【作者】 谢涛；

【导师】 王维平；

【作者基本信息】 河北医科大学 ， 神经病学， 2012， 博士

【摘要】 癫痫是常见的神经系统慢性疾病，全球发病率约为1%。癫痫给社会、家庭、个人带来了沉重的负担。在全球约五千万癫痫患者中，其中约有30%为难治性癫痫，这些难治性癫痫患者中，大部分为颞叶癫痫。癫痫可以导致认知、情感、行为方面异常。据报道，约有半数以上的癫痫患者伴有认知功能障碍。癫痫发作类型、病灶部位及社会心理因素均可影响认知，近几年研究发现抗癫痫药物本身也有导致认知损伤的副作用。因此寻求一种既可以有效抑制癫痫发作同时不会影响认知功能的新型抗癫痫药物成为当务之急。氧化应激参与了许多神经变性疾病的生理病理过程，如肌萎缩侧索硬化（Amyotrophic Lateral Sclerosis,ALS）、帕金森病（Parkinson’sdisease, PD）、阿尔茨海默病（Alzheimer’s disease, AD）。近来研究发现氧化应激同样存在于癫痫的发病机制之中。癫痫发作过程伴有氧自由基生成增多，而脑组织抗氧化系统相对薄弱，当自由基的生成超过抗氧化能力时，导致大分子物质如脂类、蛋白和DNA的损伤。在不同的癫痫模型中均证实了氧化损伤的存在。颞叶癫痫伴有海马部位神经元损伤，这既是癫痫发作的结果，又可导致潜伏期神经网络环路重建，继而出现反复惊厥发作。氧化应激与产生的大量活性氧簇能通过脂质过氧化反应，激活非选择性钙通道，调节细胞凋亡相关基因诱导海马神经元发生凋亡。PI3K/Akt信号通路具有调节细胞凋亡、增殖、代谢和分化的生理作用, Akt活化对氧化应激诱导的细胞凋亡有很好的保护作用。研究发现抗氧化剂维生素C、维生素E可以有效抑制动物模型中癫痫发作。近年来，具有抗氧化作用的中药单体被广泛用于抗癫痫药物筛选。在动物试验中积雪草（centella asiatica）、姜黄素（curcumin）、马齿苋（bacopa monnieri）均证明可以有效抑制癫痫发作及减轻氧化损伤。(-)表没食子儿茶素没食子酸酯（epigallocatechin-3-gallate，EGCG），是绿茶中主要的儿茶素组分，具有抗氧化、抗炎、抗凋亡等多种功效。EGCG抑制脂质过氧化功效比维生素E和维生素C分别高16倍和18倍。EGCG可以通过直接作用于自由基、与金属离子络合、激活体内抗氧化酶活性等多种方式起到抗氧化作用。研究发现EGCG可以透过血脑屏障而直接发挥神经保护作用。同时，EGCG可以改善酒精、糖尿病及铅中毒所导致的认知功能损伤，正常大鼠给予EGCG同样可以有效提高认知功能。EGCG还可以对脑缺血、脊髓损伤起到神经保护作用。而EGCG对于癫痫的保护作用未见报道，有鉴于此，本研究通过建立戊四氮（pentylenetetrazoloe，PTZ）点燃慢性癫痫大鼠模型，给予EGCG进行干预，观察EGCG对戊四氮致痫大鼠行为学、认知能力及对海马神经元的影响，评价EGCG的保护作用并进一步探讨其可能的作用机制。第一部分EGCG对戊四氮致痫大鼠的抗癫痫作用及机制探讨目的：建立戊四氮（pentylenetetrazoloe，PTZ）点燃慢性癫痫大鼠模型，观察EGCG对戊四氮致痫大鼠行为学的影响及大鼠脑内谷胱甘肽（glutathione，GSH）和丙二醛（malondialdehyde，MDA）的变化情况，评价EGCG的抗癫痫作用并对其机制进行初步探讨。方法：7-8周龄清洁级健康雄性Sprague-Dawley(SD)大鼠随机分为五组：模型组（PTZ组）；正常对照组（CON组）；25mg/kg EGCG+PTZ组；50mg/kg EGCG+PTZ组；50mg/kg EGCG组。EGCG和PTZ于每次给药前溶于生理盐水新鲜配制。每日于08：00-09：00之间进行药物注射。PTZ组大鼠隔日腹腔注射1%PTZ(35mg/kg),共13次，注射PTZ前给予0.9%生理盐水3.5ml/kg腹腔注射；CON组大鼠隔日腹腔注射生理盐水3.5ml/kg，共13次；PTZ+25mg/kg EGCG组大鼠除隔日腹腔注射1%PTZ35mg/kg外，每日给予25mg/kg EGCG腹腔注射；PTZ+50mg/kg EGCG组大鼠除隔日腹腔注射1%PTZ35mg/kg外，每日给予50mg/kg EGCG腹腔注射；EGCG于PTZ给药前30分钟腹腔注射；50mg/kg EGCG组：每日给予EGCG50mg/kg腹腔注射。注射结束后观察半小时。行为学分级标准依据Racine分级，分为5级:0级：无任何反应，呈正常的行为状态；1级：湿狗样抖动、面肌痉挛如眨眼、动须及节律性咀嚼；2级：颈部肌肉痉挛表现为点头和（或）甩尾；3级：一侧前肢阵挛；4级：双侧前肢阵挛伴站立；5级：全身阵挛，失去平衡，跌倒。同时观察肌阵挛潜伏期、癫痫大发作潜伏期和癫痫大发作持续时间。给药结束后24小时，各组大鼠断头处死，全脑组织-80℃冻存，测定全脑GSH和MDA含量。结果：动物一般情况：PTZ组死亡率为30%，PTZ+25mg/kg EGCG组死亡率为20%，正常对照组、PTZ+50mg/kg EGCG组和50mg/kgEGCG组大鼠无死亡。重复给予低剂量PTZ腹腔注射后，大鼠发作等级逐渐升高，最终导致5级大发作。EGCG干预可有效降低癫痫大鼠发作等级（F(2,22)=25.073, p<0.001）且呈剂量相关性（F(1,16)=7.586,P<0.05），在对照组和单纯EGCG给药组大鼠无癫痫发作。癫痫组大鼠肌阵挛潜伏期为114.38±12.99s，PTZ+25mg/kgEGCG组和PTZ+50mg/kgEGCG组肌阵挛潜伏期分别为：142.20±20.59s和164.51±22.88s。EGCG显著延长了癫痫大鼠肌阵挛潜伏期(F(2,24)=13.07, p<0.001）且呈剂量相关性。癫痫组大鼠癫痫大发作潜伏期为184.55±52.51s, PTZ+25mg/kg EGCG组和PTZ+50mg/kgEGCG组癫痫大发作潜伏期分别为：243.38±33.30s和307.76±49.60s。EGCG剂量依赖性的延长了癫痫大鼠癫痫大发作潜伏期（F(2,20)=13.056,P<0.001）。癫痫组大鼠癫痫大发作持续时间为21.82±5.85s, PTZ+25mg/kg EGCG组和PTZ+50mg/kg EGCG组癫痫大发作持续时间分别为16.67±2.35s和14.29±1.38s。EGCG显著缩短了癫痫大发作持续时间(p<0.05)，PTZ+25mg/kg EGCG和PTZ+50mg/kgEGCG两组间无明显统计学差异（p>0.05）。GSH在癫痫组和正常对照组含量分别为：3.42±0.20mg/g prot和6.89±0.17mg/g prot。癫痫组大鼠与正常对照组比较，GSH水平明显降低（p<0.05）。MDA在癫痫组和正常对照组含量分别为：4.66±0.17nmol/mg prot和1.99±0.13nmol/mg prot。癫痫组大鼠与正常对照组比较，MDA水平显著升高（p<0.05）。GSH含量在PTZ+25mg/kg EGCG组和PTZ+50mg/kgEGCG组分别为:4.79±0.11mg/g prot和5.36±0.30mg/g prot。与癫痫组比较，EGCG干预后剂量相关性的显著升高了GSH水平（F(4,38)=382.881, p<0.001）。MDA含量在PTZ+25mg/kg EGCG组和PTZ+50mg/kg EGCG组分别为：3.23±0.12nmol/mg prot和2.57±0.27nmol/mg prot。与癫痫组大鼠相比，EGCG干预后剂量相关性的降低了MDA水平(F(4,38)=287.498, p<0.001)。单纯给予EGCG组与对照组相比MDA与GSH水平无明显统计学意义（p>0.05）。结论：本实验采用戊四氮点燃方法成功建立了大鼠慢性癫痫模型，EGCG干预后可明显降低癫痫发作级别，延长癫痫发作潜伏期和缩短癫痫持续时间，并且EGCG可以显著改善癫痫导致的氧化损伤。