他克莫司对癫痫持续状态大鼠海马PKCδ表达的影响
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摘要
背景及目的：癫痫(Epilepsy, EP)是大脑神经元过度同步异常放电导致的神经系统功能异常综合征。癫痫持续状态(Status Epilepticus, SE)是癫痫连续发作之间意识未完全恢复又频繁再发,或发作持续30分钟以上不自行停止,可导致急性或持续性的认知及行为改变,是神经内科常见急症之一。
蛋白激酶C(Protein kinase C, PKC)参与神经元的生长、分化和凋亡等生理病理过程。PKCδ是一种新型的PKC亚型的成员,在各种刺激因素的作用下,PKCδ可促使caspase3活化,引起细胞凋亡。钙调神经磷酸酶(Calcineurin,CaN)为丝/苏氨酸蛋白磷酸酶家族成员,我们既往研究结果表明,CaN可引起难治性癫痫,他克莫司(Tacrolimus, FK506)可通过特异性抑制CaN控制癫痫发生,逆转神经元损伤。已有研究发现,在脑缺血模型中,CaN活化后激活PKC,引起细胞内信号传导。在癫痫的病理生理过程中,关于PKC各亚型与CaN的相互作用,以及对于神经元损伤的影响,尚缺乏相关研究。
本研究应用Wistar大鼠建立氯化锂-匹鲁卡品癫痫持续状态模型,观察海马形态学变化、PKCδ的表达及FK506对其的影响,探究CaN与依赖PKCδ的细胞凋亡途径之间的关系。
方法：
1.动物模型：将108只健康成年雄性大鼠随机分为对照组(Control组)、癫痫组(SE组)和FK506处理组(FK506组),每组36只。SE组与FK506组各分为6组,分别于SE后3、6、12、24、72h及7d处死取材,对照组大鼠不建立癫痫模型,于对应时间点处死。
2.行为学观察：根据Racine分级观察大鼠癫痫发作行为变化,记录癫痫发作潜伏期及发作程度。
3.脑组织病理学观察：HE染色观察SE后7d海马区神经元的损伤。
4. RT-PCR法：检测SE后不同时点(3、6、12、24、72h)海马PKCδmRNA的表达。
5.免疫组织化学SP法：检测SE后不同时点(3、6、12、24、72h)海马PKCδ蛋白的表达。
结果：
1.FK506抑制癫痫发作：SE组和FK506组大鼠在注射匹鲁卡品后均出现癫痫发作,开始发作程度较轻,逐渐加重,直至出现癫痫持续状态。两组发作潜伏期和发作程度不同。与SE组相比,FK506组大鼠潜伏期明显延长(P<0.01),发作级别降低(P<0.05)：Control组大鼠无痫性发作。
2.FK506减轻SE后海马神经元损伤：在光镜下,SE后7dControl组大鼠海马CA3区神经元排列整齐,形态正常,无神经元缺失；SE组大鼠海马CA3区神经元排列紊乱,失去正常结构,与对照组相比神经元缺失严重(P<0.05)；FK506组大鼠海马CA3区神经元数目较对照组减少,但较SE组存活神经元明显增多(P<0.05)。
3.FK506抑制PKC8的表达：Control组PKC8mRNA和蛋白仅有少量表达；与Control组相比,SE组各个时点PKC8表达均显著增多(P<0.05),SE后6h PKC8mRNA表达最高,PKC8蛋白的平均光密度值SE后12h达高峰(P<0.01),以后二者均逐渐降低,但72h仍高于对照组水平(P<0.05)；FK506组降低其表达,同时可减轻海马神经元损伤(P<0.05)。
结论：
FK506可能通过特异性抑制CaN间接调节依赖PKC8的细胞凋亡途径,减少SE后神经元损伤,发挥抗癫痫和脑保护作用,为临床应用免疫抑制剂治疗癫痫提供新的理论依据。
Backgroud and Objective:Epilepsy (EP) is a nervous system dysfunction syndrome,which is due to cerebral neurons excessive synchronous abnormal discharge. Status epilepticus (SE) refers to frequent clinical seizures without complete recovery of consciousness or the occurrence of a single unremitting seizure lasting more than30minutes to stop on its own,which can lead to acute or persistent changes of cognition and behavior and is a medical emergency of department of neurology.
Protein kinase C (PKC) participates in physiological and pathological processes,such as the growth、differentiation and apoptosis of neurons. PKCδ,a member of a new kind of PKC isoforms,can activate caspase3under the effects of various stimulus,resulting in apoptosis. Calcineurin (CaN) is a member of serine/threonine protein phosphatase families. Our previous research shows that CaN can cause refractory epilepsy,and tacrolimus (FK506,a specific inhibition of CaN) is able to control epilepsy development、reverse neuronal injury. It has been found that in the model of cerebral ischemia,CaN can activate PKC,causing intracellular signal transduction. In the pathophysiological process of epilepsy,the interaction between PKC and CaN and neuronal damage is still short of related research.
