新型抗癫痫药对MDR的影响及MDR1逆转剂在癫痫治疗中的应用价值研究
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摘要
研究背景
癫痫(epilepsy)是大脑神经元突发性异常放电导致短暂大脑功能障碍的一种慢性脑部疾患。癫痫的患病率约为5‰,在儿童和青少年期发病率较高。其中大约有25%的癫痫患者对多种抗癫痫药物耐药而发展为难治性癫痫。
目前对于难治性癫痫耐药机制的研究主要集中在3个方面:(1)神经病理学研究发现大部分患者存在AHS(Ammon's horn scelrosis,Ammon's角硬化),其主要表现为海马CA1、CA3、CA4区及齿状回颗粒细胞层存在神经元缺失和反应性胶质细胞增生,近年认为这种改变可能与线粒体的功能失调有关,并参与耐药机制的形成;(2)生理和药理学研究发现药物靶点的变化可以引起药物敏感性的改变,一线抗癫痫药物的主要作用位点是海马区神经元的电压门控通道,同时电压门控钠离子通道的改变是多种癫痫的共同致病机制;(3)免疫学和分子遗传学研究发现多药耐药基因和蛋白参与耐药的形成,这是近年来研究的热点。其中以ATP结合的蛋白质超家族(ATP-Binding Cassette,ABC)运输蛋白研究最多。越来越多的学者认为难治性癫痫的耐药机制与多种药物运输蛋白过量表达而导致的抗癫痫药物不能有效进入脑细胞有关,其中MDR(Multidrug resistance gene,多药耐药基因)及其表达产物P-糖蛋白(P-gp)倍受关注。
有研究表明mdr1在癫痫模型脑中的过度表达可能是由反复癫痫发作引起。但癫痫患者,尤其是难治性癫痫患者需要长期服用抗癫痫药物,抗癫痫药物尤其抗癫痫新药是否对多药耐药基因的表达产生影响已引起学者们的关注,但相关的研究仍较少。
近年学者们对多药耐药基因的研究多以成年大鼠急性期癫痫持续状态为模型,对慢性癫痫及幼鼠模型的研究较少,但许多研究表明,在个体发育的不同阶段,癫痫的产生、维持及对抗癫痫药物的反应亦不尽相同。众所周知,儿童癫痫的发生率远远高于成人。因此,研究抗癫痫药对慢性癫痫幼年大鼠模型海马中mdr1表达的影响,更能贴近临床,从而为临床治疗提供新的依据。
如上所述,多药耐药的主要发病机制是由于多药耐药基因及其表达产物P-gp蛋白在脑组织中的过度表达,导致抗癫痫药物不能有效的进入脑细胞,从而使抗癫痫药物在脑内及神经元内不能达到有效的浓度,是引起癫痫治疗失败的重要原因。因此,逆转多药耐药基因的过表达对于促进抗癫痫药物进入脑内、提高癫痫的疗效将会具有很大的临床应用价值。目前对于多药耐药基因逆转剂的研究主要集中在肿瘤领域,在癫痫领域的研究较少。因此,研究逆转剂对难治性癫痫多药耐药基因的逆转作用将会为难治性癫痫的治疗提供更广阔的前景。
目的
1.以海人酸致癫痫持续状态后形成的慢性自发性颞叶癫痫幼年大鼠模型为研究对象,探讨多药耐药基因及其表达产物在大鼠海马中的表达及抗癫痫新药拉莫三嗪对其表达的影响。
2.利用海人酸制备的慢性自发性颞叶癫痫幼年大鼠模型探讨左乙拉西坦对多药耐药基因及其表达产物在大鼠海马中表达的影响,从而指导临床用药。
3.探讨多药耐药基因逆转剂盐酸地尔硫卓对癫痫幼鼠海马中多药耐药基因表达的影响。为难治性癫痫的临床治疗开辟新的途径。
4.探讨难治性癫痫患儿外周血中多药耐药基因的表达及多药耐药基因逆转剂氟桂利嗪在小儿难治性癫痫辅助治疗中的作用。
方法
1.将出生后7d的雄性Wistar大鼠65只随机分为海人酸(kainic acid,KA)组33只和对照组32只,KA组给予KA1mg/kg(浓度0.5mg/ml)腹腔注射,对照组只腹腔注射相同剂量生理盐水。按照Lado幼鼠癫痫发作分级标准,腹腔注射后连续观察8h,癫痫发作达5级以上癫痫持续状态的大鼠若两周后出现自发性反复惊厥发作则为造模成功。
将KA组中造模成功的26只存活大鼠随机分为癫痫未治疗(EP)组13只、癫痫拉莫三嗪治疗(EP+LTG)组13只,对照组随机分为生理盐水(NS)组、生理盐水拉莫三嗪治疗(NS+LTG)组各16只。治疗组均于自发性发作出现一周后给予抗癫痫新药拉莫三嗪治疗8周,然后将所有大鼠断头取海马,用RT-PCR法测定多药耐药基因mdr1a和mdr1b mRNA的表达。