说明EGCG可以有效抑制戊四氮诱导的癫痫发作，EGCG抗癫痫作用可能是通过抗氧化作用来实现的。第二部分EGCG对戊四氮致痫大鼠认知功能的改善作用及机制探讨目的：观察EGCG对戊四氮致痫大鼠认知功能及海马突触超微结构的影响，评价EGCG对戊四氮致痫大鼠认知功能的改善作用并对其机制进行初步探讨。方法：7-8周龄清洁级健康雄性Sprague-Dawley(SD)大鼠随机分为五组：模型组（PTZ组）；正常对照组（CON组）；25mg/kg EGCG+PTZ组；50mg/kg EGCG+PTZ组；50mg/kg EGCG组。EGCG和PTZ于每次给药前溶于生理盐水新鲜配制。每日于08：00-09：00之间进行药物注射。PTZ组大鼠隔日腹腔注射1%PTZ(35mg/kg)，共13次，注射PTZ前给予0.9%生理盐水3.5ml/kg腹腔注射；CON组大鼠隔日腹腔注射生理盐水3.5ml/kg，共13次；PTZ+25mg/kg EGCG组大鼠除隔日腹腔注射1%PTZ35mg/kg外，每日给予25mg/kg EGCG腹腔注射；PTZ+50mg/kg EGCG组大鼠除隔日腹腔注射1%PTZ35mg/kg外，每日给予50mg/kg EGCG腹腔注射；EGCG于PTZ给药前30分钟腹腔注射；50mg/kg EGCG组：每日给予EGCG50mg/kg腹腔注射。最后一次注射后24h，行Morris水迷宫检测，实验包括（1）定位航行实验：用于测试大鼠的学习能力。正式实验历时5天。每天分上、下午两个时间段，每段训练4次，中间间隔>2h。训练时随机选取一个象限中心作为入水点，将大鼠面向池壁轻轻放入水中，每个时间段4次训练分别从4个不同的入水点入水。记录大鼠从入水到爬上水下平台的时间，即逃避潜伏期。如120s大鼠仍未找到平台，由操作者将大鼠引上平台休息30s，并将潜伏期记录为120s，两次训练中间间隔60s；（2）空间探索实验：用于测试大鼠的空间记忆能力。定位航行实验完毕的第二天，将平台撤除，选定和平台区域相对的象限中点为入水点，记录大鼠在120s内为搜寻平台而在平台象限的游泳时间；（3）可视平台实验：为排除实验处理因素对动物实验感觉、视觉、知觉及运动功能的差异对空间学习记忆的影响，采用可视平台对大鼠进行测试。平台升至液面上2cm，每只大鼠释放4次，分别从不同的4个入水点入水，记录大鼠逃避潜伏期及游泳速度。行为学测试结束后，每组大鼠各取3只，10%水合氯醛麻醉，4%多聚甲醛（含2.5%戊二醛）灌注固定，利用透射电子显微镜观察各组大鼠海马CA1区突触超微结构变化。结果：水迷宫结果：各组大鼠随着训练天数的增加，逃避潜伏期逐渐缩短，说明各组大鼠对隐藏平台形成了记忆。各组间及训练天数间均有统计学意义(F(4,40)=21.20, p<0.001),(F(4,160)=301.015,p<0.001)。癫痫组大鼠与正常对照组大鼠相比，逃避潜伏期明显延长（p<0.05）；与癫痫组相比，EGCG干预后显著缩短了潜伏期（p<0.05），且与正常对照组比较无显著差异（25mg/kg EGCG+PTZ vs CON,p=0.177;50mg/kg EGCG+PTZ vs CON, p=0.192）。在平台试验中，各组比较有显著统计学意义（F (4,44)=6.071, p<0.05）。癫痫组大鼠与正常对照组大鼠相比，目标平台停留时间明显缩短（p<0.05）；与癫痫组相比，EGCG干预后明显提高目标象限百分比（p<0.05），且与正常对照组相比无明显统计学意义（25mg/kg EGCG+PTZ vs CON,p=0.065, EGCG+PTZ vs CON, p=0.489）。PTZ+25mg/kg EGCG组与PTZ+50mg/kg EGCG组间相比，逃避潜伏期及目标象限百分比无明显统计学意义（p>0.05）。各组游泳速度无明显统计学意义（p>0.05）。单纯给予EGCG组与正常对照组比较，无明显统计学意义（p>0.05）。在可视平台试验中，各组间逃避潜伏期和游泳速度无明显统计学差异（p>0.05）。海马超微结构结果：突触间隙宽度结果：PTZ：26.79±1.92nm; CON:21.60±2.29nm；PTZ+25mg/kg EGCG:23.25±1.90nm;PTZ+50mg/kg EGCG:22.52±2.80nm；50mg/kg EGCG：21.81±1.41nm。各组比较有显著统计学差异F(4,99)=19.903, p<0.001。癫痫组大鼠与正常对照组相比，突触间隙明显增宽（p<0.05）；EGCG干预后显著缩短了突触间隙（p<0.05）；PTZ+25mg/kg EGCG、PTZ+50mg/kg EGCG和正常对照组间无显著差异（p>0.05）。活性带长度结果： PTZ:257.58±17.58nm； CON:319.00±10.66nm；PTZ+25mg/kg EGCG：308.94±17.24nm；PTZ+50mg/kg EGCG:313.06±15.06nm；50mg/kg EGCG：314.11±13.86nm。各组比较有显著统计学差异：F(4,99)=56.591, p<0.001。癫痫组大鼠与正常对照组相比，活性带长度明显减少（p<0.05）；EGCG干预后显著增加了活性带长度（p<0.05）；PTZ+25mg/kg EGCG、PTZ+50mg/kg EGCG和正常对照组间无显著差异（p>0.05）。突触后致密物厚度结果：PTZ:24.28±2.63nm；CON:32.14±3.32nm；PTZ+25mg/kg EGCG:29.99±3.93nm；PTZ+50mg/kg EGCG：31.14±3.32nm；50mg/kgEGCG：30.32±2.01nm。各组比较有显著统计学差异：F(4,99)=21.150,p<0.001。癫痫组大鼠与正常对照组相比，突触后致密物厚度明显降低（p<0.05）；EGCG干预后显著增加了突触后致密物厚度（p<0.05）；PTZ+25mg/kg EGCG、PTZ+50mg/kg EGCG和正常对照组间无显著差异（p>0.05）。界面曲率结果：PTZ：1.07±0.04; CON:1.11±0.05;PTZ+25mg/kg EGCG：1.09±0.07; PTZ+50mg/kg EGCG:1.1±0.05；50mg/kg EGCG：1.10±0.06。各组之间无统计学意义（p>0.05）。单纯给予EGCG组与对照组相比，各项指标无明显统计学差异（p>0.05）。结论：戊四氮致痫大鼠的认知能力明显受损且伴有海马超微结构改变。EGCG干预后明显改善了癫痫大鼠学习记忆能力，同时对海马超微结构起到了保护作用，而EGCG本身对认知功能无明显损伤。说明EGCG可以有效改善癫痫大鼠的认知功能，这种保护作用可能是通过对海马超微结构保护来实现的。第三部分：EGCG对戊四氮致痫大鼠海马神经元的保护作用及机制探讨目的：观察EGCG对癫痫后大鼠海马神经元及PI3K/Akt信号通路，凋亡相关蛋白Bax、Bcl-2、caspase-3的影响，评价EGCG对戊四氮致痫大鼠海马神经元的保护作用并对其机制进行初步探讨。方法：7-8周龄清洁级健康雄性Sprague-Dawley(SD)大鼠随机分为四组：模型组（PTZ组）；正常对照组（CON组）；25mg/kg EGCG+PTZ组；50mg/kg EGCG+PTZ组。EGCG和PTZ于每次给药前溶于生理盐水新鲜配制。每日于08：00-09：00之间进行药物注射。PTZ组大鼠隔日腹腔注射1%PTZ(35mg/kg)，共13次，注射PTZ前给予0.9%生理盐水3.5ml/kg腹腔注射；CON组大鼠隔日腹腔注射生理盐水3.5ml/kg，共13次；PTZ+25mg/kg EGCG组大鼠除隔日腹腔注射1%PTZ35mg/kg外，每日给予25mg/kg EGCG腹腔注射；PTZ+50mg/kg EGCG组大鼠除隔日腹腔注射1%PTZ35mg/kg外，每日给予50mg/kg EGCG腹腔注射；EGCG于PTZ给药前30分钟腹腔注射。