颞叶癫痫中医证候研究及柴贝止痫汤对癫痫大鼠GABA_AR、NMDAR1表达的影响

【作者】 李淑芳；

【导师】 刘金民；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2009， 博士

【摘要】 研究背景及目的:癫痫是神经系统常见疾病之一,癫痫患者中75%通过常规的一线抗癫痫药物治疗可获得满意疗效,约25%的癫痫即使科学正规地应用常规抗癫痫药物,发作仍难以控制而成为难治性癫痫(Intractable Epilepsy,IE)。颞叶癫痫(Temporal Lobe Epilepsy,TLE)是常见的难治性癫痫之一,占难治性癫痫的50%以上。所以以颞叶癫痫为切入点,研究新的有效的治疗难治性癫痫的途径和方法有很重要的意义。临床实践中发现,中医药在治疗癫痫尤其是难治性癫痫方面有较好改善症状,控制发作的特色和疗效优势。目前癫痫的中医药临床研究存在标准混乱、证型不统一、证候描述不一致、评价指标不合理、评价方法不科学等问题,因此制定一个新的、符合临床要求的中医癫痫病证诊治标准成为必要,而证候研究是中医癫痫诊治标准化的核心。但是目前关于颞叶癫痫的中医证候研究尚未见到。本课题作为癫痫中医研究的起步工作,初步总结颞叶癫痫的中医证候分布,为癫痫的证候规范化、客观化及中医药治疗癫痫诊疗标准的建立奠定基础。同时通过动物实验探讨中药复方柴贝止痫汤干预癫痫发作的机理,以期为中医药治疗癫痫提供理论依据。方法:临床研究通过文献调研和专家咨询制定颞叶癫痫临床证候调查表,采用横断面调查的研究方法,收集北京中医药大学东方医院及首都医科大学附属北京天坛医院的102例颞叶癫痫患者中医四诊信息等相关资料,并采用因子分析的方法对颞叶癫痫中医证候分布作出初步的归纳总结。实验研究第一部分:建立匹罗卡品癫痫持续状态后颞叶癫痫模型,以中药、西药干预,观察大鼠机体状态、癫痫发作的次数、每次发作持续时间、平均持续时间、癫痫发作级别及进行体重检测,判定、分析药物的疗效。采用HE染色和尼氏染色,光学显微镜下,对颞叶癫痫模型大鼠脑组织海马和颞叶皮层进行病理形态学观察。第二部分:采用免疫组化和Western-blot的方法,检测癫痫大鼠脑组织γ-氨基丁酸A受体α1亚单位(GABA_ARα1)和NMDA受体NR1亚单位(NMDAR1)的表达。观察癫痫模型大鼠脑组织海马和颞叶皮层GABA_ARα1和NMDAR1的表达和分布,探讨中药复方柴贝止痫汤对癫痫大鼠脑组织GABA_ARα1和NMDAR1表达的影响及其治疗癫痫的机理。结果:临床研究运用因子分析的方法得到5个颞叶癫痫的中医临床证候分类:风痰内阻兼郁证、痰火内闭证、心脾两虚证、瘀阻脑络证、肝肾阴虚证。其中风痰内阻兼郁证为颞叶癫痫发作间期的主要证型,其次是痰火内闭证、心脾两虚证、瘀阻脑络证和肝肾阴虚证。性别、病程及发作频率在证候分布上没有显著性差异(P＞0.05),而年龄及发病年龄在证候分布上有显著差异(P＜0.05)。实验研究第一部分实验结果1各致痫组大鼠发作总次数、总持续时间变化与空白模型组比较,柴贝止痫汤联合卡马西平组、柴贝止痫汤组、卡马西平组的发作总次数减少,有显著性差异(P＜0.05);与柴贝止痫汤联合卡马西平组比较,柴贝止痫汤组的发作总次数增加,有显著性差异(P＜0.05)。与空白模型组比较,柴贝止痫汤联合卡马西平组发作总时间减少,有显著性差异(P＜0.05);与柴贝止痫汤联合卡马西平组比较,柴贝止痫汤组、卡马西平组发作总时间增加,均有显著性差异(P＜0.05)。2各致痫组大鼠治疗前后发作平均持续时间的变化:治疗后,与空白模型组比较,柴贝止痫汤联合卡马西平组、柴贝止痫汤组、卡马西平组发作平均持续时间减少,有显著性差异(P＜0.05);与柴贝止痫汤联合卡马西平组比较,柴贝止痫汤组发作平均持续时间增加,有显著性差异(P＜0.05)。与治疗前比较,柴贝止痫汤联合卡马西平组发作平均持续时间减少,有高度显著性差异(P＜0.01);柴贝止痫汤组、卡马西平组发作平均持续时间均减少,有显著性差异(P＜0.05)。3各致痫组大鼠治疗前后癫痫发作级别的变化:治疗后,与空白模型组比较,柴贝止痫汤联合卡马西平组、卡马西平组癫痫发作级别有明显降低,有显著性差异(P＜0.05)。与治疗前比较,柴贝止痫汤联合卡马西平组癫痫发作级别明显降低,有高度显著性差异(P＜0.01);卡马西平组癫痫发作级别降低,有显著性差异(P＜0.05)。4各组大鼠体重变化比较:治疗前,与正常组大鼠比较,空白模型组、柴贝止痫汤联合卡马西平组、柴贝止痫汤组、卡马西平组体重均减轻,有显著性差异(P＜0.05)。治疗后,与正常组大鼠比较,空白模型组、柴贝止痫汤联合卡马西平组、柴贝止痫汤组、卡马西平组体重均减轻,有显著性差异(P＜0.05);与空白模型组比较,柴贝止痫汤联合卡马西平组体重增加,有显著性差异(P＜0.05)。与治疗前比较,各组大鼠体重均有增加,有显著性差异(P＜0.05)。5病理形态学观察结果氯化锂—匹罗卡品颞叶癫痫大鼠模型脑组织海马CA1区、CA2区、CA3区、齿状回和颞叶皮层神经元出现明显损伤,其病变以海马CA1区、CA3区最明显,其次为CA2区、齿状回、颞叶皮层。中药复方柴贝止痫汤、卡马西平能够在一定程度上减轻癫痫大鼠脑组织海马CA1区、CA2区、CA3区、齿状回和颞叶皮层神经元的损伤,提示二者神经元保护作用的存在。第二部分实验结果采用免疫组化和Western-blot的方法检测GABA_ARα1和NMDAR1的表达结果相一致。1 GABA_ARα1的表达与空白模型组比较,柴贝止痫汤组、柴贝止痫汤联合卡马西平组癫痫大鼠海马CA1、CA3区及颞叶皮层的GABA_ARα1亚单位表达均明显增强,有高度显著性差异(P＜0.01),但柴贝止痫汤组与柴贝止痫汤联合卡马西平组之间无显著性差异(P＞0.05)。与正常组比较,空白模型组和卡马西平组癫痫大鼠海马CA1、CA3区及颞叶皮层的GABA_ARα1表达均明显减少,有高度显著性差异(P＜0.01)。2 NMDAR1的表达与正常组比较,柴贝止痫汤组、中柴贝止痫汤联合卡马西平组、卡马西平组、空白模型组海马CA1、CA3区及颞叶皮层的NMDAR1表达均明显增高,有显著性差异(P＜0.05),但柴贝止痫汤组、中柴贝止痫汤联合卡马西平组、卡马西平组、空白模型组各组之间无显著性差异(P＞0.05)。结论:临床研究颞叶癫痫的主要证候包括风痰内阻兼郁证、痰火内闭证、心脾两虚证、瘀阻脑络证和肝肾阴虚证。其中风痰内阻兼郁证为颞叶癫痫发作间期的主要证型,其次是痰火内闭证、心脾两虚证、瘀阻脑络证和肝肾阴虚证。颞叶癫痫的证候与性别、病程和发作频率无明显关系,而与年龄及发病年龄有一定关系,并且青中年以风痰内阻兼郁证及痰火内闭证为著,而中老年人以肝肾阴虚证突出。实验研究氯化锂—匹罗卡品颞叶癫痫模型操作简单、成功率高、稳定性好。中药柴贝止痫汤对癫痫发作有一定的抑制作用,柴贝止痫汤联合卡马西平的治疗方法对于癫痫的行为学改变优于单独应用卡马西平及柴贝止痫汤。柴贝止痫汤可以提高GABA_ARα1的表达,而对NMDAR1无明显影响。对GABA_ARα1表达的调节是柴贝止痫汤控制癫痫的可能机理之一。更多还原