2.选用生后7d(P7)的Wistar大鼠70只,随机分为癫痫观察组38只和正常对照组32只。癫痫观察组给予海人酸KA1mg/kg(0.5ml/kg),腹腔注射致痫,对照组应用相同方法给予相同剂量的生理盐水。待慢性癫痫模型建立后,癫痫观察组随机分为慢性癫痫(EP)观察组13只,慢性癫痫左乙拉西坦治疗(EP+LEV)组15只;对照组分为生理盐水(NS)组和生理盐水左乙拉西坦治疗(NS+LEV)组各16只。治疗组给予左乙拉西坦(80mg/kg)灌胃,治疗8周后,断头取海马,称重,RT-PCR法测定mdr1a和mdr1b mRNA的表达。
3.将出生后7d的雄性Wistar大鼠75只随机分为海人酸组39只和对照组36只,KA组给予KA腹腔注射,对照组腹腔注射相同剂量生理盐水。造模成功后将海人酸组中存活大鼠31只随机分为癫痫未治疗(EP)组10只、癫痫拉莫三嗪治疗(EP+LTG)组11只,癫痫加拉莫三嗪及盐酸地尔硫卓治疗(EP+LTG+D)组10只。对照组随机分为生理盐水(NS)组、生理盐水拉莫三嗪治疗(NS+LTG)及生理盐水拉莫三嗪加盐酸地尔硫卓治疗(NS+LTG+D)组各12只。治疗组均于自发性发作出现1周后给予拉莫三嗪或拉莫三嗪加盐酸地尔硫卓治疗8周,然后将所有大鼠断头取海马,用RT-PCR法测定mdr1a和mdr1b mRNA的表达。
4.应用RT-PCR技术检测MDR1在22例正常健康查体儿童和辅助治疗前后64例难治性癫痫患儿(其中氟桂利嗪组36例,安慰剂组28例)外周血中的表达情况,同时,进行临床疗效和药物不良反应观察。
结果
1.EP组、EP+LTG组的mdr1a和mdr1b mRNA表达均比NS组明显增高(P＜0.001);EP+LTG组及NS+LTG组的mdr1a和mdr1b mRNA表达分别较EP组及NS组增高,但无统计学意义(P＞0.05)。
2.EP组、EP+LEV组的mdr1a和mdr1b mRNA表达明显增高于NS组和NS+LEV,差异有显著统计学意义(P＜0.01);EP组mdr1a和mdr1b mRNA表达高于EP+LEV组,差异有统计学意义(P＜0.05);NS+LEV组与NS组相比,mdr1a和mdr1b mRNA表达有降低的趋势,但无统计学意义(P＞0.05)。
EP组大鼠脑重下降15.3%(EP组0.137±0.018g;NS组0.158±0.015g,P＜0.01);EP+LEV与EP组相比,升高8.1%(EP+LEV组0.149±0.013g,EP组0.137±0.018g,P＜0.05);NS+LEV组较NS下降3.3%(NS+LEV组0.153±0.017g;NS组0.158±0.015g,P＞0.05)。
3.EP+LTG+D组的mdr1a和mdr1b mRNA表达低于EP组及EP+LTG组(P＜0.05),NS+LTG+D组与NS+LTG组及NS组相比无统计学差异(P＞0.05)。
4.难治性癫痫组患儿未进行辅助治疗前MDR1mRNA的表达水平明显高于正常对照组(P＜0.01)。辅助治疗后,氟桂利嗪组MDR1 mRNA的表达低于安慰剂组(P＜0.01),但高于正常对照组(P＜0.05);安慰剂组MDR1 mRNA的表达比正常对照组高1.14倍。与辅助治疗前相比,氟桂利嗪组MDR1 mRNA表达降低(P＜0.01),安慰剂组增高(P＜0.05)。氟桂利嗪组与安慰剂组治疗有效率分别为55.56%和3.57%。氟桂利嗪不良反应发生率为8.33%。
结论
1.反复癫痫发作可使癫痫幼鼠海马中mdr1a、mdr1b mRNA表达增加。
2.拉莫三嗪对癫痫及正常幼鼠海马中mdr1a、mdr1b mRNA的表达无明显影响。
3.左乙拉西坦可使大鼠海马多药耐药基因的表达减少,使癫痫大鼠海马的重量增加,但使正常大鼠海马重量减轻。
4.盐酸地尔硫卓可以逆转mdr1 mRNA的表达。
5.检测外周血MDR1 mRNA的表达可以评估难治性癫痫患儿对抗癫痫药物的敏感程度。
6.氟桂利嗪对MDR1mRNA的表达有逆转作用,用于小儿难治性癫痫辅助治疗时,能减轻癫痫的发作,且小剂量氟桂利嗪副作用小,耐受性好。
Background
Epilepsy is a common chronic disease of nervous system.The prevalence rate of epilepsy patients is about 5‰,The adolescent and children's incidence rate is higher than others.About 25%of patients with epilepsy suffer from intractable seizures,which cannot be properly controlled by antiepileptic drugs.