最后一次给药后24小时，每组取3只大鼠灌注取脑，Nissl染色观察大鼠海马神经元损伤情况，测定CA1区和CA3区神经元存活数；同时每组另取3只大鼠，断头取脑，分离海马，用Wester blot方法检测海马Bcl-2、Bax、Caspase-3、p-PI3K、p-Akt蛋白水平变化。结果：正常对照组大鼠海马CA1和CA3区神经细胞结构完整，染色质分布均匀，核仁清晰，胞浆内尼氏小体丰富，没有明显的神经元丢失；与对照组相比，癫痫组大鼠神经元丢失明显，表现为细胞皱缩，染色质凝集成块、核固缩、尼氏小体数量明显减少；EGCG干预组海马细胞边缘清晰，结构正常，仅少量染色质凝集，尼氏小体数量较癫痫组显著增加。各组神经元存活数：CA1区：PTZ:137.7±25.36;CON:235.8±20.43； PTZ+25mg/kg EGCG:217.9±11.14;PTZ+50mg/kg EGCG：223.5±20.82；CA3区： PTZ:142.2±23.63;CON:252.6±17.02；PTZ+25mg/kg EGCG：233.8±13.96; PTZ+50mg/kg EGCG:240.7±18.0。与对照组相比，癫痫组大鼠海马CA1区和CA3区海马神经元数量明显减少（p<0.05）；EGCG干预后明显增加了神经元数量（p<0.05）；PTZ+25mg/kg EGCG组、PTZ+50mg/kgEGCG组及对照组之间无明显统计学意义（p>0.05）。凋亡相关蛋白结果：Bcl-2、Bax及caspase3蛋白表达水平以β-actin作为参照。Bcl-2：CON：0.72±0.03；PTZ：0.50±0.04; PTZ+25mg/kg EGCG：0.65±0.07；PTZ+50mg/kg EGCG：0.70±0.08。癫痫组大鼠与正常对照组相比，Bcl-2水平明显降低（p<0.05）；EGCG干预后可显著升高Bcl-2蛋白水平（p<0.05）；PTZ+25mg/kg EGCG组、PTZ+50mg/kg EGCG组及对照组之间无明显统计学意义(p>0.05)。Bax蛋白：CON：0.48±0.02；PTZ：0.60±0.03； PTZ+25mg/kg EGCG：0.52±0.01; PTZ+50mg/kgEGCG：0.50±0.03。癫痫组大鼠与正常对照组相比，Bax水平明显升高（p<0.05）；EGCG干预后可显著降低Bax蛋白水平（p<0.05）；PTZ+25mg/kg EGCG组、PTZ+50mg/kg EGCG组及对照组之间无明显统计学意义(p>0.05)。caspase3蛋白：CON：0.28±0.02；PTZ：0.54±0.03；PTZ+25mg/kg EGCG：0.35±0.03; PTZ+50mg/kg EGCG：0.30±0.01。癫痫组大鼠与正常对照组相比，caspase3蛋白水平明显升高（p<0.05）；EGCG干预后可显著降低caspase3蛋白水平（p<0.05）；PTZ+25mg/kg EGCG组、PTZ+50mg/kg EGCG组及对照组之间无明显统计学意义(p>0.05)。PI3K/Akt信号通路变化：p-PI3K、p-AKt蛋白水平表达分别以磷酸化蛋白与总蛋白比值表示。p-PI3K蛋白：CON：0.74±0.05; PTZ:0.45±0.08；PTZ+25mg/kg EGCG：0.64±0.05;PTZ+50mg/kg EGCG：0.70±0.07。癫痫组大鼠与正常对照组相比，p-PI3K p85水平明显降低（p<0.05）；与癫痫组相比，EGCG干预组p-PI3K p85水平显著升高（p<0.05）；PTZ+25mg/kg EGCG组、PTZ+50mg/kg EGCG组及对照组之间无明显统计学意义（p>0.05）。PI3K p85总蛋白水平各组间无明显统计学差异(p>0.05)。p-Akt蛋白：CON：0.78±0.02；PTZ：0.46±0.04；PTZ+25mg/kg EGCG：0.72±0.03；PTZ±50mg/kg EGCG：0.75±0.04。癫痫组大鼠与正常对照组相比，p-Akt水平明显降低(p<0.05)；与癫痫组相比，EGCG干预组可显著升高p-Akt水平（p<0.05）；PTZ+25mg/kg EGCG组、PTZ+50mg/kgEGCG组及对照组之间无明显统计学意义（p>0.05）。Akt总蛋白水平各组间无明显统计学意义(p>0.05)。结论：戊四氮点燃慢性癫痫模型很好模拟了人类海马神经元损伤，EGCG对癫痫大鼠海马神经元具有保护作用，这种保护作用可能是通过激活PI3K/Akt信号通路进而抑制线粒体凋亡途径来实现的。更多还原

【Abstract】 Epilepsy, one of the most common neurological disorders, affects1%ofthe world population. Among the50million epilepsy patients, about30%suffering with intractable epilepsy, most of which were temple lobe epilepsy.Many patients with epilepsy have been diagnosed with affective andpersonality disorders. Clinical investigations have shown that over half of theepileptic patients suffer from cognitive impairment. A variety of factory canadversely affect the cognition in patients with epilepsy, including the etiologyof the seizures, seizure type, and psychosocial problems. Another factor thatmay affect cognition is antiepileptic drugs (AEDs). Thus there is an urgentneed to find new drugs that can suppress seizures effectively and preventcognitive decline in the meantime.Oxidative stress, which has has been recognized to play an importantrole in pathophysiology of neurological diseases such as Amyotrophic LateralSclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD)，aswell as in epilepsy pathophysiolocy. Reactive oxygen species have beenshown in the initial phases of seizure. When the free radical generationexceeds the antioxidant defence, oxidative damage of cellular maromoleculesincluding DNA, cell lipids, protein will be happen. Lots studies havedemonstrated oxidative damage in epileptic rat model.Temporal lobe epilepsy is most commonly associated with hippocampusneuron loss. This is a result of