Nrf2-ARE信号通路在癫痫脑损伤中的作用及莱菔硫烷的神经保护机制研究
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摘要
癫痫是最常见的神经系统疾病之一，是多种病因导致的慢性脑部病变,以大脑神经元过度地、反复超同步化放电为特征，临床表现为短暂性中枢神经系统功能失常的综合征。癫痫反复发作可导致脑部神经元选择性损伤，甚至坏死丢失，从而引起胶质细胞增生、突触重构等脑结构和功能的可塑性变化；而大脑这些可塑性变化又使癫痫反复发作，是癫痫难治的主要原因之一，反复癫痫发作的患者常伴有认知功能损伤，其发生率约为30％-40％。
癫痫的发病机制非常复杂，迄今尚未完全阐明，氧化应激损伤、谷氨酸兴奋性毒性、钙超载等多种因素都能诱导神经元异常放电触发癫痫。其中氧化应激产生的氧自由基连锁反应是神经元受损的核心病理环节。近年的研究结果证实,许多慢性疾病都与人体内氧化应激发生，累积过多的自由基(Free Radical, FR)有关，癫痫与氧化应激之间的关系日益成为科研工作者研究的热点。无论是癫痫动物模型还是癫痫患者的脑内均存在着活跃的氧化应激反应，癫痫发作时FR含量明显增加,远远超过机体对FR的清除能力。大量的FR可以直接使脂质、蛋白质及DNA等大分子物质发生氧化损伤，从而破坏细胞膜及其它细胞结构，同时FR还能通过抑制线粒体功能引起细胞凋亡。因此，针对氧自由基在癫痫脑损伤病理机制中重要作用，抑制氧化应激和清除氧自由基可能是治疗癫痫的重要策略。
转录因子NF-E2相关因子（NF-E2-related factor2，Nrf２）是细胞氧化应激反应中的关键因子，通过与抗氧化反应元件（antioxidant responseelement，ARE）相互作用调节抗氧化蛋白和Ⅱ相解毒酶的表达。Nrf2是机体调节抗氧化反应的重要转录因子，正常生理情况下位于细胞浆中，在细胞浆中与Keapl结合形成复合体，且被泛素蛋白酶迅速降解，从而保持其低活性的生理状态。当机体受到氧自由基和内源性毒素等攻击后Nrf2与Keapl解离，其半衰期明显延长，然后转位进入细胞核，与抗氧化反应元件ARE结合后诱导抗氧化蛋白和Ⅱ相解毒酶基因的表达。到目前为止，已证实经Nrf2-ARE信号路径调节的可编码内源性保护基因超过200个。它们在增强组织抗氧化能力、保护组织细胞免受毒物损伤、抗肿瘤、抗炎症和抗凋亡中起着重要的作用，其中血红素氧合酶1(hemeoxygenase1，HO-1)和依赖还原型辅酶/Ⅱ醌氧化还原酶1(NADPH:quinone oxidoreductase1，NQO1)是该通路编码的重要的抗氧化酶。在中枢神经系统上调HO-1和NQO1的表达，能够减少氧自由基和内源性毒素对神经元的毒性作用。既往研究证实，激活Nrf2-ARE信号通路上调其基因产物的表达对神经系统疾病如帕金森病、脑出血、脑梗死和脑外伤等，具有很强的神经保护功能。但是，Nrf2-ARE信号通路在癫痫发病中表达改变，及其对海马神经元的保护作用还未见明确相关报道。
莱菔硫烷（Sulforaphane，SF）是一种广泛存在于十字花科蔬菜的异硫氰酸盐，具有抗氧化、抗肿瘤和免疫调节等多种生物学特性，作为化学预防药物已引起广大研究者的关注。既往研究证实，SF是Nrf2-ARE信号通路重要的激活剂，能够诱发Nrf2磷酸化转位入核，与ARE结合，正向调控抗氧化蛋白和Ⅱ相解毒酶的表达，清除自由基从而对机体细胞起到保护作用。但SF对癫痫的神经保护作用及其具体分子机制尚未见明确报道。
本实验主要采用杏仁核电点燃癫痫大鼠模型，观察癫痫大鼠模型海马组织中氧化应激参数（丙二醛和谷胱甘肽）的表达改变；Nrf2及其编码的基因产物HO-1和NQO1的表达改变；同时进一步验证以Nrf2-ARE信号通路为靶点的药物SF对海马组织氧化应激的改善情况及对神经元的保护作用。为阐明癫痫的病理生理机制提供实验依据，同时为癫痫治疗提供新的靶点，为抗癫痫药物的选择提供新的思路。
第一部分Nrf2-ARE信号通路在急性电点燃癫痫大鼠海马的表达及意义
目的：观察急性电点燃癫痫大鼠海马组织中氧化应激参数（丙二醛和谷胱甘肽）及Nrf2-ARE信号通路的表达改变，以期为癫痫的治疗提供新的靶点。
方法：采用杏仁核快速电点燃制备急性癫痫大鼠模型（每天刺激20次，刺激间隔时间10min，连续刺激2d，刺激电流为恒流，单向方波。），通过分光光度法检测大鼠海马组织中氧化应激参数（丙二醛和谷胱甘肽）表达改变；通过免疫组化和Western blot方法观察Nrf2、HO-1和NQO1在海马组织中蛋白水平的表达改变；同时通过Real-time PCR方法观察Nrf2mRNA、HO-1mRNA和NQO1mRNA在海马组织中基因水平的表达改变。
结果：1、氧化应激参数的改变：与对照组相比，癫痫组大鼠海马组织中谷胱甘肽（Glutathione, GSH）含量明显降低（P<0.01）。与对照组相比，癫痫组大鼠海马组织中丙二醛（malondialdehyde, MDA）水平明显升高（P<0.01）。假手术组与对照组相比GSH与MDA差别无明显统计学意义（P>0.05）。
2、免疫组化结果：Nrf2免疫反应在海马的锥体细胞和神经胶质细胞的胞浆和胞核均有表达；HO-1免疫反应主要在海马锥体细胞和神经胶质细胞的胞浆。对三组大鼠海马CA1、CA2和CA3区Nrf2和HO-1表达的平均光密度值（AOD）测量，癫痫组比对照组AOD值明显增高(P﹤0.01)；假手术组和对照组相比，AOD值差别无明显统计学意义（P>0.05）。
3、Western-blot结果:3.1、电点燃大鼠海马组织胞核内的Nrf2的表达明显增强，而在对照组和假手术组Nrf2表达相对较弱，Nrf2与H3免疫印迹条带相对吸光度比值电点燃组比对照组明显增加，具有明显统计学差异（P<0.01）。假手术组和对照组相比没有明显统计学差异（P>0.05）。3.2、电点燃大鼠海马组织胞浆内的HO-1的表达明显增强，而在对照组和假手术组HO-1表达相对较弱，HO-1与β-actin免疫印迹条带相对吸光度比值，电点燃组比对照组明显增加，具有统计学差异（P<0.01）。假手术组的吸光度比值和对照组相比没有明显统计学差异（P>0.05）。
4、Real-time PCR实验结果:各组大鼠海马组织中Nrf2、HO-1和NQO1mRNA的表达水平以三种物质mRNA／GAPDHmRNA表示，与对照组Nrf2、HO-1和NQO1mRNA表达水平相比，电点燃组Nrf2、HO-1和NQO1mRNA表达水平明显增高，均具有统计学意义(P<0.01)；假手术组Nrf2、HO-1和NQO1mRNA表达水平和对照组表达水平相比无明显统计学差异(P>0.05)。
结论：急性癫痫发作可以导致海马组织发生氧化应激，导致脂质氧化代谢产物MDA含量增高和自由基清除剂GSH含量降低；急性癫痫发作可以诱导海马组织中Nrf2和其编码的基因产物HO-1和NQO1在蛋白和基因水平表达明显增强。说明Nrf2-ARE信号通路与癫痫发病关系密切，可能是癫痫治疗的新的靶点。
第二部分莱菔硫烷对慢性电点燃癫痫大鼠的脑保护作用研究
目的：观察莱菔硫烷对慢性电点燃癫痫大鼠的抗癫痫作用及对认知功能的影响；同时观察其对海马神经元的保护作用。
方法：采用慢性杏仁核电点燃制备癫痫大鼠模型（每天电刺激1次，连续刺激15d，刺激电流为恒流，单向方波。），在点燃过程中给予大鼠腹腔注射SF（5mg/kg/day）。通过对大鼠点燃过程中发作等级和后放电持续时间（afterdischarge duration，ADD）比较，观察SF的抗癫痫作用；通过Morris水迷宫监测癫痫大鼠的认知功能及SF对其的改善作用；通过尼氏染色和透射电镜观察癫痫大鼠海马神经元形态学改变及SF的神经保护作用。
结果：1SF对慢性癫痫模型点燃过程的影响在连续给与15次亚惊厥电刺激的过程中，所有的大鼠均出现了不同程度的癫痫发作。连续15次电刺激后，在电点燃组中所有的大鼠均达到了完全点燃的标准（连续出现3次4-5级发作），而在电点燃+SF组的大鼠中仅有4只达到了点燃标准。对两组大鼠的发作等级比较后发现在第9-15天的时候具有明显的统计学差异（在第9-14天时P<0.05,在第15天时P<0.01）。同时，连续给予15次电刺激过程中所有大鼠的ADD逐渐延长，比较电点燃组和电点燃+SF组发现，在第7-15天的时候具有明显的统计学差异（在第9-10天时P<0.05,在第11-15天时P<0.01）。对照组和SF组没有出现癫痫发作。
2Morris水迷宫检测结果
2.1定位航行实验结果：每日逃避潜伏期取其平均值进行比较，结果显示，(1)对照组、电点燃组、SF+电点燃组和SF组四组大鼠的逃避潜伏期在第1和2天无统计学意义(P>0.05)。在第1和2天，与对照组大鼠的逃避潜伏期相比较，电点燃组大鼠及SF+电点燃组大鼠的逃避潜伏期均有延长趋势。(2)实验第3、4、5天，各组逃避潜伏期均比第1、2天有缩短趋势，在第3、4和5天，与对照组大鼠的逃避潜伏期相比较，电点燃组大鼠的逃避潜伏期均明显延长(P<0.05)；SF+电点燃组大鼠的逃避潜伏期较电点燃组大鼠均明显缩短(P<0.05)，但与对照组比较仍明显延长(P<0.05)；SF组大鼠的逃避潜伏期和对照组没有明显变化(P>0.05)。
2.2空间探索试验结果：电点燃组大鼠120s内穿越平台区域的次数明显低于对照组(P<0.01)；SF+电点燃组大鼠120s内穿越平台区域的次数与电点燃组比较次数均明显增多(P<0.01)。在平台象限的游泳时间为：与对照组相比，电点燃组明显减少(P<0.01)，SF+电点燃组在平台象限的游泳时间与电点燃组比较明显增多(P<0.01)，SF组和对照组相比无显著性差异(P>0.05)。