The current research of mechanisms of drug resistance in refractory epilepsy have focused on three fields:(1) the research of Neuropathology find that most of the patients have Ammon's horn sclerosis,The main features included that the neuron loss and reactive proliferation happened in the CA1、CA3、CA4 region of Hippocampus and the granule cell layer of the dentdate gyrus,recently it is thought that this change maybe associated with mitochondria dysfunction and participated in forming resistance mechanism.(2)Physiology and pharmacology studies found that the change of drug target could be induced the change of drug sensitivity,the important target point of first-line antiepileptic drug is the voltage-gate channel of hippocampus neuron,at the same time the change of voltage-gated sodium channels is the common pathogenic mechanism to many kinds of epilepsy;(3) Immunology and molecular genetics studies found that multidrug resistant gene and protein take part in the form of drug resistant.This is the research hotspot in recent years.The most popular research in this field is about ATP-binding cassette transport protein. More and more researchers believe that the over expression of many drug transport protein cause the antiepileptic drug enter the brain cells ineffectively,which is closely related to the mechanisms of drug resistance in refractory epilepsy. Multidrug resistance gone and its expression product have attracted more attention.
The present study showed the over expression of mdr1 in the brain of epileptic model is caused by recurrent epilepsy.Epileptic patients,especially intractable epilepsy patients need take antiepileptic drugs for a long time,whether the antiepileptic drugs,especially new antiepileptic drugs affect the express of multidrug resistance gone has attracted the attention of scholars,but related research is still less.
Recently,many scholars used the murine model of acute stage status epilepticus to study the multidrug resistant gene,the research of chronic model is less,but many studies find that in the different stage of ontogeny,the production and maintenance of epilepsy,the reaction of antiepileptic drug are both different.Everyone knows that the incidence of epilepsy is higher in children than in adult.So the research of new antiepileptic drug affect mdr1 in childhood spontaneous seizures rat model is more closed to clinical,and provide a new method for clinical treatment.
In a word,the main mechanism of drug resistance is that the over expression of many drug transport protein cause the antiepileptic drug to enter the brain cells ineffectively,thus the antiepileptic drug can not reach effective concentration in the brain and neurons,which is the important reason of poor clinical outcomes. Therefore reversing the multidrug resistance gene overexpression would have a important clinical value in improving the curative effect.At present,the research of MDR modulator mainly focuses in tumor field,less in the epilepsy field.So to study the effect of MDR modulator on multidrug resistance will bring more broad prospects for epilepsy.
Objective
1.Childhood rat model of spontaneous seizures induced by kainic acid was used to explore whether the new antiepileptic drug-lamotrigine(LTG) affects the expression of mdr1a,mdr1b in the hippocampus of spontaneous seizures rat.
2.Childhood rat model of spontaneous seizures induced by kainic acid was used to explore whether the new antiepileptic drug-levetiractam(LEV) affects the expression of mdr1a,mdr1b in the hippocampus of spontaneous seizure rat.
3.Explore whether the MDR modulator Diltiazem affects the expression of mdr1a, mdr1b in the hippocampus of spontaneous seizure childhood rat to provide a new way for clinical treatment of intractable epilepsy.
4.To explore the expression of mdr1a,mdr1b in the epileptic children's peripheral blood and the effect of flunarizine,which used as an add-on treatment for drug-resistent epilepsy in pediatrics.
Methods
1.The number of 65 male young rats of 7 days old were randomly divided into experimental group and control group.Rats in the experimental group were received intraperitoneal injection of 1mg/kg kainic acid to induce seizures,and control rats were injected with the same dose of sodium chloride.According to Lado standard classification of seizures,those young rats whose seizure degree were beyond five and became status epilepticus after intraperitoneal injection were used as successful seizure models if they caught the spontaneous seizures after two weeks.
When spontaneous seizures were developed,the 26 survived epileptic rats were divided into EP group and EP+LTG group.Rats of control group were divided into NS group and NS+LTG group.Rats of EP+LTG group and NS+LTG group were treated with therapeutic dose of lamotrigine after the spontaneous seizures developed for a week.All rats were killed at the 56th day of administration.Mdr1a and mdr1b mRNA in the hippocampus was measured by RT-PCR.
