【作者】 曹丽丽；

【导师】 迟兆富；

【作者基本信息】 山东大学 ， 儿科学， 2009， 博士

【摘要】 癫痫是严重危害人类健康的慢性脑部疾病之一,我国目前约有900万癫痫患者,给社会、家庭和个人都带来了沉重负担。约有20%～30%的癫痫患者用现有的抗癫痫治疗方法不能很好地控制发作,演变为难治性癫痫,颞叶癫痫是其中最常见的一种类型。近年来,对颞叶癫痫的病因、病理及治疗等研究已经取得了很大进展,但它的发病机制仍未完全阐明。颞叶癫痫一个重要的病理变化是海马硬化,表现为神经元脱失和胶质细胞增生。癫痫发作能造成神经元损伤已被广泛认可,一般认为癫痫发作可诱导谷氨酸过度释放,激活谷氨酸N-甲基-D-天冬氨酸(N-methyl-D-asparate,NMDA)受体,触发钙离子向神经元内流,激活一氧化氮合酶,产生大量自由基,导致氧化应激损伤,活化多种与细胞死亡相关的蛋白,最终造成神经元发生程序性死亡。目前,癫痫发作致神经元损伤的死亡形式及分子机制仍不十分清楚,对癫痫发作后的神经元保护也缺乏有效手段。细胞稳定状态的维持取决于细胞内大分子物质生物合成与代谢的平衡,这一平衡的打破往往导致肿瘤细胞生长或诱导细胞发生自我调节以适应环境。真核细胞存在两大主要的蛋白质降解途径—蛋白酶体和溶酶体途径。自噬作为溶酶体降解途径的形式之一,对于长寿命蛋白降解和细胞器更新发挥了必不可少的作用。根据细胞物质运到溶酶体内的途径不同,哺乳动物细胞内的自噬主要有三种不同的类型:①巨自噬(macroautophagy),是最主要的一类自噬;多数人认为是由内质网来源的单层膜凹陷形成杯状双层膜样的分隔膜,进而完全包绕待降解物形成自噬体,接着与溶酶体融合,自噬体内细胞物质如细胞器被溶酶体酶溶解;该途径主要降解不必要的或功能异常的细胞器和蛋白;②小自噬(microautophagy):是溶酶体的膜直接包裹如长寿命蛋白并在溶酶体内降解;③分子伴侣介导的自噬(Chaperone mediated autophagy,CMA):胞浆内蛋白结合到分子伴侣后被转运到溶酶体腔中被溶酶体酶消化。细胞为维持正常的新陈代谢,其生长过程始终都有自噬现象;而自噬的消长受多种因素的影响。在正常情况下,自噬在多数细胞内都处于一个相当低的水平。研究表明氧化应激是自噬重要的诱导因素,作为信号分子活性氧可激活自噬现象。此外,自噬参与细胞的死亡过程,自噬是凋亡之外的第二种程序性细胞死亡方式。到目前为止,自噬是否参与癫痫诱发的氧化应激反应,以及在神经元程序性死亡中发挥何种作用;自由基清除剂维生素E对自噬的影响如何;自噬抑制剂渥曼青霉素(wortmannin,WM)是否对癫痫致海马损伤具有保护作用仍未见报道。本研究建立PILO(pilocarpine,PILO)诱导的大鼠颞叶癫痫模型,探讨癫痫发作后海马神经元损伤后的自噬现象的病理特征;分别应用自由基清除剂维生素E和自噬抑制剂WM抑制剂进行干预,检测与自噬相关的蛋白和信号分子的变化,探讨自噬在大鼠癫痫致海马损伤中的作用及其分子机制。本研究分三部分。第一部分巨自噬在癫痫大鼠海马损伤中作用的研究及维生素E对巨自噬的调节作用目的探讨PILO诱导大鼠癫痫模型海马神经元损伤的病理特征;研究巨自噬在致痫大鼠海马组织的变化规律;探讨自由基清除剂维生素E对巨自噬的影响以及神经保护作用。方法1.应用腹腔注射的方法建立PILO诱导的大鼠颞叶癫痫模型。实验动物在检测自噬活性时分为对照组、PILO致痫2 h、8 h、16 h、24 h、72 h组;在检测维生素E对巨自噬抑制作用及对海马神经元保护作用时分为对照组、PILO致痫组、维生素E+生理盐水组及维生素E干预组(维生素E+PILO组)。2.观察PILO诱导的颞叶癫痫大鼠的行为学变化,致痫成功率,诱导癫痫持续状态的潜伏期(注射PILO到发生癫痫持续状态的时间)和24小时的死亡率;观察维生素E对癫痫动物行为学的保护作用。3.应用HE染色、Nissl染色观察各实验组大鼠癫痫后海马神经元的形态及数目改变4.应用电镜观察各组大鼠癫痫后海马神经元的形态特征及自噬泡的变化5.应用蛋白免疫印迹法(Western blot)检测巨自噬活性指标微管相关蛋白轻链3(LC3)Ⅱ/LC3Ⅰ的比例、beclin l在癫痫发作后不同时程的变化。应用逆转录酶聚合酶链反应(RT-PCR)检测beclin l mRNA在癫痫发作后不同时程的变化。6.应用Western blot检测维生素E对癫痫发作后巨自噬活性变化的作用结果1.注射PILO后5～30min出现刻板行为:表现为凝视不动、咀嚼、吸鼻或探索行为、湿狗样震颤、反复头颈上仰等,随后出现面肌痉挛、点头、单侧或双侧前肢阵挛、伴直立、跌倒或翻转,最后可发展至全身强直痉挛持续状态发作。本组50只大鼠,有45只出现Ⅲ-Ⅴ级发作,并最终呈SE(其中存活41只,死亡3只),另外5只未达到Ⅲ级反作。待大鼠出现SE发作后2 h,以安定终止后,大鼠处于抑制与消耗状态,反应与活动减少。PILO致痫组15只大鼠14只出现SE,致痫率为93.33%。维生素E干预组的致痫率为80%,两组间无显著性差异。注射PILO到癫痫持续状态的潜伏期从14分钟到62分钟,平均为(35.64±13.77 min,n=14),维生素E干预组的潜伏期为(54.27±22.16 min,n=12)(t=0.027,p＜0.05)。致痫24h时,14只PILO致痫组动物死亡7只,死亡率为50%;维生素E干预组中12只致痫动物死亡1只,死亡率8.3%;两组间比较有显著性差异(x~2=5.26,p＜0.05)。维生素E+生理盐水组动物无一例发生癫痫发作。2.正常大鼠海马CAl区锥体细胞结构清晰完整,细胞核结构正常,染色质分布均匀,胞浆内尼氏小体丰富。癫痫发作后大鼠海马神经元肿胀、排列紊乱,膜破裂,胞浆尼氏小体减少。维生素E干预组大鼠海马大部分锥体细胞边缘清晰,形态正常,仅少量神经元染色质凝聚、轮廓模糊。电镜下,正常大鼠海马CAl区锥体细胞结构清晰完整,细胞核结构正常,染色质分布均匀,胞浆内尼氏小体丰富;细胞内自噬泡少见。癫痫发作后大鼠海马受损神经元表现为胞核内染色质凝聚、边集,线粒体嵴消失、肿胀,细胞器减少,胞浆内形成大小不等的空腔和空泡,细胞膜破裂,细胞内容物泄漏等;细胞内自噬泡明显增多。维生素E干预组CAl区形态损伤明显减轻,细胞内自噬泡少见,。3.