epilepsy but also can reorganize the networkand induce seizures recrudescence. Seizures induce a mixed pattern of celldeath that includes features consistent with both apoptosis and necrosis.Mitochondrion linked apoptosis like signaling pathways has been describedafter seizures. The Bcl-2family comprises proapoptotic and antiapoptoticproteins. The disorder of the ratio of Bcl-2to Bax will increase the mitochondrial membrane permeability and release cytochrome c frommitochondria, and then activate caspase-3to induce apoptosis.Researches have shown that antioxidative agent such as VitC, VitE couldeffectively suppress epilepsy in animal models. As a result, medicinal plantsrecently have given particular attention as a protective agent against epilepsyand oxidative stress. Centella asiatica, curcumin and Emblica officinalis havebeen shown could suppress epilepsy effectively and mitigate oxidative damage.Epigallocatechin-3-gallate (EGCG), the main polyhenol of green tea, has beencharacterized as having anti-oxidant, anti-inflammatory and anti-apoptoticproterties. EGCG anti-oxidant property may be stronger than VitC and Vit E16times and18times. EGCG could demonstrate the antioxidative propertythrough act on the free radical, iron-chelating and increasing the body’sendogenous antioxidants to reduce oxidative damage. In vivo, EGCG couldcross the blood-brain barrier, direct exerting neuroprotective effects. Recentresearches demonstrated that EGCG could attenuate cognitive impairmentinduced by lead, alcohol and diabetes mellitus. Furthermore, EGCG couldenhance the normal rats’ leanring ability. EGCG also could showneuroprotection effect on cerebral ischemia and spinal cord injury. In our study,we used PTZ kindled rat to evaluate the effects of EGCG on seizures,seizure-induced neuron loss and cognitive impairment and furether explore theunderling mechanisms.Part Ⅰ Effects of EGCG on pentylenetetrazole-induced kindling and theunderlying mechanismsObjective: To establish a chronic epileptic rat model kindled by PTZ andobserve the effects of EGCG on pentylenetetrazole-induced kindling and thechanges of oxidative stress parameter (malondialdenhyde and glutathione) inkindled rats, evaluate the antiepilepsy property of EGCG and further explorethe underlying mechanisms.Methods: Adult male Spraque-Dawley (SD) rats weighting180-220g,obstained from Hebei Medical University, were housed in groups of four tofive per cage in a room that was maintained at a constant temperature and humidity. Prior to the experiments, EGCG and PTZ were dissolved inphysiological saline. Then, PTZ was injected intraperitoneally on alternate dayin a dose of35mg/kg (13injections total), while EGCG was injectedintraperitoneally daily. The administration work was conducted between08:00-09:00AM. The animals were randomly divided into five groups of tenanimals each group. GroupⅠ(control group) received0.9%saline i.p. everyother day (3.5ml/kg,13injections total), GroupⅡ (PTZ group) receivedsaline pretreatment along with PTZ every other day, Group Ⅲ Ⅳ(PTZ+EGCG group) received EGCG pretreatment in doses of25and50mg/kg, respectively in addition to alternate-day PTZ for13injections. In thesegroups, EGCG was given30min before PTZ; GroupⅤ,(EGCG group)EGCG50mg/kg was administered alone. Animals were observed for30minafter each PTZ administration. The latency to myoclonic jerks