2.Spontaneous recurrent seizures were induced by intraperitoneal injection of 1mg/kg kainic acid at postnatal day 7.Control rats were injected with sodium chloride.
Then all rats were classified as 4 groups after spontaneous seizures developed: spontaneous seizures(EP,n=13) group,spontaneous seizures treated with LEV (EP+LEV,n=15) group,control(n=16) group and control treated with LEV(NS +LEV,n=16) group.The treated rats were given dose of LEV(80mg/kg),dissolving in 0.9%Sodium Chloride for 10mg/mL,twice a day.All rats were killed at the 56th day of intragastric administration and separate the hippocampus to weigh.The expression of mdr1a and mdr1b mRNA in the hippocampus were measured by RT-PCR.
3.The number of 75 male young rats of 7 days old were randomly divided into experimental group and control group.Rats in the experimental group were received intraperitoneal injection of 1 mg/kg kainic acid to induce seizures,and control rats were injected with the same dose of sodium chloride.
When spontaneous seizures were developed,the 31 survived epileptic rats were divided into EP group,EP+LTG group and EP+LTG+D group.Rats of control group were divided into NS group,NS+LTG group and NS+LTG+D group.Treatment group were treated with therapeutic dose of lamotrigine and(or) Diltiazem after the spontaneous seizures developed for a week.All rats were killed at the 56th day of administration.Mdr1a and mdr1b mRNA in the hippocampus was measured by RT-PCR.
4.The expression of mdr in peripheral blood of 86 subjects were also tested by PT-PCR.All subjects were divided into intractable epilepsy group and control group, and the intractable epilepsy was made up of flunarizine treatmet and placebo treatment,the clinical effect and adverse drug reaction were observed at the same time.
Results
1.Compared with the control groups,expression of mdr1a and mdr1b mRNA in the hippocampus increased significantly in EP and EP+LTG group(P＜0.001). Mdr1a and mdr1b mRNA expression level of EP+LTG group was higher than that of EP group,and NS+LTG group higher than NS group,but there was no statistical difference(P＞0.05).
2.Expression of mdr1a and mdr1b mRNA in the hippocampus were increased significantly in the EP+LEV group and EP group compared with control group (P＜0.001).The EP group was increased compared with EP+LEV group(P＜0.05). NS+LEV group have little affect on the expression of mdr1a and mdr1b mRNA in the hippocampus than control group(P＞0.05).
Recurrent seizures causes significant reduction in brain weight:the weight of hippocampus was decreased in mean brain weight of 15.3%(0.137±0.018 vs 0.158±0.015,P＜0.01).LEV led to a increase in weight of brain of 8.1%(0.149±0.013 vs 0.137±0.018) in EP group,but it also resulted in a slight decrease in weight of brain of 3.5%(0.153±0.017 vs 0.158±0.015,P＞0.05) in NS+LEV group.
3.The expression of mdr1a and mdr1b of EP+LTG+D group was lower than EP group and EP+LTG group(P＜0.05).The mdr1a and mdr1b mRNA expression level of NS+LTG+D group,NS group and NS+LTG+D group had no statistical difference.
4.The expression level of MDR1mRNA was elevated in the intractable epilepsy is higher than control group(P＜0.01).After treatment with flunarizine,The expression of MDR1 mRNA was decreased in flunarizine group,and increased in placebo group,compared with control group.The expression of MDR1 mRNA in placebo group is 1.14 times than control group,the effective rate of treatment of lunarizine group and placebo group is respectively 55.56%and 3.57%,the incidence of adverse effect is 8.33%.
Conclusion
1.Frequent seizures results in over expression of mdr1a,mdr1b mRNA in the hippocampus.
2.Lamotrigine dose not enhance the expression of mdr1a,mdr1b mRNA in the hippocampus.
3.The expression of mdr1a and mdr1b mRNA in the hippocampus descreased after treatment with levetiractam,so levetiractam could probably lower the level of mdr mRNA.It can promote the development of hippocampus in epileptic rats,but slightly reduces the weight of hippocampus in normal rats.
4.Diltiazem can decrease the expression of mdr1 mRNA.
5.The expression of MDR1 mRNA in peripheral blood is parallel to that in brain,so it can be regarded as an index to evaluate the expression of MDR1 mRNA.
6.Flunarizine is effective,as an add-on therapy in intractable epilepsy with MDR1.It's mechanism to treat intractable epilepsy may involve in reversing the expression of MDR1 mRNA.At the same time,we find the side effects of flunarizine in low dose are very small,and it can be used as add-on therapy in children intractable epilepsy.