大鼠海马CAl区锥体细胞数目在癫痫发作24h(75.5±5.92)后较对照组(110.67±18.56)明显减少(n=6,P＜0.05);维生素E干预组大鼠CAl区锥体细胞数目(94.67±13.03)较PILO组显著增加(n＝6,P＜0.05)4.Western blot研究发现,大鼠致痫后2 h海马组织LC3Ⅱ/LC3Ⅰ的比值以及beclin l表达升高(P＜0.05),在24 h达高峰(P＜0.05),并至少持续增高到72 h(P＜0.05)。应用RT-PCR研究发现,大鼠致痫后2 h海马组织beclin l mRNA升高(P＜0.05),在24 h达高峰(P＜0.05),并至少持续增高到72 h(P＜0.05)。5.Western blot研究显示,大鼠致痫后24h,维生素E干预组较PILO组大鼠海马LC3Ⅱ/LC3Ⅰ的比值以及beclin l的表达显著降低(P＜0.05);RT-PCR研究显示,大鼠致痫后24h,维生素E干预组较PILO组大鼠海马beclin l的合成显著降低(P＜0.05)。结论1.PILO致痫大鼠海马神经元受损明显、数目减少,受损神经元存在巨自噬激活现象。2.LC3Ⅱ/LC3Ⅰ的比值以及beclin l表达活性在癫痫发作后表现出时间依赖性增高,该变化可被维生素E部分抑制。3.维生素E可减轻癫痫发作造成的海马神经元损伤,延长致痫潜伏期,提高致痫动物的存活率。第二部分分子伴侣介导的自噬在癫痫大鼠海马损伤中作用的研究及维生素E的调节作用目的探讨PILO致痫大鼠海马组织分子伴侣介导的自噬(Chaperone mediatedautophagy,CMA)随时间的变化规律及维生素E对其的调节作用方法1.应用腹腔注射的方法建立PILO诱导的大鼠颞叶癫痫模型。实验动物在检测CMA活性时分为对照组、PILO致痫2 h、8 h、16 h、24 h组;在检测维生素E对CMA抑制作用及对海马神经元保护作用时分为对照组、PILO致痫组、维生素E+生理盐水组及维生素E干预组(维生素E+PILO组)。2.溶酶体的分离和溶酶体膜的提取分离:采用速度离心和密度梯度离心的方式。3.应用Western blot检测各组大鼠海马组织溶酶体膜上和溶酶体基质内2a型溶酶体膜相关蛋白(lysosome-associated membrane protein type 2a,LAMP2a)的表达;应用RT-PCR检测各组大鼠海马组织LAMP2a的mRNA的水平。结果1.大鼠致痫后8 h海马溶酶体膜上和基质内LAMP2a表达增加(P＜0.05),72 h时仍持续升高(P＜0.05);溶膜体膜上和基质内LAMP2a的比值无明显变化。2.应用维生素E干预后,24h时,癫痫大鼠海马溶酶体膜上和基质内LAMP2a表达明低于PILO+盐水组大鼠(P＜0.05);溶膜体膜上和基质内LAMP2a的分布无明显变化(p＜0.05)。3.RT-PCR检测结果显示,大鼠海马组织LAMP2a的mRNA的水平在致痫后8 h开始升高(P＜0.05),24小时仍持续增高(P＜0.05)。应用维生素E干预后,大鼠海马组织LAMP2a的mRNA的水平低于PILO组(P＜0.05)。结论1.癫痫发作可诱发海马组织CMA明显激活,但晚于巨自噬约6小时,维生素E能明显抑制癫痫诱发CMA的激活。2.癫痫发作诱发的CMA的激活是通过LAMP2a的合成增加来实现的,而不是通过LAMP2a由溶酶体基质向膜上转移米实现。维生素E不影响LAMP2a在溶酶体膜上和基质内的分配。第三部分Wortmannin抑制癫痫大鼠海马巨自噬活性及神经保护作用的研究目的探讨巨自噬抑制剂渥曼青霉素(wortmannin,WM)对PILO致痫大鼠的行为学变化,以及对癫痫大鼠海马组织巨自噬活性即LC3Ⅱ/LC3Ⅰ比值的影响,并且探讨了磷脂酰肌醇3激酶(phosphoinositide 3-kinase,PI3K)在巨自噬中的作用。方法1.应用腹腔注射的方法建立PILO诱导的大鼠颞叶癫痫模型。实验动物在检测自噬活性时分为对照组、PILO致痫2 h、8 h、16 h、24 h、72 h组;在检测WM对巨自噬抑制作用及对海马神经元保护作用时分为对照组、PILO致痫24h组及WM干预组(WM+PILO组)。2.观察PILO诱导的颞叶癫痫大鼠的行为学变化,致痫成功率,诱导癫痫持续状态的潜伏期(注射PILO到发生癫痫持续状态的时间)和24小时的死亡率;观察WM对癫痫动物行为学的保护作用。3.应用Western blot检测各组大鼠海马组织LC3Ⅱ/LC3Ⅰ比值和磷酸化PI3K的表达。结果1.PILO致痫组15只大鼠中14只出现SE,致痫率为93.33%。WM干预组的致痫率显著下降为60%,两组间有显著性差异(p＜0.05)。PILO致痫组潜伏期为(34.75±12.76 min,n=9),WM干预组的潜伏期显著延长为(64.33±16.67min,n=9)(t=0.007,p＜0.01)。致痫24h时,WM干预组中9只致痫动物死亡0只,死亡率0;两组间比较有显著性差异(p＜0.01)。2.WM干预组大鼠海马CAl区锥体细胞边缘清晰,形态正常,仅少量神经元染色质凝聚、轮廓模糊。3.WM干预组大鼠海马CAl区存活神经元数目(100.88±18.73)较PILO组(70.16±5.09)显著增加(P＜0.05)。4.在大鼠致痫后24h,WM干预组大鼠海马组织中LC3Ⅱ/LC3Ⅰ的比值即LC3转化较PILO组显著下降(P＜0.05)。5.PI3K的激活即磷酸化PI3K的表达在大鼠致痫后2h开始显著升高(P＜0.05),8h达到高峰(P＜0.05),16h和24h下降(P＜0.05),72h基本接近正常(P＞0.05)。结论1.癫痫发作所激活得海马组织LC3Ⅰ向LC3Ⅱ转化可被WM部分抑制。2.WM抑制自噬的激活,减轻海马神经元损伤,降低致痫率,延长癫痫发作的潜伏期,并显著降低死亡率,具有神经保护作用。3.磷脂酰肌醇3激酶在癫痫发作海马损伤和自噬激活中发挥作用。研究意义本研究应用大鼠颞叶癫痫模型,首次在成体动物水平证实自噬参与癫痫发作致海马神经元损伤过程,并进一步证实了自由基清除剂维生素E和自噬抑制剂WM可以通过调解自噬发挥神经保护作用。尽管自噬对神经元损伤的具体机制仍需进一步探讨,但本研究的结果仍有助于阐明癫痫致海马神经元损伤的机制,也有助于寻找新的药物作用靶点,研发新的神经保护药物。更多还原

