TCM combined with the modern scientific medhod to uncover the theory and experience about migraine,discussing the special feature of TCM,providing reference for the clinical usage of Chinese materia medica reasonably,safely and efficiently.
1.Study of literature
We collected considerable literature of migraine from ancient period to present period, and collected clinical reports published in medical magazines in 2000-2007 which treat migraine.Then discussed the special feature about the name,etiopathogenisis,diagnosis and treatment by diferentiation of syndromes of TCM about migraine.
The result of analysis is that wind, stagnated blood,and phlegm and deficiency are the primal etiological factor.Wind blocking the meridian, excessive rising of liver,and stagnated blood blocking meridian is the basic pathogenesis.Expelling wind, calm the liver and stop endogenous wind and promote blood circulation to remove meridian obstruction is the main treating method.
According to the foundamental theory of TCM,combining the clinical experience of my advisor and the result of analysis,we make up a prescription named of TianQing Zhitong Capsule to treat migraine to aim directly at the common pathogenesis of migraine,which excessive rising of liver-YANG,wind-syndrome resulting from exogenous or endogenous evils,stagnated blood blocking meridian.The prescription is the combination of differential diagnosis of diseases with differential diagnosis of diseases,local characteristic drugs combining with traditional drugs,and make up a prescription-Tianqiong Zhitong Capsule(TQ) to treat migraine.
2.Empirical study
On the basic of searching the newest advancement about migraine, empirical study about Chinese materia medica and complex prescription preventing and cureing migraine,we design the following experiments:
2.1 The basic pharmacodynamic experiments
2.1.1 Experimental study of analgesic effect:We apply hot plate test and writhing test to approach the effects of TQ.The results demonstrate TQ can raise the pain threshold of mouse, prolong the latency and decrease the writhing times of the mouse intraperitoneal injected of acetic acid.It suggest that TQ have analgesic effect
2.1.2 Experimental study of sedative effect:We observe the effect of TQ on the autonomic activities of the normal mouse in 5 min.The results demonstrate TQ have the inhibitive effect on the central nervous system..It profits to improve crankous emotion of the migraineurs.
2.2 Experimental study of mechanism of action
2.2.1 Effects on the content of neurotransmitter in the brain:We apply high- performance liquid chromatography(HPLC) detect the content of 5-HT,DA and NE in the brain of injection NTG animal models of migraine.The results discover that TQ can increase content of 5-HTin the brain,but have no effects on the content of DA,NE in the brain.The results suggest that TQ can inhibit the attack of migraine by promote the effect of 5-HT on the vasoconstriction and the modulation of algesia.
2.2.2 Effects on the vasoactive substance:We apply method of radioimmunity(RI) to detect the content of CGRP,ET in the blood plasma of hypodermic injection NTG animal models of migraine. The results discover that TQ can decrease the content of CGRP,ET in the blood plasma. The results suggest that TQ can inhibit blood vessel dilatation and plasma protein extravasation and inhibit the neurogenic inflammation to prevent and cure migraine.
2.2.3 Effects on algesia thregulatory substance:We apply method of radioimmunity (RI) to detect the content of CGRP,ET andβ-EPin the blood plasma of animal models of migraine stimulated the dural matter near SSS by electrition.The results discover that TQ can decrease the content of CGRP,ET in the blood plasma and increase the content ofβ-EP in the blood plasma. The results suggest that TQ can inhibit the transmition of nociceptive stimulus from primary esthesioneure to CNS.
2.2.4 Effects on CGRPmRNA expression,CRLRmRNA expression and RAMP1mRNA expression:We apply method of reverse transcriptase polymerase chain reaction (RT-PCR) to detect CGRPmRNA expression,CRLRmRNA expression and RAMP1mRNA expression in PAG of animal models of migraine stimulated the dural matter near SSS by electrition.The results discover that TQ can decrease CGRPmRNA expression, CRLRmRNA expression and RAMP1mRNA expression.The results suggest that TQ can inhibit the function of CGRP and the compound made up of its receptor and regulate the function of PAG that can inhibit the transmission of nociceptive stimulus. Based on these above results, we speculate that X can modulate the vasoactive substance to influence the vascular function of dilation and contraction,inhibit neurogenic inflammation which can bring about painful stimulation.It can affect the balance of pain-producing substance and pain-relieving substance, modulate the process of nociceptive stimulus is transmited to central nerve system.