and thegeneralized tonic clonic seizures (GTCS) as well as duration of GTCS wererecorded. Seizure stage was evaluated using the following scale stage0: noresponse; Stage1: hyperactivity, vibrissae twitching; Stage2: head nodding,head clonus and myoclonic jerk; Stage3: unilateral forelimb clonus; Stage4:rearing with bilateral forelimb clonus; Stage5: generalized tonic-clonicseizure (GTCS) with loss of postural control.24h after the last administion,the animals were sacrificed and the brains were removed and stored at-80℃to evaluate MDA and GSH levels.Results: In the PTZ group and the EGCG25mg/kg+PTZ group,mortality rate was30%and20%respectively. There was no mortality in thegroups administered50mg/kg EGCG group and control animals. The repeatedadministration of subconvulsive PTZ (35mg/kg) induced severe seizuresduring the13kindling injection. Pretreatment with EGCG dose dependentlydecreased the mean seizure stage as compared to the PTZ group (F (2,22)=25.073, p<0.001). In the control and EGCG group, there was no seizureactivity. The latency to myoclonic jerk (F (2,24)=13.07, p<0.001) and GTCS(F (2,20)=13.056, p<0.001) were dose-dependent increased in PTZ+EGCGgroup as compared to the PTZ group. The latency to myoclonic jerk increased from114.38±12.99s in PTZ group to142.20±20.59and164.51±22.88s inthe groups administered EGCG25mg/kg and50mg/kg respectively,meanwhile the latency to GTCS increased from184.55±52.51in the PTZgroup to243.38±33.30and307.76±49.60in the groups administered EGCG25mg/kg and50mg/kg respectively. Our research also showed that EGCGmarkedly decreased the duration of GTCS from21.82±5.85in the PTZ groupto16.67±2.35and14.29±1.38in the PTZ+EGCG25mg/kg and50mg/kgrespectively. The content of GSH in the PTZ group was significantly lowercompared with that of control group(GSH: control:6.89±0.17mg/g prot,n=10; PTZ:3.42±0.20mg/g prot n=7, p<0.05), and the level of MDA in PTZgroup was much higher than that of control group (MDA: control:1.99±0.13nmol/mg prot, n=10; PTZ:4.66±0.17nmol/mg prot, n=7p<0.05), whilepretreatment with EGCG led to a significant increase in GSH level in adose-dependent manner.(PTZ:3.42±0.20mg/g prot, n=7; PTZ+25mg/kgEGCG:4.79±0.11mg/g prot, n=8; PTZ+50mg/kg EGCG:5.36±0.30mg/gprot n=10). A noticeable decrease in the concentration of MDA was also notedwith the application of EGCG as compared to the PTZ group.(PTZ:4.66±0.17nmol/mg prot, n=7; PTZ+25mg/kg EGCG:3.23±0.12nmol/mg prot,n=8; PTZ+50mg/kg EGCG:2.57±0.27nmol/mg prot n=10). EGCG per secaused no change as compared to the control group (p>0.05).Conclusion: PTZ induced kindling provides a useful model of postseizuredysfunction. The present study showed that EGCG could effectively decreasethe mean seizure stage and the duration of GTCS, EGCG also could increasethe latency to myoclonic jerk and latency to GTCS. We also found that EGCGcould ameliorate the oxidative damage induced by seizures. Thus, EGCGcould successfully suppress PTZ induced kindling and this may be through itsanti-oxidative property.PartⅡ Effects of EGCG on pentylenetetrazole-induced cognitive impairmentand the underling mechanismsObjective: To observe the effects of EGCG on pentylenetetrazole inducedcognitive impairment and synaptic ultrasturcture changes, evaluate the anti-dementia property of EGCG and further explore the underlyingmechanisms.Methods: Adult male Spraque-Dawley (SD) rats weighting180-220g,obstained from Hebei Medical University, were housed in groups of four tofive per cage in a room that was maintained at a constant temperature andhumidity. Prior to the experiments, EGCG and PTZ were dissolved inphysiological saline. Then, PTZ was injected intraperitoneally on alternate dayin a dose of35mg/kg (13injections total). While EGCG was injectedintraperitoneally daily. The administration work was conducted between08:00-09:00AM. The animals were randomly divided into five groups of tenanimals each group. GroupⅠ(control group) received0.9%saline i.p. everyother day (3.5ml/kg,13injections total), GroupⅡ (PTZ group) receivedsaline pretreatment along with PTZ every other day, Group Ⅲ Ⅳ(PTZ+EGCG group) received EGCG pretreatment in doses of25and50mg/kg, respectively in addition to alternate-day PTZ for13injections. In thesegroups, EGCG was given30min before PTZ; GroupⅤ,(EGCG group)EGCG50mg/kg was administered alone.24h after the last administration,Morris water maze test was performed. Place navigation test was to test therats’ learning ability. Before the traning started, rats were allowed to swimfreely in the pool for120s without platform. Rats were given two sessions perday for5days. Each session comprised four trials, with an intertribal intervalof60s, and the intersession interval was>2h. In each trail, the rat was gentlyplaced into the pool at the middle of the circular edge in a randomly selectedquadrant, with the nose pointing toward the wall. If rats could not find escapeto the platform within120s by themselves, they were placed on the platformby hand and allowed to remain there for30s and their escape latency wasaccepted as120s. After climbing onto the platform, the animal remained therefor30s before the commencement of the next trial. Spatial probe test: on thesixth day, a probe trial without the platform was assessed, and the time spentin the target quadrant where the platform had been located was recorded.Visible platform trial: to exclude the sensorimotor or motivational factors in rats on learning performance, we added the visible trial on the last day. Ratswere given four trials per day similar to those described above for the hiddenplatform trial, but the escape platform was elevated above water surface2cm,the escape latency and the swimming speed were recorded. After the behaviortest, three rats from each group were anesthetized by10%chloral hydrate andperfused with4%paraformaldehyde solution (containing2.5%glutaraldehyde). The synaptic ultrastructure of the hippocampal CA1are wereobserved through the electron microscopy.Results: In the Morris water maze, all animals showed a progressivedecline in the escape latency with training, and main effects for day (F (4,160)=301.015, p<0.001) and for group (F (4,40)=21.10, p<0.001) were significant.Rats in PTZ group exhibited a significant prolonged of escape latency duringall sessions compared with control group (p<0.05). However, the poorperformance was mitigated by the pretreated with EGCG. These two groupsperformed equivalently to the control group (25mg/kg EGCG+PTZ vs CON,p=0.177;50mg/kg EGCG+PTZ vs CON, p=0.192). On the probe trial, withthe platform removed, there was a significant difference among groups (F (4,44)=6.071, p<0.05). The PTZ group spent significantly less time in the targetquadrant than the control group (p<0.05). In25mg/kg and50mg/kgpre-treatment EGCG groups, the deficits were significantly improved andshowed no difference from control group (25mg/kg EGCG+PTZ vs CON,p=0.065, EGCG+PTZ vs CON, p=0.489). For swimming speed, no significantdifferences were found among the five groups (F (4,38)=1.066, p>0.05). Inaddition, EGCG per se had no effect on cognition. There was no significantdifference in the visible plat form among the groups (p>0.05). The results ofthe width of synaptic cleft: there was a significant difference among thegroups (F(4,99)=19.903, p<0.001), kindled rats significantly enlarged thewidth of synaptic cleft compared to the normal group (P<0.05)(PTZ:26.79±1.92nm; CON:21.6045±2.29nm), while EGCG significantly reduced thesynaptic cleft compared to the PTZ group(p<0.05)(PTZ+25mg/kg EGCG:23.25±1.90nm; PTZ+50mg/kg EGCG:25.52±2.80nm), there was no statistic difference among PTZ+25mg/kg EGCG, PTZ+50mg/kgEGCG andcontrol group; The results of the length of active zone: there was a significantdifference among the group (F(4,99)=56.591, p<0.001), kindled ratssignificantly reduced the active zone compared to the controlgroup(p<0.05)(PTZ:257.17.58±17.58nm; CON:319.00±10.66nm), whileEGCG significantly increased the active zone compared to the PTZgroup(p<0.05)(PTZ+25mg/kg EGCG:308.94±17.24nm; PTZ+50mg/kgEGCG:313.06±15.06nm), there was no statistic difference amongPTZ+25mg/kg EGCG, PTZ+50mg/kgEGCG and control group. Results of thethickness of PSD: there was a significant difference among the groups(F(4,99)=21.150, p<0.001), kindled rats significantly reduced the thickness ofPSD compared to the control group(p<0.05)(PTZ:24.28±2.63nm; CON:32.14±3.32nm), while EGCG significantly increase the PSD thicknesscompared to the PTZ group (p<0.05)(PTZ+25mg/kg EGCG:29.99±3.93nm;PTZ+50mg/kg EGCG:31.14±3.32nm), there was no statistic differenceamong PTZ+25mg/kg EGCG, PTZ+50mg/kgEGCG and control group.Theresults of the synaptic curvature: there was no significant difference among thegroup (p>0.05).(PTZ:1.07±0.41; CON:1.11±0.52; PTZ±25mg/kg EGCG:1.09±0.79; PTZ+50mg/kg EGCG:1.1±0.50). Furthermore, there was nostatistic difference between the EGCG group and control group among thesynaptic parameter (p>0.05).Conclusion: PTZ kindled rat impaired spatial learning and memory withthe abnormal changes in synaptic ultrastructure in hippocampal CA1area.EGCG could ameliorate the cognitive impairment and protect the synapticultrastructure. There might be a close relationship between protective effectsof synaptic ultrastructure and anti-dementia property of EGCG. Furthermore,EGCG per se had no significant effects on cognition.PartⅢ Effects of EGCG on PTZ induced neuron loss and the underlyingmechanisms.Objective: To observe the effects of EGCG on hippocampal neuron lossinduced by PTZ and the effects of EGCG on the PI3K/Akt signaling pathway and the mitochondrial apoptosis-related protein, then evaluate theneuroprotective effects of EGCG and further explore the underlyingmechanisms.Methods: Adult male Spraque-Dawley (SD) rats weighting180-220g,obstained from Hebei Medical University, were housed in groups of four tofive per cage in a room that was maintained at a constant temperature andhumidity. Prior to the experiments, EGCG and PTZ were dissolved inphysiological saline. Then, PTZ was injected intraperitoneally on alternate dayin a dose of35mg/kg (13injections