【Abstract】 BackgroundEpilepsy is one of the most common chronic neurological disorders affecting people of all ages.The striking characteristic of epilepsy is a tendency to recurrent, unprovoked seizures.At present,there are about 9 million of epilepsy patients in China.They suffer a lot from this disease and become big burdens to the family and society.As many as 20%～30%of medicated epilepsy patients are still far from seizures-free control.In the end,they will become patients with intractable epilepsy. Temporal epilepsy usually turns out to be intractable epilepsy syndrome.Although great progress has been made on the etiology,pathology and treatment of temporal epilepsy,the mechanism of the disease is still far from clear.The main pathology feature of temporal epilepsy is hippocampus sclerosis including the loss of neurons and hyperplasia of astrocytes.As known,seizures are associated with a variety of pathophysiological alterations and can result in neuronal degeneration in hippocampus.It has been indicated that seizures-induced neuronal death is associated with glutamate release and N-methyl-D-asparate(NMDA) receptor activation.When NMDA receptor is stimulated,calcium influxes into cells and activates nitric-oxide synthase,which elevates oxygen-free radical levels and causes oxidative damage. Until now,the mechanism underlying the neuron death is still unclear.Moreover, there are still limited strategies to protect neuronal death and the subsequent epileptogenesis.Autophagy is the regulated process by which cytoplasmic organelles and long-lived proteins are delivered for lysosomal degradation.There are 3 major forms of "autophagy".These forms of autophagy share in common the delivery of intracellular cargo for degradation within lysosomes.Macroautophagy involves the sequestration of cytoplasmic proteins and organelles into a double-membrane vesicle,followed by stepwise maturation involving dissolution of the inner membrane,acidification, delivery to and fusion with lysosomes.Chaperone mediated autophagy(CMA) involves recognition of protein motifs by trans-membrane lysosomal proteins,and their direct import across the lysosomal membrane.Microautophagy involves rearrangement of the lysosomal membrane to engulf portions of adjacent cytoplasm or nucleus.Under normal condition,autophagy is at basal level in most cells.Autophagy is induced ten times under starvation,differentiation,and hormone stimulation to maintain cellular homeostasis and survival.The reported studies showed that oxidative stress is an important inducer of autophagy.As a signal molecule,active oxygen can activate autophagy.Simultaneously,it has also been evident that autophagy can trigger a form of cell death distinct from apoptosis in neurons,known as typeⅡprogrammed cell death.To our knowledge,the role of autophagy has not been