EGCG对戊四氮致痫大鼠的保护作用及机制探讨

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英文题名：Protective Effects and Mechanisms of EGCG on Pentylenetetrazole-induced Seizures in Rats 作者：谢涛 论文级别：博士 学科专业名称：神经病学 中文关键词：戊四氮 ; 癫痫 ; 氧化应激 ; EGCG ; 学习记忆 ; 神经保护作用 英文关键词：PTZ ; epilepsy ; oxidative stress ; EGCG ; learning and 英文关键词：memory ; neuroprotective effect 学位年度：2012 导师：王维平 学科代码：100204 学位授予单位：河北医科大学 论文提交日期：2012-04-01

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。
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 mechanisms
     Objective: 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 mechanisms
     Objective: 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.

引文

1Prilipko L, de Boer MH, Dua T, et al. Epilepsy Care-TheWHO/ILAE/IBE Global Campaign Against Epilepsy. US NeurologicalDisease,2006:39-40
    2Patel M. Mitochondrial dysfunction and oxidative stress: cause andconsequence of epileptic seizures. Free Radic Biol Med,2004,37:1951-1962
    3Baron M, Kudin AP, Kunz WS. Mitochondrial dysfunction inneurodegenerative disorders. Biochem Soc Trans,2007,35:1228-1231
    4Gao J, Chi ZF, Liu XW, et al. Mitochondrial dysfunction and ultrastructuraldamage in the hippocampus of pilocarpine-induced epileptic rat. NeurosciLett,2007,411:152-157
    5Frantseva MV, Perez Velazquez JL, Tsoraklidis G, et al. Oxidative stress isinvolved in seizure-induced neurodegeneration in the kindling model ofepilepsy. Neuroscience2000,97:431-435
    6Rola R, Swiader M, Czuczwar SJ. Electroconvulsions elevate the levels oflipid peroxidation products in mice. Pol J Pharmacol,2002,54:521-524
    7El-Abhar HS, El. Gawad HMA Modulation of cortical nitric oxide synthase,glutamate, and redox state by nifedipine and taurine in PTZ-kindled mice.Epilepsia,2003,44:276-281
    8Marini H, Costa C, Passaniti M, et al. Levetiracetam protects against kainicacid-induced toxicity. Life Sci,2004,74:1253-1264
    9Gulati K, Ray A, Pal G, et al. Possible role of free radicals intheophylline-induced seizures in mice. Pharmacol Biochem Behav,2005,82:241-245
    10Rajasekaran K. Seizure-induced oxidative stress in rat brain regions:blockade by nNOS inhibition. Pharmacol Biochem Behav,2005,80:263-272
    11Tejada S, Sureda A, Roca C, et al. Antioxidant response and oxidativedamage in brain cortex after high dose of pilocarpine. Brain Res Bull,2007,71:372-375
    12Xavier SM, Barbosa CO, Barros DO, et al. Vitamin C antioxidant effects inhippocampus of adult Wistar rats after seizures and status epilepticusinduced by pilocarpine. Neurosci Lett,2007,420:76-79
    13Barros DO, Xavier SML, Barbosa CO, et al. Effects of the vitamin E incatalase activities in hippocampus after status epilepticus induced bypilocarpine in Wistar rats,2007,416:227-230
    14Reznichenko L, Amit T, Zheng H, et al. Reduction of iron-regulatedamyloid precursor protein and beta-amyloid peptide by(-)-epigallocatechin-3-gallate in cell cultures: implications for ironchelation in Alzheimer’s disease. J Neurochem,2006,97:527-536
    15Higuchi A, Yonemitsu K, Koreeda A, et al. Inhibitory activity ofepigallocatechin gallate(EGCG) in paraquat-induced microsomal lipidperoxidation a mechanism of protective effects of EGCG against paraquattoxicity. Toxicology,2003,183:143-149
    16Nane CL, Shearer WT. Is green tea good for HIV-1infection? Journal ofallergy and clinical immunology,2003,112:851-853
    17Nurulain TZ. Green tea and its polyphenolic catechins: Medicinal uses incancer and noncancer applications. Life Sciences,2006,78:2073-2080
    18Mandel SA, Amit T, Weinreb O, et al. Simultaneous manipulation ofmultiple brain targets by green tea catechins: a potential neuroprotectivestrategy for Alzheimer and Parkinson disease. CNS Neurosci Ther,2008,14:352-365
    19Kang KS, Wen Y, Yamabe N, et al. Dual beneficial effects of(-)-epigallocatechin-3-gallate on levodopa methylation and hippocampalneurodegeneration: in vitro and in vivo studies. Plos One,2010,5:11951
    20Weinreb O, Amit T, Mandel S, et al. Neuroprotective molecularmechanisms of (-)-epigallocatechin-3-gallate: a reflective outcome of itsantioxidant, iron chelating and neuritogenic properties. Genes Nutr2009
    21Racine RJ. Modification of seizure activity by electrical stimulation: ⅡMotor seizure. Electroencephalogr. Clin. Neurophysiol,1972,32:281-294
    22Okhawa H, Ohishi N, Yagi K. Assay of lipid peroxides in animal tissue bythiobarituric acid reaction. Ann. Biochem,1979,95:351-358
    23Ellman GL. Tissue sulphydryl groups. Arch. Biochem. Biophys,1959,82:70-77
    24Goddard GV. Development of epileptic seizures through brain stimulationat low intensity. Nature,1967,214:1020-1021
    25Coulter DA, Mclntyre DC, Loscher W. Animal models of limbi epilepsies:what can they tell us? Brain Pathol,2002,12:240-256
    26Loscher W. Animal models of intractable epilepsy. Prog Neurobiol,1997,53:239-258
    27Park KK, Reuben JS, Soliman KFA. The role of inducible-nitricoxide in cocaine induced kindling. Exp Biol Med,2001,226:185-190
    28Yokoi I, Kabuto H, Akiyama K, et al. Tannins inhibit the occurrence ofepileptic focus induced by Fecl3injection in rats. Jan. J. Psychiatr. Neurol,1989,43:552-553
    29Liu CS, Wu HM, Kao SH, et al. Phenytoin-mediated oxidative stress inserum of female epileptics: a possible pathogenesis in the fetal hydantoinsyndrome Human and Experimental Toxicology,1997,16:1152-1156
    30Ono H, Sakamoto A, Sakura N. Plasma total glutathione concentrations inepileptic patients taking anti-convulsants. Clinica Chimia Acta,2000,298:135-143
    31Tourandokht B, Mehrdad R. Chronic epigalloatechin-3-gallate ameliorateslearning and memory deficits in diatetic rats via modulation of nitric oxideand oxidative stress. Behav Brain Res,2011,224:305-310
    32Tiwari V, Kuhad A, Chopra K. Epigallocatechin-3-gallate amelioratesalcohol-induced cognitive dysfunctions and apoptotic neurodegeneration inthe developing rat brain. Int J Neuropsychoph,2010,13:1053-1066
    33Agarwal NB, Jain S, Agarwak NK. Modulation of pentylenetetrazole-indued kindling and oxidative stress by curcumin in mice. Phytomedicine,2011,18:756-759
    34Mehla J, Reeta KH, Gupta P. Protective effect of curcumin against seizuresand cognitive impairment in a pentylenetetrazole-kindled epileptic ratmodel. Life Sciences,2010,87：596-603
    35Morin F, Beaulieu C, Lacaille JC. Selective loss of GABA neurons in areaCA1of the rat hippocampus after intraventricular entate. Epilepsy Res,1998,32:363-369
    36Loscher W. Animal models of epilepsy for the development ofantiepileptogenic and disease-modifying drugs. A comparison of thepharmacology of kindling and post-status epilepticus models of temporallobe epilepsy. Epilepsy Res,2002,50:105-123
    37Huang LT, Yang SN, Liou CW, et al. Pentylenetetrazol-induced recurrentseizures in rat pups: time course on spatial learning and long-term effects.Epilepsia,2002,43:567-573
    38Dringen R. Metabolism and functions of glutathione in brain. Progress InNeurobiology,2000,62:649-671
    39Vyawahare NS, Khandelwal AR, Batra VR, et al. Herbal anticonvulsants. JHerb Med Toxicol,2007,1:9-14
    40Mathew J, Paul J, Nandhu MS, et al. Bacopa monnieri and Bacoside-A forameliorating epilepsy associated behavioral deficits. Fitoterapia,2010,81:315-322
    41Golechha M, Bhatia J, Arya DS. Hydroalcoholic extract of Emblicaofficinalis Gaertn. Affords protection against PTZ-induced seizures,oxidative stress and cognitive impairment in rats. Indian J Exp Biol,2010,48:474-478
