草果知母汤对戊四唑慢性诱导癫痫模型大鼠脑内海马区凋亡调控因子Bcl-2、Bax蛋白表达的影响

Effects of tsaoko-anemarrhenae decoction on hippocampal Bcl-2,Bax protein expression in epileptic rat models induced by pentylenetetrazol

目的:动态观察具有调理中焦脾胃气机作用的草果知母汤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:实验于2005-03/12在广西中医学院基础医学院中医实验中心完成。选择雄性SD大鼠82只,按随机数字表法分为4组:模型组26只,中药治疗组及西药治疗组各25只,正常对照组6只。除正常对照组外,其余各组均以戊四唑亚惊厥剂量35mg/kg腹腔注射诱导癫痫模型,1次/d,持续4周。正常对照组同法给予相同剂量的生理盐水。中药治疗组予草果知母汤(由草果、知母、厚朴、半夏、黄芩等组成)5g/(kg·d)灌胃;西药治疗组给予苯巴比妥混悬液60mg/(kg·d)灌胃;模型组和正常对照组胃饲双蒸水,2mL(kg·d),1次/d,持续5周。采用免疫组化PV-600二步法标记bcl-2、bax蛋白阳性细胞,动态观察各组大鼠海马区Bcl-2、Bax蛋白表达值在癫痫形成过程中的变化。结果:模型组2只因惊厥大发作抽搐致死,中、西药治疗组各死亡1只,解剖无特殊发现,死亡原因不明,共78只大鼠进入结果分析。①模型组大鼠1周后逐渐出现癫痫发作,并随着点燃次数的增加而不断增强,至第4周出现6级大发作;中、西药治疗组发作次数和级别明显降低,最高级别为4级,且两组间发作级别、次数相比差异不显著(P>0.05)②随着点燃周次的增加,Bcl-2蛋白表达呈不断减少的趋势,模型组与正常对照组比较,在实验第3,4,5周,明显低于正常对照组(P<0.05～0.01),中、西药治疗组大鼠海马区Bcl-2蛋白表达数量均明显高于模型组(P<0.05～0.01);在相同时间段(2、3、4、5周),中、西药治疗组间比较,差异不显著(P>0.05)。③bax蛋白表达数随着点燃次数增加呈不断增多的趋势,至实验第3,4,5周,模型组大鼠海马区Bax蛋白表达数量明显高于正常对照组(P<0.05～0.01),中、西药治疗组大鼠海马区Bax蛋白表达数量均明显高于模型组(P<0.05～0.01);在相同时间段(2、3、4、5周),中、西药治疗组间比较,差异不显著(P>0.05)结论:草果知母汤可有效地阻断癫痫发作,其抗痫作用可能与其干预癫痫形成中大鼠脑内凋亡调控因子Bcl-2、Bax蛋白表达有关。

AIM： To dynamically observe the effects of tsaoko-anemarrhenae decoction （TAD）, which could recuperate the middle-jiao （the spleen and stomach）, on the Bcl-2, Bax protein expression in the hippocampus of epileptic model rats, and investigate the molecular biological mechanism of the anti-epileptic effect of TAD. METHODS： The experiment was performed in the experimental center of Guangxi College of Traditional Chinese Medicine from March to December 2005. Totally 82 male SD rats were selected and then randomly assigned into 4 groups： model group （n =26）, TAD group （n =25）, western medicine group （n =25） in and normal control group （n =6）. Except the control group, all the groups were intraperitoneally injected with 35 mg/kg pentylenetetrazol （PB）, once a day for 4 weeks to induce the models of epilepsy. The normal group was injected with matching saline by the same way. The TAD group was intragastrically infused with 5 g/kg TAD daily （composed of tsaoko, anemarrhenae, magnolia bark, pinelliae tuber, baical skullcap root and so on）. The western medicine （PB） group received 60 mg/kg phenobarbital suspension daily by gastric perfusion. The normal group and model group were drenched with distilled water, 2 mUkg daily, once a day for 5 weeks. The Bd-2, Bax protein positive calls were labeled with immunohistochemical PV-600 second footwork to dynamically observe the changes of Bcl-2, Bax protein expression in the rats＇ hippocampus during epileptic formation. RESULTS： A total of 78 rats were involved in the result analysis except 2 rats in model group died of onset of eclampsia, 1 in the TAD group and 1 in the PB group died of unknown causes. （1）The rats in the model group appeared the epileptic behavior at the 1^st week, and it became more and more serious with kindle times; the rats appeared grade 6 behavior at the 4^th week. The break-out times and break-out grade of the TAD group and PB group were fewer and lower, and the highest grade were only 4; moreover, there were no significant differences in the break-out grade and times between the two groups （P 〉 0.05）. （2）With the increase of kindle times, the expression was continuously decreased; at the 3^rd, 4^th and 5^th weeks, the number of the Bcl-2 protein expression at hippocampus in the model group was obviously less than the normal control group （P 〈 0.05-0.01）, and the TAD group and PB group were obviously more than the model group （P 〈 0.05-0.01）; there were no significant differences between the TAD group and PB group in the same period （2^nd, 3^rd, 4^th and 5^th weeks, P 〉 0.05）. （3）Bax protein expression was gradually increased with the increase of kindle times; at the 3^rd, 4^th and 5^th weeks, the number of the Bax protein expression at cerebral hippocampus in the model group was obviously more than the normal control group （P 〈 0.05-0.01）, and the TAD group and western medicine group were obviously less than the model group （P 〈 0.05-0.01）. There were no significant differences between TAD group and western medicine group in the same period （the 2^nd, 3^rd, 4^th and 5^th weeks, P 〉 0.05）. CONCLUSION： TAD shows an anti-epileptic effect, which may be associated with the interruption of Bcl-2, Bax protein expression in the rats＇ hippocampus during epileptic formation.