定痫丸对难治性癫痫大鼠抗癫痫作用及机制

目的:探讨定痫丸对难治性癫痫大鼠的抗痫作用及可能的分子生物学机制。方法:将健康雄性Wistar大鼠90只随机分成2组,分为正常组10只,造模组80只,然后将造模成功的64只大鼠,随机分成模型组、定痫丸组、卡马西平组、中西药联用组,每组各16只。定痫丸组灌服定痫丸混悬剂1. 5 g·kg~(-1)·d~(-1),卡马西平组灌服卡马西平溶液30 mg·kg~(-1)·d~(-1),中西药联用组同时给予定痫丸混悬剂和卡马西平溶液,正常组及模型组灌服蒸馏水15 m L·kg~(-1)·d~(-1),连续给药1月。通过行为学及脑电图指标评价定痫丸的抗痫作用,通过高效液相色谱评价定痫丸对卡马西平脑内药物浓度的影响,通过免疫组化、蛋白质免疫印迹法(Western blot)及实时荧光定量聚合酶链式反应(Real-time PCR)方法观察大鼠脑内多药耐药基因1(multidrug resistance gene 1,MDR1)及P-糖蛋白(P-glycoprotein,P-gp)蛋白及mRNA表达情况及定痫丸对其干预作用。结果:正常组大鼠无癫痫发作,脑内仅有少量MDR1,P-gp表达。与正常组比较,模型组大鼠癫痫发作级别升高(P 0. 05),持续时间延长(P 0. 05),脑皮质及海马MDR1,P-gp表达增加(P 0. 05);与模型组比较,各实验组大鼠癫痫发作级别及持续时间降低(P 0. 05),大鼠脑内MDR1,P-gp表达减少(P 0. 05);与定痫丸组比较,中西药联用组大鼠癫痫发作级别更低(P 0. 05),持续时间更短(P 0. 05),MDR1,P-gp表达减少更加明显(P 0. 05);与卡马西平组比较,定痫丸组及中西药联用组大鼠脑内MDR1,P-gp表达减少(P 0. 05),中西药联用组大鼠癫痫发作级别更低(P 0. 05),持续时间更短(P 0. 05),且大鼠脑内卡马西平药物浓度升高(P 0. 05)。结论:定痫丸对难治性癫痫具有一定的抗痫作用,且能逆转卡马西平的耐药,二者具有协同增效作用。其逆转耐药作用可能与阻断脑内MDR1,P-gp表达有关。

Antiepileptic Effect of Dingxianwan on Antiepileptic and Its Mechanism in Rats with Refractory Epilepsy

Objective: To explore the antiepileptic effect of Dingxianwan in rats with refractory epilepsy and investigate its possible molecular mechanism. Method: The 90 healthy male Wistar rats were randomly divided into two groups:normal control group (n=10) and refractory epilepsy model group (n=80). Then, 64 successful modeling rats were randomly divided into model group, Dingxianwan group, carbamazepine group and integrated Chinese and western medicine group, with 16 rats in each group. The rats in Dingxianwan group were ig administered with Dingxianwan suspension 1.5 g·kg^-1·d^-1; rats in carbamazepine group were ig administered with carbamazepine solution 30 mg·kg^-1·d^-1; rats in integrated Chinese and western medicine group were ig administered with Dingxianwan suspension 1.5 g·kg^-1·d^-1 +carbamazepine solution 30 mg·kg^-1·d^-1,while rats in normal control group and model group were ig administered with normal saline 15 mL·kg^-1·d^-1. Administration lasted for one month. Then the antiepileptic effects of Dingxianwan were evaluated by behavior and electroencephalogram (EEG). The effects of Dingxianwan on carbamazepine concentration in rat brains were evaluated by high performance liquid chromatography (HPLC) method. The multi-drug resistance gene 1 (MDR1) and P-glycoprotein (P-gp) protein and mRNA expression levels in rat brains were detected by immunohistochemistry, Western blot and real time polymerase chain reation (Real-time PCR) techniques. Result: In normal control group, there was no epileptic attack and only a small amount of MDR1,P-gp expressed in rats brain. As compared with normal control group, the epileptic seizure grade was higher (P<0.05); the duration was longer (P<0.05) and MDR1,P-gp expression levels were higher (P<0.05) in the cortex and hippocampus of model group rats. As compared with model group, the grade and duration of epileptic seizures in experimental groups were lower (P<0.05), and the expression levels of MDR1,P-gp in brains were significantly lower (P<0.05). As compared with Dingxianwan group, the seizure grade was lower (P<0.05); the duration was shorter (P<0.05) and the expression levels of MDR1,P-gp were lower in the integrated Chinese and western medicine group (P<0.05). As compared with carbamazepine group, the expression levels of MDR1,P-gp in brains were lower in Dingxianwan group and integrated Chinese and western medicine group; the seizure grade was lower (P<0.05) and duration was shorter (P<0.05) while the concentration of carbamazepine in the brain of rats was higher in rats of integrated Chinese and western medicine group (P<0.05). Conclusion: Dingxianwan has certain antiepileptic effect on refractory epilepsy and can reverse carbamazepine resistance, showing synergistic effect. The reversal of drug resistance may be related to the blocking of MDR1,P-gp expression in rat brains.