3.Peculiarity of the research
Tianqing Zhitong Capucal is the combination of differential diagnosis of diseases with differential diagnosis of diseases,local characteristic drugs combining with traditional drugs.
The model of electrical stimulation of the superior sagittal sinus is a feasible and advanced animal models of migraine.It has been applied to approach the pathophysiological mechanisms of migraine and the therapeutical effect of the western anti-medicine.It have not been to research the mechanisms of Chinese materia medica.Our study apply the model to discuss the effect of Chinese materia medica first time .
CGRP is involved in the pathogenesis of migraine the,having effects on neurogenic inflammation and transmition of algesia.CGRP has been the focus of reseach the pathogenesis and treatment of migraine abroad,but little reseach have done about it in domestic field.The scientific investigation of the CGRP and migraine is significant.
1.文献研究
查阅从秦汉时期到清代的古代中医学家对偏头痛的文献记载,检索2000-2007年发表在医学期刊上治疗偏头痛的最新临床报道,既系统全面地分析总结历代医家对偏头痛的名称、病因病机、证候分型、治则治法、处方用药的传统特色,又反映治疗偏头痛的时代用药特点,为进一步开展中医药防治偏头痛研究奠定文献基础,为寻找有效的防治偏头痛提供思路与方法。
文献研究分析发现,偏头痛主要是由于风、瘀、痰、虚杂合而致,风邪侵袭、肝阳上扰、瘀血阻滞是基本病机特点。治疗方法常是数法联用,以祛风止痛、平肝熄风、搜风通络法、活血祛瘀为主。疏散外风药、平熄内风药、活血化瘀药等是治疗偏头痛的常用药物。川芎“上行头目,下行血海,行血中之气,祛血中之风,走而不守”成为治疗偏头痛的第一要药。
本课题以上述研究结果为基础,结合导师临床用药经验,针对风邪外袭、肝阳上扰、瘀血阻络所致的常见型偏头痛,以平肝息风、活血通络、祛风止痛为治则,挖掘地方特色药物资源,选取传统药物与专治神经性头痛的汉桃叶相结合,拟订治疗偏头痛的中药复方——天穹止痛胶囊。
2.实验研究
在全面检索国内外最新的有关偏头痛研究进展以及实验性偏头痛动物模型研究概况的基础上,进行以下实验研究内容:
2.1基础药效研究
2.1.1镇痛实验:采用小鼠热板实验和醋酸扭体实验,通过行为学反应观察天穹止痛胶囊的镇痛作用。实验结果显示天穹止痛胶囊能提高小鼠热板实验的痛阈值,延长小鼠腹腔注射醋酸所致扭体反应潜伏期,减少扭体次数,提示天穹止痛胶囊具有一定的镇痛作用。
2.1.2镇静实验:观察天穹止痛胶囊对正常小鼠自主活动的影响,探讨其镇静作用。实验结果显示天穹止痛胶囊能够减少正常小鼠的自主活动数,提示其对中枢神经系统具有一定的抑制作用,有利于改善偏头痛患者发病时的急躁情绪。
2.2作用机制研究
2.2.1对神经递质的影响:采用皮下注射硝酸甘油偏头痛大鼠模型,以高效液相法检测给药后脑组织中5-HT、NE、DA的含量。实验结果显示,天穹止痛胶囊可以升高脑组织中5-HT水平,但对NE、DA无明显影响,提示天穹止痛胶囊可能通过促进5-HT对血管收缩和痛觉调节作用,抑制偏头痛的发作。
2.2.2对血管活性物质的影响:采用皮下注射硝酸甘油偏头痛大鼠模型,以放免法检测给药后血浆中CGRP、ET的含量。实验结果显示,天穹止痛胶囊可以降低血浆中CGRP、ET水平,提示天穹止痛胶囊可以通过抑制CGRP介导的血管扩张及CGRP对ET收缩血管的抑制作用,有利于抑制神经源性炎症,对偏头痛起到防治作用。
2.2.3对痛觉调节物质的影响:采用电刺激上矢状窦(SSS)硬脑膜偏头痛大鼠模型,以放免法检测给药后血浆中CGRP、SP、β-EP的含量。实验结果显示,天穹止痛胶囊可以降低血浆中CGRP、SP含量,升高β-EP含量,提示天穹止痛胶囊可以通过抑制伤害性刺激信息向中枢的传递过程,抑制偏头痛的发作。
2.2.4对CGRP及其受体mRNA表达的影响:采用电刺激上矢状窦(SSS)硬脑膜偏头痛大鼠模型,以RT-PCR方法检测中脑导水管周围灰质(PAG)区CGRP及其受体复合物CRLR/RAMP1 mRNA的表达。实验结果显示,天穹止痛胶囊可以抑制PAG中CGRP mRNA、CRLRm RNA、RAMP1 mRNA的过多表达,并且CGPR表达与RAMP1表达有一定的相关性,提示天穹止痛胶囊不仅可以抑制CGRP mRNA的过度表达,而且可以抑制其受体复合物CRLR/RAMP1 mRNA的过度表达,影响功能性CGRP的表达,发挥对疼痛信息传导的调节作用,抑制三叉神经节传入冲动的发放。