    42Gupta YK, Veerendra Kumar MH, Srivastava AK. Effect of Centellaasiatica on pentylenetetrazole-induced kindling, cognition and oxidativestress in rats. Paharmaol Biochem Behav,2003,74:579-585
    43Young JF, Dragstedt LO, Haraldsdottir J, et al. Green tea extract onlyaffects markers of oxidative status postprandially: lasing antioxidant effectof flavonoid-free diet. Br J Nutr,2002,87:343-355
    44Matsuo N, Yamada K, Shoji K, et al. Effect of tea polyphenols onhistamine release from rat basophilic leukemia (RBL-2H3) cells: thestructure-inhibitory activity relationship. Allergy,1997,52:58-64
    45Guo Q, Zhao B, Li M, et al. Studies on protective mechanisms of fourcomponents of green tea polyphenols against lipid peroxidation insynaptosomes. Biochim. Biophys. Acta,1996,1304:210-222
    46Sano, M, Takahashi Y, Yoshino K, et al. Effect of tea on lipid peroxidationin rat liver and kidney: a comparison of green and black tea feeding. BiolPharm Bull,1995,18:1006-1008
    47Aucamp J, Gaspar A, Hara Y, et al. Inhibition of xanthine oxidase bycatechins from tea (Camellia sinensis). Anticancer Res,1997,17:4381-4386
    48Suganuma M, Okabe S, Oniyama M, et al. Wide distribution of (-)epigallocatechin gallate, a cancer preventive tea polyphenol, in mousetissue. Carcinogenesis,1998,19:1771-1776
    49Schulz JB, Lindenau J, Seyfried J, et al. Glutathione, oxidative stress andneurodegeneration, Eur.J.Biochem,2000,267:4904-4911
    50Yin ST, Tang ML, Su L, et al. Effects of Epigallocatechin-3-gallate onlead-induced oxidative damage. Toxicology,2008,249:45-54
    51Halliwell B. Reactive species and antioxidants. Redox biology is afundamental theme of aerobic life. Plant Physiol,2006,141:312-322
    52Fridovich I. Superoxide anion radical, superoxide dismutases, and relatedmatters. J.Biol. Chem,1997,272:18515-18517
    53Bruce AJ, Baudry M. Oxygen free radicals in rat limbic structures afterkainite-induced seizures. Free Radic Biol Med,1995,18:993-1002
    54Sudha K, Rao AV, Rao A. Oxidative stress and antioxidants in epilepsy.Clinica Chimica Acta,2001,303:19-24
    1Mula M, Trimble MR. Antiepileptic drug-induced cognitive adverse effects.CNS Drugs，2009，23：121-137
    2Carreno M, Donaire A, Sanhez-Carpintero, S. Cognitive disordersassociated with epilepsy: diagnosis and treatment. Neurologist,2008,14:26-34
    3Ortinski P, Measor KJ. Congintive side effects of antiepileptic drugs.Epilepsy Behav,2004,5:60-65
    4Hessen E, Lossius MI, Reinvang I, et al. Influence of major antiepilepticdrugs on attention, reaction time, and speed of information processing:results from a randomized, double-blind, placebo controlled withdrawalstudy of seizure-free epilepsy patients receiving monotherapy. Epilepsia,2006,47:2038-2045
    5Tourandokht B, Mehrdad R. Chronic epigalloatechin-3-gallate ameliorateslearning and memory deficits in diatetic rats via modulation of nitric oxideand oxidative stress. Behav Brain Res,2011,224:305-310
    6Kim TI, Lee YK, Park SG, et al. L-Theanine, an amino acid in green tea,attenuates beta-amyloid-induced cognitive dysfunction and neurotoxicity:reduction in oxidative damage and inativation of ERK/p38kinase andNF-kappaB pathways. Free Radic Biol Med,2009,47:1601-1610
    7Kana KS, Wen Y, Yamabe N, et al. Dual beneficial effects of(-)-epigallocatechin-3-gallate on levodopa methylation and hippocampalneurodegeneration: in vitro and in vivo studies. PLoS One,2010,5:11951
    8Chen WQ, Zhao XL, Hou Y, et al. Protective effects of green teapolyphenols on cognitive impairments induced by psychological stress inrats. Behav Brain Res,2009,202:71-76
    9Haque AM, Hashimoto M, Katakura M, et al. Long-term administration ofgreen tea catechins improves spatial cognition learing ability in rats. J Nutr,2006,136:1043-1047
    10Vrensen G, Nunes, Cardoz, J. Changes in size and shape of synapticconnections after visual training: an ultrastructural approach of synapticplasticity. Brain Res,1981,218:79-99
    11Kong FJ, Xu LH, He DQ, et al. Effets of gestational isoflurane exposure onpostnatal memory and learning in rats. Eur J Pharmacol,2011,670:168-174
    12Yang JH, Liu QF, Zhang LF, et al. Lanthanum chloride impairs memory,decreases pCaMKⅣ, p-MAPK and p-CREB expression of hippocampus inrats. Toxicol Lett,2009,190:208-214
    13Essatarsa MB, Morley JE, Levine AS. The role of the endogenous opiatesin zinc deficiency anorexia. Physiology and Behavior,1984,32:475-478
    14Jones DG, Devon RM. An ultrastructural study into the effects ofpentobarbitone on synaptic organization. Brain Res,1978,147:47-63
    15Guldner FH, Ingham CA. Increase in postsynaptic density material in optictarget neurons of the rat suprachiasmatic nucleus after bilateral enucleation.Neuroscience letters,1980,17:27-31
    16Morris RG. Development of a water-maze procedure for studying spatiallearning in the rat. J Neurosci Methods,1984,11:47-60
    17Wang P, Wang WP, Zhang S, et al. Impaired spatial learning related withdecreased expression of calcium/calmodulin-dependent protein kinaseⅡαand Camp-response element binding protein in the pentylenetetrazol-kindled rats. Brain Res,2008,1238:108-117
    18Lamberty Y, Klitgaard H. Consequenes of pentylenetetrazole kindling onspatial memory and emotional responding in the rat. Epilepsy Behav,2000,1:256-261
    19Hamm RJ, Pike BR, Temple MD, et al. The effect of postinjury kindledseizures on cognitive performance of traumatically brain-injured rats. ExpNeurol,1995,136:143-148
    20Kennedy MB. The postsynaptic density.Curr Opin Neurobiol,1993,3:732-737
    21Ziff EB. Enlightening the postsynaptic density. Neuron,1997,19:1163-1174
    22Okabe S. Molecular anatomy of the postsynaptic density. Mol CellNeurosci,2007,34:503-518
    23Sheng M, Hoogenraad CC. The postsynaptic architecture of excitatorysynapses: a mor quantitative view. Annu Rev Biochem,2007,76:823-847
    24Petukhov VV, Popov VI. Quantitative analysis of ultrastructural changes insynapss of the rat hippocampal field CA3in vitro in different functionalstatus. Neuroscience,1986,18:823-835
    25Nyffeler M, Zhang W, Feldon J. Differential expresson of PSD proteins inage-related spatial learning impairments. Neurobiol Aging,2007,28:143-155
    26Sun QJ, Duan RS, Wang AH, et al. Alterations of NR2B and PSD-95inhippocampus of kainic acid-exposed rats with behavioral deficits. BehavBrain Res,2009,201:292-299
    27Tian Y, Wang Y, Deng Y, et al. Methylphenidate improves spatial memoryof spontaneously hypertensive rats: evidence in behavioral andultrastructural changes. Neurosci Lett,2009,461:106-109
    28Walsh MJ, Kuruc N. The postsynaptic density: constituent and associatedproteins characterized by electrophoresis, immunoblotting, and peptidesequencing. J Neurochem,1992,59:667-678
    29Peters A, Palay SL.1996. The morphology of synapse. J Neurocytol25:687-700
    30Aicardi J, Chevrie JJ. Convulsive status epilepticus in infants and children:A study of239cases. Epilepsia,1970,11:187-197
    31Aminoff MJ, Simon RP. Status epilepticus: Causes, clinical features andconsequences in98patients. Am J Med,1980,69:657-666
    32Delgado-Escueta AV, Treiman DM, Walsh GO. The treatable epilepsies. NEngl J Med,1983,308:1508-1514
    33Harrison RM, Taylor DC. Childhood seizures: a25-year follow up. Socialand medical prognosis. Lancet1976,1:948-951
    34Hodgman CH, McAnamey ER,Myers GJ, et al. Emotional complicationsof adolescent grand mal epilepsy. J Pediatr,1979,95:309-312
