【作者】 鲍勇；

【导师】 谢炜；

【作者基本信息】 南方医科大学 ， 中西医结合临床医学， 2007， 硕士

【摘要】 一、目的与意义癫痫是以大脑功能短暂失调为表现的慢性反复发作性疾病。由癫痫引起的医疗、家庭和社会问题极为显著。在世界范围内，癫痫患者大约占1％，目前治疗是以抑制惊厥发作的对症治疗为主，而并非对癫痫疾病本身的调节，而有效的预防和治愈的药理机制尚未被认识到，但癫痫是通过各种癫痫致病因素导致“正常脑”变为“痫性脑”。目前常用一线抗癫痫药(AEDs)可使大部分癫痫患者发作得以控制，但长期应用安全性较差，对于一些难治性癫痫行外科手术治疗，也受到适应症的限制，风险大，费用昂贵，还存在复发的可能，因此难治性癫痫仍然以药物治疗为主要手段，这就需要使用新的抗癫痫药，但在研究AEDs的前提是对于癫痫发病机制的深入认识。近年来癫痫的发病机制研究有了长足进步。神经元兴奋性增高和高度同步化发放是癫痫发病的两个重要特征已得到公认，国内外很多学者试图从多个方面来探索引起神经元兴奋性增加和高度同步化发放的形成机制，提出了不少有益的见解，其中主要的认识到“免疫-神经-内分泌网络”的存在以及对于星形胶质细胞与神经突触信号传递的深入认识。“免疫-神经-内分泌网络”的观点认为机体内存在免疫、神经、内分泌系统，且三大系统之间相互影响，存在互相调节的双向回路，从而构成网络，与癫痫发病机制有着密切的关系。从免疫和内分泌系统去探讨癫痫的发病机制可以说是癫痫研究的一大突破，免疫和内分泌系统可以通过神经活性物质、激素等对神经系统进行调节，其不仅能够调节正常的神经生理活动，还与癫痫的发病有着密切的关系。而包括IL-6在内的细胞因子作为重要的活性因子，在癫痫中被发现存在异常的表达，发现其在癫痫发作中有着重要的作用，可以介导神经损伤，所以对其研究得到重视。随着“免疫-神经-内分泌网络”的观点提出，作为中枢神经系统中占绝大多数的胶质细胞的作用也被逐渐认识。首先发现在癫痫发病中，往往伴随星形胶质细胞的活化，又称反应性胶质增生(Reactive gliosis)，首要特征是其重要的骨架蛋白胶质纤维酸性蛋白(GFAP)表达增加，激活的星形胶质细胞对神经突触传递施加影响，可以通过多种途径来作用于神经元，增加神经元兴奋性，进而引起癫痫的发作。柴胡皂甙具有公认的抗炎、免疫调节作用，并且在我们前期的研究基础上发现柴胡总皂甙和柴胡皂甙a具有抗实验性癫痫、抑制慢性点燃大鼠GFAP的表达，调节海马Glu和GABA的表达，故我们推测柴胡皂甙a可以通过抑制细胞因子对神经系统作用和激活星形胶质细胞来发挥抗实验性癫痫。所以在本课题研究中主要研究目的是研究中枢神经系统过量表达IL-6对大鼠脑电活动以及对星形胶质细胞活化的影响，并进一步观察SSa在其中的作用。二、方法与内容1、IL-6对大鼠脑电活动的影响及柴胡皂甙a的干预作用随机将24只SD大鼠分成4组，雌雄各半，每组6只：A组为正常组(生理盐水＋生理盐水)、B组为模型组(生理盐水＋IL-6)、C组为SSa高剂量组(1.81mg／kg，SSa＋IL-6)、D组为SSa低剂量组(0.91mg／kg，SSa＋IL-6)。大鼠腹腔注射给于预处理药(A组、B组：生理盐水3ml／kg；C组：SSa溶液1.81mg／kg；D组：0.91mg／kg)30min后IL-6侧脑室注射，并进行电极埋置。埋置电极后分时间点(1h、2h、4h、8h、12h、24h)采用单极导联在麻醉状态下运用多道生理仪采集脑电图(记录5分钟)。对各组脑电图结果进行分析，计算尖波个数，高频(100Hz)脑电的功率和波幅的变化。2、IL-6对大鼠海马GFAP表达的影响及柴胡皂甙a的干预作用随机将44只SD大鼠分成3组，雌雄各半，分5个时间点：A组为正常组(生理盐水＋生理盐水)4只、B组为模型组(生理盐水＋IL-6)20只、C组为SSa组(1.81mg／kg，SSa＋IL-6)20只。大鼠腹腔注射给于预处理药(A组、B组：生理盐水3ml／kg；C组：SSa溶液1.81mg／kg)。30min后，麻醉固定后使用微量进样器于大鼠侧脑室A组注射生理盐水、B组和C组注射IL-6，之后取脑组织标本，B组和C组动物按时间点(1h、4h、8h、12h、24h，每个时间点4只，雌雄各半)取标本，运用western-blot技术检测海马GFAP表达的变化，对结果进行灰度分析，计算GFAP与GAPDH的比值。三、结果1、IL-6对大鼠脑电活动的影响及柴胡皂甙a的干预作用(1) A、B、C、D组脑电图中尖波在1h、4h、8h、12h时，A组、C组与B组比较有显著性差异，而D组与B组比较无显著性差异；在时间点上的变化中尖波在A组以12h显著低于1h时(P＜0.05)其他时间点变化不显著；而在B组4h、8h、12h、24h显著高于1h(P＜0.05)；C组变化不显著(P＞0.05)；D组以2h显著高于1h(P＜0.05)。(2) A、B、C、D组高频(100Hz)脑电波的功率在4h、8h、12h时，除D组外，A组、C组与B组比较有显著性差异。在时间点上的变化中高频脑电(100Hz)的功率，A组在4h、8h、12h与1h比较存在显著性差异(P＜0.05)；而在B组4h、8h、12h、24h显著高于1h(P＜0.05)；C组在4h、8h与1h比较显著降低(P＜0.05)；D组以8h显著高于1h(P＜0.05)。(3) A、B、C、D组高频(100Hz)脑电波的波幅在1h、4h、8h、12h时，除D组外，A组、C组与B组比较有显著性差异。在时间点上的变化中高频脑电(100Hz)的幅度，A组在2h、4h、12h、24h与1h比较存在显著性差异(P＜0.05)；而在B组4h、8h、12h显著高于1h(P＜0.05)；C组在4h、24h与1h比较显著降低(P＜0.05)；D组各时点与1h相比较，变化不显著(P＜0.05)。(4)脑电图形态A组、B组(D组于B组波形类似)、C组在24小时内的脑电动态变化过程，正常(A组)大鼠脑电图以α、β波为主，波幅介于20～100μv之间，且在24小时内脑电图(波幅、频率)无明显改变；模型组(B组)大鼠痫性波(棘波、尖波)大量出现，多为高频、高幅(频率为70-200Hz，波幅为100～200μv)，且呈阵发性出现；柴胡皂甙a组(C组)也存在散在的痫性放电，与B组比较，痫波样改变明显减少，频率波幅较低，接近A组。2、IL-6对大鼠海马GFAP表达的影响及柴胡皂甙a的干预作用在实验组中大鼠海马GFAP的表达量以B组最高，A组、C组与之比较，在4小时、8小时、12小时、24小时存在显著性差异(P＜0.05)。GFAP的表达在各时点存在的动态变化，在4h、8h、12h、24h时B组表达高于A、C组，GFAP条带颜色较浓，而A、C组的颜色较淡。四、结论(1) IL-6可以增加尖波的数量，提高大鼠高频(100Hz)脑电波的功率和幅度。证实IL-6可以诱导大鼠痫性放电，并且其作用强度与作用时间存在密切关系。(2) IL-6可以诱导星形胶质细胞GFAP蛋白的表达增加。证实IL-6可以激活星形胶质细胞，从而影响痫性活动的产生。(3) SSa高剂量在IL-6侧脑室注射诱发的脑电活动异常中可以显著抑制大鼠脑电尖波数，抑制高频(100Hz)脑电波的功率和幅度，从而抑制IL-6侧脑室注射诱发的脑电活动异常，而低剂量组抑制作用不显著，提示SSa作用强度可能与剂量大小有关。在IL-6侧脑室注射可以诱导大鼠星形胶质细胞GFAP的表达，实验中SSa可以抑制GFAP表达(4h、8h、12h、24h)，反映SSa可以抑制由IL-6诱导的星形胶质细胞的激活。SSa抗癫痫作用可能在于抑制IL-6诱导的痫性脑电活动和星形胶质细胞的激活。更多还原