癫痫(epilepsy)是大脑神经元突发性异常放电导致短暂大脑功能障碍的一种慢性脑部疾患。癫痫的患病率约为5‰,在儿童和青少年期发病率较高。其中大约有25%的癫痫患者对多种抗癫痫药物耐药而发展为难治性癫痫。
目前对于难治性癫痫耐药机制的研究主要集中在3个方面:(1)神经病理学研究发现大部分患者存在AHS(Ammon's horn scelrosis,Ammon's角硬化),其主要表现为海马CA1、CA3、CA4区及齿状回颗粒细胞层存在神经元缺失和反应性胶质细胞增生,近年认为这种改变可能与线粒体的功能失调有关,并参与耐药机制的形成;(2)生理和药理学研究发现药物靶点的变化可以引起药物敏感性的改变,一线抗癫痫药物的主要作用位点是海马区神经元的电压门控通道,同时电压门控钠离子通道的改变是多种癫痫的共同致病机制;(3)免疫学和分子遗传学研究发现多药耐药基因和蛋白参与耐药的形成,这是近年来研究的热点。其中以ATP结合的蛋白质超家族(ATP-Binding Cassette,ABC)运输蛋白研究最多。越来越多的学者认为难治性癫痫的耐药机制与多种药物运输蛋白过量表达而导致的抗癫痫药物不能有效进入脑细胞有关,其中MDR(Multidrug resistance gene,多药耐药基因)及其表达产物P-糖蛋白(P-gp)倍受关注。
有研究表明mdr1在癫痫模型脑中的过度表达可能是由反复癫痫发作引起。但癫痫患者,尤其是难治性癫痫患者需要长期服用抗癫痫药物,抗癫痫药物尤其抗癫痫新药是否对多药耐药基因的表达产生影响已引起学者们的关注,但相关的研究仍较少。
近年学者们对多药耐药基因的研究多以成年大鼠急性期癫痫持续状态为模型,对慢性癫痫及幼鼠模型的研究较少,但许多研究表明,在个体发育的不同阶段,癫痫的产生、维持及对抗癫痫药物的反应亦不尽相同。众所周知,儿童癫痫的发生率远远高于成人。因此,研究抗癫痫药对慢性癫痫幼年大鼠模型海马中mdr1表达的影响,更能贴近临床,从而为临床治疗提供新的依据。
如上所述,多药耐药的主要发病机制是由于多药耐药基因及其表达产物P-gp蛋白在脑组织中的过度表达,导致抗癫痫药物不能有效的进入脑细胞,从而使抗癫痫药物在脑内及神经元内不能达到有效的浓度,是引起癫痫治疗失败的重要原因。因此,逆转多药耐药基因的过表达对于促进抗癫痫药物进入脑内、提高癫痫的疗效将会具有很大的临床应用价值。目前对于多药耐药基因逆转剂的研究主要集中在肿瘤领域,在癫痫领域的研究较少。因此,研究逆转剂对难治性癫痫多药耐药基因的逆转作用将会为难治性癫痫的治疗提供更广阔的前景。
目的
1.以海人酸致癫痫持续状态后形成的慢性自发性颞叶癫痫幼年大鼠模型为研究对象,探讨多药耐药基因及其表达产物在大鼠海马中的表达及抗癫痫新药拉莫三嗪对其表达的影响。
2.利用海人酸制备的慢性自发性颞叶癫痫幼年大鼠模型探讨左乙拉西坦对多药耐药基因及其表达产物在大鼠海马中表达的影响,从而指导临床用药。
3.探讨多药耐药基因逆转剂盐酸地尔硫卓对癫痫幼鼠海马中多药耐药基因表达的影响。为难治性癫痫的临床治疗开辟新的途径。
4.探讨难治性癫痫患儿外周血中多药耐药基因的表达及多药耐药基因逆转剂氟桂利嗪在小儿难治性癫痫辅助治疗中的作用。
方法
1.将出生后7d的雄性Wistar大鼠65只随机分为海人酸(kainic acid,KA)组33只和对照组32只,KA组给予KA1mg/kg(浓度0.5mg/ml)腹腔注射,对照组只腹腔注射相同剂量生理盐水。按照Lado幼鼠癫痫发作分级标准,腹腔注射后连续观察8h,癫痫发作达5级以上癫痫持续状态的大鼠若两周后出现自发性反复惊厥发作则为造模成功。
将KA组中造模成功的26只存活大鼠随机分为癫痫未治疗(EP)组13只、癫痫拉莫三嗪治疗(EP+LTG)组13只,对照组随机分为生理盐水(NS)组、生理盐水拉莫三嗪治疗(NS+LTG)组各16只。治疗组均于自发性发作出现一周后给予抗癫痫新药拉莫三嗪治疗8周,然后将所有大鼠断头取海马,用RT-PCR法测定多药耐药基因mdr1a和mdr1b mRNA的表达。
2.选用生后7d(P7)的Wistar大鼠70只,随机分为癫痫观察组38只和正常对照组32只。癫痫观察组给予海人酸KA1mg/kg(0.5ml/kg),腹腔注射致痫,对照组应用相同方法给予相同剂量的生理盐水。待慢性癫痫模型建立后,癫痫观察组随机分为慢性癫痫(EP)观察组13只,慢性癫痫左乙拉西坦治疗(EP+LEV)组15只;对照组分为生理盐水(NS)组和生理盐水左乙拉西坦治疗(NS+LEV)组各16只。治疗组给予左乙拉西坦(80mg/kg)灌胃,治疗8周后,断头取海马,称重,RT-PCR法测定mdr1a和mdr1b mRNA的表达。
