PTZ诱导的癫痫急性发作大鼠大脑神经元中F-actin、Calponin3和ROCK2的表达及其意义

doi:10.13481/j.1671-587x.20150218

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (02): 299-303.doi: 10.13481/j.1671-587x.20150218

PTZ诱导的癫痫急性发作大鼠大脑神经元中F-actin、Calponin3和ROCK2的表达及其意义

程春旭^1,2, 李树蕾³, 古小云³, 黄可欣⁴, 李艳超³, 张舒岩⁵, 杨立彬¹

1. 吉林大学第一医院儿科, 吉林长春 130021;
2. 吉林省长春市传染病医院传染科, 吉林长春 130123;
3. 吉林大学基础医学院组织学与胚胎学系, 吉林长春 130021;
4. 吉林大学基础医学院生物学实验中心, 吉林长春 130021;
5. 吉林大学第一医院神经外科, 吉林长春 130021

收稿日期:2014-06-17 出版日期:2015-03-28 发布日期:2015-04-04
通讯作者: 张舒岩, 副教授(Tel:0431-81875891, E-mail:syzh27@yahoo.com.cn);杨立彬, 副教授, 硕士研究生导师(Tel:0431-88782412, E-mail:yanglibin19681126@tom.com) E-mail:syzh27@yahoo.com.cn;yanglibin19681126@tom.com
作者简介:程春旭(1975-), 女, 吉林省长春市人, 副主任医师, 在读医学硕士, 主要从事神经系统疾病方面的研究。
基金资助:
吉林省科技厅自然科学基金资助课题(20130101138JC);吉林大学白求恩医学部青年科研基金资助课题(2069)

Expressions of F-actin,Calponin3,and ROCK2 in cerebral neurons of rats with acute epileptic seizure induced by PTZ

CHENG Chunxu^1,2, LI Shulei³, GU Xiaoyun³, HUANG Kexin⁴, LI Yanchao³, ZHANG Shuyan⁵, YANG Libin¹

1. Department of Pediatrics, First Hospital, Jiliin University, Changchun 130021, China;
2. Department of Infection, Infectious Disease Hospital, Jilin Province, Changchun 130123, China;
3. Department of Histology and Embryology, School of Basic Medical Sciences, Jilin University, Changchun 130021, China;
4. Center for Biological Experiment, School of Basic Medical Sciences, Jilin University, Changchun 130021, China;
5. Department of Neurosurgery, First Hospital, Jiliin University, Changchun 130021, China

Received:2014-06-17 Online:2015-03-28 Published:2015-04-04

摘要/Abstract

摘要：

目的:探讨戊四氮(PTZ) 诱发的大鼠癫痫急性发作对大脑神经元中F-actin、Calponin3和ROCK2蛋白表达水平的影响,阐明癫痫急性发作时阻断F-actin异常解聚、触发F-actin重构的可能机制。方法:3周龄Wistar 幼鼠56只分为对照组(n=6)和癫痫组(n=50)。对照组大鼠给予腹腔注射生理盐水;癫痫组大鼠经腹腔注射60 mg·kg^-1 PTZ建立癫痫急性发作模型,造模成功的24只大鼠分别在癫痫急性发作后的相应时间点(1、2、3和7 d时)随机处死6只用于取材。采用Alex-488标记的phalloidine进行荧光染色观察大鼠大脑海马组织中分子层的F-actin荧光强度;采用免疫荧光染色法观察大鼠大脑皮层和海马神经元中Calponin3和ROCK2的分布;采用Western blotting法检测大鼠海马组织中Calponin3、ROCK2和磷酸化ROCK2蛋白的相对表达水平。结果:与对照组比较,癫痫急性发作1 d后,癫痫组大鼠大脑树突棘密集的海马中分子层的F-actin荧光强度降低(P<0.05),点状聚集结构消失。免疫荧光染色,对照组大鼠Calponin3在神经元胞质中弥漫性分布,而癫痫急性发作7 d后,癫痫组大鼠神经元中Calponin3则聚集在细胞皮质;对照组大鼠ROCK2仅在少量神经元突起中分布,而癫痫急性发作7 d后,癫痫组大鼠大量神经元胞体和突起中均可见ROCK2分布。Western blotting检测,与对照组比较,癫痫组大鼠各时间点海马组织中Calponin3相对表达水平显著降低(P<0.05),但是随着时间延长,1周内逐渐升高并趋向正常水平。与对照组比较,癫痫组大鼠海马组织中ROCK2蛋白相对表达水平从癫痫急性发作后3 d开始显著增加并持续至造模后7 d(P<0.05);磷酸化ROCK2蛋白相对表达水平则从癫痫急性发作后1 d就开始显著增加并持续到7 d(P<0.05),且随着时间延长逐渐降低。结论:PTZ诱导幼鼠癫痫急性发作导致F-actin异常解聚,同时激活RhoA/ROCK2信号途径,上调ROCK2和Calponin3蛋白表达水平。