    35Williams J, Griebel ML, Dykman RA. Neuropsychological patterns inpediatric epilepsy. Seizure,1998,7:223-228
    36Abrahams S, Pickering A, Polkey C, et al. Spatial memory deficits inpatients with unilateral damage to right hippocampal formation.Neuropsychologia,1997,35:11-24
    37Gripo A, Pelosi L, Metha V, et al. Working memory in temporal lobeepilepsy: an enent related potential study. Electroencephalogr ClinNeurophysiol,1996,99:200-213
    38Kandinov B, Giladi N, Korczyn AD. Smoking and tea consumption delayonset of Parkinson’s disease. Parkinsonism Relat Disord,2009,15:41-46
    39Rasoolijazi H, Joghataie MT, Roghani M, et al. The beneficial effect of(-)-epigallocatechin-3-gallate in an experimental model of Alzheimer’sdisease in rat: a behavioral analysis. Iranian Biome Journal,2007,11:237-243
    40Mandel SA, Amit T, Weinreb O, et al. Simultaneous manipulaton ofmultiple brain targets by green tea catechins: a potential neuroprotectivestrategy for Alzheimer and Parkinson diseases. CNS Neurosci Ther,2008,14:352-365
    41Packard MG, McGaugh JL. Double dissociation of fornix and caudatenucleus lesions on acquisition of two water maze tasks: further evidencefor multiple memory systems. Behav. Neurosci,1992,106:439-446
    42Gupta YK, Veerendra Kumar MH, Srivastava AK. Effect of Centellaasiatica on pentylenetetrazole-induced kindling, cognition and oxidativestress in rats. Paharmaol Biochem Behav,2003,74:579-585
    43Rezai-Zadeh K, Arendash GW, Hou H, et al. Green tea epigallocatechin-3-gallate (EGCG) reduces beta-amyloid mediated cognitive impairment andmodulates tau pathology in Alzheimer transgenic mice. Brain Res,2008,1214:177-187
    44Tiwari V, Kuhad A, Chopra K. Epigalloatechin-3-gallate amelioratesalcohol induced cognitive dysfunctions and apoptotic neurodegeneration inthe developing rat brain. Int J Neuropsychopharmacol,2010,13:1053-1066
    45He M, Zhao L, Wei MJ, et al. Neuroprotective effects of (-)-epigallocatechin-3-gallate on aging mice induced by D-galactose. Biol.Pharm. Bull,2009,32:55-60
    46Jing YH, Wang ZR, Song YF. Quantitative study of aluminum-inducedchanges in synaptic ultrastructure in rats. Synapse,2004,52:292-298
    47Xu XH, Ye, LJ, Ruan Q. Environmental enrichment induces synapticstructural modification after transient focal cerebral ischemia in rats. ExpBiol Med,2009,234:296-305
    1Bertram EH, Lothman EW, Lenn NJ. The hippocampus in experimentalchronic epilepsy. Annals of Neurol,1990,27:43-48
    2DeGiorgio, Chr M, Tomiyasu U, et al. Hippocampal pyramidal cell loss inhuman status epilepticus. Epilepsia,1992,33:23-27
    3Meldrum B. Excitotoxicity and epileptic brain damage. Epilepsy Research,1991,10:55-61
    4Holems GL. Seizure-induced neuronal injury. Neurology,2002,59:3-6
    5Borges K, Gearing M, Mcdermott DL, et al. Neuronal and glialpathological changes during epileptogenesis in the mouse pilocarpinemodel. Exp Neurol,2003,182:21-34
    6Brandt C, Glien M, Potschka H, et al. Epileptogenesis and neuropathologyafter different types of status epilepticus induced by prolonged electricalstimulation of the basolateral amygdale in rats. Epilepsy Res,2003,55:83-103
    7Mikati MA, Bi-Habib RJ, Sabban ME, et al. Hippocampal programmedcell death after status epilepticus: evidence for NMDA-receptor andceramide-mediated mechanisms, Epilepsia,2003,46:282-291
    8Henshall DC, Araki T, Schindler CK, et al. Activation of Bcl-2associateddeath protein and counter-response on AKt within cell populations duringseizure-induece neuronal death, J. Neurosci,2002,22:8458-8465
    9Asnaghi L, Calastretti A, Bevilacqua A, et al. Bcl-2phosphorylation andapoptosis activated by damaged microtubules require Mtor and areregulated by Akt. Oncogene,2004,23:5781-5791
    10Wang XT, Pei DS, Xu J, et al. Oppositing effects of Bad phosphorylation attwo distinct sites by Akt1and JNK1/2on ischemic brain injury. Cell Signal,2007,19:1844-1856
    11Lee SR, Suh S, Kim SP. Protective effects of the green tea polyphenol(-)-epigallocatechin gallate against hippocampal neuronal damage aftertransient global ischemia in gerbils. Neurosci Lett,2000,287:191-194
    12Khalatbary AR, Tiraihi T, Boroujeni MB. Effects of epigallocatechingallate on tissue protection and functional recovery after contusive spinalcord injury in rats. Brain Res,2010,1306:168-175
    13Nie G, Jin C, Cao Y, et al. Distinct effects of tea catechins on6-hydroxydopamine-induced apoptosis in PC12cells. Arch. Biochem.Biophysics,2002,397:84-90
    14Koh SH, Kim SH, Kwon H, et al. Phosphatidylinositol-3kinase/AKt andGSK-3mediated cytoprotetive effect of epigallocatechin gallate onoxidative stress-injured neuronal-differentiated N18D3cells.Neurotoxicoloty,2004,25:793-802
    15Katada T, Kurosu H, Okada T, et al. Synergistic activation of a family ofphosphoinositide3-kinase via G-protein coupled and tyrosinekinase-related receptors. Chem, phys. Lipids,1990,98:79-86
    16Vanhaesebroeck B, Watedfield MD. Signaling by distince classes ofphosphoinositide3-kinase. Exp Cell Res,1999,253:239-254
    17Vanhaesebroeck B, Leevers SJ, Ahmadi K, et al. Synthesis and function of3-phosphorylated inositol lipids. Annu Rev Biochem,2001,70:535-602
    18Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts.Genes Dev,1999,13:2905-2927
    19Hemmings BA. Structure, regulation and function of PKB/AKt-a majortherapeutic target. Cell Mol Biol Lett,203,8:527
    20Burgering BM, Coffer PJ. Protein kinase B (c-AKt) inphosphatidylinositol-3-OH kinas signals transduction. Nature,1995,17:599-602
    21Franke T, Yang S, Chan T, et al. The protein kinase encoded by the Aktprotooncogene is a target of the PDGF-activated phosphatidylinositol3-kinase. Cell,1995,81:727-736
    22Vivanco I, Sawyers CL. The phosphatidylinositol3-kinase AKT pathwayin human cancer. Nat Rev Cancer,2002,2:489-501
    23Pohle W, Becher A, Grecksch G, et al. Piracetam prevents pentylenetetrazolkindling-induced neuronal loss and learning deficits. Seizure,1997,6:467-474
    24Lee SH, Chun W, Kong PJ, et al. Sustained activation of Akt by melatonincontributes to the protection against kainic acid-induced neuronal deathhippocampus. J. Pineal Res,2006,40:79-85
    25Piermartiri TCB, Vandresen-Filho S, Araujo Herculano B, et al.Atorvastatin prevents hippocampal cell death due to quinolinicacid-induced seizures in mice by increasing Akt phosphorylation andglutamate uptake.Neurotox Res,2009,16:106-115
    26Xu JJ, Wang SZ, Lin YT, et al. Ghrelin protects against cell death ofhippocampal neurons in pilocarpine-induced seizures in rats. NeuroscienceLett,2009,453:58-61
    27Xue Y, Xie NC, Lin YT, et al. Role of PI3K/Akt in diazoxidepreconditioning against rat hippocampal neuronal death inpilocarpine-induced seizures. Brain Res,2011,1383:135-140
    28Zhang B, Wong M. Pentylenetetrazole-induced seizures cause acute, butnot chronic, Mtor pathway activation in rat. Epilepsia,2012,53:506-511
    29Kluck RM, Bossy-Wetzel E, Green DR, et al. The release of cytochrome cfrom mitochondria: a primary site for Bcl-2regulation of apoptosis.Science,2997,275:1132-1136
    30Pavlova TV, Yakovlev AA, Stepanichev MY, et al. Pentylenetetrazolekindling induces activation of caspase-3in the rat brain. Neuroscience andBehav Physio,2004,34:45-47
    31Cantley LC. The phosphoinositide3-kinase pathway. Science,2002,296:1655-1657
    32Fruman DA, Cantley LC. Phosphoinositide3-kinase in immunologicalsystems,2002,14:7-18