3.将出生后7d的雄性Wistar大鼠75只随机分为海人酸组39只和对照组36只,KA组给予KA腹腔注射,对照组腹腔注射相同剂量生理盐水。造模成功后将海人酸组中存活大鼠31只随机分为癫痫未治疗(EP)组10只、癫痫拉莫三嗪治疗(EP+LTG)组11只,癫痫加拉莫三嗪及盐酸地尔硫卓治疗(EP+LTG+D)组10只。对照组随机分为生理盐水(NS)组、生理盐水拉莫三嗪治疗(NS+LTG)及生理盐水拉莫三嗪加盐酸地尔硫卓治疗(NS+LTG+D)组各12只。治疗组均于自发性发作出现1周后给予拉莫三嗪或拉莫三嗪加盐酸地尔硫卓治疗8周,然后将所有大鼠断头取海马,用RT-PCR法测定mdr1a和mdr1b mRNA的表达。
4.应用RT-PCR技术检测MDR1在22例正常健康查体儿童和辅助治疗前后64例难治性癫痫患儿(其中氟桂利嗪组36例,安慰剂组28例)外周血中的表达情况,同时,进行临床疗效和药物不良反应观察。
结果
1.EP组、EP+LTG组的mdr1a和mdr1b mRNA表达均比NS组明显增高(P＜0.001);EP+LTG组及NS+LTG组的mdr1a和mdr1b mRNA表达分别较EP组及NS组增高,但无统计学意义(P＞0.05)。
2.EP组、EP+LEV组的mdr1a和mdr1b mRNA表达明显增高于NS组和NS+LEV,差异有显著统计学意义(P＜0.01);EP组mdr1a和mdr1b mRNA表达高于EP+LEV组,差异有统计学意义(P＜0.05);NS+LEV组与NS组相比,mdr1a和mdr1b mRNA表达有降低的趋势,但无统计学意义(P＞0.05)。
EP组大鼠脑重下降15.3%(EP组0.137±0.018g;NS组0.158±0.015g,P＜0.01);EP+LEV与EP组相比,升高8.1%(EP+LEV组0.149±0.013g,EP组0.137±0.018g,P＜0.05);NS+LEV组较NS下降3.3%(NS+LEV组0.153±0.017g;NS组0.158±0.015g,P＞0.05)。
3.EP+LTG+D组的mdr1a和mdr1b mRNA表达低于EP组及EP+LTG组(P＜0.05),NS+LTG+D组与NS+LTG组及NS组相比无统计学差异(P＞0.05)。
4.难治性癫痫组患儿未进行辅助治疗前MDR1mRNA的表达水平明显高于正常对照组(P＜0.01)。辅助治疗后,氟桂利嗪组MDR1 mRNA的表达低于安慰剂组(P＜0.01),但高于正常对照组(P＜0.05);安慰剂组MDR1 mRNA的表达比正常对照组高1.14倍。与辅助治疗前相比,氟桂利嗪组MDR1 mRNA表达降低(P＜0.01),安慰剂组增高(P＜0.05)。氟桂利嗪组与安慰剂组治疗有效率分别为55.56%和3.57%。氟桂利嗪不良反应发生率为8.33%。
结论
1.反复癫痫发作可使癫痫幼鼠海马中mdr1a、mdr1b mRNA表达增加。
2.拉莫三嗪对癫痫及正常幼鼠海马中mdr1a、mdr1b mRNA的表达无明显影响。
3.左乙拉西坦可使大鼠海马多药耐药基因的表达减少,使癫痫大鼠海马的重量增加,但使正常大鼠海马重量减轻。
4.盐酸地尔硫卓可以逆转mdr1 mRNA的表达。
5.检测外周血MDR1 mRNA的表达可以评估难治性癫痫患儿对抗癫痫药物的敏感程度。
6.氟桂利嗪对MDR1mRNA的表达有逆转作用,用于小儿难治性癫痫辅助治疗时,能减轻癫痫的发作,且小剂量氟桂利嗪副作用小,耐受性好。
Background
Epilepsy is a common chronic disease of nervous system.The prevalence rate of epilepsy patients is about 5‰,The adolescent and children's incidence rate is higher than others.About 25%of patients with epilepsy suffer from intractable seizures,which cannot be properly controlled by antiepileptic drugs.