3尼氏染色结果：对照组大鼠海马神经元形态完整、核仁清晰，胞浆内尼氏体丰富，锥体细胞排列规则紧密，没有明显神经元丢失；与对照组相比，电点燃组大鼠海马神经元丢失明显，排列紊乱，可见细胞皱缩，染色质凝集成块、核固缩、尼氏体数量减少；SF治疗后海马组织神经元边缘清晰，神经元没有明显丢失，结构正常，仅少量染色质凝集、尼氏体数量较癫痫组显著增加。SF单独用药组海马组织神经元和对照组没有明显改变。
4透射电极观察海马CA1区超微结构变化：对照组电镜观察：神经元形态完整，结构清晰。胞核呈圆形，染色质均匀分布。胞质内细胞器丰富，可见线粒体、粗面内质网、滑面内质网和大量的多核糖体。线粒体呈圆形或椭圆形，嵴清晰可见。粗面内质网和高尔基体发达。轴突中神经微丝微管清晰可见。突触前、后膜结构清晰，突触前膜靠近突触间隙一侧有较多圆形的突触囊泡，突触间隙清晰。电点燃组电镜观察：海马神经元胞核肿胀、形态不规则，核内异染色质减少分散，胞核疏松、透明，有些出现核固缩现象。线粒体空泡化或均质化。神经微丝微管结构不清，可见聚集。突触前、后膜结构模糊不清，突触间隙增大，靠近突触前膜的突触囊泡减少。SF+电点燃组电镜观察：海马神经元核膜完整，核形态基本正常，染色质均匀分布，胞质中细胞器形态基本正常，有髓神经髓鞘完整。突触数量较多，形态接近于正常。突触前、后膜结构清楚，突触间隙清晰，突触前膜的突触囊泡较多。SF单组用药组电镜观察和正常对照组超微结构没有明显改变。
结论：本研究通过给慢性电点燃大鼠腹腔注射SF后发现其具有明显的抗癫痫效果，同时对癫痫大鼠认知损伤也有一定的改善作用。进一步对癫痫模型海马组织的大体形态和超微结构观察发现，SF能有效防止癫痫发作对海马神经元的损伤。
第三部分莱菔硫烷对慢性电点燃癫痫大鼠脑保护作用的机制研究
目的：观察SF对癫痫大鼠海马组织中氧化应激和对Nrf2-ARE信号通路及其基因产物HO-1和NQO1表达的影响，探讨SF的脑保护作用机制。
方法：采用杏仁核慢电点燃制备慢性癫痫大鼠模型（每天电刺激1次，连续刺激15d，刺激电流为恒流，单向方波），同时在点燃过程中给予SF（5mg/kg/day）腹腔注射，通过分光光度法检测大鼠海马组织中氧化应激参数（GSH和MDA）表达改变；通过Western blot实验方法观察Nrf2、HO-1和NQO1在海马组织中蛋白水平的表达改变；同时通过Real-time PCR实验方法观察Nrf2mRNA、HO-1mRNA和NQO1mRNA在海马组织中基因水平的表达改变。
结果：1氧化应激参数GSH和MDA含量比较与对照组相比，电点燃组大鼠海马组织中GSH含量明显降低（P<0.01）;与正常对照组相比，电点燃组大鼠海马组织中MDA水平明显升高（P<0.01）。SF治疗后明显升高了电点燃组大鼠海马组织中GSH水平（P<0.01），明显降低了电点燃组大鼠海马组织中MDA水平(P<0.01)。SF单独用药组与对照组相比MDA明显降低（P<0.01）， GSH明显增高（P<0.01）。
2Western blot检测Nrf2、HO-1和NQO1在海马组织中的表达改变
Nrf2蛋白表达水平以H3作为参照，电点燃组大鼠与正常对照组相比，Nrf2水平没有明显统计学差异（P>0.05）；SF治疗后可显著升高癫痫大鼠海马组织中Nrf2蛋白水平（P<0.01）；与对照组相比，单独SF用药组也使Nrf2蛋白表达水平明显增高(P<0.01)。
HO-1和NQO1蛋白表达水平以β-actin作为参照，癫痫组大鼠与正常对照组相比，HO-1和NQO1表达水平无明显统计学差异（P>0.05）；SF治疗后可显著增高癫痫大鼠海马组织中HO-1和NQO1蛋白水平（p<0.01）；与对照组相比，单独SF组也使大鼠海马组织中HO-1和NQO1蛋白表达水平明显增高(P<0.01)。
3Real-time PCR检测Nrf2、HO-1和NQO1mRNA在海马组织的表达改变
Nrf2、HO-1和NQO1mRNA表达水平以GAPDHmRNA作为参照。电点燃组大鼠与正常对照组相比， Nrf2mRNA、 HO-1mRNA和NQO1mRNA水平没有明显统计学差异（P>0.05）；SF治疗后可显著升高癫痫大鼠海马组织中Nrf2mRNA、HO-1mRNA和NQO1mRNA表达（P<0.01）；与对照组相比，单独SF组也使大鼠海马组织中Nrf2mRNA、HO-1mRNA和NQO1mRNA表达水平明显增高(P<0.01)。
结论：SF能有效激活海马组织中Nrf2-ARE信号通路，同时改善癫痫发作导致的氧化应激状态，这可能是其脑保护的作用机制。该研究为阐明SF的神经保护作用机制提供实验依据，同时为抗癫痫药物的选择提供新的思路。
Epilepsy, one of the most common diseases of the nervous system, is thecause of excessive brain neurons repeatedly synchronization discharge, and isa transient of the central nervous system dysfunction clinical syndromeinduced by a variety of chronic brain diseases. Epilepsy can lead to brainneuron selective damage even necrosis, and cause glial cell hyperplasia,reconstructing synaptic plasticity of the brain structure and function change.These brain plasticity changes and repeated seizure are the leading causes ofrefractory epilepsy. At the same time, the patients often accompaniedcognitive dysfunction with repeated seizures, its incidence is about30%-40%.The pathogenesis of epilepsy is very complex, and has not been fullyelucidated, oxidative stress injury, glutamate toxicity of excitatory, calciumoverload and other factors can induce the abnormal discharge of neuronsinducing seizures. The oxidative stress induced oxygen free radical (FR) chainreaction is the core of the damaged neurons pathological link. The resultsconfirmed that many chronic diseases are linked to oxidative stress inducedexcessive free radicals in the human body. The relationship between epilepsyand oxidative stress have become the research focus. There are oxidative stressresponse in the brain of epilepsy animal models or people with epilepsy.Seizure induce FR increased significantly and far more than the body’s abilityto remove FR. Lots of FR can directly make the macromolecular substancessuch as lipid, protein and DNA oxidative damage damaging cell membraneand other cell structures, and FR also can cause apoptosis by inhibiting themitochondrial function. Therefore, in view of the important role of the oxygenfree radicals in the pathological mechanism of epileptic brain damage, theinhibition of oxidative stress and remove oxygen free radicals may be the important strategy for the treatment of epilepsy.