关键词: 癫痫, 戊四氮, 丝状肌动蛋白, 钙调节蛋白3, ROCK2

Abstract:

Objective To disscuss the effect of the acute epileptic seizure induced by pentetrazol (PTZ) on the expressions of F-actin,Calponin3,and ROCK2 in the cerebral neurons of the rats,and to illuminate the possible mechanism of preventing the F-actin from abnormal depolymerization and triggering the rearrangement of F-actin.Methods 56 immature rats aged 3 weeks were divided into control (n=6) and epilepsy (n=50) groups. The rats in control group were injected with physiological saline introperitoneally.The rats in epilepsy group were introperitoneally injected with 60 mg·kg^-1 PTZ to establish the acute epileptic seizure models.And 6 rats from successful acute epileptic seizure models were sacrificed at different time points (1,2,3,and 7 d) after modeling.The fluorescence intensity of F-actin in the internal molecular layer of hippocampus of the rats was observed by phalloidine staining labeled by Alex-488; the distributions of Calponin3 and ROCK2 in the cerebral neurons in the pallium and hippocampus of the rats were detected by immunofluorescence method; the relative expression levels of Calponin3,ROCK2,and phosphorylated ROCK2 were analyzed by Western blotting method.Results Compared with control group,the fluorescence intensity of F-actin in the hippocampal internal molecular layer of the rats in epilepsy group was decreased (P<0.05),and the dot-shaped aggregation of F-actin was disappeared 1 d after modeling.The immunofluorescence results showed that Calponin3 dispersed in the cytoplasm of the neurons of the rats in control group;however,it aggregated in the cell cortex of the neurons 7 d after modeling in epilepsy group.ROCK2 was located in a small amount of neuritis of the rats in control group,whereas a great quantity of ROCK2 was found in both of the cell body and neuritis in epilepsy group 7 d after modeling.The Western blotting results showed that the relative expression levels of Calponin3 protein in the epilepsy group were markedly decreased at different time points after modeling compared with control group (P<0.05);however,it was increased gradually with the prolongation of time and closed to the level in control group within 1 week.The relative expression levels of ROCK2 protein in the hippocampus of the rats in epilepsy group began to increase steadily from 3 to 7 d after modeling compared with control group (P<0.05);however,the relative expression levels of phosphorylated ROCK2 protein in epilepsy group were increased sustainably from 1 to 7 d after modeling compared with control group (P<0.05),and were decreased gradually in a time-dependent manner.Conclusion The acute epileptic seizure of the immature rats induced by PTZ not only leads to the abnormal depolymerization of F-actin,but also actives the RhoA/ROCK2 signal pathway simultaneously and up-regulates the expression levels of ROCK2 and Calponin3 proteins.

Key words: epilepsy, pentetrazol, filiment-actin, Calponin3, ROCK2

中图分类号:

R742.1

程春旭, 李树蕾, 古小云, 黄可欣, 李艳超, 张舒岩, 杨立彬. PTZ诱导的癫痫急性发作大鼠大脑神经元中F-actin、Calponin3和ROCK2的表达及其意义[J]. 吉林大学学报(医学版), 2015, 41(02): 299-303.