The current research of mechanisms of drug resistance in refractory epilepsy have focused on three fields:(1) the research of Neuropathology find that most of the patients have Ammon's horn sclerosis,The main features included that the neuron loss and reactive proliferation happened in the CA1、CA3、CA4 region of Hippocampus and the granule cell layer of the dentdate gyrus,recently it is thought that this change maybe associated with mitochondria dysfunction and participated in forming resistance mechanism.(2)Physiology and pharmacology studies found that the change of drug target could be induced the change of drug sensitivity,the important target point of first-line antiepileptic drug is the voltage-gate channel of hippocampus neuron,at the same time the change of voltage-gated sodium channels is the common pathogenic mechanism to many kinds of epilepsy;(3) Immunology and molecular genetics studies found that multidrug resistant gene and protein take part in the form of drug resistant.This is the research hotspot in recent years.The most popular research in this field is about ATP-binding cassette transport protein. More and more researchers believe that the over expression of many drug transport protein cause the antiepileptic drug enter the brain cells ineffectively,which is closely related to the mechanisms of drug resistance in refractory epilepsy. Multidrug resistance gone and its expression product have attracted more attention.
The present study showed the over expression of mdr1 in the brain of epileptic model is caused by recurrent epilepsy.Epileptic patients,especially intractable epilepsy patients need take antiepileptic drugs for a long time,whether the antiepileptic drugs,especially new antiepileptic drugs affect the express of multidrug resistance gone has attracted the attention of scholars,but related research is still less.
Recently,many scholars used the murine model of acute stage status epilepticus to study the multidrug resistant gene,the research of chronic model is less,but many studies find that in the different stage of ontogeny,the production and maintenance of epilepsy,the reaction of antiepileptic drug are both different.Everyone knows that the incidence of epilepsy is higher in children than in adult.So the research of new antiepileptic drug affect mdr1 in childhood spontaneous seizures rat model is more closed to clinical,and provide a new method for clinical treatment.
In a word,the main mechanism of drug resistance is that the over expression of many drug transport protein cause the antiepileptic drug to enter the brain cells ineffectively,thus the antiepileptic drug can not reach effective concentration in the brain and neurons,which is the important reason of poor clinical outcomes. Therefore reversing the multidrug resistance gene overexpression would have a important clinical value in improving the curative effect.At present,the research of MDR modulator mainly focuses in tumor field,less in the epilepsy field.So to study the effect of MDR modulator on multidrug resistance will bring more broad prospects for epilepsy.
Objective
1.Childhood rat model of spontaneous seizures induced by kainic acid was used to explore whether the new antiepileptic drug-lamotrigine(LTG) affects the expression of mdr1a,mdr1b in the hippocampus of spontaneous seizures rat.
2.Childhood rat model of spontaneous seizures induced by kainic acid was used to explore whether the new antiepileptic drug-levetiractam(LEV) affects the expression of mdr1a,mdr1b in the hippocampus of spontaneous seizure rat.
3.Explore whether the MDR modulator Diltiazem affects the expression of mdr1a, mdr1b in the hippocampus of spontaneous seizure childhood rat to provide a new way for clinical treatment of intractable epilepsy.
4.To explore the expression of mdr1a,mdr1b in the epileptic children's peripheral blood and the effect of flunarizine,which used as an add-on treatment for drug-resistent epilepsy in pediatrics.
Methods
1.The number of 65 male young rats of 7 days old were randomly divided into experimental group and control group.Rats in the experimental group were received intraperitoneal injection of 1mg/kg kainic acid to induce seizures,and control rats were injected with the same dose of sodium chloride.According to Lado standard classification of seizures,those young rats whose seizure degree were beyond five and became status epilepticus after intraperitoneal injection were used as successful seizure models if they caught the spontaneous seizures after two weeks.
When spontaneous seizures were developed,the 26 survived epileptic rats were divided into EP group and EP+LTG group.Rats of control group were divided into NS group and NS+LTG group.Rats of EP+LTG group and NS+LTG group were treated with therapeutic dose of lamotrigine after the spontaneous seizures developed for a week.All rats were killed at the 56th day of administration.Mdr1a and mdr1b mRNA in the hippocampus was measured by RT-PCR.