studied in oxidative stress induced by seizures in animal model of epilepsy produced by pilocarpine(PILO).The effects of antioxidant vitamin E and autophagy inhibitor wortmanin(WM) in on activated autophagy in seizures have not been determined.Thus,in this study we try to investigate whether seizures promote autophagy including macroautophagy and CMA.In addition,we also examined whether vitamin E and wortmannin might attenuate hippocampal neuron damage after seizures induced by pilocarpine. PartⅠThe role of macroautophagy in rat hippocampus injury induced by seizures and the effect of vitamin E on macrautophagyObjectiveTo explore the pathology features of rat hippocampus injury caused by PILO-induced seizures.To investigate the changing patterns of autophgy and neuroprotective effects of antioxidant vitamin E on rat hippocampus injury induced by seizures.Method1.Seizures were induced by PILO through intraperitoneal injection.To detect the time courses of autophagy activity,rats were randomly divided into control group and groups at 2,8,16,24 and 72 h after PILO treatment.Next,to study the neuroprotective effects of vitamin E or pathology features of hippocampus injury,rats were randomly divided into control,PILO 24h,vitamin E+saline and vitamin E+PILO groups.2.The animal behaviors were observed including the incidence rate of animals with status epilepticus(SE),the latent period from PILO injection to initiation of SE,and mortality rate of animals with SE at 24h after induction of SE.The neuroprotective effects of vitamin E on animal behavior were also studied.3.To evaluate pathology features of hippocampal neurons of every experiment groups,Haematoxylin & Eosin staining and Nissl staining by Toluidine Blue were performed.4.Electron microscope was used to detect the ultrastructure of damaged neurons and the changes of autophagic vacuole.5.Western blot analysis was used to detect the ratio of light chain(LC3)Ⅱto LC3Ⅰand beclin 1 which were respectively related to the macroautophagy activity at different experiment group.Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect beclin 1 mRNA at different experiment group.6.Furthermore,the effect of vitamin E on macroautophagy was investigated by Western blot examination. Result1.The incidence rate of SE induced by pilocarpine was 93.33%.The vitamin E pretreatment only slightly decreased the incidence rate of SE(the incidence rate of SE in vitamin E+PILO group was 80%(p＞0.05).The latent period from PILO injection to initiation of SE was(35.64±13.77 min,n=14),but the latent period in vitamin E+PILO group was(54.27±22.16 min,n=12),which was significantly longer than PILO group(t=0.027,p＜0.05).The mortality rate of animals with SE at 24h after