Study On The Influence Of New Antiepileptic Drugs On Multidrug Resistance Gene(MDR) Expression And The Therapy Value Of Reverse Drugs Of MDR1 In Children Intractable Epilepsy

BackgroundEpilepsy 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.Objective1.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.Methods1.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.Results1.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%.Conclusion1.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.