【Abstract】 Background and Objective:Epilepsy is one of common neurological diseases.Now there are about 50 million epileptic all of the world,and 9 million in our country.About 25%of patients are refractory to treatment normally using more than one AEDs,which is called medically intractable epilepsy. It has poor prognosis with increased morbidity and mortality.Temporal lobe epilepsy(TLE) is one of common intractable epilepsy,and account for above 50%.Therefore TLE as starting point,it is important to develope new effective therapeutics for IE.With Traditional Chinese Medicine(TCM) to therapy epilepsy especially intractable epilepsy shows good therapeutic effect in clinical practice.But nowadays TCM syndrome of epilepsy research is seldom,there are some questions in clinical research of epilepsy,such as chaos of standard,pattern of syndrome and syndrome described disunity,the method of curative effect evaluation is irrational.so it’s necessary to institute a new and popular standard of epileptic diagnosis and therapy,and the TCM syndrome research is a center in standardized of epileptic diagnosis and therapy.This study is beginning of the epileptic TCM research,and adoption factor analysis to analyse and sum up the distributing of TLE TCM main syndrome,to establish base for normalization of epileptic syndrome and setting up diagnosis and treatment standard of epilepsy.At the same time,by animal experiment to investigate the molecular mechanisms of the chai bei zhi xian tang anti-epilepsy.methods:clinical researchA prospectively cross-sectional investigation is carried out in this part.the questionnaire of the TLE clinical information was established by investigating the related literature and specialist opinion.The included TLE patients were all from Dongfang Hospital affiliated to Beijing university of Chinese Medicine and Beijing tiantan hospital of the study.The general conditions,four diagnostic information of TCM and relative information were collected. Adoption factor analysis to analyse and Sum up the distributing of TLE TCM main syndrome.experimental studyIn the first section,the model of medically TLE was established by PILO,and it’s praxiology and brain tissue histomorphlogy was observed at 5 groups:the normal control group,the blank model group,the herbs and western medicine group,the herbs groups,the western medicine group.We observe the organism status of rats,the praxiology of rats,such as seizure frequency,time length,average time length,grade of epileptic seizure and body weight to analyses the curative effect.We observed the rats brain tissue histomorphlogy by campeachy-eosin staining and Nissl’s staining.We investigate the change of neuron morphosis and the survivorship state of neuron and Nissl’s body on the hippocampus and the cortex of temporal lobe.In the second section,the expression and distrbution of GABA_ARα1,NMDAR1 protein was observed in hippocampus and cortex of temporal lobe of rats by immunohistochemical method and Western blotting.We investigate the effect on GABA_ARα1 and NMDAR1 by the herbs of complex prescription of thai