基于上述研究结果,我们推测天穹止痛胶囊防治偏头痛的作用途径可能是:①抑制神经源性炎症反应产生的疼痛刺激。②调节致痛物质与镇痛物质的释放平衡,调节CGRP及其受体复合物mRNA的表达,抑制伤害性信息向中枢的传递过程。
3.课题的创新性天穹止痛胶囊的组方是采用辨证与辨病相结合,传统药物与地方药物相结合而成。处方中的天麻、川芎等是中医辨证治疗偏头痛的传统用药;汉桃叶为广西常用药物,是治疗偏头痛的地方优势药物。这种组方思路有利于挖掘地方优势药材资源,丰富临床用药品种。
电刺激SSS硬脑膜作为一种可行的、先进的偏头痛动物模型,已在偏头痛的实验研究中逐渐推广使用。但是该模型目前在国内外主要是用于探讨偏头痛产生的病理机制以及西药对偏头痛的治疗作用,尚未应用于探讨中药复方治疗偏头痛的作用途径,本课题首次将该模型引入中药复方防治偏头痛的实验研究中,进行了有意义的开创研究,并取得了相应结果。
CGRP作为一种参与偏头痛病理过程的重要因子,在神经源性炎症反应和伤害性信息传递中发挥重要作用。其生物学效应不是通过结合单一受体实现,而是由受体复合物所介导,即CRLR与RAMP1组成的受体复合物与CGRP有高度亲和力,二者共同构建功能性的CGRP受体,目前防治偏头痛的药物尚未涉及相关内容的研究工作。本实验不仅探讨了天芎止痛胶囊对偏头痛动物模型CGRP的基因表达水平,还进一步探讨其受体复合物CRLR/RAMP1的基因表达水平,以及三者的相关性,进行了有意义的探索研究,并取得了相应结果。
Migraine is a common and complex neurological disorder that often attacks recurrently and is difficult to cure completely. An investigation made by WHO reveals that migraine is one of the chronic disease which seriously influence the quality of the patient’s life.It affects a significant proportion of the adult population worldwide.It represents an enormous socio-economic burden to the individual as well as to the socity. Although a great variety western medicine have been prepared,it is short of drugs which have no side effects and have positive prostecdtive efficacy.The effects of Chinese materia medica is mildly and have little side effects.This research aims at applying the theory of TCM combined with the modern scientific medhod to uncover the theory and experience about migraine,discussing the special feature of TCM,providing reference for the clinical usage of Chinese materia medica reasonably,safely and efficiently.
1.Study of literature
We collected considerable literature of migraine from ancient period to present period, and collected clinical reports published in medical magazines in 2000-2007 which treat migraine.Then discussed the special feature about the name,etiopathogenisis,diagnosis and treatment by diferentiation of syndromes of TCM about migraine.
The result of analysis is that wind, stagnated blood,and phlegm and deficiency are the primal etiological factor.Wind blocking the meridian, excessive rising of liver,and stagnated blood blocking meridian is the basic pathogenesis.Expelling wind, calm the liver and stop endogenous wind and promote blood circulation to remove meridian obstruction is the main treating method.
