【作者】 闫禹竹；

【导师】 程为平；

【作者基本信息】 黑龙江中医药大学 ， 中西医结合临床， 2014， 博士

【摘要】 目的通过观察戊四氮致痫大鼠在补脑止痫散治疗前后,实验大鼠一般状态、行为学、海马组织病理改变,治疗后大鼠海马组织中GABAARα1、 NMDAR1、IL-1β及I1-6表达的变化,探讨补脑止痫散抗痫机制,为该方临床治疗及预防癫痫疾病提供理论依据。方法成年Wistar大鼠55只,在造模前7天随机选取15只大鼠作为补脑止痫散防治组(简称防治组),并进行补脑止痫散灌胃,每日一次。7天后,除防治组外,从40只大鼠中随机选取10只大鼠作为空白组,防治组及其余30只大鼠进行癫痫模型点燃,造模后将30只大鼠中点燃成功的大鼠随机平均分配到模型组、补脑止痫散组。在开始统一灌胃治疗前1天及治疗21天时观察记录4组大鼠的一般状态,称取体质量,记录痫性发作潜伏期和痫性发作级别。通过HE染色及尼氏染色观察海马组织病理学变化情况。免疫组化法检测海马GABAARα1、NMDAR1、IL-1β及I1-6阳性细胞数,计算阳性细胞的平均灰度值。实验数据均输入SPSS17.0进行统计学处理,P<0.05为有显著差异,P<0.01为有高度显著性差异。结果共有33只大鼠成功点燃。在灌胃治疗过程中,模型组死亡4只,防治组及补脑止痫散组各死亡1只。1.一般状态及体质量变化模型组大鼠一般状态明显较空白组差,防治组、补脑止痫散组与模型组相比均有改善。灌胃治疗前,与空白组比较,模型组、防治组、补脑止痫散组大鼠体质量均明显减小,有高度显著性差异(P<0.01)；灌胃治疗21天后,与模型组比较,空白组、防治组、补脑止痫散组大鼠的体质量均明显增加,差异具有高度显著性差异(P<0.01)；空白组、模型组、防治组、补脑止痫散组大鼠治疗后的体质量与治疗前各组体质量相比,均有高度显著差异性(P<0.01)。2.行为学观察空白组无痫性发作。灌胃治疗前,模型组、防治组、补脑止痫散组间痫性发作潜伏期及发作级别比较无显著差异(P>0.05)。治疗21天后,与模型组比较,防治组、补脑止痫散组大鼠的发作潜伏期均明显延长,具有高度显著性差异(P<0.01)：防治组、补脑止痫散组大鼠治疗后的发作潜伏期与治疗前相比,均有高度显著差异性(P<0.01),2组发作潜伏期均显著延长。治疗21天后,防治组、补脑止痫散组大鼠的发作级别与模型组比较均明显降低,具有高度显著性差异(P<0.01)；防治组、补脑止痫散组大鼠治疗后的发作级别与治疗前相比,均有高度显著差异性(P<0.01),2组发作级别均显著降低。3.病理学观察空白组大鼠海马细胞数量和结构均正常。模型组大鼠海马细胞数量明显减少,层次紊乱,细胞肿胀变形,胞浆染色加深,胞核固缩、碎裂。防治组、补脑止痫散组较模型组均有改善。4.GABAARα1的表达情况与空白组比较,模型组大鼠海马的GABAARα1阳性细胞数目明显减少,平均灰度值明显增高,且均有高度显著性差异(P<0.01)；与模型组比较,防治组、补脑止痫散组癫痫大鼠海马的GABAARα1阳性细胞数均明显增加,平均灰度值均明显降低,防治组与模型组比较GABAARα1阳性细胞数及平均灰度值均有高度显著性差异(P<0.01),补脑止痫散组与模型组比较GABAARα1阳性细胞数及平均灰度值均有显著性差异(P<0.05)。5.NMDAR1的表达情况与空白组比较,模型组大鼠海马的NMDAR1阳性细胞数明显增高,平均灰度值明显降低,均有高度显著性差异(P<0.01)；与模型组比较,防治组、补脑止痫散组癫痫大鼠海马的NMDAR1阳性细胞数均明显降低,平均灰度值均明显增高,防治组与模型组比较NMDAR1阳性细胞数及平均灰度值均有高度显著性差异(P<0.01),补脑止痫散组与模型组比较NMDAR1阳性细胞数及平均灰度值均有显著性差异(P<0.05)。6.IL-1p的表达情况与空白组比较,模型组大鼠海马的IL-1p阳性细胞数明显增高,平均灰度值明显降低,且均有高度显著性差异(P<0.01)。与模型组比较,防治组、补脑止痫散组癫痫大鼠海马的IL-1β阳性细胞数均明显降低,平均灰度值均明显增高,防治组与模型组比较IL-1p阳性细胞数及平均灰度值均有高度显著性差异(P<0.01),补脑止痫散组与模型组比较IL-1p阳性细胞数及平均灰度值均有显著性差异(P<0.05)。7.IL-6的表达情况与空白组比较,模型组大鼠海马的IL-6阳性细胞数明显增高,平均灰度值明显降低,且均有高度显著性差异(P<0.01)。与模型组比较,防治组、补脑止痫散组癫痫大鼠海马的IL-6阳性细胞数均明显降低,平均灰度值均明显增高,防治组与模型组比较IL-6阳性细胞数及平均灰度值均有高度显著性差异(P<0.01),补脑止痫散组与模型组比较IL-6阳性细胞数及平均灰度值均有显著性差异(P<0.05)。结论1.中药补脑止痫散可明显改善戊四氮致痫大鼠的一般状态,纠正其体质量减轻。2.补脑止痫散能明显延长癫痫大鼠发作潜伏期,降低痫性发作级别。3.中药补脑止痫散可明显减轻海马神经元的病理损伤。4.中药补脑止痫散能增强GABAARα1的表达,防治组作用强于补脑止痫散组。5.中药补脑止痫散抑制NMDARl的表达,防治组作用强于补脑止痫散组。6.中药补脑止痫散抑制IL-1β的表达,防治组作用强于补脑止痫散组。7.中药补脑止痫散抑制IL-6的表达,防治组作用强于补脑止痫散组。更多还原