The nuclear factor erythroid2-related factor2(Nrf2) is the key factor incell oxidative stress reaction, Nrf2through reaction with the antioxidantresponse element (ARE), which regulates the expression of a group ofcytoprotective enzymes. Nrf2is an important transcription factor ofadjustment oxidation reaction. Nrf2is located in the cytoplasm combined withKeap1in the physiological state, and is rapidly degraded by ubiquitinproteasome to maintain the low activity. When the body was attacked by suchas oxygen free radicals and endogenous toxin, the Nrf2dissociated with Keapl,and the half-life of Nrf2extended obviously, then Nrf2transposition into thenucleus, and combined with the ARE inducing the expression of a group ofcytoprotective enzymes. So far, it was confirmedn that the Nrf2-ARE signalpathway encoded endogenous protection more than200species. Theendogenous protection can strengthen antioxidant capacity of cell, protectcells from the toxic injury, in anti-tumor, anti-inflammatory and anti-apoptosisHeme oxygenase1(HO-1) and NADPH: quinone oxidoreductase1(NQO1)are important antioxidant enzymes of the signal pathway coding. The previousstudy confirmed that activation of Nrf2-ARE signaling pathways increasesthe expression of the gene products play nerve protection function in nervoussystem diseases such as Parkinson’s disease, cerebral hemorrhage, cerebralinfarction and cerebral trauma and so on. However, to our knowledge, theNrf2-ARE signal pathway and two Nrf2-regulated gene products (HO-1andNQO1) have not been studied after seizure.
Sulforaphane (SF) is isothiocyanates of a widely exists in cruciferousvegetables which have the biological characteristics of anti-oxidation,anti-tumor and immune regulation and so on. SF as a chemical preventiondrug has aroused the attention of the researchers. The previous study confirmsthat SF is a important activator of Nrf2-ARE signal pathway, can induce Nrf2phosphorylation transposition into the nucleus, and combine ARE, regulatingthe expression of antioxidants and detoxifying enzyme, scavenging freeradicals to protect the body’s cells. However, to our knowledge, the neuroprotective effect and the molecular mechanism of SF have not beenstudied after seizure.
The present study was aimed to examine oxidative stress parameters(malondialdehyde and glutathione) and determine the expression of Nrf2,HO-1and NQO1at protein or gene levels in hippocampus of amygdalakindling rats. And further to evaluating if activation of Nrf2-ARE signalpathway with sulforaphane (SF) in hippocampus can suppress the progressionof chronic amygdala kindling, and ameliorate the cognitive impairment andoxidative stress induced by epileptic seizure. The present study was aimed toclarify that the pathophysiology of epilepsy, and to provide new targets forepilepsy treatment.
Part Ⅰ The expression and significance of Nrf2-ARE signaling pathwayin hippocampus of rapid amygdala kindling rats
Objective: To observe oxidative stress parameters(malondialdehyde andglutathione) in hippocampus of rapid amygdala kindling rats and theexpression of Nrf2-ARE signal pathway.
Methods: In the present study, Wistar rats were rapidly kindled in theamygdala. Twenty-four hours after the last seizure, the hippocampus of control,sham and kindled rats were examined for oxidative stress parameters(malondialdehyde and glutathione) by spectrophotometry, the expression ofNrf2, heme oxygenase-1(HO-1) and NAD(P)H:quinone oxidoreductase-1(NQO1) were determined using immunohistochemistry, Western blot andreal-time fluorescence quantitative polymerase chain reaction (PCR).
Results:1The level of GSH in kindling group was significantly lowercompared to sham group (P <0.01) and the level of MDA was much higherthan that of sham group (P <0.01). There was no difference between controland sham groups(P>0.05).
2To localize Nrf2and HO-1expression, immunohistochemical studywas performed. In kindling group, the immunoreactive intensity of Nrf2andHO-1significantly increased in hippocampus, and Nrf2expressed both atcytoplasm and nucleus. In addition, similar localization was found between Nrf2and HO-1in neurons and glial cells. The differences were shownbetween sham and kindling groups by AOD measurement in the expression ofNrf2(P <0.01) and HO-1(P <0.01). There was no difference between controland sham groups (P>0.05).