induction of SE was 50%(7 from 14),vitamin E pretreatment significantly increased the mortality rate of animals with SE at 24h,which was 8.3%(1 out of 12)(x~2=5.26, p＜0.05).2.Neurons in CA1 of control rats were clear and complete with normal nucleolus, well-distributed karyotin and rich nissl bodies in kytoplasm.PILO-induced seizures led to obvious neuron damage in rat hippocampus.The surviving neurons showed round and palely stained nuclei,meanwhile,the dead neurons in hippocampus showed pyknotic nuclei and shrunken plasma body.There were small and irregular chromatin clumps in the dead cells.Most pyramid cells were normal and only a few neurons showed fuzzy outline and agglomerated karyotin in vitamin E+PILO group.Under electron microscope,the dead neurons displayed rupture of nuclear and cytoplasmic membrane,organelle swelling and a high number of vacuoles in plasma.There were numerous and irregular clumps distributed throughout the nucleus that was associated with cytoplasmic hyperchromasia,rough endoplasmic reticulum dispersion, mitochondrial swelling and cristaeolysis.Many autophagic vacuoles were seen obviously.On the contrary,autophagic vacuoles were rarely seen in neurons from hippocampus CA1 of rats in control group under electron microscope.Vitamin E also reduced autophagic vacuoles obviously.3.The surviving neuron number at 24h was sharply decreased in the PILO-treated group(75.5±5.92) compared with control(110.67±18.56)(P＜0.05).Moreover, vitamin E significantly decreased the neuron loss induced by seizures(94.67±13.03) compared with the PILO group(P＜0.05). 4.By Western blot,the ratio of LC3Ⅱto LC3Ⅰbegan to increase at 2 h,peaked at 24 h and kept at a high level until 72 h after seizures(P＜0.05).The immunoreactivity of beclin 1 was weak in control group and began to increase at 2h,peaked at 24 h and kept at a high level until 72 h after seizures(P＜0.05).In addition,the time-course change of beclin 1 mRNA was the same as beclin 1 protein.Vitamin E could effectively inhibit the overactivation of macroautophagy induced by seizures by inhibiting the transformation of LC3Ⅰto LC3Ⅱand the synthesis of beclin 1 in vivo (P＜0.05).Conclusion1.PILO-induced seizures lead to severe hippocampal neuron damage.The damaged neuron mainly shows a feature of necrosis.Macroautophagy was activated in damaged neuron.2.PILO-induced seizures can trigger the transformation of LC3Ⅰto LC3Ⅱand the up-regulation of beclin 1 in hippocampus that is partly inhibited by vitamin E.3.Vitamin E prolongs the latent period of SE and improves the survival rate of rats with SE,and has the neuroprotective effect on hippocampus injury induced by seizures.PartⅡThe role of CMA on rat hippocampus injury induced by