CHENG Chunxu, LI Shulei, GU Xiaoyun, HUANG Kexin, LI Yanchao, ZHANG Shuyan, YANG Libin. Expressions of F-actin,Calponin3,and ROCK2 in cerebral neurons of rats with acute epileptic seizure induced by PTZ[J]. Journal of Jilin University Medicine Edition, 2015, 41(02): 299-303.

参考文献

[1] Zhang W,Benson DL.Developmentally regulated changes in cellular compartmentation and synaptic distribution of actin in hippocampal neurons [J].J Neurosci Res,2002,69 (4):427-436.

[2] Capani F,Martone ME,Deerinck TJ,et al.Selective localization of high concentrations of F-actin in subpopulations of dendritic spines in rat central nervous system:a three-dimensional electron microscopic study [J].J Comp Neurol,2001,435(2):156-170.

[3] 宁薇,徐淑君,罗建红.培养大鼠海马神经元树突发育的活细胞成像和量化分析[J].浙江大学学报:医学版,2007,36(2):155-160.

[4] Isokawa M.Remodeling dendritic spines of dentate granule cells in temporal lobe epilepsy patients and the rat pilocarpine model [J].Epilepsia,2000,41 (Suppl 6):S14-S17.

[5] Mizrahi A,Crowley JC,Shtoyerman E,et al.High-resolution in vivo imaging of hippocampal dendrites and spines [J].J Neurosci,2004,24(13):3147-3151.

[6] Zeng LH,Xu L,Rensing NR,et al.Kainate seizures cause acute dendritic injury and actin depolymerization in vivo [J].J Neurosci,2007,27(43):11604-11613.

[7] Ouyang Y,Yang XF,Hu XY,et al.Hippocampal seizures cause depolymerization of filamentous actin in neurons independent of acute morphological changes [J].Brain Res,2007,1143:238-246.

[8] Kurz JE,Moore BJ,Henderson SC,et al.A cellular mechanism for dendritic spine loss in the pilocarpine model of status epilepticus [J].Epilepsia,2008,49(10):1696-1710.

[9] Zhang YF,Li SL,Xiong TQ,et al.The differential rearrangement of filamentous actin in mossy fiber synapses in Pentylenetetrazol-kindled C57BL/6 mice [J].Epilepsy Res,2014,108 (1):20-28.

[10] Zhang YF,Xiong TQ,Tan BH,et al.Pilocarpine-induced epilepsy is associated with actin cytoskeleton reorganization in the mossy fiber-CA3 synapses [J].Epilepsy Res,2014,108 (3):379-389.

[11] 陈雅岚,李杰,徐馨,等.黑皮质素受体-4在癫痫模型大鼠脑组织中的表达及相关机制[J].第三军医大学学报,2014, 36(24):2445-2450.

[12] 张军,黄晓元,王凌峰,等.调宁蛋白的研究进展与展望[J].中华临床医师杂志:电子版,2013, 7(2):739-741.

[13] Rami G,Caillard O,Medina I,et al.Change in the shape and density of dendritic spines caused by overexpression of acidic calponin in cultured hippocampal neurons [J].Hippocampus,2006,16 (2):183-197.

[14] Shibukawa K,Yamazaki N,Kumasawa K,et al.Calponin 3 regulates actin cytoskeleton rearrangement in trophoblastic cell fusion [J].Mol Biol Cell,2010,21 (22):3973-3984.

[15] 肖保国.Rho激酶抑制剂的研究现状及其在神经系统疾病中的应用前景[J].重庆医科大学学报,2008,33 (Suppl 1):99-112.

[16] Schofield AV,Bernard O.Rho-associated coiled-coil kinase (ROCK) signaling and disease [J].Crit Rev Biochem Mol Biol,2013,48(4):301-316.

PTZ诱导的癫痫急性发作大鼠大脑神经元中F-actin、Calponin3和ROCK2的表达及其意义

Expressions of F-actin,Calponin3,and ROCK2 in cerebral neurons of rats with acute epileptic seizure induced by PTZ

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