【Abstract】 Objective and significanceEpilepsy is chronic and disorders of recurrent seizures, which due to neuron discharge abruptly and intermittently, induce transient Cerebral Functional disorder that recurrent attacks. It can’t be ignore that problem of medical treatment, family and social problem induced by epilepsy.The epilepsies, affect about 1% of the population worldwide. Available therapy is symptomatic in that drugs inhibit seizures but are not disease-modifying; that is, no effective pharmacological prevention or cure has been identified. The term epileptogenesis refers to the process by which a normal brain becomes epileptic.In now, epileptic treatment still dependent on chemicals, general anti-epileptic drug(AEDs) can control epileptic attack from 70% to 80%,but security is a problem in treatment of long-term, and the method to treat refractoriness epilepsy by surgery be confined by indication, high risk, expensive, and possible to recur. So epileptic treatment still dependent on chemicals, and the original AEDs is needed urgently. But it must know clearly that is pathogenesy of epilepsy before develop new AEDs.For the past few years, research of reepileptic pathogenesy has been advanced. Increase in neuronal excitability and synchronize firing is two important characteristic of epileptic, which had be generally accepted. Many scholars try them best to study mechanism of form, and introduced many view. Recognition of exist of "the network of immuno-neuro-endocrine" and relation of astrocyte with synaptic signal transmission have been gradually clear.The view of "the network of immuno-neuro-endocrine" proposed that system of immune, nerves and endocrine exist in organism of human, and the three systems have influence and bidirectional modulatory circuit in each other. Thus, the network was constituted, which closely related with pathogenesy of epilepsy. Research on epilepsy from the system of immune and endocrine can be regard as breakthrough. The system of immune and endocrine can modulate nervous system by the neuronal active compound, hormone and so on, and it can modulate normal neuronal physiological functions or epileptic morbidity. IL-6 is cytokine and very importent active factors, which be found abnormally express in epilepsy. Thus, the importent effects of IL-6 on epilepsy were found. IL-6 can modulate nerve injury, so it was emphasized. With the view of "the network of immuno-neuro-endocrine" introduced, the effects of astrocyte that majority in central nervous system have been gradually clear, Astrocytes can become reactive gliosis when nervous system have been damaged. Histologic characteristics of reactive gliosis is greaten and hyperplasia of astrocyte, biochemical marker is increase in content of glial fibrillary acidic protein(GFAP)of astrocyte. The reactive gliosis have effect on synaptic transmission that exciting neuronal by many kinds of pathway, and is epileptogenous.It is well known that Pharmacodynamic action of saikosaponin a (SSa) have anti-inflammatory, immunological regulation, promoting secrete of corticosterone. In the past research, we have found that saikosaponins and saikosaponin a can inhibit experimental epilepsy; saikosaponins