【Abstract】 Objective:Observe the general state, behavior changes, hippocampal tissue pathological changes of PTZ induced epilepsy rats before and after treatment.Observe GABAARα1, NMDAR1, IL-1β and IL-6of hippocampal tissue of PTZ induced epilepsy rats after treatment. Discuss the mechanism of the resistance to epilepsy of Bunaozhixian power.Provide the theory basis to prevent and treat epilepsy with Bunaozhixian power.Methods:7days before rats model establish, take15rats as Bunaozhixian power prevention group(be called prevention group for short) randomly from55adult Wistar rats.Give a gavage of Bunaozhixian power to prevention group rats once a day.After7days,start to establish epilepsy model rats with the other30rats and prevention rats.Divide randomly model rats into model group and Bunaozhixian power group.Observe the general state,weight, seizure latency and seizure level of before and after treatment.Observe hippocampal tissue pathological changes of PTZ induced epilepsy rats after treatment in HE staining and Nissl’s staining.Detect number of positive cells and its’average gray value of GABAARal,NMDARl,IL-1β,Il-6by immunohistochemical.Input the data into SPSS17.0,P<0.05means significant difference.P<0.01means highly significant difference.Results:There are33rats were established successfully.During the gavage, there were4rats dead in model group,1rat dead in prevention group and1rat dead in Bunaozhixian power group.1. The change of general state and weightModel group rats’ general state is worse than blank group rats. Compare with model group,prevention group and Bunaozhixian power group rats have improvement.Compare with blank group, before gavage,all the experiment rats’ weight decrease obviously,has highly significant difference(P<0.01).Compare with model group, after gavage therapy,all other group rats’weight increase obviously,has highly significant difference(P<0.01).All rats’ after treatment weight are obviously different from before treatment weight,has highly significant difference(P<0.01).2. The behavioral observationBlank group has no epileptic seizure.There is no difference between model group,Bunaozhixian power group and prevention group before treatment(P>0.05).Compare with model group,prevention group and Bunaozhixian power group rats’seizure latency and seizure level are obviously prolong,has highly significant difference(P<0.01).Prevention group and Bunaozhixian power group rats’before treatment seizure latency and seizure level are highly significant difference(P<0.01) from after treatment seizure latency and seizure level.The seizure latency all obviously prolong.The seizure level all obviously decrease.3. The pathological observationBlank group rats hippocampal cells number and structure are all normal.Number of model group rats hippocampal cells decreased significantly structure disordered, cell swelling deformation, cytoplasm staining, cell nucleus pycnosis, fracture.Prevention group and Bunaozhixian power group were improved.4. The expression of GABAAR alpha1Blank group GABAAR alpha1positive cells number is more,AGV is lower.Compare with blank group,number of model group decreased significantly, AGV increased significantly,highly significant difference (P<0.01);Compared with model group, prevention group, Bunaozhixian power group epilepsy rat hippocampal GABAAR alpha1expression were significantly increased, prevention group has highly significant difference (P <0.01);Bunaozhixian power group has significant difference (P<0.05).5.The expression of NMDAR1 Compared with the blank group, model group rats hippocampal NMDAR1expression increased obviously, there are highly significant difference (P<0.01);Compared with model group, prevention group, Bunaozhixian power group were significantly reduced in the rat hippocampus of NMDAR1expression, prevention and treatment group compared with model group was highly significant difference (P<0.01), Bunaozhixian power group compared with model group had significant difference (P<0.05).6. The expression of IL-1betaCompared with the blank group, model group rats hippocampal NMDAR1expression increased obviously, there are highly significant difference (P<0.01);Compared with model group, prevention group, Bunaozhixian power group were significantly reduced in the rat hippocampus of NMDAR1expression, prevention and Bunaozhixian power group compared with model group was highly significant difference (P<0.01), Bunaozhixian power group compared with model group had significant difference (P<0.05).7.The expression of IL-6Compared with the blank group, model group rats hippocampal significantly higher, IL-6expression was highly significant difference (P<0.01).Compared with model group, prevention group, Bunaozhixian power group rat hippocampus of IL-6expression were significantly lower, prevention and treatment group compared with model group was highly significant difference (P<0.01), Bunaozhixian power group compared with model group had significant difference (P<0.05).Conclusion:1. Bunaozhixian power can obviously improve the PTZ epilepsy rats’general status, correct its body quality loss.2. Bunaozhixian power can prolong seizure latency and decrease seizure level. 3. Bunaozhixian power can reduce the pathological damage of hippocampal neurons.4.Bunaozhixian power can strengthen the expression of GABAARα1.Prevention group is more effective than Bunaozhixian power group.5. Bunaozhixian power can restrain the expression of NMDAR1.Prevention group is more effective than Bunaozhixian power group.6. Bunaozhixian power can restrain the expression of IL-1β.Prevention group is more effective than Bunaozhixian power group.7. Bunaozhixian power can restrain the expression of IL-6.Prevention group is more effective than Bunaozhixian power group.更多还原