【作者】 李长征；

【导师】 谢炜；

【作者基本信息】 第一军医大学 ， 中西医结合临床医学， 2007， 硕士

【摘要】 一、目的与意义癫痫(epilepsy)是神经系统常见病，是由于脑部神经元突然、间歇性痫样放电导致反复发作的以短暂大脑功能失调为特征的一种慢性疾病。我国约有900万癫痫患者，其中约600万人每年都会发作。正是这些癫痫患者及其家人在社会生活中承担了巨大的心理压力，而由癫痫引起的医疗、家庭和社会问题不容忽视。对于癫痫的发病，到目前为止其机制仍未完全阐明，但癫痫发病的两个重要特征是神经元兴奋性增高和高度同步化发放，这已经得到了学者们的公认，在这一过程中，星形胶质细胞的作用得到了越来越多的重视。有证据显示，在癫痫的发病机制中，除了神经元病理及功能方面的明显改变外，星形胶质细胞存在着不可忽视的功能和形态方面的异常，其结构和功能的变化会影响神经元的功能。而细胞因子可能在激活胶质细胞的过程中发挥重要作用。有研究发现癫痫患者和癫痫大鼠模型存在细胞因子水平的升高；白细胞介素(IL-1β、IL-6)、肿瘤坏死因子(TNF-α)、表皮生长因子(EGF)、碱性成纤维生长因子(bFGF)等细胞因子对神经干细胞诱导成胶质细胞、对胶质细胞的活性及细胞周期均有影响，认为其参与了胶质细胞的激活。目前控制癫痫仍以化学药物为主，常用一线抗癫痫药(AEDs)可使70％-80％各类型的癫痫患者发作得以控制，但长期应用会产生镇静、嗜睡、注意力分散、失眠、眩晕、影响发育、认知功能下降、诱发早期骨质疏松等，安全性较差。这就需要使用新的抗癫痫药，或合理地使用新老抗癫痫药。柴胡为临床常用中药，其主要药理成分有柴胡皂甙(saikosaponins，SS)、挥发油、有机酸、甾醇类、黄酮类、多糖等，其中柴胡皂甙又可分为柴胡皂甙a(saikosaponin a，SSa)、柴胡皂甙b2、柴胡皂甙c、柴胡皂甙d等单体成分。我科著名老中医陈宝田教授提出了癫痫从肝论治的观点，并运用自拟方“柴胡疏肝汤”临床治疗癫痫取得肯定的疗效。本课题组在前期研究中发现，柴胡疏肝汤、方中君药“柴胡”、柴胡总皂甙具有抗癫痫的作用，继而发现柴胡皂甙a为其抗癫痫作用的主要有效成分。为了进一步研究柴胡皂甙a的抗癫痫作用及探讨其是否通过细胞因子途径发挥抗癫痫作用，本课题组进行了柴胡皂甙a对戊四氮致痫大鼠脑脊液细胞因子表达的影响的研究，从而在细胞因子水平讨论柴胡皂甙a的抗癲痫作用机制，为筛选新的抗癲痫药物提供实验依据和理论基础。二、方法与内容1．SSa对PTZ致痫大鼠行为学的影响应用公认的PTZ致痫动物模型，将大鼠分为模型组、SSa组和VPA组，采用腹腔注射60mg／kg的PTZ造大鼠痫性发作模型，观察SSa与VPA对大鼠痫性发作潜伏期和强制性惊厥率的影响。2．SSa对PTZ致痫大鼠脑脊液细胞因子表达的影响将96只健康成年SD大鼠随机分为4组，即空白对照组(A组、6只)、模型组(B组、30只)、SSa组(C组、30只)和VPA组(D组、30只)，除空白对照组采用腹腔注射生理盐水处理外，其他各组均采用腹腔注射60mg／kg的PTZ造大鼠痫性发作模型，并给予以上不同处理因素处理。其中B组、C组和D组各随机分为5个小组，分别于观察大鼠痫性发作后0小时、2小时、4小时、8小时和12小时采集脑脊液，检测细胞因子IL-1B、IL-6和TNF-α的含量。三、结果1．SSa对PTZ致痫大鼠行为学的影响结果显示，与模型组相比，SSa组与VPA组均能够延长大鼠阵发性痉挛的潜伏期(P＜0.05)，并且能够延长大鼠发生强直性惊厥的潜伏期(P＜0.05)，提示：SSa与VPA可以显著延长PTZ诱发的大鼠痫性发作的潜伏期。经对PTZ致痫的各组大鼠强直性惊厥发作率的数据进行比较，发现三组之间差异有显著性意义(P＜0.05)。其中模型组大鼠在PTZ诱导下强直性惊厥的发作率为100％，SSa组与VPA组的强直性惊厥发作率分别为80％与90％，均低于模型组，提示：SSa与VPA均可显著降低实验性癫痫大鼠强直性惊厥发作率。2．SSa对PTZ致痫大鼠脑脊液细胞因子表达的影响2．1 SSa对PTZ致痫大鼠脑脊液细胞因子IL-1β表达的影响大鼠经PTZ致痫后12小时的脑脊液细胞因子IL-1β水平存在动态变化，先显著性升高后降低并逐步接近正常水平；在这一过程中，SSa与VPA对IL-1β水平的变化存在一定抑制作用。2．2 SSa对PTZ致痫大鼠脑脊液细胞因子IL-6表达的影响在PTZ急性致痫后，大鼠脑脊液中IL-6的水平先经历下降的过程，在4h后存在迅速上升而且持续地高表达；SSa对于其前期的降低阶段作用趋势不显著，而对于其上升存在抑制的趋势；而在观察的时间范围内VPA存在升高和维持IL-6水平的趋势。2．3 SSa对PTZ致痫大鼠脑脊液细胞因子TNF-α表达的影响在PTZ急性致痫后大鼠脑脊液中TNF-α含量迅速升高，并在8小时内逐步下降之后缓慢恢复正常水平。而SSa和VPA对其在PTZ急性致痫后含量的迅速升高有一定的抑制作用。四、结论1．本研究通过实验性大鼠癫痫模型研究SSa的抗癫痫效应，发现SSa不但能够明显延长痫性发作模型大鼠的阵发性痉挛潜伏期和强直性惊厥潜伏期；而且能够降低大鼠的强直性惊厥的发生率，进一步说明了SSa的抗癫痫作用。2．在PTZ致痫后CSF中细胞因子水平(IL-1β、IL-6、TNF-α)存在显著的动态变化，其中IL-1β、TNF-α在致痫后迅速升高随之下降并逐步恢复至正常水平，而IL-6在致痫后并不是迅速升高，其在4h内先下降后才逐步升高(12h)。SSa和VPA对于IL-6的作用有所不同，虽然SSa和VPA的作用不显著，但VPA在2h内可以升高IL-6并维持在一个较为平稳的水平直到观察时间结束，而SSa对于致痫后的IL-6的升高可能存在抑制作用。SSa和VPA可以对致痫后IL-1β的迅速升高存在显著的抑制作用，对于致痫后TNF-α的迅速升高也存在一定的抑制作用。更多还原

【Abstract】 Objective and significanceEpilepsy is a common chronic disease in nervous system, which is characterized by transient disorder of cerebral function and caused by suddenly and recurrently seizure of neuron in brain. There are about 9 million epileptic patients in our country, among which about 6 million epileptic patients attacked every year. These patients and their families suffer great mental stress in their social lives. So, it cannot be ignored that there are a lot of medical, domestic and social problems caused by epilepsy.So far, the mechanism of epilepsy has not been fully uncovered. Nevertheless, many experts of epilepsy have generally accepted that there are two important characters of epilepsy: the increase of neuronal excitability and the firing of highly synchronization. The functionality