total), while EGCG was injectedintraperitoneally daily. The administration work was conducted between08:00-09:00AM. The animals were randomly divided into four groups of tenanimals each group. GroupⅠ(control group) received0.9%saline i.p. everyother day (3.5ml/kg,13injections total), GroupⅡ (PTZ group) receivedsaline pretreatment along with PTZ every other day, Group Ⅲ Ⅳ(PTZ+EGCG group) received EGCG pretreatment in doses of25and50mg/kg, respectively in addition to alternate-day PTZ for13injections. In thesegroups, EGCG was given30min before PTZ.24h after the last adiminstration,Nissl staining was performed to examine the number of surving neurons inhippocampal CA1and CA3regions in rats. The changes of phosphor-PI3Kp85, phosphor-Akt, Bax, Bcl-2and caspase-3in hippocampus were test bywestern blot.Results: Nissl staining showed that: neurons in CA1and CA3of controlgroup were clear with normal nucleolus, well-distributed karyotin and richnissl bodies in kytoplasm, there was no significantly neuron loss. While in thekindled rats, neuron loss was obviously, with shrunken plasma body andpyknotic nuclei. In the EGCG group, most pyramid cells were normal andonly a few showed chromatin condensation. The number of surviving neurons:compared to the control group, the number of surving neurons in the PTZgroup was significantly decreased (p<0.05)(CA1: PTZ:137.7±25.36; CON:235.8±20.43)(CA3: PTZ:142.2±23.63; CON:252.6±17.02), comparedwith the PTZ gourp, EGCG significantly reduced the neuron loss(p<0.05) (CA1: PTZ+25mg/kg EGCG:217.9±11.14; PTZ+50mg/kg EGCG;223.5±20.82)(CA3: PTZ+25mg/kg EGCG:233.8±13.96; PTZ+50mg/kg EGCG:240.7±18.03). There was no statistic difference among PTZ+25mg/kg EGCG,PTZ+50mg/kgEGCG and control group. Bcl-2protein level: CON:0.72±0.03; PTZ:0.50±0.04; PTZ+25mg/kg EGCG:0.65±0.07; PTZ+50mg/kgEGCG=0.70±0.08. Bax protein level: CON:0.48±0.02; PTZ:0.60±0.03;PTZ+25mg/kg EGCG:0.52±0.01; PTZ+50mg/kg EGCG:0.50±0.03;caspase3protein level: CON:0.28±0.02; PTZ:0.54±0.03; PTZ+25mg/kgEGCG:0.35±0.03; PTZ+50mg/kg EGCG:0.30±0.01. Compared with thecontrol group, the protein level of Bcl-2was significantly lower, while theprotein levels of Bax and caspase-3were significantly higher in the PTZgroup(p<0.05). Compared with the PTZ group, the protein level of Bcl-2wasmarkedly increased, while the protein levels of Bax and caspase-3weremarkedly decreased in the EGCG group (p<0.05). There was no statisticdifference among the PTZ+25mg/kg EGCG, PTZ+50mg/kg EGCG andcontrol group. p-PI3K protein level: CON:0.74±0.05; PTZ:0.45±0.08;PTZ+25mg/kg EGCG:0.64±0.05; PTZ+50mg/kg EGCG:0.70±0.07. p-Aktprotein level: CON:0.78±0.02; PTZ:0.46±0.04; PTZ+25mg/kg EGCG:0.72±0.03; PTZ±50mg/kg EGCG:0.75±0.04. Compared to the controlgroup, the protein levels of p-PI3K and p-Akt were significantlty decreased inthe PTZ group(p<0.05), while the protein levels of p-PI3K and p-Akt weremarkedly increased after EGCG adiminstration (p<0.05), there was no statisticdifference among the group PTZ+25mg/kg EGCG, PTZ+50mg/kg EGCGand control group. There was no significant dfference of the total protein ofPI3K and Akt among the groups.Conclusion: There was a significant neuron loss in PTZ kindled rats.EGCG could attenuate the deficits, and the neuroprotective effects may bethrough promoting the PI3K/Akt signaling pathway and inhibiting themitochondrial apoptosis pathway.更多还原