Our this study,we use Wistar rats to build lithium chloride-pilocarpine-induced status epilepticus model to observe the morphologic changes of the hippocampus, the expression of protein kinase Cδ and the influence of FK506on it,and explore the relationship between CaN and PKCδ-dependent apoptotic pathway.
Methods:
1. Animal models:One hundred and eight healthy adult male Wistar rats were randomly divided into the control group,the epilepsy group and the FK506group (36rats in each group). SE group and FK506group were divided into six groups, espectively3,6,12,24,72hours and7days after SE were sacrificed. At the same time, control group rats without being established epilepsy model, were sacrificed at corresponding time points.
2. Behavioral observation:Observe the epileptic seizure of rats on the basis of Racine classification, and record seizures incubation period and the onset level.
3. Observation of brain tissue pathology:Observe the hippocampal neuronal damage7days after SE by HE staining.
4. RT-PCR:Detect the expression of PKCδ mRNA in the hippocampus at different time points (3,6,12,24,72h) after SE.
5. Immunohistochemistry(SP):Detect the expression of PKCδprotein in the hippocampus at different time points (3,6,12,24,72h) after SE.
Results:
1. FK506inhibited seizures:After injection of pilocarpine,the SE group and the FK506rats had seizures,the onset at a lesser extent and gradually increased until the status epilepticus. Incubation period and the onset level of the two groups were different. Compared with the SE group,incubation period of the FK506group was significantly longer (P<0.01), and the onset level lower (P<0.05). The control group did not have seizures.
2. FK506reduced SE hippocampal neurons injuries:Under the light microscope, the control group neurons arranged in neat rows, had normal morphology and had no neuronal loss in the hippocampal CA3region of rats7days after SE. In the SE group, the neurons arranged in disorder and lost normal structure; compared with the control group,the neurons lost more (P<0.05). Compared with the control group,the neurons number of the FK506group decreased,but compared with the SE group,survival neurons increased significantly (P<0.05).
3. FK506suppressed the expression of PKC8:There were a few expressions of PKCδ mRNA and protein in the control group. Compared with the control group, the expressions of PKC8in the epilepsy group were increased observably (P<0.05),the expression of PKCδ mRNA arrived at a peak6h after SE,the expression of PKCδ protein arrived at a peak12h after SE(P<0.01), and both of PKCδ mRNA and protein were still more than the level of the control group72h after SE (P<0.05). Compared with the epilepsy group,pre-treatment with FK506could remarkably decrease the expression of PKCδ and also obviously alleviate hippocampal neuronal damage (P<0.05)
Conclusions:
FK506may regulate the PKCδ-dependent apoptotic pathway, reduce neuronal damage after SE and exert anti-epileptic and cerebral protection effects by inhibiting the CaN specifically,which provide a new drug target for the clinical application of immunosuppressive to treat epilepsy.
蛋白激酶C(Protein kinase C, PKC)参与神经元的生长、分化和凋亡等生理病理过程。PKCδ是一种新型的PKC亚型的成员,在各种刺激因素的作用下,PKCδ可促使caspase3活化,引起细胞凋亡。钙调神经磷酸酶(Calcineurin,CaN)为丝/苏氨酸蛋白磷酸酶家族成员,我们既往研究结果表明,CaN可引起难治性癫痫,他克莫司(Tacrolimus, FK506)可通过特异性抑制CaN控制癫痫发生,逆转神经元损伤。已有研究发现,在脑缺血模型中,CaN活化后激活PKC,引起细胞内信号传导。在癫痫的病理生理过程中,关于PKC各亚型与CaN的相互作用,以及对于神经元损伤的影响,尚缺乏相关研究。
本研究应用Wistar大鼠建立氯化锂-匹鲁卡品癫痫持续状态模型,观察海马形态学变化、PKCδ的表达及FK506对其的影响,探究CaN与依赖PKCδ的细胞凋亡途径之间的关系。
方法：
1.动物模型：将108只健康成年雄性大鼠随机分为对照组(Control组)、癫痫组(SE组)和FK506处理组(FK506组),每组36只。SE组与FK506组各分为6组,分别于SE后3、6、12、24、72h及7d处死取材,对照组大鼠不建立癫痫模型,于对应时间点处死。
2.行为学观察：根据Racine分级观察大鼠癫痫发作行为变化,记录癫痫发作潜伏期及发作程度。
3.脑组织病理学观察：HE染色观察SE后7d海马区神经元的损伤。
4. RT-PCR法：检测SE后不同时点(3、6、12、24、72h)海马PKCδmRNA的表达。
5.免疫组织化学SP法：检测SE后不同时点(3、6、12、24、72h)海马PKCδ蛋白的表达。
结果：
1.FK506抑制癫痫发作：SE组和FK506组大鼠在注射匹鲁卡品后均出现癫痫发作,开始发作程度较轻,逐渐加重,直至出现癫痫持续状态。两组发作潜伏期和发作程度不同。与SE组相比,FK506组大鼠潜伏期明显延长(P<0.01),发作级别降低(P<0.05)：Control组大鼠无痫性发作。
2.FK506减轻SE后海马神经元损伤：在光镜下,SE后7dControl组大鼠海马CA3区神经元排列整齐,形态正常,无神经元缺失；SE组大鼠海马CA3区神经元排列紊乱,失去正常结构,与对照组相比神经元缺失严重(P<0.05)；FK506组大鼠海马CA3区神经元数目较对照组减少,但较SE组存活神经元明显增多(P<0.05)。
3.FK506抑制PKC8的表达：Control组PKC8mRNA和蛋白仅有少量表达；与Control组相比,SE组各个时点PKC8表达均显著增多(P<0.05),SE后6h PKC8mRNA表达最高,PKC8蛋白的平均光密度值SE后12h达高峰(P<0.01),以后二者均逐渐降低,但72h仍高于对照组水平(P<0.05)；FK506组降低其表达,同时可减轻海马神经元损伤(P<0.05)。
结论：
FK506可能通过特异性抑制CaN间接调节依赖PKC8的细胞凋亡途径,减少SE后神经元损伤,发挥抗癫痫和脑保护作用,为临床应用免疫抑制剂治疗癫痫提供新的理论依据。