3Western blot analysis was used to measure in protein level of Nrf2innuclear and HO-1in cytoplasm extracted from hippocampus. Groupdifferences were found between sham and kindling groups with the expressionof Nrf2(P <0.01) and HO-1(P <0.01). There was no difference betweencontrol and shamgroups (P>0.05).
4The expression of Nrf2, HO-1and NQO1mRNA in hippocampus wasstudied by real-time fluorescence quantitative PCR. Group differences werefound between sham and kindling groups in the expression of Nrf2mRNA (P<0.01), HO-1mRNA (P <0.01) and NQO1mRNA (P <0.01). There was nodifference between control and sham groups (P>0.05).
Conclusion:In conclusion, the results of the present study indicate thatthe seizure can induce oxidative stress in hippocampus of rats, which activateNrf2-ARE signal pathway to result in up-regulation of antioxidative anddetoxifying enzymes. Therefore, to activate Nrf2-ARE signal pathway may beone of the strategic targets for epilepsy therapies.
Part ⅡThe brain protection of sulforaphane in chronic amygdalakindling rats
Objective: To observe the role of of sulforaphane in the kindlingprogression and the cognitive impairment of chronic amygdala kindling rats.At the same time, to observe the neuroprotective effect for hippocampalneurons.
Methods: The rats were kindled by one daily electric stimulation of theamygdala, Electric stimulation was delivered15times for the kindling. Thesulforaphane (5mg/kg/day) was injected intraperitoneally at30min beforeevery electric stimulation. The mean seizure stage and the after dischargeduration in the course of kindling were detected for sulforaphane antiepilepticeffect. Morris water maze (MWM) test assessed the cognitive function of kindled rats, and detected the role of sulforaphane. Using Nissal’s staining andtransmission electron microscope detected the role of sulforaphane inhippocampal neuron of kindled rats.
Results:
1The repeated administration of subconvulsive electric stimulationinduced severe seizures during the15kindling stimulation. Electricstimulation was terminated on the15th day. By this time, all of the kindlinggroup rats reached the kindling criterion, i.e.,ageneralized stage4or5at leastthree times, while8out of12rats treated with SF did not reach kindlingcriterion over this time. There were significant differences in seizure stagebetween the kindling and kindling+SF groups from the9th day to the15th day(P<0.05from the9th day to the14th dayand P<0.01on the15th day).Meanwhile, pretreatment with SF markedly decreased the after dischargeduration (ADD) as compared to the kindling group from the7th day to the15th day (P<0.05from the7th day to the10th day and P<0.01from the11thday to the15th day). In the control and SF groups, there were no seizureactivities.
2In the Morris water maze, all animals showed a progressive decline inthe escape latency with training. Rats in the kindling group exhibitedsignificantly prolonged escape latency as compared to the control group(P<0.01). However, the poor performance was mitigated by pretreatment withSF(P<0.01). In the probe trial, the kindling group spent significantly less timein the target quadrant than the control group (P<0.01), while pretreatment withSF significantly improved the performance (P<0.01). The number of crossingthe plat form in the kindling group obviously decreased as compared to thecontrol group(P<0.01), while pretreatment with SF markedly increased thenumber of crossing(P<0.01). Inaddition, SF per se had no significant effectoncognition.
3Nissl staining showed that: neurons in hippocampus of control groupwere clear with normal nucleolus, well-distributed karyotin and rich nisslbodies in kytoplasm, there was no significantly neuron loss. While in the kindled rats, neuron loss was obviously, with shrunken plasma body andpyknotic nuclei. In the kindling+SF group, most pyramid cells were normaland only a few showed chromatin condensation. The hippocampus neurons ofSF group were no significant change as compared to the control group.
4In hippocampal CA1area of the rats in NC group，there were intactneurons，distinct structure，uniform chromatin，abundant apparatus，andintegrate myelin．In PTZ group，however，there were condensed nucleus，edematous neuron，swollen perikaryon with vacuole，reduced mitochondriaand Golgi apparatus and polyribosome；there was also Myelin-splitting nervefiber．In kindling+SF group，the neuron and neuropile were less swollen,karyotheca were normally intact，chromatin were uniform，the apparatus wererich and basically normal，and the myelin was complete and integrate．Incontral group，the synapses in hippocampal CA1area were abundant withdistinct pre-and-post synaptic membranes and rich synaptic vesicles．Thesynaptic cleft was clear．In kindling group，there were reduced synapses withindistinct pre-and-post synaptic membranes．In kindling+SF group，there weremore synapses than that in kindling group. The ultrastructure of hippocampusneurons in SF group were no significant change as compared to the controlgroup.
Conclusions：In summary, the results from the present study demonstratethat SF could suppress the progression of amygdala kindling, also amelioratethe cognitive impairment induced by epileptic seizure. SF also can protecthippocampal neuron damage induced by seizures.
Part Ⅲ The neuroprotection mechanism research of sulforaphane in inchronic amygdala kindling rats
Objective: The present study was aimed to evaluate if activation ofNrf2-ARE signal pathway with SF in hippocampus can ameliorate oxidativestress induced by epileptic seizure, to explore the mechanism of brainprotection.
Methods: The rats were kindled by one daily electric stimulation of theamygdala, Electric stimulation was delivered15times for the kindling. The sulforaphane (5mg/kg/day) was injected intraperitoneally at30min beforeevery electric stimulation. Twenty-four hours after the last seizure, thehippocampus of control, kindled, kindling+SF and SF rats were examined foroxidative stress parameters (malondialdehyde and glutathione) byspectrophotometry, the expression of Nrf2, heme oxygenase-1(HO-1) andNAD(P)H:quinone oxidoreductase-1(NQO1) were determined usingimmunohistochemistry, Western blot and real-time fluorescence quantitativepolymerase chain reaction (PCR).
Results:
1The level of Malondialdehyde (MDA) in the kindling group wasmuch higher as compared to the control group (P<0.01), and the level ofreduced glutathione (GSH) in the kindling group was significantly lower ascompared to the control group (P<0.01). However, pretreatment with SF ledto a noticeable decrease in the concentration of MDA (P<0.01) and asignificant increase in GSH level (P<0.01) as compared to the kindling group.Although SF treatment ameliorated theoxidative damage induced by seizures, the oxidative stress level wasstill higher than the control group (P<0.01). SF per se caused a decrease inthe oxidative stress as indicated by the significant decrease in MDA levelsand the significant increase in GSH levels as compared to the control group(P<0.05).
2Western blot analysis of protein level of Nrf2in nuclear and proteinlevels of HO-1and NQO1in cytoplasm extracted from hippocampus. Theexpression of Nrf2in nuclear and the expression of HO-1and NQO1incytoplasm were not significantly different between control and kindlinggroups(P>0.05). However, pretreatment with SF led to significant increase inthe expression of Nrf2, HO-1and NQO1at protein levels (P<0.01) ascompared to the control and kindling groups. SF per se also causedsignificant increase in the expression of Nrf2, HO-1and NQO1at proteinlevels (P<0.01) as compared to the control group.
3The expression of Nrf2, HO-1and NQO1mRNA in hippocampus was studied by real-time fluorescence quantitative PCR. The expressionof Nrf2, HO-1and NQO1mRNA were not significantly different betweencontrol and kindling groups(P>0.05). However, pretreatment with SF led tosignificant increase in Nrf2, HO-1and NQO1mRNA levels (P<0.01) ascompared to the control and kindling groups. SF per se also causedsignificant increase in Nrf2, HO-1and NQO1mRNA levels (P<0.01) ascompared to the control group.
