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柴贝止痫汤影响难治性癫痫耐药蛋白表达及海马GLU、γ-GABA含量的研究
【作者】 鄢泽然；
【导师】 刘金民；
【作者基本信息】 北京中医药大学 ， 中医内科学， 2014， 博士
【摘要】 难治性癫痫患者的主要特征是对绝大多数抗癫痫药物(AEDs)耐药,即使这些抗癫痫药的作用途径不同。近年来许多研究认为多药耐药基因及其编码蛋白的高表达可能是耐药机制之一。P糖蛋白、乳腺癌耐药蛋白(BCRP)、主穹窿蛋白(MVP)等耐药蛋白在难治性癫痫患者及动物模型脑部的高表达限制了病灶内AEDs的浓度。因此,抑制多药耐药蛋白的高表达来提高AEDs在病变脑组织的浓度已成为IE领域研究的热点。前期临床观察发现柴贝止痫汤联合抗癫痫药物治疗难治性癫痫可以减少患者的发作次数,减轻发作程度,而且联合使用抗癫痫药物的治疗效果优于单用柴贝止痫汤,说明柴贝止痫汤可能增敏了抗癫痫药物的疗效。根据中药多靶点效应特点,柴贝止痫汤可能通过抑制脑内多种耐药蛋白的高表达来增加脑内抗癫痫药物的浓度,抑或通过调节脑内病灶部位兴奋性神经递质和抑制性神经递质的平衡来协同抗癫痫药物提高疗效。[目的]观察柴贝止痫汤对血脑屏障耐药蛋白P-gp、BCRP、MVP表达的影响；观察柴贝止痫汤对难治性癫痫大鼠海马CA3区细胞外液GLU、γ-GABA含量的影响。[方法]本研究离体实验采用大鼠脑微血管内皮细胞,用谷氨酸和地塞米松孵育的方法,分别引起P-gp和BCRP表达的升高,经中药柴贝止痫颗粒水溶液干预后观察细胞P-gp和BCRP表达的变化；整体实验采用侧脑室注射海人酸的方法建立癫痫持续状态后慢性难治性癫痫大鼠模型,分设模型组、西药组、中药组、中西药组、正常组和假手术组,给药3月后观察各组大鼠脑内BCRP及MVP表达的情况并比较组间差异；同时采用微透析结合HPLC技术,检测干预后各组大鼠海马CA3区细胞外液GLU、γ-GABA的水平,比较组间差异。[结果](1)大鼠脑微血管内皮细胞经2mM的L-谷氨酸作用3h后,P-gp及MDR1mRNA的表达较正常组升高,柴贝止痫颗粒水溶液100μg/mL、500μg/mL干预细胞后P-gp的表达较模型组降低(P<0.05),MDRlamRNA的表达较模型组降低(P<0.05)：500μg/mL干预组MDRlb mRNA的表达较模型组降低(P<0.05)；(2)细胞经100nM的地塞米松作用24h后,BCRP的表达较正常组升高,经柴贝止痫颗粒水溶液10μg/ml.100μg/ml干预后BCRP蛋白及mRNA的表达较模型组降低(P<0.05)；(3)海人酸致痫大鼠脑内BCRP的表达升高,经柴贝止痫汤及卡马西平干预后,中药组及中西药组BCRP的表达较模型组降低(P<0.05)；(4)中药组、中西药组GLU/y-GABA的值较模型组降低(P<0.05)。[结论]柴贝止痫汤通过下调海人酸致痫大鼠脑内BCRP的表达,降低海马CA3区细胞外液中GLU/y-GABA的值,增敏抗癫痫药物,提高协同治疗难治性癫痫的疗效。
【Abstract】 The most important feature of intractable epilepsy (IE) is resistant to many kinds of antiepileptic drugs. Some studies suggested that multidrug resistance (MDR) genes and their coding proteins excessive expression should be one of the mechanisms of drug resistance. The up-regulation of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in IE patients and animals limit the AEDs concentration in epileptic focus. Therefore, the inhibitions of multidrug resistance proteins expression increasing the concentration of AEDs in the lesions brain tissue are becoming more and more popular in clinical domain. Chaibei Zhixian Decoction improves the efficacy of anti-epileptic drugs and the integrative treatment can reduce seizure frequencies and severity. So we conclude that Chaibei Zhixian Decoction down-regulate the levels of multidrug resistance protein at the blood-brain barrier and increase the concentration of antiepileptic drugs in intractable patients’brains. On the other hand, Chaibei Zhixian Decoction may regulate the balance between excitatory neurotransmitters and inhibitory neurotransmitters. Therefore, Chaibei Zhixian Decoction enhance the antiepileptic effects of antiepileptic drugs.Objective:In order to observe the antiepileptic effect of Chaibei Zhixian Decoction and investigate the mechanisms, this study will detect the protein and gene levels of BCRP and MVP after administrating with Chaibei Zhixian Decoction. Then, GLU concentration, y-GABA concentration, and the GLU/y-GABA ratio at rat hippocampal CA3region are our aims.Methods:Primary cultures of brain microvascular endothelial cells are prepared from rats, then the3rd passage cells are exposed to L-glutamate and dexamethasone to up-regulate the expression of P-gp/MDRl and BCRP respectively, which are up-regulation as multi-drug resistant epilepsy. After treatments, the solutions were replaced with a concentration (10μg/mL,100μg/mL,500μg/mL and1mg/mL) of Chaibei Zhixian Decoction for24h. Western blot is used for the measurement of P-gp and BCRP expression and real-time PCR is used for the determination of Mdrl mRNA and BCRP mRNA amplifications.This study rebuilt chronic intractable epileptic animal models with the method of injecting kainic acid in rat intracerebroventricular. Rats are divided into6groups randomly:normal group, sham group, model group, vehicle group, Chinese medicine group and integrative group. After3months treatment, Western blot are used to assess the expression of P-gp and BCRP in brain cortex and Realtime PCR are used to detect the amplifications of MDR1mRNA and BCRP mRNA.Microdialysis and HPLC are used to measure the concentration of GLU and γ-GABA. Results:(1) The P-gp expression of a concentration (100μg/mL、500μg/mL) of Chaibei Zhixian Decoction group is significant lower than that of model cell group(P<0.05); the Mdrla mRNA level of a concentration (100μg/mL,500μg/mL) of Chaibei Zhixian Decoction group is lower than model group(.P<0.05); the Mdrlb mRNA amplification of a concentration (500μg/mL) of Chaibei Zhixian Decoction group is lower than model group(P <0.05).(2) Exposure to Chaibei Zhixian Decoction(10μg/ml,100μg/ml)resulted in significant decrease in protein and mRNA levels of BCRP (P<0.05); While BCRP was down-regulated, NF-κBp65expression was decreased by Chaibei Zhixian Decoction(1mg/ml)(P<0.05).(3) Injecting kainic acid in rat intracerebroventricular can up-regulate the levels of BCRP and BCRP mRNA, MVP and MVP mRNA. Treatment with Chaibei Zhixian Decoction can decrease the expression of BCRP and BCRP mRNA (P<0.05). Chinese medicine group and integrative group can decrease the level of GLU/y-GABA ratio in CA3hippocampus (P <0.05).Conclusions:Chaibei Zhixian Decoction down-regulate the expression of BCRP at the blood-brain barrier and decrease the hippocampal CA3extracellular GLU/y-GABA ratio. These are the possible mechanisms of collaborative treatment.