二氮嗪抑制癫痫大鼠海马神经元凋亡的研究

Diazoxide protects hippocampal neurons from apoptosis in epileptic rats

目的观察线粒体ATP敏感性钾离子通道(mitoKATP)开放剂二氮嗪(DZ)对氯化锂-匹鲁卡品致痫大鼠海马神经元线粒体凋亡通路相关凋亡因子的影响,探讨DZ对癫痫大鼠神经保护作用的机制。方法采用腹腔注射的方法,建立氯化锂-匹鲁卡品(PILO)诱导的大鼠癫痫持续状态(SE)模型。在检测大鼠海马凋亡相关蛋白水平变化时分为对照组,SE后24 h、72 h、5 d组;在检测DZ对海马神经元保护作用时分为对照组、PILO致痫72 h组(PILO组)、DZ预处理组(PILO+DZ组)、DZ+5-羟基癸酸(5-HD)预处理组(PILO+DZ+5-HD组)。SE后分别在相应时间点灌注取脑,采用TUNEL法检测细胞的凋亡;采用Western blot法检测大鼠海马Bcl-2、Bax、活性半胱氨酸天冬氨酸特异性蛋白酶-3(Caspase-3)蛋白的水平及细胞色素C(cytC)由线粒体到胞浆的释放。结果与正常对照组比较,SE组大鼠在SE后各相应时间点TUNEL染色阳性细胞明显增加(P<0.05),海马Bcl-2蛋白、线粒体中的细胞色素C(mito-cytC)的表达显著降低(P<0.05),海马Bax、活性Caspase-3蛋白及胞浆中的细胞色素C(cyto-cytC)的表达明显增高(P<0.05)。与PILO组及PILO+DZ+5-HD组比较,PILO+DZ组降低的海马Bcl-2蛋白水平显著升高(P<0.05),而升高的海马Bax、活性Caspase-3蛋白的水平明显降低(P<0.05),cytC由线粒体到胞浆的释放减少(P<0.05),TUNEL染色阳性细胞明显减少(P<0.05)。DZ的保护作用能被5-HD阻断。结论 DZ可通过上调Bcl-2/Bax水平,减少cytC的释放,抑制Caspase-3的激活,从而通过线粒体凋亡通路抑制氯化锂-匹鲁卡品致痫大鼠SE后海马神经元的凋亡,对癫痫发作所致的脑损伤具有神经保护作用,为癫痫的神经保护治疗提供新的治疗靶点。

Objective To investigate the effect of diazoxide（DZ）,a mitochondrial ATP-sensitive potassium channel（mitoKATP） opener,on the mitochondrial apoptosis-related protein in the hippocampus in epileptic rats induced by lithium-pilocarpine,and further explore the mechanism of the neuroprotective effect of DZ on epileptic rats.Methods The status epilepticus（SE） model were induced by lithium-pilocarpine through intraperitoneal injection.To detect the changes of apoptosis-related protein levels at different times in the hippocampus,rats were randomly divided into the control group,and SE 24 h,72 h,5 d groups.To study the neuroprotective effect of DZ against hippocampus injury,rats were randomly divided into the control group,the pilocarpine 72 h group（PILO group）,the PILO＋DZ group,and the PILO＋DZ＋5-hydroxydecanoate（5-HD） group.Neuron apoptosis was detected by TUNEL technology.Expressions of Bcl-2,Bax,and activated caspase-3 proteins in rat′s hippocampus and release of cytochrome c（cytC） from mitochondria to cytoplasm were detected by Western blot.Results Compared with the control group,TUNEL staining-positive cells in SE 24 h,72 h,5 d groups were significantly increased（P0.05）.Expression of the Bcl-2 protein in the rat hippocampus and cytC in the mitochondrial fraction（mito-cytC） in SE 24 h,72 h,5 d groups was markedly decreased（P0.05）.In contrast,the levels of Bax and activated caspase-3 proteins in the hippocampus and cytC in the cytosolic fraction（cyto-cytC） in SE 24 h,72 h,5 d groups were significantly increased（P0.05）.72 h after SE,compared with the PILO group and PILO＋DZ＋5-HD group,the decrease in the Bcl-2 protein level induced by seizures was markedly reversed in the PILO＋DZ group（P0.05）,while increased Bax and activated caspase-3 protein levels were reversed in the PILO＋DZ group（P0.05）.The release of cytC from mitochondria to the cytoplasm was suppressed（P0.05）.TUNEL staining-positive cells were markedly decreased in the PILO＋DZ group（P0.05）.The protective effect of DZ could be abolished by 5-HD treatment.Conclusions DZ may protect hippocampal neurons from apoptosis in epileptic rats induced by lithium-pilocarpine by up-regulating Bcl-2/Bax expression,suppressing cytC release and inhibiting caspase-3 activation,and thus inhibiting the mitochondrial apoptosis pathway.These beneficial effects suggest that the mitoKATP opener could be a possible new target for neuroprotective treatment of epilepsy.