Backgroud and Objective:Epilepsy (EP) is a nervous system dysfunction syndrome,which is due to cerebral neurons excessive synchronous abnormal discharge. Status epilepticus (SE) refers to frequent clinical seizures without complete recovery of consciousness or the occurrence of a single unremitting seizure lasting more than30minutes to stop on its own,which can lead to acute or persistent changes of cognition and behavior and is a medical emergency of department of neurology.
Protein kinase C (PKC) participates in physiological and pathological processes,such as the growth、differentiation and apoptosis of neurons. PKCδ,a member of a new kind of PKC isoforms,can activate caspase3under the effects of various stimulus,resulting in apoptosis. Calcineurin (CaN) is a member of serine/threonine protein phosphatase families. Our previous research shows that CaN can cause refractory epilepsy,and tacrolimus (FK506,a specific inhibition of CaN) is able to control epilepsy development、reverse neuronal injury. It has been found that in the model of cerebral ischemia,CaN can activate PKC,causing intracellular signal transduction. In the pathophysiological process of epilepsy,the interaction between PKC and CaN and neuronal damage is still short of related research.
Our this study,we use Wistar rats to build lithium chloride-pilocarpine-induced status epilepticus model to observe the morphologic changes of the hippocampus, the expression of protein kinase Cδ and the influence of FK506on it,and explore the relationship between CaN and PKCδ-dependent apoptotic pathway.
Methods:
1. Animal models:One hundred and eight healthy adult male Wistar rats were randomly divided into the control group,the epilepsy group and the FK506group (36rats in each group). SE group and FK506group were divided into six groups, espectively3,6,12,24,72hours and7days after SE were sacrificed. At the same time, control group rats without being established epilepsy model, were sacrificed at corresponding time points.
2. Behavioral observation:Observe the epileptic seizure of rats on the basis of Racine classification, and record seizures incubation period and the onset level.
3. Observation of brain tissue pathology:Observe the hippocampal neuronal damage7days after SE by HE staining.
4. RT-PCR:Detect the expression of PKCδ mRNA in the hippocampus at different time points (3,6,12,24,72h) after SE.
5. Immunohistochemistry(SP):Detect the expression of PKCδprotein in the hippocampus at different time points (3,6,12,24,72h) after SE.
Results:
1. FK506inhibited seizures:After injection of pilocarpine,the SE group and the FK506rats had seizures,the onset at a lesser extent and gradually increased until the status epilepticus. Incubation period and the onset level of the two groups were different. Compared with the SE group,incubation period of the FK506group was significantly longer (P<0.01), and the onset level lower (P<0.05). The control group did not have seizures.
2. FK506reduced SE hippocampal neurons injuries:Under the light microscope, the control group neurons arranged in neat rows, had normal morphology and had no neuronal loss in the hippocampal CA3region of rats7days after SE. In the SE group, the neurons arranged in disorder and lost normal structure; compared with the control group,the neurons lost more (P<0.05). Compared with the control group,the neurons number of the FK506group decreased,but compared with the SE group,survival neurons increased significantly (P<0.05).
3. FK506suppressed the expression of PKC8:There were a few expressions of PKCδ mRNA and protein in the control group. Compared with the control group, the expressions of PKC8in the epilepsy group were increased observably (P<0.05),the expression of PKCδ mRNA arrived at a peak6h after SE,the expression of PKCδ protein arrived at a peak12h after SE(P<0.01), and both of PKCδ mRNA and protein were still more than the level of the control group72h after SE (P<0.05). Compared with the epilepsy group,pre-treatment with FK506could remarkably decrease the expression of PKCδ and also obviously alleviate hippocampal neuronal damage (P<0.05)
Conclusions:
FK506may regulate the PKCδ-dependent apoptotic pathway, reduce neuronal damage after SE and exert anti-epileptic and cerebral protection effects by inhibiting the CaN specifically,which provide a new drug target for the clinical application of immunosuppressive to treat epilepsy.
引文
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