    33Shi Y. Critical regulation of CD4+T cell survival and autoimmunity bybeta-arrestin1. Nat Immunol,2007,8:817-824
    34Zhang H. Diazoxide preconditioning alleviates caspase-dependent andcaspase-indpendent apoptosis induced by anoxia-reoxygenation of PC12cells. J. Biochem,2010,148:413-421
    35Reed JC, Jurgensmeier JM, Matsyyama S. Bcl-2family proteins andmitochondria. Biophys. Acta,1998,1366:127-137
    36Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2heterodimerizes in vivowith a conserved homolog, Bax, that accelerates programmed cell death.Cell,1993,74:609-619
    37Zha H, Aime-Sempe, Sato T, et al. Proapoptotic protein Baxheterodimerizes with Bcl-2and homodimerizes with Bax via a noveldomain (BH3) distinct from BH1and BH2. J.Biol. Chem,1996,271:7440-7744
    38Sutherland BA, Rahman RM, Appleton I. Mechanisms of ation of green teacatechins, with a focus on ischemia induced neurodegeneration. J. Nutr.Biochem,2006,17:291-306
    39Koh, SH, Lee SM, Kim HY, et al. The effect of epigallocatechin gallate onsuppressing disease progression of ALS model mice. Neurosci. Lett,2006,395:103-107
    40He M, Zhao L, Wet MJ, et al. Neuroprotective effects of(-)-epigallocatechin-3-gallate on aging mie induced by D-galactose. Biol.Pham. Bull,2009,32:55-60
    41Korsmeyer SJ, Wei MC, Saito M, et al. Pro-apoptotic cascade activate BID,which oligomerizes BAK or BAX into pores that result in the release ofcytochrome c. Cell Death Differ,2000,7:1166-1173
    42von Ahsen O, Renken C, Perkins G, et al. Preservation of mitochondrialstructure and function after Bid-or–Bax-mediated cytochrome c release. JCell Biol,2000,150:1027-1036
    43Daata SR, Katsov A, Hu L, et al.14-3-3proteins and survival kinasescooperate to inactivate BAD by BH3domain phosphorylation. Mol Cell,2000,6:41-51
    44Tan Y, Demeter MR, Ruan H, et al. BAD Ser-155phosphorylationregulates BAD/Bcl-XL interaction and cell survival. J Biochemistry,1998,37:14317-14325
    45Wu HT, Lu DY, Jiang H, et al. Increase in phosphorylation of Akt and itsdownstream signaling targets and suppression of apoptosis by simvastatinafter traumatic brain injury. J Neurosurg,2008,109:691-698
    46Jiang SJ, Jeong HS, Park JS, et al. Neuroprotective effects of(-)-epigallocatechin-3-gallate against quinolinic acid-induced excitotoxicityvia PI3K pathway and NO inhibition. Brain Res,2010,1313:25-33
    47Mortazavi F, Erison M, Story D, et al. Spatial learning deficits andemotional impairments in pentylenetetrazole-kindled rats. Epilepsy Behav,2005,7:629-638
    48Asnaghi L, Calastretti A, Bevilacqua A, et al. Niolin, Bcl-2phosphorylation and apoptosis activated by damaged microtubules requireMtor and are regulated by Akt. Oncogene,2004,23:5781-5791
    49Jin YP, Fishbein MC, Said JW, et al. Anti-HLA classⅠantibody-mediatedactivation of the PI3K/Akt signaling pathway and induction of Bcl-2andBcl-xL expression in endothelial cells. Hum. Immunol,2004,65:291-302
    50Slovitor RS, Dempster DW. Epileptic brain damage is replicatedqualitatively in the rat hippocampus by central injection of glutamate oraspartate but not by GABA or acetylcholine. Brain Res. Bull,1985,15:39-60
    51Olney JW. Inciting excitotoxic cytocide among central neurons. In:Ben-Ari Y, ed. Advances in experimental medicine and biology. New York:Plenum Press,1986,203:632-645
    52Meldrum BS, Chapman AG. Excitatory amino acid receptors andantiepileptic drug development. Adv. Neurol,1999,79:965-978
    53Bruton CJ. The neuropathology of temporal lobe epilepsy. New York:Oxford Univ. Press.1988
    54Nakilahti S, Pirttila TJ, Lukasiuk K, et al. Expression and activation ofCaspase-3following status epilepticus in the rat. Eur J Neurosci,2003,18:1486-1496
    55Gilman SC, Bonner MJ, Pellmar C. Free radicals enhance basal release ofD-(3H) aspartate from cerebral cortical synaptosomes. Journal ofNeurochemistry,1994,62:1757-1763
    56Bouchelouche P, Belhage B, Frandsen A, et al. Glutamate receptoractivation in cultured cerebellar cells increased cytosolic free Ca2+bymobilization of cellular Ca2+and activation of Ca2+influx. ExperimentalBrain Research,1989,76:281-291
    57Dubinsky JM, Rothman SM. Intracellular calcium concentration duringchemical hypoxia and excitotoxic neuronal injury. Journal of Neuroscience,1991,11:1545-2551
    58Etus V, Etus T, Bele A, et al. Green tea polyphenol (-)-epigallocatechingallate prevents oxidative damage on periventricular white matter ofinfantile rats with hydrocephalus. Tohoku J. Exp. Med,2003,200:203-209
    59Meldrum BS, Bruton CJ. Epilepsy. In: Adams JH, Duchen LW, eds.Greenfield’s neuropathology,5thed. New York: Oxford University Press,1992:1246-1283
    60Kalviainen R, Salmenpera T, Partanen K, et al. Recurrent seizures maycause hippocampal damage in temporal lobe epilepsy. Neurology,1998,50:1377-1382
    61Tashch E, Cendes F, Li LM, et al. Neuroimaging evidence of progressiveneuronal loss and dysfunction in temporal lobe epilepsy. Ann Neurol,1999,45:568-576
    62Zhang L, Qu Y, Tang J, et al. PI3K/Akt signaling pathway is required forneuroprotection of thalidomide on hypoxic-ischemic cortical neurons invitro. Brain Res,2010,1357:157-165
    63Wang XQ, Yao RQ, Liu X, et al. Querctin protects oligodendrocyteprecursor cells from oxygen/glucose deprivation injury in vitro via theactivation of the PI3K/Akt signaling pathway. Brain Res Bull,2011,86:277-284
    64Jang S, Jeong HS. Neuroprotective effects of (-)-epigallocatechin-3-gallateagainst quinolinic acid-induced excitotoxicity via PI3K pathway and NOinhibition. Brain Res,2010,1313:25-33
    65Henshall DC, Clark RSB, Adelson PD, et al. Alterations in bcl-2andcaspase gene family protein expression in human temporal lobe epilepsy,Neurology,2000,55:250-257
    66Sagar HJ, Oxbury JM. Hippocampal neuron loss in temporal lobe epilepsy:correlation with early childhood convulsions. Ann Neurol,1987,22:334-340
    1Mac TL, Tran DS, Quet F, et al. Epidemiology, aetiology, and clinicalmanagement of epilepsy in Asia: a systematic review. Lancer Neurol,2007,6:533-543
    2Patel M. Mitochondrial dysfunction and oxidative stress: cause andconsequence of epileptic seizures. Free Radic Biol Med,2004,37:1951-1962
    3Baron M, Kudin AP, Kunz WS. Mitochondrial dysfunction inneurodegenerative disorders. Biochem Soc Trans,2007,35:1228-1231
    4Gao J, Chi ZF, Liu XW, et al. Mitochondrial dysfunction and ultrastructuraldamage in the hippocampus of pilocarpine-induced epileptic rat. NeurosciLett,2007,411:152-157
    5Sas K, Robotka H, Toldi J, et al. Mitochondria, metabolic disturbances,oxidative stress and the kynurenine system, with focus onneurodegenerative disorders. J Neurol Sci,2007,257:221-239
    6Brue AJ, Baudry M. Oxygen free radicals in rat limbic structures afterkainite-induced seizures. Free Radic Biol Med,1995,18:993-1002
    7Frantseva MV, Perez Velazquez JL, Tsoraklidis G, et al. Oxidative stress isinvolved in seizure-induced neurodegeneration in the kindling model ofepilepsy. Neuroscience2000,97:431-435
    8Rola R, Swiader M, Czuczwar SJ. Electroconvulsions elevate the levels oflipid peroxidation products in mice. Pol J Pharmacol,2002,54:521-524
    9El-Abhar HS, El Gawad HMA. Modulation of cortical nitric oxidesynthase, glutamate, and redox state by nifedipine and taurine inPTZ-kindled mice. Epilepsia,2003,44:276-281
    10Marini H, Costa C, Passaniti M, et al. Levetiracetam protects against kainicacid-induced toxicity. Life Sci,2004,74:1253-1264