According to the foundamental theory of TCM,combining the clinical experience of my advisor and the result of analysis,we make up a prescription named of TianQing Zhitong Capsule to treat migraine to aim directly at the common pathogenesis of migraine,which excessive rising of liver-YANG,wind-syndrome resulting from exogenous or endogenous evils,stagnated blood blocking meridian.The prescription is the combination of differential diagnosis of diseases with differential diagnosis of diseases,local characteristic drugs combining with traditional drugs,and make up a prescription-Tianqiong Zhitong Capsule(TQ) to treat migraine.
2.Empirical study
On the basic of searching the newest advancement about migraine, empirical study about Chinese materia medica and complex prescription preventing and cureing migraine,we design the following experiments:
2.1 The basic pharmacodynamic experiments
2.1.1 Experimental study of analgesic effect:We apply hot plate test and writhing test to approach the effects of TQ.The results demonstrate TQ can raise the pain threshold of mouse, prolong the latency and decrease the writhing times of the mouse intraperitoneal injected of acetic acid.It suggest that TQ have analgesic effect
2.1.2 Experimental study of sedative effect:We observe the effect of TQ on the autonomic activities of the normal mouse in 5 min.The results demonstrate TQ have the inhibitive effect on the central nervous system..It profits to improve crankous emotion of the migraineurs.
2.2 Experimental study of mechanism of action
2.2.1 Effects on the content of neurotransmitter in the brain:We apply high- performance liquid chromatography(HPLC) detect the content of 5-HT,DA and NE in the brain of injection NTG animal models of migraine.The results discover that TQ can increase content of 5-HTin the brain,but have no effects on the content of DA,NE in the brain.The results suggest that TQ can inhibit the attack of migraine by promote the effect of 5-HT on the vasoconstriction and the modulation of algesia.
2.2.2 Effects on the vasoactive substance:We apply method of radioimmunity(RI) to detect the content of CGRP,ET in the blood plasma of hypodermic injection NTG animal models of migraine. The results discover that TQ can decrease the content of CGRP,ET in the blood plasma. The results suggest that TQ can inhibit blood vessel dilatation and plasma protein extravasation and inhibit the neurogenic inflammation to prevent and cure migraine.
2.2.3 Effects on algesia thregulatory substance:We apply method of radioimmunity (RI) to detect the content of CGRP,ET andβ-EPin the blood plasma of animal models of migraine stimulated the dural matter near SSS by electrition.The results discover that TQ can decrease the content of CGRP,ET in the blood plasma and increase the content ofβ-EP in the blood plasma. The results suggest that TQ can inhibit the transmition of nociceptive stimulus from primary esthesioneure to CNS.
2.2.4 Effects on CGRPmRNA expression,CRLRmRNA expression and RAMP1mRNA expression:We apply method of reverse transcriptase polymerase chain reaction (RT-PCR) to detect CGRPmRNA expression,CRLRmRNA expression and RAMP1mRNA expression in PAG of animal models of migraine stimulated the dural matter near SSS by electrition.The results discover that TQ can decrease CGRPmRNA expression, CRLRmRNA expression and RAMP1mRNA expression.The results suggest that TQ can inhibit the function of CGRP and the compound made up of its receptor and regulate the function of PAG that can inhibit the transmission of nociceptive stimulus. Based on these above results, we speculate that X can modulate the vasoactive substance to influence the vascular function of dilation and contraction,inhibit neurogenic inflammation which can bring about painful stimulation.It can affect the balance of pain-producing substance and pain-relieving substance, modulate the process of nociceptive stimulus is transmited to central nerve system.
3.Peculiarity of the research
Tianqing Zhitong Capucal is the combination of differential diagnosis of diseases with differential diagnosis of diseases,local characteristic drugs combining with traditional drugs.
The model of electrical stimulation of the superior sagittal sinus is a feasible and advanced animal models of migraine.It has been applied to approach the pathophysiological mechanisms of migraine and the therapeutical effect of the western anti-medicine.It have not been to research the mechanisms of Chinese materia medica.Our study apply the model to discuss the effect of Chinese materia medica first time .
CGRP is involved in the pathogenesis of migraine the,having effects on neurogenic inflammation and transmition of algesia.CGRP has been the focus of reseach the pathogenesis and treatment of migraine abroad,but little reseach have done about it in domestic field.The scientific investigation of the CGRP and migraine is significant.
引文
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