Conclusions： In summary, the results from the present studydemonstrate that SF could activate the Nrf2-ARE singal pathway, increasethe expression of antioxidative enzymes(HO-1and NQO1) and alsoameliorate oxidative stress induced by epileptic seizure. Therefore, toactivate Nrf2-ARE signal pathway maybe one of the strategic targets forepilepsy therapies.
癫痫的发病机制非常复杂，迄今尚未完全阐明，氧化应激损伤、谷氨酸兴奋性毒性、钙超载等多种因素都能诱导神经元异常放电触发癫痫。其中氧化应激产生的氧自由基连锁反应是神经元受损的核心病理环节。近年的研究结果证实,许多慢性疾病都与人体内氧化应激发生，累积过多的自由基(Free Radical, FR)有关，癫痫与氧化应激之间的关系日益成为科研工作者研究的热点。无论是癫痫动物模型还是癫痫患者的脑内均存在着活跃的氧化应激反应，癫痫发作时FR含量明显增加,远远超过机体对FR的清除能力。大量的FR可以直接使脂质、蛋白质及DNA等大分子物质发生氧化损伤，从而破坏细胞膜及其它细胞结构，同时FR还能通过抑制线粒体功能引起细胞凋亡。因此，针对氧自由基在癫痫脑损伤病理机制中重要作用，抑制氧化应激和清除氧自由基可能是治疗癫痫的重要策略。
转录因子NF-E2相关因子（NF-E2-related factor2，Nrf２）是细胞氧化应激反应中的关键因子，通过与抗氧化反应元件（antioxidant responseelement，ARE）相互作用调节抗氧化蛋白和Ⅱ相解毒酶的表达。Nrf2是机体调节抗氧化反应的重要转录因子，正常生理情况下位于细胞浆中，在细胞浆中与Keapl结合形成复合体，且被泛素蛋白酶迅速降解，从而保持其低活性的生理状态。当机体受到氧自由基和内源性毒素等攻击后Nrf2与Keapl解离，其半衰期明显延长，然后转位进入细胞核，与抗氧化反应元件ARE结合后诱导抗氧化蛋白和Ⅱ相解毒酶基因的表达。到目前为止，已证实经Nrf2-ARE信号路径调节的可编码内源性保护基因超过200个。它们在增强组织抗氧化能力、保护组织细胞免受毒物损伤、抗肿瘤、抗炎症和抗凋亡中起着重要的作用，其中血红素氧合酶1(hemeoxygenase1，HO-1)和依赖还原型辅酶/Ⅱ醌氧化还原酶1(NADPH:quinone oxidoreductase1，NQO1)是该通路编码的重要的抗氧化酶。在中枢神经系统上调HO-1和NQO1的表达，能够减少氧自由基和内源性毒素对神经元的毒性作用。既往研究证实，激活Nrf2-ARE信号通路上调其基因产物的表达对神经系统疾病如帕金森病、脑出血、脑梗死和脑外伤等，具有很强的神经保护功能。但是，Nrf2-ARE信号通路在癫痫发病中表达改变，及其对海马神经元的保护作用还未见明确相关报道。
莱菔硫烷（Sulforaphane，SF）是一种广泛存在于十字花科蔬菜的异硫氰酸盐，具有抗氧化、抗肿瘤和免疫调节等多种生物学特性，作为化学预防药物已引起广大研究者的关注。既往研究证实，SF是Nrf2-ARE信号通路重要的激活剂，能够诱发Nrf2磷酸化转位入核，与ARE结合，正向调控抗氧化蛋白和Ⅱ相解毒酶的表达，清除自由基从而对机体细胞起到保护作用。但SF对癫痫的神经保护作用及其具体分子机制尚未见明确报道。
本实验主要采用杏仁核电点燃癫痫大鼠模型，观察癫痫大鼠模型海马组织中氧化应激参数（丙二醛和谷胱甘肽）的表达改变；Nrf2及其编码的基因产物HO-1和NQO1的表达改变；同时进一步验证以Nrf2-ARE信号通路为靶点的药物SF对海马组织氧化应激的改善情况及对神经元的保护作用。为阐明癫痫的病理生理机制提供实验依据，同时为癫痫治疗提供新的靶点，为抗癫痫药物的选择提供新的思路。
第一部分Nrf2-ARE信号通路在急性电点燃癫痫大鼠海马的表达及意义
目的：观察急性电点燃癫痫大鼠海马组织中氧化应激参数（丙二醛和谷胱甘肽）及Nrf2-ARE信号通路的表达改变，以期为癫痫的治疗提供新的靶点。
方法：采用杏仁核快速电点燃制备急性癫痫大鼠模型（每天刺激20次，刺激间隔时间10min，连续刺激2d，刺激电流为恒流，单向方波。），通过分光光度法检测大鼠海马组织中氧化应激参数（丙二醛和谷胱甘肽）表达改变；通过免疫组化和Western blot方法观察Nrf2、HO-1和NQO1在海马组织中蛋白水平的表达改变；同时通过Real-time PCR方法观察Nrf2mRNA、HO-1mRNA和NQO1mRNA在海马组织中基因水平的表达改变。
结果：1、氧化应激参数的改变：与对照组相比，癫痫组大鼠海马组织中谷胱甘肽（Glutathione, GSH）含量明显降低（P<0.01）。与对照组相比，癫痫组大鼠海马组织中丙二醛（malondialdehyde, MDA）水平明显升高（P<0.01）。假手术组与对照组相比GSH与MDA差别无明显统计学意义（P>0.05）。
2、免疫组化结果：Nrf2免疫反应在海马的锥体细胞和神经胶质细胞的胞浆和胞核均有表达；HO-1免疫反应主要在海马锥体细胞和神经胶质细胞的胞浆。对三组大鼠海马CA1、CA2和CA3区Nrf2和HO-1表达的平均光密度值（AOD）测量，癫痫组比对照组AOD值明显增高(P﹤0.01)；假手术组和对照组相比，AOD值差别无明显统计学意义（P>0.05）。
3、Western-blot结果:3.1、电点燃大鼠海马组织胞核内的Nrf2的表达明显增强，而在对照组和假手术组Nrf2表达相对较弱，Nrf2与H3免疫印迹条带相对吸光度比值电点燃组比对照组明显增加，具有明显统计学差异（P<0.01）。假手术组和对照组相比没有明显统计学差异（P>0.05）。3.2、电点燃大鼠海马组织胞浆内的HO-1的表达明显增强，而在对照组和假手术组HO-1表达相对较弱，HO-1与β-actin免疫印迹条带相对吸光度比值，电点燃组比对照组明显增加，具有统计学差异（P<0.01）。假手术组的吸光度比值和对照组相比没有明显统计学差异（P>0.05）。
4、Real-time PCR实验结果:各组大鼠海马组织中Nrf2、HO-1和NQO1mRNA的表达水平以三种物质mRNA／GAPDHmRNA表示，与对照组Nrf2、HO-1和NQO1mRNA表达水平相比，电点燃组Nrf2、HO-1和NQO1mRNA表达水平明显增高，均具有统计学意义(P<0.01)；假手术组Nrf2、HO-1和NQO1mRNA表达水平和对照组表达水平相比无明显统计学差异(P>0.05)。
结论：急性癫痫发作可以导致海马组织发生氧化应激，导致脂质氧化代谢产物MDA含量增高和自由基清除剂GSH含量降低；急性癫痫发作可以诱导海马组织中Nrf2和其编码的基因产物HO-1和NQO1在蛋白和基因水平表达明显增强。说明Nrf2-ARE信号通路与癫痫发病关系密切，可能是癫痫治疗的新的靶点。
第二部分莱菔硫烷对慢性电点燃癫痫大鼠的脑保护作用研究
目的：观察莱菔硫烷对慢性电点燃癫痫大鼠的抗癫痫作用及对认知功能的影响；同时观察其对海马神经元的保护作用。
方法：采用慢性杏仁核电点燃制备癫痫大鼠模型（每天电刺激1次，连续刺激15d，刺激电流为恒流，单向方波。），在点燃过程中给予大鼠腹腔注射SF（5mg/kg/day）。通过对大鼠点燃过程中发作等级和后放电持续时间（afterdischarge duration，ADD）比较，观察SF的抗癫痫作用；通过Morris水迷宫监测癫痫大鼠的认知功能及SF对其的改善作用；通过尼氏染色和透射电镜观察癫痫大鼠海马神经元形态学改变及SF的神经保护作用。
结果：1SF对慢性癫痫模型点燃过程的影响在连续给与15次亚惊厥电刺激的过程中，所有的大鼠均出现了不同程度的癫痫发作。连续15次电刺激后，在电点燃组中所有的大鼠均达到了完全点燃的标准（连续出现3次4-5级发作），而在电点燃+SF组的大鼠中仅有4只达到了点燃标准。对两组大鼠的发作等级比较后发现在第9-15天的时候具有明显的统计学差异（在第9-14天时P<0.05,在第15天时P<0.01）。同时，连续给予15次电刺激过程中所有大鼠的ADD逐渐延长，比较电点燃组和电点燃+SF组发现，在第7-15天的时候具有明显的统计学差异（在第9-10天时P<0.05,在第11-15天时P<0.01）。对照组和SF组没有出现癫痫发作。
2Morris水迷宫检测结果
2.1定位航行实验结果：每日逃避潜伏期取其平均值进行比较，结果显示，(1)对照组、电点燃组、SF+电点燃组和SF组四组大鼠的逃避潜伏期在第1和2天无统计学意义(P>0.05)。在第1和2天，与对照组大鼠的逃避潜伏期相比较，电点燃组大鼠及SF+电点燃组大鼠的逃避潜伏期均有延长趋势。(2)实验第3、4、5天，各组逃避潜伏期均比第1、2天有缩短趋势，在第3、4和5天，与对照组大鼠的逃避潜伏期相比较，电点燃组大鼠的逃避潜伏期均明显延长(P<0.05)；SF+电点燃组大鼠的逃避潜伏期较电点燃组大鼠均明显缩短(P<0.05)，但与对照组比较仍明显延长(P<0.05)；SF组大鼠的逃避潜伏期和对照组没有明显变化(P>0.05)。
2.2空间探索试验结果：电点燃组大鼠120s内穿越平台区域的次数明显低于对照组(P<0.01)；SF+电点燃组大鼠120s内穿越平台区域的次数与电点燃组比较次数均明显增多(P<0.01)。在平台象限的游泳时间为：与对照组相比，电点燃组明显减少(P<0.01)，SF+电点燃组在平台象限的游泳时间与电点燃组比较明显增多(P<0.01)，SF组和对照组相比无显著性差异(P>0.05)。