seizures and the effect of vitamin E on CMAObjectiveTo investigate changes of CMA in rat hippocampus injury induced by seizures and effects of vitamin E on CMA.Method1.Seizures were induced by PILO through intraperitoneal injection.To detect the time courses of CMA activity,rats were randomly divided into control group and groups at 2,8,16,and 24 h after PILO treatment.Next,to study the effects of vitamin E on CMA injury,rats were randomly divided into control,PILO 24h,vitamin E+saline and vitamin E+PILO groups.2.The isolation of lysosomes and the preparation of lysosomal membranes and matrices.3.Western blot analysis was used to detect LAMP2a expression on lysosomal membranes and in lysosomal matrices at various experiment groups.Next,the mRNA of LAMP2a was evaluated by RT-PCR.Lastly,the effects of vitamin E on synthesis and expressions of LAMP2a were also detected by RT-PCR and Western blot examination.Result1.The levels of LAMP2a both on lysosomal membranes and in lysosomal matrices increased significantly at 8h after seizues,and continued to increase up to 24 h after PILO treatment(P＜0.05).The distribution of LAMP2a between on lysosomal membranes and in lysosomal matrices was not affected by seizures(p＞0.05).2.Vitamin E inhibited the increase of LAMP2a both on lysosomal membranes and in lysosomal matrices(P＜0.05),but did not affect the distribution of LAMP2a (p＞0.05)。3.RT-PCR showed that mRNA LAMP2a began to increase significantly at 8h after seizures,and continued to increase up to 24 h after PILO treatment(P＜0.05).Vitamin E also inhibited the synthesis of LAMP2a in hippocampus in vivo at 24h after seizures(P＜0.05).Conclusion1.PILO-induced seizures lead to activation of CMA that began at 8h,6h later than macroautophagy.Vitamin E can partly inhibit activated CMA by seizures.2.The activation of LAMP2a by seizures was achieved by increase of LAMP2a syhthesis in vivo,not by the redistribution of LAMP2a between on lysosomal membranes and in lysosomal matrices.Vitamin E did not affect the distribution of LAMP2a between on lysosomal membranes and in lysosomal matrices. PartⅢThe neuroprotective effect of wortmannin on epileptic rat by inhibiting macroautophagyObjectiveTo investigate the effects of wortmannin(WM) on the activation of macroautophagy in epileptic rat hippocampus,and the roles of phosphoinositide 3-kinase(PI3K) in macroautophagy and neuroprotective effects of WM on epileptic rats.Method1.Seizures were induced by PILO through intraperitoneal injection.To detect the time courses of macroautophagy activity,rats were randomly divided into control group and groups at 2,8,16,24 and 72 h after PILO treatment.Next,to study the neuroprotective effects of WM on hippocampus injury and