can inhibit expression of glial fibrillary acidic protein and modulate expression of Glu and GABA in hippocampus. So, We suppose that saikosaponin a may inhibit experimental epilepsy by inhibit the effect of cytokine on nervous system and reactive gliosis.Based on the above, the objective of this research is to study on the effect of IL-6 that excesssive express in the central nervous system on electrical activity of rats brain and reactive gliosis and to observe the role of SSa.Method and content1. The effect of IL-6 on electrical activity of rats brain and the role of SSa.Twenty four healthy Sprague-Dawley rats were randomly divided into four groups with six in each group: Group A, Normal Sodium (NS+NS) group; Group B blank group(NS+IL-6); Group C, group of High-dose Saikosaponin a(1.81mg/kg, SSa+IL-6); Group D, group of Small-dose Saikosaponin a(0.91mg/kg, SSa+IL-6). Rats were injected the pretreatment medicine by intraperitoneal (Group A and Group B: NS 3ml/kg; Group C: the solution of SSa 1.81mg/kg; Group D: the solution of SSa 0.91mg/kg), IL-6 was injected by lateral cerebral ventricle after 30 minutes, and imbedment electrode in rats brain. Multichannel physiograph was applied in record rats electroencephalogr(EEG), unipolar lead, narcotism(record time 1h, 2h, 4h, 8h, 12h, 24h), land counted the sharp wave, computed the power and amplitude of the high-frequency(100Hz)wave.2. The effect of IL-6 on expression of rats hippocampal glial fibrillary acidic protein (GAFP) and the role of SSa.Fourty four healthy Sprague-Dawley rats were randomly divided into three groups: Group A, Normal Sodium (NS+NS) group, four rats; Group B, blank group(NS+IL-6), twenty rats; Group C, group of Saikosaponin a(1.81mg/kg, SSa+IL-6), twenty rats. Rats were injected the pretreatment medicine by intraperitoneal(Group A and Group B: NS 3ml/kg; Group C: the solution of SSa 1.81mg/kg), In group B and group C, IL-6 was injected by lateral cerebral ventricle after 30 minutes, and to obtain the sample of brain tissue on time 1h, 4h, 8h, 12h, 24h, four rats on each time, the technique of western-blot was applied in detect the expression of GFAP, and last, the result of western-blot was analyzed, and demonstrated in value of gray scale, and calculate the ratio of the value of gray scale of GFAP with the value of gray scale of GAPDH.Result1.The effect of IL-6 on electrical activity of rat’s brain and the role of SSa(1) In four groups, the sharp wave were induced by IL-6, and the Group A and Group C have significant difference compare with the Group B at times of lh, 4h, 8h, 12h(P＜0.05), but except Group D. The dynamic change of the four groups sharp wave, Group A have significant difference compare with the 1h only at time of 12h (P＜0.05), Group B have significant difference compare with the lh at times of 4h, 8h, 12h, 24h (P＜0.05), the change of Group C wasn’t significant (P＜0.05), Group D have significant difference compare with the 1h only at times of 2h (P＜0.05).