of astrocytes has been thought to become stronger and stronger. It is proved that besides the ch.anges of neuron during the process of epilepsy, the astrocytes also have some important abnormality of structure and function, which can affect the function of neuron. Furthermore, the cytokines may have some important effect in the progress of the activation of astrocytes. It is discovered that the expression of cytokines increased in the epileptic patients and rats model. And it is also discovered that interleukins(IL-1β、IL-6), tumor necrosis factor(TNF-α), epidermal growth factor(EGF), basic fibroblast growth factor(bFGF), and so on, have participated in the activation of astrocytes and have some effects on the activity and cell cycle of astrocytes and on the progress of nerve stem cell becoming astrocytes.At present, the main method of controlling epilepsy is to use chemicals. However the mainly used anti-epileptic drugs (AEDs) can only control the seizure of about 70 to 80 percent of epileptic drugs while the safety of the AEDs is not satisfactory because of the side effects accompanied with long term administration, such as distractibility, insomnia, dizziness and etc. So the new anti-epileptic drug should be used or the old anti- epileptic drugs should be used in a proper way in order to increase the curative effects and decrease the side effects.Bupleurum chinense is a widely used Chinese herb, which contain some pharmaco-ingredients such as saikosaponins (SS), aetheroleas, organic acids, sterins, flavonoids, polyose etc. And saikosaponins can be divided into some monomer such as saikosaponin A (SSa), saikosaponin b2, saikosaponin c, saikosaponin d and so on. Professor Chen Bao-tian is one of the famous veteran doctors of TCM in our department. He has proposed the theory that epileptic patients should be treated from liver (of the TCM definition), then prescribed "Chai Hu Shu Gan (CHSG) Soup" to treat epileptic patients and resulted in significant curative effect. We have found in our early research that "CHSG Soup", Bupleurum chinense and SS have anti-epileptic effects and SSa is the main active component. In order to investigate the anti-epileptic effect of SSa and to see if the anti-epileptic effect of SSa is obtained by the mechanism of cytokines, the effects of Saikosaponin A on Cerebrospinal Fluid Cytokines Expression are studied on the Epileptic Rats induced by Pentylenetetrazol".Method and content1. Effect of saikosaponin A on the etiology of PTZ induced epileptic rats Used the widely received PTZ induced epileptic rats as animal model, the experimental animals are divided into three groups: model group, SSa group and VPA group. The acute epileptic rat model were made by intraperitoneal injecting 60mg/kg PTZ, the effect of SSa and VPA were observed on the incubation period of seizure and the rate oftetanic convulsion of PTZ induced epileptic rats.2. Effect of saikosaponin A on cerebrospinal fluid cytokines expression of PTZ induced epileptic rats96 health grown-up rats were randomly divided into four groups: normal control group (group A, 6 rats), model group (group B, 30 rats), SSa group (group SSa, 30 rats), VPA group (group D, 30 rats). The normal control group was induced by intraperitoneal injecting isotonic sodium chloride