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

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60mg·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7d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1d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7d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7d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3d开始显著增加并持续至造模后7d(P<0.05);磷酸化ROCK2蛋白相对表达水平则从癫痫急性发作后1d就开始显著增加并持续到7d(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。

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 3weeks 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 60mg·kg-1 PTZ to establish the acute epileptic seizure models.And 6rats from successful acute epileptic seizure models were sacrificed at different time points（1,2,3,and 7d）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 ROCK2in 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 1dafter 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 7dafter 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 7dafter 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 1week.The relative expression levels of ROCK2 protein in the hippocampus of the rats in epilepsy group began to increase steadily from 3to 7dafter modeling compared with control group（P〈0.05）;however,the relative expression levels of phosphorylated ROCK2 protein in epilepsy group were increased sustainably from 1to 7dafter 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 Calponin3proteins.