    11Gulati K, Ray A, Pal G, et al. Possible role of free radicals intheophylline-induced seizures in mice. Pharmacol Biochem Behav,2005,82:241-245
    12Rajasekaran K. Seizure-induced oxidative stress in rat brain regions:blockade by nNOS inhibition. Pharmacol Biochem Behav,2005,80:263-272
    13Tejada S, Sureda A, Roca C, et al. Antioxidant response and oxidativedamage in brain cortex after high dose of pilocarpine. Brain Res Bull,2007,71:372-375
    14Gibbs JE, Walker MC, Cock HR. Levetiracetam: antiepileptic propertiesand protective effects on mitochondrial dysfunction in experimental statusepilepticus. Epilepsia,2006,47:469-478
    15Tokumaru J, Ueda Y, Yokoyama H, et al. In vivo evaluation ofhippocampal anti-oxidant ability of zonisamide in rats. Neurochem. Res,2000,25:1107-1111
    16Barros DO, Xavier SM, Barbosa CO, et al. Effects of the vitamin E incatalase activities in hippocampus after status epileptius induced bypiloarpine in Wistar rats. Neurosci. Lett,2007,416:227-230
    17Xavier SM, Barbosa CO, Barros DO, et al. Vitamin C antioxidant effects inhippocampus of adult Wistar rats after seizures and status epilepticusinduced by pilocarpine. Neurosci. Lett,2007,420:76-79
    18Santos LF, Freitas RL, Xavier SM, et al. Neuroprotective actions ofvitamin C released to decreased lipid peroxidation and increased catalaseactivity in adult rats after pilocarpine-induced seizures. Pharmacol.Biochem. Behav,2008,89:1-5
    19Ayyildiz M, Cosku S, Yildirim M, et al. The effects of ascorbic acid onpenicillin-induced epileptiform activity in rats. Epilepsia,2007,48:1388-1395
    20Leon J, Acuna-castroviejo D, Sainz RM, et al. Melatonin andmitochondrial function. Life Sci,2004,75:765-790
    21Kabuto H, Yokoi I, Ogawa N. Melatonin inhibits iron induced epilepticdischarges in rats by suppressing peroxidation. Epilepsia,1998,39:237-243
    22Yildirim M, Marangoz C. Anticonvulsant effects of melatonin onpenicillin-induced epileptiform activity in rats. Brain Res,2006,1099:183-188
    23Xu K, Stringer JL. Antioxidants and free radical scavengers do notconsistently delay seizure onset in animal models of acute seizures. EpilepyBehav,2008,13:77-82
    24Mathew J, Paul J, Nandhu MS, et al. Bacopa monnieri and Bacoside-A forameliorating epilepsy associated behavioral deficits. Fitoterapia,2010,81:315-322
    25Golechha M, Bhatia J, Arya DS. Hydroalcoholic extract of Embliaofficinalis Gaertn affords protection against PTZ-induced seizures,oxidative stress and cognitive impairment in rats. Indian J Exp Biol,2010,48:474-478
    26Gupta YK, Veerendra Kumar MH, Srivastava AK. Effect of Centellaasiatica on pentylenetetrazole-induced kindling, cognition and oxidativestress in rats. Pharmacol Biochem Behav,2003,74:579-585
    27Mehla J, Reeta KH, Gupta p, et al. Protective effect of curcumin againstseizures and cognitive impairment in a pentylenetetrazole-kindledepileptic rat model. Life Sci,2010,87:596-603
    28Agarwal NB, Jain S, Agarwal NK, et al. Modulation of pentylenetetrazole-induced kindling and oxidative stress by curcumin in mice. Phytomedicine,2010,18:756-759
    29Schwartzkroin PA. Mehanisms underlying the anti-epileptic efficacy of theketogenic diet. Epilepsy Res,1999,37:171-180
    30Bough KJ, Valiyil R, Han FT, et al. Seizure resistance is dependent uponage and calorie restriction in rats fed a ketogenic diet. Epilepsy Res,1999,35:21-28
    31Stafstrom CE. Animal models of the ketogenic diet: what have we learned,what can we learn? Epilepsy Res,1999,37:241-259
    32Noh HS, Hah YS, Nilufar R, et al. Acetoaetate protects neuronal cells fromoxidative glutamate toxicity. J Neurosci Res,2006,83:702-709
    33Kim do Y, Davis LM, Sullivan PG, et al. Ketone bodies are protectiveagainst oxidative stress in neocortical neurons. J Neurochem,2007,101:1316-1326
    34Schwechter EM, J. V, L.V. Correlation between extracellular glucose andseizure susceptibility in adult rats. Ann Neurol,2002,53:91-101
    35Garriga-Canut M, Schoenike B, Qazi R, et al.2-Deoxy-D-glucose reducesepilepsy progression by NRSF-CtBP-dependent metabolic regulation ofchromatin structure. Nat Neurosci,2006,9:1382-1387
    36Ma W, Berg J, Yellen G. Ketogenic diet metabolites reduce firing in centralneurons by opening K (ATP) channels. J Neurosci,2007,27:3618-3625
    37Thio LL, Wong M, Yamada KA. Ketone bodies do not directly alterexcitatory or inhibitory hippocampal synaptic transmission. Neurology,2000,54:325-331
    38Bough KJ, Wetherington J, Hassel B, et al. Mitochondrial biogenesis in theanticonvulsant mechanism of the ketogeni diet. Ann Neurol,2006,60:223-235
    39Maalouf M, Sullivan PG, Davis L, et al. Ketones inhibit mitochondrialproduction of reactive oxygen species production following glutamateexcitotoxicity by increasing NADH oxidation. Neuroscience,2007,145:256-264
    40Sullivan PG, Rippy NA, Dorenbos K, et al. The ketogenic diet increasesmitochondrial uncoupling protein levels and activity. Ann Neurol,2004,55:576-580
    41Jiang Y, Yang Y, Wang S, et al. Ketogenic diet protects againstepileptogenesis as well as neuronal loss in amygdaloid-kindlingseizures.Neurosci Lett,2012,508:22-26
    42Hansen SL, Nielsen AH, Knudsen KE, et al. Ketogenic diet isantiepileptogenic in pentylenetetrazole kindled mice and decrease levels ofN-acylethanolamines in hippocampus. Neurochem. Int,2009,54:199-204
    43Tritschler HJ, Medori R. Mitochondrial DNA alterations as a source ofhuman disorders. Neurology,1993,43:280-288
    44Ames BN. Endogenous oxidative DNA damage, aging, and cancer. FreeRadic Res Commun,1989,7:121-128
    45Dizdaroglu M. Quantitative determination of oxidative base damage inDNA by stable isotope-dilution mass spectrometry. FEBS Lett,1993,315:1-6
    46Dizdaroglu M. Chemical dtermination of free radical-induced damage toDNA. Free Radic Biol Med,1991,10:225-242
    47Jarrett SG, Liang LP, Hellier JL. Mitochondrial DNA damage and impairedbase excision repair during epileptogenesis. Neurobiol Dis,2008,30:130-138
    48Mecocci P, MaGarvey U, Kaufman AE, et al. Oxidative damage tomitochondrial DNA shows marked age-dependent increases in human brain.Ann Neurol,1993,34:609-616
    49Kudin AP, Kudina TA, Seyfried J, et al. Seizure-dependent modulation ofmitochondrial oxidative phosphorylation in rat hippocampus. Eur JNeurosci,2002,15:1105-1114
    50Yamamoto HA, Mohanan PV. Effect of alpha-ketoglutarate and oxaloactateon brain mitochondrial DNA damage and seizures induced by kainic acidin mice. Toxicol Lett,2003,143:115-122
    51Patel M, Day BJ. Metalloporphyrin class of therapeutic catalyticantioxidant. Trends Pharmacol Sci,1999,20:359-364
    52Liang LP, Ho YS, Patel M. Mitochondrial superoxide production inkainite-induced hippocampal damage. Neuroscience,2000,101:563-570
    53Mackensen GB, Patel M, Sheng H, et al. Neuroprotection from delayedpostishemic administration of a metalloporphyrin catalytic antioxidant. JNeurosci,2001,21:4582-4592
    54Crow JP, Calingasan NY, Chen J, et al. Manganese porphyrin given atsymptom onset markedly extends survival of ALS mice. Ann Neurol,2005,58:258-265
    55Bruce AJ, Baudry M. Oxygen free radicals in rat limbic structures afterkainite-induced seizures. Free Radic Biol Med,1995,18:993-1002
    56Cock HR, Tong X, Hargreaves IP, et al. Mitochondrial dysfunctionassociated with neuronal death following status epilepticus in rat. EpilepsyRes,2002,48:157-168
    57Mariani E, Polidori MC, Cherubini A, et al. Oxidative stress in brain aging,neurodegenerative and vascular disease: an overview. J. Chromatogr. B,2005,827:65-75