bei zhi xian tang and carbamazepine intervention,and partly intended to study the molecular mechanisms of the chai bei zhi xian tang anti-epilepsy.Result:clinical researchUsing the factor analysis method,5 TCM syndromes of TLE were summarised:wind phlegm with stagnation of liver-Qi type,phlegm fire type,deficiency in heart and spleen type, stagnation of blood stasis type,deficiency of yin in liver and kidney type.The wind phlegm with stagnation of liver-Qi type is the main TCM syndrome of TLE,and the second one is phlegm fire type.There was no significant difference in the sex,course of disease and seizure frequency over the distribution of the 5 TCM syndromes(P＞0.05).There was a significant difference in the age group and age of onset over the distribution of the 5 TCM syndromes (P＜0.05).experimental studyThe results of first section:1 The change of seizure frequency and time length of rats with TLEThe seizure frequency in the herbs and western medicine group,the herbs group and the western medicine group was significantly decreased compared with that of the blank model group(P＜0.05);Compared with that of the herbs and western medicine group,the seizure frequency in the herbs group was significantly increased(P＜0.05).The seizure time length in the herbs and western medicine group was significantly decreased compared with that of the blank model group(P＜0.05);Compared with that of the herbs and western medicine group,the seizure time length in the herbs group and the western medicine group was significantly increased(P＜0.05).2 The seizure average time length of rats with TLEAfter treatment,the seizure average time length in the herbs and western medicine group, the herbs group and the western medicine group was significantly decreased compared with that of the blank model group(P＜0.05);Compared with that of the herbs and western medicine group,the seizure average time length in the herbs group was significantly decreased(P＜0.05).Compared with that of pretherapy,the seizure average time length in the herbs and western medicine group was very significantly decreased(P＜0.01),and in the herbs group and the western medicine group was significantly decreased(P＜0.05).3 The seizure grade of rats with TLEAfter treatment,the seizure grade in the herbs and western medicine group,the western medicine group was significantly decreased compared with that of the blank model group (P<0.05).Compared with that of pretherapy,the seizure grade in the herbs and western medicine group was very significantly decreased(P<0.01),and in the western medicine group was signifycantly decreased(P<0.05).4 The change of body weight of rats with all groupsAfter treatment,the body weight in the blank model group,the herbs and western medicine group,the herbs group and the western medicine group was significantly decreased compared with that of the normal control group(P<0.05).Before treatment,compared with that of the normal control group,the body weight in the blank model group,the herbs and western medicine group,the herbs group and the western