macroautophagy,rats were randomly divided into control,PILO 24h,WM+PILO groups.2.The animal behavior were observed including the incidence rate of animals with status epilepticus(SE) induced by pilocarpine,the latent period from PILO injection to initiation of SE,and mortality rate of animals with SE at 24h after induction of SE. The neuroprotective effects of wortmannin on animal behavior were also studied.3.Western blot analysis was used to detect the ratio of LC3Ⅱto LC3Ⅰand phospho-PI3K at various experiment groups.Result1.WM pretreatment significantly decreased the incidence rate of SE(the incidence rate of SE in WM+PILO group was 60%)(p＜0.05).The latent period from PILO injection to initiation of SE was(34.75±12.76 min,n=14),but the latent period in WM+PILO group was(64.33±16.67min,n=9)(t=0.007,p＜0.01),which was significantly longer than PILO group.WM pretreatment significantly increased the mortality rate of animals with SE at 24h,which was 0(0 out of 9)(p＜0.01).2.Most pyramid cells were normal and only a few neurons showed fuzzy outline and agglomerated karyotin in WM+PILO group.3.The surviving neuron number at 24h was sharply decreased in the PILO-treated group(70.16±5.09) compared with control(110.67±18.56)(P＜0.05).Moreover, wortmannin significantly decreased the neuron loss induced by seizures (100.88±18.73) compared with PILO group(P＜0.05).4.The ratio of LC3Ⅱto LC3Ⅰand beclin 1 were significantly lower in WM+PILO group than in PILO group(P＜0.05).5.PI3K phosphorylation or activation temporally increased at 2 h and 8h(P＜0.05), began to decrease at 16h(P＜0.05),and returned to normal level at 72 h after seizures (P＜0.05).Conclusion1.WM can inhibit macroautophagy activated by seizures by reducing the transformation of LC3Ⅱto LC3Ⅰ.2.WM have neuroprotective effect on epileptic rats by reducing incidence rate of SE,prolonging the latent period of SE,decreasing the mortality rate of animals with SE and increasing the survival neurons in CA1 of rats with SE.3.PI3K may play a important role in activated macroautophagy induced by seizures.SignificanceUsing a rodent epilepsy model induced by PILO,we investigated and confirmed that autophagy played an important role in seizures-induced hippocampal neuronal death.Furthermore,we showed that antioxidant vitamin E and macroautophagy inhibitor,WM could regulate autophagy and play neuroprotective roles on epileptic rats.Although the mechanism underlying the effects of autophagy on neuron injury needs further study,studies on autophagy will undoubtedly help us to know more about the mechanism of seizures-induced neuronal injury.Thus,this study may provide new insights into therapeutic advances of vitamin E and WM that may be neuroprotective.更多还原