丙酮酸乙酯对癫痫持续状态大鼠神经元凋亡及XIAP、Caspase-3表达的影响
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摘要
目的:丙酮酸乙酯为丙酮酸的酯化物,是一种安全、稳定且极易被细胞摄取的物质,有报道显示丙酮酸乙酯可以减轻急性全脑缺血/再灌注时脑组织损伤,起到脑保护作用。但丙酮酸乙酯对癫痫持续状态导致的脑损伤是否有保护作用尚未见研究报道。本研究用丙酮酸乙酯预处理,采用氯化锂-匹罗卡品腹腔注射的方法制备大鼠癫痫持续状态动物模型,观察癫痫持续状态后不同时间点大鼠海马神经元凋亡相关基因XIAP、Caspase-3的表达情况,探讨丙酮酸乙酯干预对癫痫持续状态导致的神经元损伤的影响及可能的神经保护机制,并为丙酮酸乙酯应用于临床治疗癫痫持续状态提供动物实验依据。
方法:选用健康成年雄性Wistar大鼠54只,随机分为对照组(n=6只)、模型组(n=24只)和丙酮酸乙酯组(n=24只),后二组再分为6h、12h、24h、48h四个亚组,每个亚组6只。丙酮酸乙酯组在造模前提前15分钟腹腔注射丙酮酸乙酯50mg/kg,对照组和模型组在造模前提前15分钟给予等容积的生理盐水。15分钟后模型组、丙酮酸乙酯组按127mg/kg给予腹腔注射氯化锂, 18小时后再腹腔注射新鲜配制的匹罗卡品30 mg/kg,制备癫痫持续状态模型,同时给对照组大鼠腹腔注射等量的生理盐水。观察大鼠出现痫性发作的时间和行为表现,在癫痫持续状态后相应时间点断头取脑,对海马组织进行研究,HE染色,光镜下观察形态学变化,TUNEL染色检测大鼠海马神经元凋亡,免疫组化法检测XIAP和Caspase- 3蛋白的表达。数据采用SPSS13.0进行统计分析,P<0.05差异有统计学意义。结果:对照组TUNEL、XIAP和Caspase-3阳性细胞仅有微量的基础表达。模型组和丙酮酸乙酯组癫痫持续状态后6h海马组织可见TUNEL、XIAP和Caspase-3阳性细胞表达增多,TUNEL和Caspase-3表达高峰均在24h,XIAP表达高峰在12h。丙酮酸乙酯组各时间点TUNEL、Caspase-3阳性细胞数较模型组明显减少(除6h时外,均为P<0.05),而XIAP阳性细胞数较模型组则明显增加(除6h外,均为P< 0.05)。
结论:(1)癫痫持续状态后XIAP和Caspase-3蛋白的表达均出现变化,证实这两种蛋白均参与癫痫持续状态后神经元的损伤过程。(2)丙酮酸乙酯可通过下调脑组织Caspase-3和上调XIAP的表达,减轻癫痫持续状态脑损伤。(3)丙酮酸乙酯对大鼠癫痫持续状态后神经元损伤的保护作用与抗凋亡有关。
Objective: Ethyl pyruvate is a new type of brain protective agent. Reports have suggested that ethyl pyruvate can reduce the brain injury after Acute Global Cerebral Ischemia/Reperfusion, so as to protect the cerebral.However, the protective effection of Penehyclidine on brain damage which is led by status epilepticus( SE) have not been reported. In this research, after ethyl pyruvate pretreatment, the rats abdominal cavity are injected lithium-pilecarpine to make status epilepticus model. Observe the expression of apoptosis related genes Caspase-3,XIAP in rats hippocampus at different time points after SE, and compare it with anisodamine, discuss the effect of ethyl pyruvate intervention on neuronal damage after SE and its possible mechanism, with a view to ethyl pyruvate used in clinical treatment of SE to provide a possible basis for animal experiments.
Methods: 54 healthy adult male Wistar rats were randomly divided into control group(n = 6 rats), model group(n = 24 rats), ethyl pyruvate group(n = 24 rats), the last two groups were divided into four sub-groups which are used at 6 hours, 12 hours, 24 hours, 48 hours after status epilepticus, each sub-group has 6 rats. The rats abdominal cavity of ethyl pyruvate group were injected ethyl pyruvate (50mg/kg) before 15 minutes ahead of making model. The control group were injected the same amount of saline at the corresponding time points. After 15 minutes, the model group and ethyl pyruvate group were given intraperitoneal injection of lithium chloride according to (127mg/kg). After 18 hours,the two groups were injected fresh pilocarpine (30mg/kg), making the model of status epilepticus.At the same time, the control group were injected the same amount of saline. Observe the performance and time when seizures rats appears . Model control group and ethyl pyruvate t group rats were sacrificed, respectively, at 6h, 12h, 24h, 48h after SE. The control group rats were killed together with rats that were sacrificed 6h after SE .then do some research on hippocampus. After HE staining, observe morphological changes under light microscope. Using the method of TUNEL to detect the apoptosis of rat hippocampal neurons, and immunohistochemical staining was used to test the expression of XIAP and Caspase-3. SPSS 13.0 version was used for statistical analysis and P<0.05 was considered statistically significant.
Results: The positive cells of Caspase-3 ,XIAP and TUNEL in the control group only have a little basal expression. The expression of Caspase-3 , XIAP and TUNEL positive cells of hippocampus in the model group and ethyl pyruvate group is increasing after 6h of status epilepticus , Caspase-3 and TUNEL expression peaks are at 24h; XIAP expression reached its peak at 12h. Caspase-3 and TUNEL positive cells in ethyl pyruvate group were significantly reduced compared with model group (except for 6h, P <0.05), at different time points ,while the XIAP positive cells was significantly increased (except for 6h, P <0.05).