3尼氏染色结果：对照组大鼠海马神经元形态完整、核仁清晰，胞浆内尼氏体丰富，锥体细胞排列规则紧密，没有明显神经元丢失；与对照组相比，电点燃组大鼠海马神经元丢失明显，排列紊乱，可见细胞皱缩，染色质凝集成块、核固缩、尼氏体数量减少；SF治疗后海马组织神经元边缘清晰，神经元没有明显丢失，结构正常，仅少量染色质凝集、尼氏体数量较癫痫组显著增加。SF单独用药组海马组织神经元和对照组没有明显改变。
4透射电极观察海马CA1区超微结构变化：对照组电镜观察：神经元形态完整，结构清晰。胞核呈圆形，染色质均匀分布。胞质内细胞器丰富，可见线粒体、粗面内质网、滑面内质网和大量的多核糖体。线粒体呈圆形或椭圆形，嵴清晰可见。粗面内质网和高尔基体发达。轴突中神经微丝微管清晰可见。突触前、后膜结构清晰，突触前膜靠近突触间隙一侧有较多圆形的突触囊泡，突触间隙清晰。电点燃组电镜观察：海马神经元胞核肿胀、形态不规则，核内异染色质减少分散，胞核疏松、透明，有些出现核固缩现象。线粒体空泡化或均质化。神经微丝微管结构不清，可见聚集。突触前、后膜结构模糊不清，突触间隙增大，靠近突触前膜的突触囊泡减少。SF+电点燃组电镜观察：海马神经元核膜完整，核形态基本正常，染色质均匀分布，胞质中细胞器形态基本正常，有髓神经髓鞘完整。突触数量较多，形态接近于正常。突触前、后膜结构清楚，突触间隙清晰，突触前膜的突触囊泡较多。SF单组用药组电镜观察和正常对照组超微结构没有明显改变。
结论：本研究通过给慢性电点燃大鼠腹腔注射SF后发现其具有明显的抗癫痫效果，同时对癫痫大鼠认知损伤也有一定的改善作用。进一步对癫痫模型海马组织的大体形态和超微结构观察发现，SF能有效防止癫痫发作对海马神经元的损伤。
第三部分莱菔硫烷对慢性电点燃癫痫大鼠脑保护作用的机制研究
目的：观察SF对癫痫大鼠海马组织中氧化应激和对Nrf2-ARE信号通路及其基因产物HO-1和NQO1表达的影响，探讨SF的脑保护作用机制。
方法：采用杏仁核慢电点燃制备慢性癫痫大鼠模型（每天电刺激1次，连续刺激15d，刺激电流为恒流，单向方波），同时在点燃过程中给予SF（5mg/kg/day）腹腔注射，通过分光光度法检测大鼠海马组织中氧化应激参数（GSH和MDA）表达改变；通过Western blot实验方法观察Nrf2、HO-1和NQO1在海马组织中蛋白水平的表达改变；同时通过Real-time PCR实验方法观察Nrf2mRNA、HO-1mRNA和NQO1mRNA在海马组织中基因水平的表达改变。
结果：1氧化应激参数GSH和MDA含量比较与对照组相比，电点燃组大鼠海马组织中GSH含量明显降低（P<0.01）;与正常对照组相比，电点燃组大鼠海马组织中MDA水平明显升高（P<0.01）。SF治疗后明显升高了电点燃组大鼠海马组织中GSH水平（P<0.01），明显降低了电点燃组大鼠海马组织中MDA水平(P<0.01)。SF单独用药组与对照组相比MDA明显降低（P<0.01）， GSH明显增高（P<0.01）。
2Western blot检测Nrf2、HO-1和NQO1在海马组织中的表达改变
Nrf2蛋白表达水平以H3作为参照，电点燃组大鼠与正常对照组相比，Nrf2水平没有明显统计学差异（P>0.05）；SF治疗后可显著升高癫痫大鼠海马组织中Nrf2蛋白水平（P<0.01）；与对照组相比，单独SF用药组也使Nrf2蛋白表达水平明显增高(P<0.01)。
HO-1和NQO1蛋白表达水平以β-actin作为参照，癫痫组大鼠与正常对照组相比，HO-1和NQO1表达水平无明显统计学差异（P>0.05）；SF治疗后可显著增高癫痫大鼠海马组织中HO-1和NQO1蛋白水平（p<0.01）；与对照组相比，单独SF组也使大鼠海马组织中HO-1和NQO1蛋白表达水平明显增高(P<0.01)。
3Real-time PCR检测Nrf2、HO-1和NQO1mRNA在海马组织的表达改变
Nrf2、HO-1和NQO1mRNA表达水平以GAPDHmRNA作为参照。电点燃组大鼠与正常对照组相比， Nrf2mRNA、 HO-1mRNA和NQO1mRNA水平没有明显统计学差异（P>0.05）；SF治疗后可显著升高癫痫大鼠海马组织中Nrf2mRNA、HO-1mRNA和NQO1mRNA表达（P<0.01）；与对照组相比，单独SF组也使大鼠海马组织中Nrf2mRNA、HO-1mRNA和NQO1mRNA表达水平明显增高(P<0.01)。
结论：SF能有效激活海马组织中Nrf2-ARE信号通路，同时改善癫痫发作导致的氧化应激状态，这可能是其脑保护的作用机制。该研究为阐明SF的神经保护作用机制提供实验依据，同时为抗癫痫药物的选择提供新的思路。
Epilepsy, one of the most common diseases of the nervous system, is thecause of excessive brain neurons repeatedly synchronization discharge, and isa transient of the central nervous system dysfunction clinical syndromeinduced by a variety of chronic brain diseases. Epilepsy can lead to brainneuron selective damage even necrosis, and cause glial cell hyperplasia,reconstructing synaptic plasticity of the brain structure and function change.These brain plasticity changes and repeated seizure are the leading causes ofrefractory epilepsy. At the same time, the patients often accompaniedcognitive dysfunction with repeated seizures, its incidence is about30%-40%.The pathogenesis of epilepsy is very complex, and has not been fullyelucidated, oxidative stress injury, glutamate toxicity of excitatory, calciumoverload and other factors can induce the abnormal discharge of neuronsinducing seizures. The oxidative stress induced oxygen free radical (FR) chainreaction is the core of the damaged neurons pathological link. The resultsconfirmed that many chronic diseases are linked to oxidative stress inducedexcessive free radicals in the human body. The relationship between epilepsyand oxidative stress have become the research focus. There are oxidative stressresponse in the brain of epilepsy animal models or people with epilepsy.Seizure induce FR increased significantly and far more than the body’s abilityto remove FR. Lots of FR can directly make the macromolecular substancessuch as lipid, protein and DNA oxidative damage damaging cell membraneand other cell structures, and FR also can cause apoptosis by inhibiting themitochondrial function. Therefore, in view of the important role of the oxygenfree radicals in the pathological mechanism of epileptic brain damage, theinhibition of oxidative stress and remove oxygen free radicals may be the important strategy for the treatment of epilepsy.