    58Jellinger KA. General aspects of neurodegeneration. J.Neurol. Transm,2003,65:101-144
    59Stadtman ER. Protein oxidation in aging and age-related diseases. Ann.N.Y.Acad.Sci,2001,928:22-38
    60Wong-ekkabut J, Xu Z, Triampo W. Effect of lipid peroxidation of lipidbilayers: a molecular dynamic study. Biophys,2007,93:4225-4236
    61Ashrafi MR, Shams S, Nouri M, et al. A probable causative factor for anold problem: selenium and glutathione peroxidase appear to play importantroles in epilepsy pathogenesis. Epilepsia,2007,48:1750-1755
    62Packer L. Antioxidant properties of lipoid acid and its therapeutic effects inprevention of diabetes complications and cataracts. Ann.N.Y.Acad.Sci,1994,738:257-264
    63Militao GC, Ferreira PM, de Freitas RM. Effects of lipoic acid on oxidativestress in rat striatum after pilocarpine-induced seizures. Neurochem. Int,2010,56:16-20
    64De Freitas RM. Lipoic acid alters delta-aminolevulinic dehydratase,glutathione peroxidase and Na+,K+-ATPase activities and glutathione-reduced levels in rat hippocampus after pilocarpine-induced seizures. Cell.Mol. Neurobiol,2010,30:381-387
    65Rong Y, Doctrow SR, Too G, et al. EUK-134, a synthetic superoxidedismutase and catalase mimetic, prevents oxidative stress and attenuateskainite-induced neuropathology. Proc.Natl.Acad. Sci,1999,96:9897-9902
    66Liang LP, Ho YS, Patel M. Mitochondrial superoxide production inkainite-induced hippocampal damage. Neuroscience,2000,101:563-570
    67Liu W, Liu R, Schreiber SS, et al. Role of polyamine metabolism in kainicacid excitotoxicity in organotypic hippocampal slice cultures. J.Neurochem,2001,79:976-984
    68Chuang YC, Chen SD, Liou CW, et al. Contribution of nitric oxide,superoxide anion, and peroxynitrite to activation of mitochondrialapoptotic signaling in hippocampal CA3subfield following experimentaltemporal lob status epilepticus. Epilepsia,2009,50:731-746
    69Winyard PG, Mody CJ, Hacob C. Oxidative activation of antioxidantdefense. Trends Biochem. Sci,2005,30:453-461
    70Brown GC, Borutaite V. Nitric oxide, mitochondria, and cell death.IUBMB Life,2001,52:189-195
    71Manev H, Uz T, Kharlamov A, et al. Increased brain damage after stroke orexcitotoxic seizures in melatonin-deficient rats. FASEB J,1996,10:1546-1551
    72Lima FD, Souza MA, Furian AF, et al. Na+, K+-ATPase activity impairmentafter experimental traumatic brain injury: relationship to spatial learningdeficits and oxidative stress. Behav. Brain Res,2008,193:306-310
    73Shin EJ, Sun SK, Lim YK, et al. Ascorbate attenuates trimethyltin-inducedoxidative burden and neuronal degeneration in the rat hippocampus bymaintaining glutathione homeostasic. Neuroscience,2005,133:715-727
    74Veinbergs I, Mallory M, Sagara Y, et al. Vitamin E supplementationprevents spatial learning deficits and dendritic alterations in agedapolipoprotein E-deficient mice. Eur. J. Neurosci,2000,12:45414-45416
    75Sudha K, Rao AV, Rao A. Oxidative stress and antioxidants in epilepsy.Clin. Chim. Acta,2001,303:19-24
    76Kalayci R, Kaya M, Kucuk M, et al. Catalase and alpha-tocopherolattenuate blood-brain barrier breakdown in pentylenetetrazole-inducedepileptic seizures in acute hyperglycaemic rats. Pharmacol. Res,2002,45:129-133
    77Tome AR, Feng D, Fretas RM. The effets of alpha-tocopherol onhippocampal oxidative stress prior to in piloarpine-induced seizures.Neurochem. Res,2010,35:580-587
    78Shin HJ, Le JY, Son E, et al. Curumin attenuates the kainic acid-inducedhippocampal cell death in the mice. Neurosci. Lett,2007,416:49-54
    79Scapagnini G, Foresti R, Calabrese V, et al. Caffeic acid phenethyl esterand curcumin: a novel class of hemeoxygenase-1induces. Mol. Pharmacol,2002,61:554-561
    80Hong J, Bose M, Ju J, et al. Modulation of arachidonic acid metabolism bycurcumin and related beta-diketone derivatives: effects on cytosolicphospholipase A2, cyclooxygenases and5-lipoxygenase. Carcinogenesis,2004,25:1671-1679
    81Dexter DT, Carter CJ, Wells FR, et al. Basal lipid peroxidation insubstantia nigra is increased in Parkinson’s disease. J Neurochem,1989,52:381-389
    82Cini M, Moretti A. Studies on lipid peroxidation and protein oxidation inthe aging brain. Neurobiol Aging,1995,16:53-57
    83Gluck MR, Jayatilleke E, Shaw S, et al. CNS oxidative stress associatedwith the kainic acid rodent model of experimental epilepsy. Epilepsy Res,2000,39:63-71
    84Fridovich I. Superoxide anion radical (O2-), superoxide dismutases, andrelated matters. J Biol Chem,1997,272:18515-18517
    85Vielhaber S, Niessen HG, Debska-Vielhaber G, et al. Subfield specific lossof hippocampal N-acetylaspartate in temporal lobe epilepsy. Epilepsia,2008,49:40-50
    86Erakovic V, Zupan G, Varljen J, et al. Lithium plus pilocarpine inducedstatus epilepticus-biochemical changes. Neurosci Res,2000,36:157-166
    87Bellissimo MI, Amado D, Abdalla DS, et al. Superoxide dismutase,glutathione peroxidase activities and the hydroperoxide concentration aremodified in the hippocampus of epileptic rats. Epilepsy Res,2001,46:121-128
    88Ueda Y, Yokoyama H, Niwa R, et al. Generation of lipid radicals in thehippocampal extracellular space during kainic acid-induced seizures in rats.Epilepsy Res,1997,26:329-333
    89Dal-Pizzol F, Klamt F, Vianna MM, et al. Lipid peroxidation in
    hippocampus early and late after status epilepticus induced by pilocarpine
    or kainic acid in Wistar rats. Neurosci. Lett,2000,291:179-182
    1Singh BN, Shankar S, Srivastava RK. Green tea catechin,epigallocatehin-3-gallate (EGCG): Mechansims, perspectives and clinicalapplications. Biochem Pharmacol,2011,82:1807-1821
    2Matsuo N, Yamada K, Shoji K, et al. Effect of tea polyphenols onhistamine release from rat basophilic leukemia (RBL-2H3) cells: thestructure-inhibitory activity relationship. Allergy,1997,52:58-64
    3Yin ST, Tang ML, Su L, et al. Effects of epigallocatechin-3-gallate on lead–induced oxidative damage. Toxicology,2008,249:45-54
    4Ramesh E, Jayakumar T, Elanchezhian R. Green tea catechins, alleviatehepatic lipidemic-oxidative injury in Wistar rats fed an atherogenic diet.Chem Biol,2009,180:10-19
    5Twiwari V, Kuhad A, Chopra K. Epigallocatechin-3-gallate amelioratesalcohol-induced cognitive dysfunctions and apoptotic neurodegeneration inthe developing rat brain. Int J Neuropsychoph,2010,13:1053-1066
    6Guo Q, Zhao B, Li M, et al. Studies on protective mechanisms of fourcomponents of green tea polyphenols against lipid peroxidation insynaptosomes. Biochim. Biophys. Acta,1996,1304:210-222
    7Kondo K, Kurihara M, Miyata N, et al. Scavenging mechanisms of(-)-epigallocatechin gallate and (-)-epicatechin gallate on peroxyl radicalsand formation of superoxide during the inhibitory action. Free.Radic.Biol.Med,1999,27:855-863
    8Sano M, Takahashi Y, Yoshino K, et al. Effect of tea(Camellia sinensis L.)on lipid peroxidation in rat liver and kidney: a comparison of green andblack tea feeding. Biol. Pharm. Bull,1995,18:1006-1008
    9Aucamp J, Gaspar A, Hara Y, et al. Inhibition of xanthine oxidase bycatechins from tea (Camellia sinensis). Anticancer Res,1997,17:4381-4386
    10Chan MM, Fong D, Ho CT, et al. Inhibition of inducible nitric oxidesynthase gene expression and enzyme activity by epigallocatechin gallate,a natural product from green tea. Biochem. Pharmacol,1997,54:1281-1286