2.Spontaneous recurrent seizures were induced by intraperitoneal injection of 1mg/kg kainic acid at postnatal day 7.Control rats were injected with sodium chloride.
Then all rats were classified as 4 groups after spontaneous seizures developed: spontaneous seizures(EP,n=13) group,spontaneous seizures treated with LEV (EP+LEV,n=15) group,control(n=16) group and control treated with LEV(NS +LEV,n=16) group.The treated rats were given dose of LEV(80mg/kg),dissolving in 0.9%Sodium Chloride for 10mg/mL,twice a day.All rats were killed at the 56th day of intragastric administration and separate the hippocampus to weigh.The expression of mdr1a and mdr1b mRNA in the hippocampus were measured by RT-PCR.
3.The number of 75 male young rats of 7 days old were randomly divided into experimental group and control group.Rats in the experimental group were received intraperitoneal injection of 1 mg/kg kainic acid to induce seizures,and control rats were injected with the same dose of sodium chloride.
When spontaneous seizures were developed,the 31 survived epileptic rats were divided into EP group,EP+LTG group and EP+LTG+D group.Rats of control group were divided into NS group,NS+LTG group and NS+LTG+D group.Treatment group were treated with therapeutic dose of lamotrigine and(or) Diltiazem after the spontaneous seizures developed for a week.All rats were killed at the 56th day of administration.Mdr1a and mdr1b mRNA in the hippocampus was measured by RT-PCR.
4.The expression of mdr in peripheral blood of 86 subjects were also tested by PT-PCR.All subjects were divided into intractable epilepsy group and control group, and the intractable epilepsy was made up of flunarizine treatmet and placebo treatment,the clinical effect and adverse drug reaction were observed at the same time.
Results
1.Compared with the control groups,expression of mdr1a and mdr1b mRNA in the hippocampus increased significantly in EP and EP+LTG group(P＜0.001). Mdr1a and mdr1b mRNA expression level of EP+LTG group was higher than that of EP group,and NS+LTG group higher than NS group,but there was no statistical difference(P＞0.05).
2.Expression of mdr1a and mdr1b mRNA in the hippocampus were increased significantly in the EP+LEV group and EP group compared with control group (P＜0.001).The EP group was increased compared with EP+LEV group(P＜0.05). NS+LEV group have little affect on the expression of mdr1a and mdr1b mRNA in the hippocampus than control group(P＞0.05).
Recurrent seizures causes significant reduction in brain weight:the weight of hippocampus was decreased in mean brain weight of 15.3%(0.137±0.018 vs 0.158±0.015,P＜0.01).LEV led to a increase in weight of brain of 8.1%(0.149±0.013 vs 0.137±0.018) in EP group,but it also resulted in a slight decrease in weight of brain of 3.5%(0.153±0.017 vs 0.158±0.015,P＞0.05) in NS+LEV group.
3.The expression of mdr1a and mdr1b of EP+LTG+D group was lower than EP group and EP+LTG group(P＜0.05).The mdr1a and mdr1b mRNA expression level of NS+LTG+D group,NS group and NS+LTG+D group had no statistical difference.
4.The expression level of MDR1mRNA was elevated in the intractable epilepsy is higher than control group(P＜0.01).After treatment with flunarizine,The expression of MDR1 mRNA was decreased in flunarizine group,and increased in placebo group,compared with control group.The expression of MDR1 mRNA in placebo group is 1.14 times than control group,the effective rate of treatment of lunarizine group and placebo group is respectively 55.56%and 3.57%,the incidence of adverse effect is 8.33%.
Conclusion
1.Frequent seizures results in over expression of mdr1a,mdr1b mRNA in the hippocampus.
2.Lamotrigine dose not enhance the expression of mdr1a,mdr1b mRNA in the hippocampus.
3.The expression of mdr1a and mdr1b mRNA in the hippocampus descreased after treatment with levetiractam,so levetiractam could probably lower the level of mdr mRNA.It can promote the development of hippocampus in epileptic rats,but slightly reduces the weight of hippocampus in normal rats.
4.Diltiazem can decrease the expression of mdr1 mRNA.
5.The expression of MDR1 mRNA in peripheral blood is parallel to that in brain,so it can be regarded as an index to evaluate the expression of MDR1 mRNA.
6.Flunarizine is effective,as an add-on therapy in intractable epilepsy with MDR1.It's mechanism to treat intractable epilepsy may involve in reversing the expression of MDR1 mRNA.At the same time,we find the side effects of flunarizine in low dose are very small,and it can be used as add-on therapy in children intractable epilepsy.
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