medicine group was significantly decreased(P<0.05);Compared with that of the blank model group,the body weight in the herbs and western medicine group was significantly increased (P<0.05).5 result of histomorphlogyThe neuron showed obviously damage on the district of CA1,CA2,CA3 and dentate band of hippocampus,cortex of temporal lobe in the model of TLE established by PILO.The most severe diseased region was the district of CA1 and CA3 of hippocampus,then is the district of CA2 and dentate band of hippocampus,the cortex of temporal lobe.The herbs of complex prescription of chai bei zhi xian tang and carbamazepine could relieve neuron damage on the district of CA1,CA2,CA3 and dentate band of hippocampus,cortex of temporal lobe in rats with temporal lobe,which hinted the two kinds of medicine could protect neuron.The results of second section:1 the expression of GABA_ARα1The expression of GABA_ARα1 in temporal lobe cortex and CA1,CA3 of hippocampus on rats with temporal lobe epilepsy of the herbs group,the herbs and western medicine group was very significantly increased compared with that of the blank model group(P<0.01).Compared with that of the normal control group,the expression of GABA_ARα1 in the western medicine group and the blank model group was very significantly increased(P<0.01).2 the expression of NMDAR1The expression of NMDAR1 in temporal lobe cortex and CA1,CA3 of hippocampus on rats with TLE of the herbs group,the herbs and western medicine group,the western medicine group and the blank model group was significantly increased compared with that of the normal control group(P<0.05).there was no significant difference among the herbs group,the herbs and western medicine group,the western medicine group and the blank model group (P>0.05).Conclusions:clinical researchWind phlegm with stagnation of liver-Qi type,phlegm fire type,deficiency in heart and spleen type,stagnation of blood stasis type,deficiency of yin in liver and kidney type are main TCM syndrome of TLE.The wind phlegm with stagnation of liver-Qi type is the common one,and the second one is phlegm fire type.TCM syndromes of TLE have no relation with the patient’s sex,course of disease and seizure frequency.TCM syndromes are closely related to the age group and age of onset.The wind phlegm with stagnation of liver-Qi type and phlegm fire type are the main TCM syndromes among the young and middle aged patients,and deficiency of yin in liver and kidney type is common syndrome among middle and old aged patients.experimental studyThe manipulateion of the model of medically TLE established by PILO was simple and it’s achievement ratio and stability was fine.The curative effect of the way of the herbs combined western medicine is greatest than the others.The herbs of complex prescription of chai bei zhi xian tang can increase expression of GABA_ARα1 and cann’t decrease the expression of NMDAR1.The one of mechanism on the herbs of complex prescription of chai bei zhi xian tang healing epilepsy may be it’s increasing expression of GABA_ARα1.更多还原