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目的:观察平痫颗粒在阻抗癫痫形成中对大鼠海马区凋亡调控基因Bcl-2,Bax蛋白表达的影响,探讨其抗癫痫作用的分子生物学机制。方法:选择60只45 d龄Wistar大鼠,随机分为正常对照组、模型组、阳性对照组、平痫低剂量组、平痫高剂量组,每组12只。除正常对照组,其余各组大鼠均以戊四唑亚惊厥剂量35 mg.kg-1腹腔注射诱导癫痫模型。造模同时平痫高、低剂量组分别以1.66,0.42 g.mL-1的生药含量2 mL.d-1灌胃;阳性对照组给予等量3.6 g.L-1的苯巴比妥混悬液灌胃;模型组和正常对照组胃饲等量双蒸水,持续5周。采用免疫组化染色(SABC法)于3,5周末分别测定Bcl-2,Bax免疫阳性细胞。结果:①给大鼠连续注射戊四唑过程中,模型组大鼠癫痫发作级别随点燃周次增加逐渐升级,由第1周的Ⅰ～Ⅱ级至第4周时的Ⅵ级大发作。治疗组发作次数和级别相对明显降低,最高级别Ⅳ级,且组间发作级别、次数比较无显著性差异。②随着点燃周次的增加,在3,5周末Bcl-2表达模型组明显低于正常对照组(P<0.01),Bax表达模型组明显高于正常对照组(P<0.01);阳性对照组、平痫高、低剂量组Bcl-2表达均明显高于模型组(P<0.01),Bax表达阳性对照组、平痫高、低剂量组与模型组比较有明显降低(P<0.01)。结论:平痫颗粒可能是通过促进抗凋亡调控基因Bcl-2蛋白的表达,以及抑制促凋亡调控基因Bax蛋白的表达,从而减少神经元细胞的凋亡来阻抗癫痫。
OBJECTIVE: To observe the effect of Pingxian Granule on the expression of Bcl-2 and Bax in rat hippocampus during the development of impedance epilepsy, and to explore the molecular mechanism of its anti-epileptic effect. Methods: Sixty Wistar rats, 45 days old, were randomly divided into normal control group, model group, positive control group, low-dose epilepsy group and high-dose epilepsy group, with 12 rats in each group. Except the normal control group, the other rats in each group were injected intraperitoneally with pentylenetetrazole at a dose of 35 mg.kg-1 to induce epilepsy model. At the same time, the rats in model group with high and low dose of epilepsy were given gavage with the crude drug concentration of 1.66,0.42 g.mL-1 2 mL.d-1 respectively. The positive control group was given phenobarbital suspension of 3.6 gL-1 Gavage; model group and normal control group stomach equal amount of double distilled water for 5 weeks. Bcl-2 and Bax immunoreactive cells were detected by immunohistochemical staining (SABC method) at 3 and 5 weeks. Results: (1) During the continuous injection of pentylenetetrazol into rats, the severity of seizures in the model group increased gradually with the increase of ignition cycles, ranging from Ⅰ ~ Ⅱ in the first week to Ⅵ in the fourth week. The number and severity of attacks in the treatment group were significantly lower, the highest level of grade IV, and there was no significant difference in seizure levels and frequency between the two groups. (2) With the increase of lighting cycle, Bcl-2 expression was significantly lower than that of normal control (P <0.01) at 3 and 5 weeks, Bax expression was significantly higher than that of normal control (P <0.01); positive control (P <0.01). Compared with model group, the expression of Bcl-2 in high and low dose group was significantly lower than that in model group (P <0.01) . Conclusion: Pingxie granules may be through the promotion of anti-apoptotic regulatory gene Bcl-2 protein expression, as well as inhibition of apoptosis-regulating gene Bax protein expression, thereby reducing neuronal apoptosis to impedance epilepsy.