低频率电刺激通过Ras同源基因/Rho相关蛋白激酶途径调节自噬对癫痫大鼠的作用研究

Effect of LFS regulating autophagy on epileptic rats through Ras homologous gene/Rho-associated coiled-coil containing protein kinase pathway

目的探究低频率电刺激(LFS)对癫痫的作用及其可能的作用机制。方法将60只SD大鼠按随机数字表法分为对照组、癫痫模型组、假刺激组和LFS治疗组,每组各15只。氯化锂-匹鲁卡品法建立癫痫大鼠模型。记录大鼠Racine评分和脑电图,Morris水迷宫实验检测大鼠空间学习记忆能力;苏木素-伊红(HE)染色和尼氏染色检测大鼠海马CA1区病理学变化;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)染色检测大鼠海马神经细胞凋亡;Western blot检测大鼠海马Ras同源基因家族成员A(RhoA)、Rho相关蛋白激酶1(ROCK1)、ROCK2、自噬和凋亡相关蛋白表达;逆转录聚合酶链反应(RT-PCR)检测大鼠海马RhoA、ROCK1和ROCK2 mRNA表达。结果与对照组比较,癫痫模型组大鼠出现连续性棘波,Racine评分[(4.23±0.29)分]、逃跑潜伏期[(49.36±4.69)s]、神经细胞凋亡率[(27.52±2.95)%]和B淋巴细胞瘤-2(Bcl-2)相关X[Bax,(0.82±0.07)]、裂解-caspase3[c-caspase3,(1.70±0.15)]、微管相关蛋白1A/1B轻链3BⅡ/微管相关蛋白1A/1B轻链3BⅠ[LC3Ⅱ/LC3Ⅰ,(5.20±0.42)]和Beclin1(0.73±0.05)蛋白表达水平明显升高(均P<0.05),RhoA、ROCK1和ROCK2 mRNA和蛋白表达水平均明显升高(均P<0.05),游泳距离[(240.68±22.91)cm]、平台停留时间[(39.89±2.20)s]、神经细胞数量(17.13±3.14)、尼氏体数量(6.75±1.09)以及Bcl-2(0.24±0.02)和p62(0.20±0.02)蛋白表达水平明显降低(P<0.05);与假刺激组比较,LFS治疗组大鼠波峰明显下降,Racine评分[(2.82±0.23)]分、逃跑潜伏期[(34.83±3.85)s]、神经细胞凋亡率[(9.25±0.91)%]及Bax(0.43±0.05)、c-caspase3(0.53±0.02)、LC3Ⅱ/LC3Ⅰ(1.17±0.11)和Beclin1(0.36±0.02)蛋白表达水平明显降低(均P<0.05),RhoA、ROCK1和ROCK2 mRNA和蛋白表达水平均明显降低(均P<0.05),游泳距离[(284.21±22.36)cm]、平台停留时间[(46.85±2.93)s]、神经细胞数量(47.00±5.07)、尼氏体数量(33.75±2.90)和Bcl-2(0.87±0.07)、p62(0.96±0.05)蛋白表达水平明显升高(均P<0.05)。结论LFS可能通过抑制Rho/ROCK途径调节自噬发挥抗癫痫作用。

Objective To explore the effect of low-frequency stimulation(LFS)on epilepsy and its possible mechanism.Methods A total of 60 SD rats were randomly divided into control group,epilepsy model group,sham stimulation group and LFS treatment group,with 15 rats in each group.A rat model of epilepsy was established by the method of lithium chloride and pilocarpine.The Racine score and electroencephalogram of rats were recorded.Morris water maze test was used to detect the spatial learning and memory ability of the rats;Hematoxylin eosin(HE)staining and Nissl staining were used to detect the pathological changes in the hippocampus CA1 area of the rats;TdT mediated dUTP nick end labeling(TUNEL)staining was used to detect the apoptosis of hippocampal neurons;Western blot was used to detect the expression of RhoA,ROCK1,ROCK2,autophagy and apoptosis-related proteins in the hippocampus of the rats;Reverse Transcription-Polymerase Chain Reaction(RT-PCR)was used to detect the mRNA expression of RhoA,ROCK1 and ROCK2 in the hippocampus of the rats.Rusults Compared with the control group,rats in the epilepsy model group showed continuous spikes,Racine score(4.23±0.29),escape latency[(49.36±4.69)s],neuronal cell apoptosis rate[(27.52±2.95)%],expression of B-lymphoma-2(Bcl-2)related X[Bax,(0.82±0.07)],c-caspase3(1.70±0.15),microtubule-associated proteins 1A/1B light chain 3BⅡ/microtubule-associated proteins 1A/1B light chain 3BⅠ[LC3Ⅱ/LC3Ⅰ,(5.20±0.42)]and Beclin1(0.73±0.05)protein were significantly increased(all P<0.05).Expression of RhoA,ROCK1 and ROCK2 mRNA and protein were significantly increased(all P<0.05).Swimming distance[(240.68±22.91)cm],platform residence time[(39.89±2.20)s],number of neurons(17.13±3.14),number of Nissl bodies(6.75±1.09),protein expression of Bcl-2(0.24±0.02)and p62(0.20±0.02)were significantly reduced(P<0.05);Compared with the sham stimulation group,the wave peak of the rats in the LFS treatment group was significantly decreased,Racine score(2.82±0.23),escape latency[(34.83±3.85)s],neuronal cell apoptosis rate[(9.25±0.91)%],expression of Bax(0.43±0.05),c-caspase3(0.53±0.02),LC3Ⅱ/LC3Ⅰ(1.17±0.11)and Beclin1(0.36±0.02)protein were significantly reduced(P<0.05).Expression of RhoA,ROCK1 and ROCK2 mRNA and protein were significantly reduced(P<0.05).Swimming distance[(284.21±22.36)cm],platform residence time[(46.85±2.93)s],the number of neurons(47.00±5.07),the number of Nissl bodies(33.75±2.90),protein expression of Bcl-2(0.87±0.07)and p62(0.96±0.05)increased significantly(P<0.05).Conclusions LFS may regulate autophagy by inhibiting the Rho/ROCK pathway to play an anti-epileptic effect.