    11Lin YL, Lin JK.(-)-Epigallocatechin-3-gallate blocks the induction ofnitric oxide synthase by down-regulating ipopolysaccha-ride-inducedactivity of transcription factor nuclear factor-Kb. Mol.Pharmacol,1997,52:465-472
    12Nie G, Jin C, Cao Y, et al. Distince effects of tea catechins on6-hydroxydopamine-induced apoptosis in PC12cells. Arch. Biochem.Biophysics,2002,397:84-90
    13Koh SH, Kim SH, Kwon H, et al. Phosphtidylinositol-3kinase/AKt andGSK-3mediated cytoprotetive effect of epigallocatechin gallate onoxidative stress-injured neuronal-differentiated N18D3cells.Neurotoxicoloty,2004,25:793-802
    14Lee SR, Suh S, Kim SP. Protective effects of the green tea polyphenol(-)-epigallocatechin gallate against hippocampal neuronal damage aftertransient global ischemia in gerbils. Neurosci Lett,2000,287:191-194
    15Khalatbary AR, Tiraihi T, Boroujeni MB. Effects of epigallocatechingallate on tissue protection and functional recovery after contusive spinalcord injury in rats. Brain Res,2010,1306:168-175
    16Koh SH, Lee SM, Kim HY, et al. The effect of epigalloatechin gallate onsuppressing disease progression of ALS model mice. Neurosci Lett,2006,395:103-107
    17He M, Zhao L, Wet MJ, et al. Neuroprotective effects of(-)-epigallocatechin-3-gallate on aging mice induced by D-galactose. Biol.Pharm. Bull,2009,32:55-60
    18Khalatbary AR, Ahmadvand H. Anti-inflammatory effect of theepigallocatechin gallate following spinal cord trauma in rat. Iran Biomed J,2011,15:31-37
    19Tsai PY, Ka SM. Epigallocatechin-3-gallate prevents lupus nephritisdevelopment in mice via enhancing the Nrf2antioxidant pathway andinhibiting NLRP3inflammasome activation. Free Radic Biol Med,2011,51:744-754
    20Shimizu M, Deguchi A, Joe AK, et al. EGCG inhibits activation of HER3and expression of cyclooxygenase-2in human colon cancer cells. J ExpTher Oncol,2005,5:69-78
    21Shimizu M, Deguchi A, Lim JT, et al.(-)-Egigallocatechin gallate andpolyphenon E inhibit growth and activation of the epidermal growth factorreceptor and human epidermal growth factor receptor-2signaling pathwaysin human colon cancer cells. Clin Cancer Res,2005,11:2735-2746
    22Peng G, Dixon DA, Muga SJ, et al. Green tea polyphenol(-)-egigallocatechin-3-gallate inhibits cyclooxygenase-2expression incolon carcinogenesis. Mol Carcinog,2006,45:309-319
    23Tedeschi E, Menegazzi M, Yao Y, et al. Green tea inhibits human induciblenitric-oxide synthase expression by down-regulating signal transducer andactivator of transcription-lalpha activation. Mol. Pharmacol,2004,65:111-120
    24Thawonsuwan J, Kiron V, Satoh S, et al. Epigallocatechin-3-gallate(EGCG) affects the antioxidant and immune defense of the rainbow trout,Oncorhynchus mykiss. Fish Physiol Biochem,2010,36:687-97
    25Higdon JV, Frei B. Tea catechins and polyphenols: health effects,metabolism, and antioxidant functions. Crit Rev Food Sci Nutr,2003,43:89-143
    26Mukhtar H, Ahmad N. Tea polyphenols: prevention of cancer andoptimizing health. Am J Clin Nutr,2000,71:698-702
    27Gupta S, Hastak K, Afaq F, et al. Essential role of caspases inepigallocatechin-3-gallate mediated inhibition of nuclear factor kappa Band induction of apoptosis. Oncogene,2004,23:2507-2522
    28Ahmed S, Wang N, Lalonde M, et al. Green tea polyphenolepigallocatechin-3-gallate(EGCG) differentially inhibits interleukin-1betainduced expression of matrix metalloproteinase-1and-13in humanchondrocytes. J Pharmacol Exp Ther,2004,308:767-773
    29Levites Y, Weinreb O, Maor G, et al. Green tea polyphenol (-)-epigallocatechin-3-gallate prevents N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration. J.Neurochem,2001,78:1073-1082
    30Rasoolijazi H, Joghataie MT, Roghani M, et al. The beneficial effect of(-)-epigallocatechin-3-gallate in an experimental model of Alzheimer’sdisease in rat: a behavioral analysis. Iranian Biome Journal,2007,11:237-243
    31Chan MM, Ho CT, Huang HI. Effects of three dietary phytochemicals fromtea, rosemary and turmeric on inflammation-induced nitrite production.Cancer Lett,1995,96:23-29
    32Tedeschi E, Menegazzi M, Yao Y, et al. Green tea inhibits human induiblenitric-oxide synthase expression by down-regulating signal transducer andactivator of transcription-lalpha activation. Mol Pharmacol,2004,65:111-120
    33Suzuki M, Tabuchi M, Ikeda M, Umegaki K, et al. Protective effects ofgreen tea catechins on cerebral ischemic damage. Med Sci Monit,2004,10:166-174
    34Sutherland BA, Shaw OM, Clarkson AN, et al. Neuroprotective effects of(-)-epigalloatechin gallate following hypoxia-ischemia induced braindamage: novel mechanism of action. Faseb J,2005,19:259-260
    35Stevens JF, Miranda CL, Wolthers KR, et al. Identifiation and in vitrobiological activities of hop proanthocyanidins: inhibition of Nnos activityand scavenging of reative nitrogen species. J Agric Food Chem,2002,50:3435-3443
    36Chan MM, Fong D, Ho CT, et al. Inhibition of inducible nitric oxidesynthase gene expression and enzyme activity by epigallocatechin gallate,a natural product from green tea. Biochem Pharmacol,1997,54:1281-1286
    37Levites Y, Amit T, Youdim MBH. Involvement of protein kinase Cactivation and cell survival/cell cycle genes in green tea polyphenol(-)-epigallocatechin-3-gallate neuroprotective action. J. Biol. Chem,2002,277:30574-30580
    38Levites Y, Youdim MBH, Maor G. Attenuation of6-hydroxydopamine(6-OHDA)-induced nuclear factor kappB (NF-kB) activation and cell deathby tea extracts in neuronal cultures. Biochem. Pharmacol,2002,63:21-29
    39Choi YT, Jung CH, Lee SR. The green tea polyphenol (-)-epigallocatechingallate attenuates β-amyloid-induced neurotoxicity in culturedhippocampal neurons. Life Sci,2001:70:603-614
    40Levites Y, Amit T, Mandel S. Neuroprotection and neurorescue againstamyloid beta toxicity and PKC dependent release of non-amyloidogenicsoluble precursor protein by green tea polyphenol (-)-epigallocatechin-3-gallate. Faseb J,2003,17:952-954
    41Matsuoka Y, Hasegawa H, Okuda S. Ameliorative effects of tea catechinson active oxygen-related nerve cell injuries. J. Pharmacol. Exp. Ther,1995,274:602-608
    42Rezai-Zadeh K, Arendash GW, Hou H, et al. Green teaepigallocatechin-3-gallate(EGCG) reduces beta-amyloid mediatedcognitive impairment and modulates tau pathology in Alzheimer transgenicmice. Brain Res,2008,1214:177-187
    43Bravo L. Polyphenols: chemistry, dietary sources, metabolism, andnutritional significance. Nutr. Rev,1998,56:317-333
    44Nakagawa K, Miyazawa T. Absorption and distribution of tea catechin,(-)-epigalloatehin-3-gallate, in the rat. J.Nutr.Sci,1997,43:679-684
    45Suganuma M, Okabe S, Oniyama M, et al. Wide distribution of [3H](-)-epigallocatechin gallate, a cancer preventive tea polyphenol, in mousetissue. Carcinogenesis,1998,19:1771-1776
    46Abd EL Mohsen MM, Kuhnle G, Rechner AR, et al. Uptake andmetabolism of epicatechin and its access to the brain after oral ingestion.Free Radic Biol Med,2002,33:1693-1702
    47Xu ZH, Chen S, Li XP, et al. Neuroprotective effects of(-)-epigallocatechin-3-gallate in a transgenic mouse model of amyotrophiclateral sclerosis. Neurochem Res,2006,31:1263-1269
    48Yang CS, Maliakal P, Meng X. Inhibition of carcinogenesis by tea. Annu.Rev. Pharmacol. Toxicol,2002,42:25-54
    49Ji BT, Chow WH, Hsing AW, et al. Green tea consumption and the risk ofpancreatic and colorectal ancers. Int. J. Cancer,1997,70:255-258
    50Adhami VM, Ahmad N, Ahmad H, et al. Molecular targets for green tea inprostate cancer prevention. J. Nutr,2003,133:2417-2424