Conclusion: (1) After status epilepticus, the protein expression of XIAP and Caspase-3 changes, confirmed that the two kinds of protein involved in the neuronal injury after status epilepticus. (2) The intervention of ethyl pyruvate can mitigate brain damage after status epilepticus by increasing the expression of XIAP and reducing the expression of Caspase-3. (3) The protective role of ethyl pyruvate against post-status epilepticus neuronal damage in rat is related with the mechanism effect of anti-apoptosis.
方法:选用健康成年雄性Wistar大鼠54只,随机分为对照组(n=6只)、模型组(n=24只)和丙酮酸乙酯组(n=24只),后二组再分为6h、12h、24h、48h四个亚组,每个亚组6只。丙酮酸乙酯组在造模前提前15分钟腹腔注射丙酮酸乙酯50mg/kg,对照组和模型组在造模前提前15分钟给予等容积的生理盐水。15分钟后模型组、丙酮酸乙酯组按127mg/kg给予腹腔注射氯化锂, 18小时后再腹腔注射新鲜配制的匹罗卡品30 mg/kg,制备癫痫持续状态模型,同时给对照组大鼠腹腔注射等量的生理盐水。观察大鼠出现痫性发作的时间和行为表现,在癫痫持续状态后相应时间点断头取脑,对海马组织进行研究,HE染色,光镜下观察形态学变化,TUNEL染色检测大鼠海马神经元凋亡,免疫组化法检测XIAP和Caspase- 3蛋白的表达。数据采用SPSS13.0进行统计分析,P<0.05差异有统计学意义。结果:对照组TUNEL、XIAP和Caspase-3阳性细胞仅有微量的基础表达。模型组和丙酮酸乙酯组癫痫持续状态后6h海马组织可见TUNEL、XIAP和Caspase-3阳性细胞表达增多,TUNEL和Caspase-3表达高峰均在24h,XIAP表达高峰在12h。丙酮酸乙酯组各时间点TUNEL、Caspase-3阳性细胞数较模型组明显减少(除6h时外,均为P<0.05),而XIAP阳性细胞数较模型组则明显增加(除6h外,均为P< 0.05)。
结论:(1)癫痫持续状态后XIAP和Caspase-3蛋白的表达均出现变化,证实这两种蛋白均参与癫痫持续状态后神经元的损伤过程。(2)丙酮酸乙酯可通过下调脑组织Caspase-3和上调XIAP的表达,减轻癫痫持续状态脑损伤。(3)丙酮酸乙酯对大鼠癫痫持续状态后神经元损伤的保护作用与抗凋亡有关。
Objective: Ethyl pyruvate is a new type of brain protective agent. Reports have suggested that ethyl pyruvate can reduce the brain injury after Acute Global Cerebral Ischemia/Reperfusion, so as to protect the cerebral.However, the protective effection of Penehyclidine on brain damage which is led by status epilepticus( SE) have not been reported. In this research, after ethyl pyruvate pretreatment, the rats abdominal cavity are injected lithium-pilecarpine to make status epilepticus model. Observe the expression of apoptosis related genes Caspase-3,XIAP in rats hippocampus at different time points after SE, and compare it with anisodamine, discuss the effect of ethyl pyruvate intervention on neuronal damage after SE and its possible mechanism, with a view to ethyl pyruvate used in clinical treatment of SE to provide a possible basis for animal experiments.
Methods: 54 healthy adult male Wistar rats were randomly divided into control group(n = 6 rats), model group(n = 24 rats), ethyl pyruvate group(n = 24 rats), the last two groups were divided into four sub-groups which are used at 6 hours, 12 hours, 24 hours, 48 hours after status epilepticus, each sub-group has 6 rats. The rats abdominal cavity of ethyl pyruvate group were injected ethyl pyruvate (50mg/kg) before 15 minutes ahead of making model. The control group were injected the same amount of saline at the corresponding time points. After 15 minutes, the model group and ethyl pyruvate group were given intraperitoneal injection of lithium chloride according to (127mg/kg). After 18 hours,the two groups were injected fresh pilocarpine (30mg/kg), making the model of status epilepticus.At the same time, the control group were injected the same amount of saline. Observe the performance and time when seizures rats appears . Model control group and ethyl pyruvate t group rats were sacrificed, respectively, at 6h, 12h, 24h, 48h after SE. The control group rats were killed together with rats that were sacrificed 6h after SE .then do some research on hippocampus. After HE staining, observe morphological changes under light microscope. Using the method of TUNEL to detect the apoptosis of rat hippocampal neurons, and immunohistochemical staining was used to test the expression of XIAP and Caspase-3. SPSS 13.0 version was used for statistical analysis and P<0.05 was considered statistically significant.
Results: The positive cells of Caspase-3 ,XIAP and TUNEL in the control group only have a little basal expression. The expression of Caspase-3 , XIAP and TUNEL positive cells of hippocampus in the model group and ethyl pyruvate group is increasing after 6h of status epilepticus , Caspase-3 and TUNEL expression peaks are at 24h; XIAP expression reached its peak at 12h. Caspase-3 and TUNEL positive cells in ethyl pyruvate group were significantly reduced compared with model group (except for 6h, P <0.05), at different time points ,while the XIAP positive cells was significantly increased (except for 6h, P <0.05).
Conclusion: (1) After status epilepticus, the protein expression of XIAP and Caspase-3 changes, confirmed that the two kinds of protein involved in the neuronal injury after status epilepticus. (2) The intervention of ethyl pyruvate can mitigate brain damage after status epilepticus by increasing the expression of XIAP and reducing the expression of Caspase-3. (3) The protective role of ethyl pyruvate against post-status epilepticus neuronal damage in rat is related with the mechanism effect of anti-apoptosis.
引文
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