The nuclear factor erythroid2-related factor2(Nrf2) is the key factor incell oxidative stress reaction, Nrf2through reaction with the antioxidantresponse element (ARE), which regulates the expression of a group ofcytoprotective enzymes. Nrf2is an important transcription factor ofadjustment oxidation reaction. Nrf2is located in the cytoplasm combined withKeap1in the physiological state, and is rapidly degraded by ubiquitinproteasome to maintain the low activity. When the body was attacked by suchas oxygen free radicals and endogenous toxin, the Nrf2dissociated with Keapl,and the half-life of Nrf2extended obviously, then Nrf2transposition into thenucleus, and combined with the ARE inducing the expression of a group ofcytoprotective enzymes. So far, it was confirmedn that the Nrf2-ARE signalpathway encoded endogenous protection more than200species. Theendogenous protection can strengthen antioxidant capacity of cell, protectcells from the toxic injury, in anti-tumor, anti-inflammatory and anti-apoptosisHeme oxygenase1(HO-1) and NADPH: quinone oxidoreductase1(NQO1)are important antioxidant enzymes of the signal pathway coding. The previousstudy confirmed that activation of Nrf2-ARE signaling pathways increasesthe expression of the gene products play nerve protection function in nervoussystem diseases such as Parkinson’s disease, cerebral hemorrhage, cerebralinfarction and cerebral trauma and so on. However, to our knowledge, theNrf2-ARE signal pathway and two Nrf2-regulated gene products (HO-1andNQO1) have not been studied after seizure.
Sulforaphane (SF) is isothiocyanates of a widely exists in cruciferousvegetables which have the biological characteristics of anti-oxidation,anti-tumor and immune regulation and so on. SF as a chemical preventiondrug has aroused the attention of the researchers. The previous study confirmsthat SF is a important activator of Nrf2-ARE signal pathway, can induce Nrf2phosphorylation transposition into the nucleus, and combine ARE, regulatingthe expression of antioxidants and detoxifying enzyme, scavenging freeradicals to protect the body’s cells. However, to our knowledge, the neuroprotective effect and the molecular mechanism of SF have not beenstudied after seizure.
The present study was aimed to examine oxidative stress parameters(malondialdehyde and glutathione) and determine the expression of Nrf2,HO-1and NQO1at protein or gene levels in hippocampus of amygdalakindling rats. And further to evaluating if activation of Nrf2-ARE signalpathway with sulforaphane (SF) in hippocampus can suppress the progressionof chronic amygdala kindling, and ameliorate the cognitive impairment andoxidative stress induced by epileptic seizure. The present study was aimed toclarify that the pathophysiology of epilepsy, and to provide new targets forepilepsy treatment.
Part Ⅰ The expression and significance of Nrf2-ARE signaling pathwayin hippocampus of rapid amygdala kindling rats
Objective: To observe oxidative stress parameters(malondialdehyde andglutathione) in hippocampus of rapid amygdala kindling rats and theexpression of Nrf2-ARE signal pathway.
Methods: In the present study, Wistar rats were rapidly kindled in theamygdala. Twenty-four hours after the last seizure, the hippocampus of control,sham and kindled rats were examined for oxidative stress parameters(malondialdehyde and glutathione) by spectrophotometry, the expression ofNrf2, heme oxygenase-1(HO-1) and NAD(P)H:quinone oxidoreductase-1(NQO1) were determined using immunohistochemistry, Western blot andreal-time fluorescence quantitative polymerase chain reaction (PCR).
Results:1The level of GSH in kindling group was significantly lowercompared to sham group (P <0.01) and the level of MDA was much higherthan that of sham group (P <0.01). There was no difference between controland sham groups(P>0.05).
2To localize Nrf2and HO-1expression, immunohistochemical studywas performed. In kindling group, the immunoreactive intensity of Nrf2andHO-1significantly increased in hippocampus, and Nrf2expressed both atcytoplasm and nucleus. In addition, similar localization was found between Nrf2and HO-1in neurons and glial cells. The differences were shownbetween sham and kindling groups by AOD measurement in the expression ofNrf2(P <0.01) and HO-1(P <0.01). There was no difference between controland sham groups (P>0.05).
3Western blot analysis was used to measure in protein level of Nrf2innuclear and HO-1in cytoplasm extracted from hippocampus. Groupdifferences were found between sham and kindling groups with the expressionof Nrf2(P <0.01) and HO-1(P <0.01). There was no difference betweencontrol and shamgroups (P>0.05).