    51Hibasami H, Komiya T, Achiwa Y, et al. Induction of apoptosis in humanstomach cancer cells by green tea catechins. Oncol. Rep,1998,5:527-529
    52Sartippour MR, Heber D, Ma J, et al. Green tea and its catechins inhibitbreast cancer xenografts. Nutr. Cancer,2001,40:149-151
    53Gao YT, Mclaughlin JK, Blot WJ, et al. Reduced risk of esophageal cancerassociated with green tea consumption. J. Natl. Cancer Inst,1994,86:855-858
    54Komatsu M, Hiramatsu M. The efficacy of an antioxidant cocktail on lipidperoxide level and superoxide dismutase activity in aged rat brain andDNA damage in iron-induced epileptogenic foci. Toxicology,2000,148:143-148
    55Lu H, Meng X, Yang CS. Enzymology of methylation of tea catechins andinhibition of catechol-O-methyltransferase by (-)-epigallocatechin gallate.Drug Metab Dispos,2003,31:572-579
    56Lee H, Bae JH, Lee SR. Protective effect of green tea polyphenol EGCGagainst neuronal damage and brain edema after unilateral cerebral ischemiain gerbils. J Neurosci Res,2004,77:892-900
    57Townsend PA, Scarabelli TM, Davidson SM, et al. STAT-1interacts withp53to enhance DNA damage-induced apoptosis. J Biol Chem,2004,279:5811-5820
    58Aktas O, Prozorovski T, Smorodhenko A, et al. Green teaepigallocatechin-3-gallate mediates T cellular NF-kappa B inhibition andexerts neuroprotetion in autoimmune encephalomyelitis. J Immunol,2004,173:5794-5800
    59Schroeter H, Spencer JP, Rice-Evans C, et al. Flavonoids protect neuronsfrom oxidized low-density-lipoprotein induced apoptosis involving c-JunN-terminal kinase (JNK), c-Jun and caspase-3. Biochem J,2001,358:547-557
    60Spener JP, Schroeter H, Kuhnle G, et al. Epicatechin and its in vivometabolite,3’-O-methylepicatechin, protet human fibroblasts fromoxidative-stress-induced cell death involving caspase-3activation.Biochem J,2001,354:493-500
    61Nobre Junior HV, Cunha GM, Maia FD, et al. Catechin attenuates6-hydroxydopamine (6-OHDA)-induced cell death in primary cultures ofmesencephalic cells. Comp Biochem Physiol,2003,136:175-180
    62Mercer LD, Kelly BL, Home MK, et al. Dietary polyphenols protectdopamine neurons from oxidative insults and apoptosis: Investigations inprimary rat mesencephalic cultures. Biochem Pharmacol,2005,69:339-345
    63Siddiqui IA, Asim M, Hafez BB, et al. Green tea polyphenol EGCG bluntsandrogen receptor function in prostate cancer. FASEB J,2010
    64Shankar S, Suthakar G, Srivastava RK. Epigallocatechin-3-gallate inhibitscell cycle and induces apoptosis in pancreatic cancer. Front Biosci,2007,12:5039-5051
    65Nandakumar V, Vaid M, Katiyar SK.(-)-Epigallocatechin-3-gallatereactivates silenced tumor suppressor genes, Clip1/p21and p161NK4a, byreducing DNA methylation and increasing histones acetylation in humanskin cancer cells. Carcinogenesis2011
    66Imai K, Suga K, Nakachi K. Caner-preventive effects of drinking green teaamong a Japanese population. Prevent Med,1997,26:769-775
    67Lotito SB, Fraga CG, Catechins delay lipid oxidation and alphatocopheroland beta-carotene depletion following ascorbate depletion in human plasma.Proc Soc Exp Biol Med,2000,225:32-38
    68Erba D, Riso P, Bordoni A, et al. Effectiveness of moderate green teaconsumption on antioxidative status and plasma lipid profile in humans. JNutr Biochem,2005,16:144-149
    69Young JF, Dragstedt LO, Haraldsdottir J, et al. Green tea extract onlyaffects markers of oxidative status postprandially: lasing antioxidant effectof flavonoid-free diet. Br J Nutr,2002,87:343-355
    70Kagaya N, Tagawa Y, Nagashima H, et al. Suppression of cytotoxin-induced cell death in isolated hepatocytes by tea catechins. Eur JPharmacol,2002,450:231-236
    71Kakuda T. Neuroprotective effects of the green tea components theanineand catechins. Biol Pharm Bull,2002,25:1513-1518
    72Loest HB, Noh SK, Koo SI. Green tea extract inhibits the lymphaticabsorption of cholesterol and alpha-tocopherol in ovariectomized rats. JNutr,2002,132:1282-1288
    73Ikeda I, Kobayashi M, Hamada T, et al. Heat-epimerized tea catechins richin gallocatechin gallate and catechin gallate are more effective to inhibitcholesterol absorption than tea catechins rich in epigallocatechin gallateand epicatechin gallate. J Agric Food Chem,2003,51:7303-7307
    74Miura Y, Chiba T, Tomita I, et al. Tea atechin prevent the development ofatherosclerosis in apoprotein E-deficient mice. J Nutr,2001,131:27-32
    75Miura S, Watanabe J, Tomita T, et al. The inhibitory effects of teapolyphenols on CU2+mediated oxidative modification of low densitylipoprotein. Biol Pharm Bull,1994,17:1567-1572
    76Locher R, Emmanuele L, Suter PM, et al. Green tea polyphenols inhibithuman vascular smooth muscle cell proliferation stimulated by nativelow-density lipoprotein. Eur J Pharmaol,2002,434:1-7
    77Shammas MA, Neri P, Koley H, et al. Specific killing of multiple myelomacells by (-)-epigallocatechin-3-gallate extracted from green tea: biologicactivity and therapeutic implications. Blood,2006,108:2804-2810
    78Umeda D, Yano s, Yamada K, et al. Green tea polyphenol epigallocatechin-3-gallate signaling pathway through67-kDa laminin receptor. J. Biol.Chem,2008,283:3050-3058
    79Khan N, Afaq F, Saleem M, et al. Targeting multiple signaling pathways bygreen tea polyphenol (-)-epigallocatechin-3-gallate. Caner Res,2006,66:2500-2505
    80Wu PP, Kuo SC, Huang WW, et al.(-)-Epigallocatechin gallate inducedapoptosis in human adrenal cancer NCL-H295cells through caspase-dependent and caspase-independent pathway. Anticancer Res,2009,29:1435-1442
    81Chung LY, Cheung TC, Kong SK, et al. Induction of apoptosis by green teacatechins in human prostate cancer DU145cells. Life Sci,2001,68:1207-1214
    82Nakazato T, Ito K, Ikeda Y, et al. Green tea component, catechin, inducesapoptosis of human malignant B cells via production of reactive oxygenspeies. Clin. Cancer Res,2005,11:6040-6049
    83Ahmad N, Gupta S, Mukhtar H. Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor kappaB in cancer cellsversus normal cells. Arch. Biochem. Biophys,2000,376:338-346
    84Nishikawa T, Nakajima T, Moriguchi M, et al. A green tea polyphenol,epigalloatechin-3-gallate, induces apoptosis of human hepatocellualrcarcinoma, possibly through inhibition of Bcl-2family proteins. J. Hepatol,2006,44:1074-1082
    85Lin AM, Chyi BY, Wu LY, et al. The antioxidative property of green teaagainst iron-induced oxidative stress in rat brain. Chin. J. Physiol,1998,41:189-194
    86Helfman C, Phillis JW. Oxypurinol administered post-ischaemia preventsbrain injury in the gerbil, Med. Sci. Res,1989,17:969-970
    87Kawai K, Tsuno NH, Kitayama J, et al. Epigallocatechin gallate attenuatesadhesion and migration of CD8+T cells by binding to CD11b. J AlergyClin Immunol,2004,113:1211-1217
    88Uchida S, Ozaki M, Akashi T, et al. Effects of (-)-epigalloatechin-3-O-gallate (green tea tannin) on the life span of stroke prone spontaneouslyhypertensive rats. Clin Exp Pharmacol Physiol Suppl,1995,22:302-303
    89Tourandokht B, Mehrdad R. Chronic epigalloatechin-3-gallate ameliorateslearning and memory deficits in diatetic rats via modulation of nitric oxideand oxidative stress. Behav Brain Res,2011,224:305-310
    90Tiwari V, Kuhad A, Chopra K. Epigallocatechin-3-gallate amelioratesalcohol-induced cognitive dysfunctions and apoptotic neurodegeneration inthe developing rat brain. Int J Neuropsychoph,2010,13:1053-1066