solution, while the other groups were induced by intraperitoneal injecting 60mg/kg PTZ and treated by different factors as above. The group B, group C and group D were then divided into five groups, gathered the cerebrospinal fluid of the rats after the rats was induced 0 hours, 2 hours, 4 hours, 8 hours and 12 hours, then detected the cytokine IL-1β, cytokine IL-6 and cytokine TNF-α.Result1. Effect of saikosaponin A on the etiology of PTZ induced epileptic rats Compared with model group, SSa group and VPA group could prolong the incubation period of clonic cramp (P＜0.05) and could prolong the incubation period of tetanic convulsion (P＜0.05). It. is suggested that SSa and VPA could prolong the incubation period of seizure of PTZ induced epileptic rats.The rates of tetanic convulsion of PTZ induced epileptic rats in the three groups were significantly different. The rate of tetanic convulsion of model group was 100%, while the rate of SSa group was 80% and of VPA group was 90%. It is evidenced that SSa and VPA could reduce the rate oftetanic convulsion of PTZ induced epileptic rats.2. Effect of saikosaponin A on cerebrospinal fluid cytokines of PTZ induced epileptic rats2.1 Effect of saikosaponin A on cerebrospinal fluid cytokine IL-1βof PTZ induced epileptic ratsWithin the first 12 hours, the expression of IL-1βin cerebrospinal fluid of epileptic rats induced by PTZ was changed in a dynamic state: increased at first, then went down to a lower level than normal and slowly raised to normal finally. In this process, SSa and VPA could inhibit the changes of the expression of IL-1βin cerebrospinal fluid of epileptic rats.2.2 Effect of saikosaponin A on cerebrospinal fluid cytokine IL-6 of PTZ induced epileptic ratsAfter induced by PTZ, the expression of IL-6 in cerebrospinal fluid of epileptic rats decreased at first, increased at 4h and then kept a high level of expression. SSa almost had no effect when the expression of IL-6 decreased at first but could restrain this tendency of its increasing. However VPA could always had the tendency of heightening, and keeping the expression of IL-6 within the experimental period.2.3 Effect of saikosaponin A on cerebrospinal fluid cytokine TNF-αof PTZ induced epileptic ratsAfter induced by PTZ, the expression of TNF-αin cerebrospinal fluid was increased rapidly, and went down to a lower level within 8 hours, and then slowly recovered back to normal. SSa and VPA could inhibit the rapidly increasing of TNF-αafter induced by PTZ.Conclusion1. We investigated the anti-epileptic effect of SSa using the epileptic rat model and discovered that SSa could either prolong the incubation period of clonic and tonic convulsion or decrease the rate of tonic convulsion. The result evidenced the anti-epileptic effect of SSa.2. The expression of cytokines (IL-1β、IL-6、TNF-α) changed in dynamic state in epileptic rats. The expression of IL-1βand TNF-αraised rapidly after induced by PTZ and decreased to normal then. While the expression of IL-6 decreased first and raised after. SSa and VPA have different effect on IL-6. VPA could increase IL-6 in 2h and keep it in a steady level, while SSa could inhibit the raising of IL-6 after treated by PTZ. And both SSa and VPA could inhibit the raising of IL-1βand TNF-αafter treated by PTZ.更多还原