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p53蛋白表达的影响

Influence of Tsaoko-Anemarrhenae Decoction on p53 protein expression in hippocampus of rat models with epilepsia chronically induced by pentylenetetrazol

目的:动态观察具有调理脾胃气机作用的草果知母汤在阻抗癫痫形成中对大鼠大脑海马区凋亡调控因子p53蛋白表达的影响,分析其抗痫作用的分子生物学途径。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。模型组及中、西药治疗组均以戊四唑亚惊厥剂量35mg/kg腹腔注射,1次/d,持续4周,诱导癫痫模型。正常对照组同法给予相同剂量的生理盐水。各组在造模同时即开始给药,中药治疗组予草果知母汤液(由草果、知母、厚朴、半夏、黄芩等组成)5g/(kg·d)灌胃;西药治疗组予苯巴比妥混悬液60mg/(kg·d)灌胃;模型组和正常组胃饲双蒸水,2mL/(kg·d);1次/d,持续5周。采用Smialowki6级评分法进行行为学观察(1级,节律性点头或头部颤搐;6级,强直性惊厥)。正常对照组在实验第5周末取材,其他各组分别在实验第2,3,4,5周末取材。采用免疫组化PV-600二步法标记p53蛋白阳性细胞,比较各组大鼠脑海马区p53蛋白表达值在癫痫慢性形成过程中的变化。结果:进入结果分析78只,模型组、中药治疗组及西药治疗组各24只,正常对照组6只。①癫痫形成不同时间段惊厥等级得分:模型组大鼠1周后逐渐出现癫痫发作,并随点燃次数不断增强,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、次数相比差异无显著性意义(P>0.05)。②凋亡调控因子p53蛋白标记的阳性细胞数:至实验第3,4,5周,模型组大鼠脑海马区p53蛋白表达数量显著高于正常对照组(P<0.05～0.01),中、西药治疗组大鼠脑海马区p53蛋白表达数量均显著低于模型组(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均无显著性意义(P>0.05)。结论:草果知母汤可有效地阻断癫痫发作,其抗痫作用可能与抑制癫痫形成过程中大鼠脑内凋亡调控因子p53蛋白表达有关。

AIM：To dynamically investigate anti-epileptic effect of Tsaoko-Anemarrhenae Decoction （TAD）which can regulate ＇spleen, stomach and activities of qi on p53 protein expression in hippocampus of epileptic model rats, and analyze the molecular biological mechanism of anti-epileptic effect. METHODS： The experiment was performed in the Center Laboratory of Traditional Chinese Medicine, Basic Medical College, Guangxi College of Traditional Chinese Medicine from March to December 2005. Totally 82 SD male rats were selected as the animal model, and then they were assigned into 4 groups at random digit schedule. There were 26 rats in model group, 25 rats in TAD group, 25 rats in western medicine group and 6 rats in normal control group. The model group, TAD group and western medicine group were all injected with 35 mg/kg pentylenetetrazol （PTZ） by intraperitoneal injection, once a day for 4 weeks to induce epileptic models. The normal group was injected with saline of the same volume by the same way. Each group was treated with medicine during establishing the epilepsy disease models. The TAD group was treated with 5 g/kg TAD （which included tsaoko, anemarrhenae, magnolia bark, pinelliae tuber, baieal skullcap root and so on） by gastric perfusion daily. The western medicine group received 60 mg/kg phenobarbital suspension daily by gastrie perfusion. The normal group and model group were drenched with distilled water, 2 mL/kg daily, once a day for 5 weeks. Behavioral observation was performed with Smialowki 6 grade score （1 grade represented rhyth- micity nod or head twitching; 6 grade represented tonic convulsion）. The materials were drawn from the normal control group at the 5^th week, the other group to be marked use at the 2^nd 3^rd, 4^th and 5^th weeks, The p53 protein positive cells were labeled with immunohistochemical PV-600 second footwork. Change of p53 protein expression in the rats＇ hippoeampus in epileptic chronic formation was compared in each group. RESULTS： A total of 78 rats were involved in the result analysis, including 24 rats in model group, 24 rats in TAD group, 24 rats in western medicine group and 6 rats in the normal control group. ①Seore of eclampsia grade in different period of epileptic formation： The model group appeared the epilepsy＇s behavior at the 1^st week, and it become more and more serous, when it came to the 4^th week, and appeared the 6 grade behavior. The break-out times and break-out grade in the TAD group and western medicine group became fewer and lower, and the highest grade were only 4. There was no significant difference in break-out times and break-out grade between the two groups （P 〉 0.05）. ②The number of the p53 protein labeled positive cells： At the 3^rd, 4^th and 5^th weeks, the number of p53 protein expression at cerebral hippocanipus in model group were more obviously than the normal control group （P 〈 0.05-0,01）, and to be compared with the TAD group and western medicine group were obviously less than the model group （P 〈 0,05-0.01） .There were no significant difference 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 can effectively inhibit epileptic attack. The antiepileptic effect may be associated with the inhibiting p53 protein expression in the rats ＇hippocampus in epileptic formation,