(2) In four groups, the power of the high-frequency(100Hz)wave were raised by IL-6, and the Group A and Group C have significant difference compare with the Group B at times of lh, 4h, 8h, 12h(P＜0.05), but except Group D. The dynamic change of the four groups power of the high-frequency(100Hz)wave, Group A have significant difference compare with the 1h at times of 4h, 8h, 12h(P＜0.05), Group B have significant difference compare with the lh at times of4h, 8h, 12h, 24h (P＜0.05), Group C have significant difference compare with the 1h at times of 4h, 8h(P＜0.05), Group D have significant difference compare with the lh only at times of 8h(P＜0.05).(3) In four groups, the amplitude of the high-frequency(100Hz)wave were raised by IL-6, and the Group A and Group C have significant difference compare with the Group B at times of 1h, 4h, 8h, 12h(P＜0.05), but except Group D. The dynamic change of the four groups amplitude of the high-frequency(100Hz)wave, Group A have significant difference compare with the lh at times of 2h, 4h, 12h, 24h (P＜0.05), Group B have significant difference compare with the lh at times of 4h, 8h, 12h (P＜0.05), Group C have significant difference compare with the lh at times of 4h, 24h(P＜0.05), the change of Group D wasn’t significant.(4) The change of EEG shape was dynamic in four groups, and the shape of Group D like as Group B. the wave of a andβwas majority in the EEG of Group A, which the range of amplitude was from 20μv tol00μv, and unchanged in 24h(amplitude of wave and frequency), epileptic wave (spike and sharp wave) of Group B appeared abundantly, the majority of that is high frequency (70-200Hz), high amplitude (100-200μv)and paroxysm. Epileptic wave of Group C is sporadic, and compare with Group B, the wave reduced significantly, the frequency and amplitude is low, just likes Group A.2.The effect of IL-6 on expression of rats hippocampal glial fibrillary acidic protein (GAFP) and the role of SSa.In four groups, the expression of GFAP was increased by IL-6, and the Group A and Group C have significant difference compare with the Group B at times of 4h, 8h, 12h, 24h(P＜0.05), but except Group D. And, the change of expression was dynamic. Group A and Group C have significant difference compare with Group B at times of 4h, 8h, 12h, 24h (P＜0.05), The band of GFAP in Group B is thicker than it inGroup A and Group C.Conclusions(1) The sharp wave were induced by IL-6, and IL-6 can raise the power and amplitude of the high-frequency (100Hz) wave. IL-6 can induced epileptic discharge of rats and the effect have relationship with action time that can be confirmed in this study. (2) The increased of GFAP’s expression can be induced by IL-6. Astrocyte can be activated by IL-6 in this study confirmed, and then, to affect the occurrence of epilepsy.(3) The high dose of SSa can inhibit the epileptic EEG of rats induced by IL-6. SSa can significantly inhibit the number of the sharp wave and can inhibit the power and amplitude of the high-frequency (100Hz) wave. But in this study, the effect of low-dose of SSa on the EEG wasn’t significant, suggested that the effect was dose-dependent. The increased of GFAP’s expression can be induced by lateral cerebral ventricle injection of IL-6, SSa can inhibit the expression of GFAP induced by IL-6 in this study. The anti-epileptic effect of SSa that may be inhibit the epileptic electrical activity of brain and astrocyte activated by IL-6.更多还原