4The expression of Nrf2, HO-1and NQO1mRNA in hippocampus wasstudied by real-time fluorescence quantitative PCR. Group differences werefound between sham and kindling groups in the expression of Nrf2mRNA (P<0.01), HO-1mRNA (P <0.01) and NQO1mRNA (P <0.01). There was nodifference between control and sham groups (P>0.05).
Conclusion:In conclusion, the results of the present study indicate thatthe seizure can induce oxidative stress in hippocampus of rats, which activateNrf2-ARE signal pathway to result in up-regulation of antioxidative anddetoxifying enzymes. Therefore, to activate Nrf2-ARE signal pathway may beone of the strategic targets for epilepsy therapies.
Part ⅡThe brain protection of sulforaphane in chronic amygdalakindling rats
Objective: To observe the role of of sulforaphane in the kindlingprogression and the cognitive impairment of chronic amygdala kindling rats.At the same time, to observe the neuroprotective effect for hippocampalneurons.
Methods: The rats were kindled by one daily electric stimulation of theamygdala, Electric stimulation was delivered15times for the kindling. Thesulforaphane (5mg/kg/day) was injected intraperitoneally at30min beforeevery electric stimulation. The mean seizure stage and the after dischargeduration in the course of kindling were detected for sulforaphane antiepilepticeffect. Morris water maze (MWM) test assessed the cognitive function of kindled rats, and detected the role of sulforaphane. Using Nissal’s staining andtransmission electron microscope detected the role of sulforaphane inhippocampal neuron of kindled rats.
Results:
1The repeated administration of subconvulsive electric stimulationinduced severe seizures during the15kindling stimulation. Electricstimulation was terminated on the15th day. By this time, all of the kindlinggroup rats reached the kindling criterion, i.e.,ageneralized stage4or5at leastthree times, while8out of12rats treated with SF did not reach kindlingcriterion over this time. There were significant differences in seizure stagebetween the kindling and kindling+SF groups from the9th day to the15th day(P<0.05from the9th day to the14th dayand P<0.01on the15th day).Meanwhile, pretreatment with SF markedly decreased the after dischargeduration (ADD) as compared to the kindling group from the7th day to the15th day (P<0.05from the7th day to the10th day and P<0.01from the11thday to the15th day). In the control and SF groups, there were no seizureactivities.
2In the Morris water maze, all animals showed a progressive decline inthe escape latency with training. Rats in the kindling group exhibitedsignificantly prolonged escape latency as compared to the control group(P<0.01). However, the poor performance was mitigated by pretreatment withSF(P<0.01). In the probe trial, the kindling group spent significantly less timein the target quadrant than the control group (P<0.01), while pretreatment withSF significantly improved the performance (P<0.01). The number of crossingthe plat form in the kindling group obviously decreased as compared to thecontrol group(P<0.01), while pretreatment with SF markedly increased thenumber of crossing(P<0.01). Inaddition, SF per se had no significant effectoncognition.
3Nissl staining showed that: neurons in hippocampus of control groupwere clear with normal nucleolus, well-distributed karyotin and rich nisslbodies in kytoplasm, there was no significantly neuron loss. While in the kindled rats, neuron loss was obviously, with shrunken plasma body andpyknotic nuclei. In the kindling+SF group, most pyramid cells were normaland only a few showed chromatin condensation. The hippocampus neurons ofSF group were no significant change as compared to the control group.
4In hippocampal CA1area of the rats in NC group，there were intactneurons，distinct structure，uniform chromatin，abundant apparatus，andintegrate myelin．In PTZ group，however，there were condensed nucleus，edematous neuron，swollen perikaryon with vacuole，reduced mitochondriaand Golgi apparatus and polyribosome；there was also Myelin-splitting nervefiber．In kindling+SF group，the neuron and neuropile were less swollen,karyotheca were normally intact，chromatin were uniform，the apparatus wererich and basically normal，and the myelin was complete and integrate．Incontral group，the synapses in hippocampal CA1area were abundant withdistinct pre-and-post synaptic membranes and rich synaptic vesicles．Thesynaptic cleft was clear．In kindling group，there were reduced synapses withindistinct pre-and-post synaptic membranes．In kindling+SF group，there weremore synapses than that in kindling group. The ultrastructure of hippocampusneurons in SF group were no significant change as compared to the controlgroup.
Conclusions：In summary, the results from the present study demonstratethat SF could suppress the progression of amygdala kindling, also amelioratethe cognitive impairment induced by epileptic seizure. SF also can protecthippocampal neuron damage induced by seizures.
Part Ⅲ The neuroprotection mechanism research of sulforaphane in inchronic amygdala kindling rats
Objective: The present study was aimed to evaluate if activation ofNrf2-ARE signal pathway with SF in hippocampus can ameliorate oxidativestress induced by epileptic seizure, to explore the mechanism of brainprotection.
Methods: The rats were kindled by one daily electric stimulation of theamygdala, Electric stimulation was delivered15times for the kindling. The sulforaphane (5mg/kg/day) was injected intraperitoneally at30min beforeevery electric stimulation. Twenty-four hours after the last seizure, thehippocampus of control, kindled, kindling+SF and SF rats were examined foroxidative stress parameters (malondialdehyde and glutathione) byspectrophotometry, the expression of Nrf2, heme oxygenase-1(HO-1) andNAD(P)H:quinone oxidoreductase-1(NQO1) were determined usingimmunohistochemistry, Western blot and real-time fluorescence quantitativepolymerase chain reaction (PCR).
Results:
1The level of Malondialdehyde (MDA) in the kindling group wasmuch higher as compared to the control group (P<0.01), and the level ofreduced glutathione (GSH) in the kindling group was significantly lower ascompared to the control group (P<0.01). However, pretreatment with SF ledto a noticeable decrease in the concentration of MDA (P<0.01) and asignificant increase in GSH level (P<0.01) as compared to the kindling group.Although SF treatment ameliorated theoxidative damage induced by seizures, the oxidative stress level wasstill higher than the control group (P<0.01). SF per se caused a decrease inthe oxidative stress as indicated by the significant decrease in MDA levelsand the significant increase in GSH levels as compared to the control group(P<0.05).
2Western blot analysis of protein level of Nrf2in nuclear and proteinlevels of HO-1and NQO1in cytoplasm extracted from hippocampus. Theexpression of Nrf2in nuclear and the expression of HO-1and NQO1incytoplasm were not significantly different between control and kindlinggroups(P>0.05). However, pretreatment with SF led to significant increase inthe expression of Nrf2, HO-1and NQO1at protein levels (P<0.01) ascompared to the control and kindling groups. SF per se also causedsignificant increase in the expression of Nrf2, HO-1and NQO1at proteinlevels (P<0.01) as compared to the control group.
3The expression of Nrf2, HO-1and NQO1mRNA in hippocampus was studied by real-time fluorescence quantitative PCR. The expressionof Nrf2, HO-1and NQO1mRNA were not significantly different betweencontrol and kindling groups(P>0.05). However, pretreatment with SF led tosignificant increase in Nrf2, HO-1and NQO1mRNA levels (P<0.01) ascompared to the control and kindling groups. SF per se also causedsignificant increase in Nrf2, HO-1and NQO1mRNA levels (P<0.01) ascompared to the control group.
Conclusions： In summary, the results from the present studydemonstrate that SF could activate the Nrf2-ARE singal pathway, increasethe expression of antioxidative enzymes(HO-1and NQO1) and alsoameliorate oxidative stress induced by epileptic seizure. Therefore, toactivate Nrf2-ARE signal pathway maybe one of the strategic targets forepilepsy therapies.
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