杏仁核注射红藻氨酸诱导大鼠边缘叶癫痫发作时海马中Smac和XIAP蛋白的表达

应用红藻氨酸(kainic acid,KA)诱导的大鼠边缘叶癫痫发作模型,检测第二个线粒体源的半胱天冬蛋白酶激活物,直接与凋亡抑制蛋白结合的低等电点蛋白(second mitochondrial activator of caspases／direct inhibitor of apoptosis protein-binding protein of low isoelectric pointPI],Smac／DIABLO)和X染色体连锁的凋亡抑制蛋白(X-chromosome-linked inhibitor of apoptosis protein,XIAP)在癫痫大鼠海马神经元表达。单侧杏仁核内注射KA诱导癫痫发作,1h后用安定终止发作,然后分别用TUNEL染色和cresyl violet染色观察海马神经元存活和凋亡的变化,用免疫荧光和Western blot检测海马Smac／DIABLO、XIAP和半胱天冬蛋白酶-9(caspase-9)的表达。结果表明,发作终止2h时KA注射同侧海马CA3区细胞浆内Smac／DIABLO蛋白表达增加,4h时caspase-9出现裂解片断,8h时出现TUNEL阳性细胞,24h时达高峰。脑室内注射caspase-9抑制剂z-LEHD-fluoromethyl ketone(z-LEHD-fmk)可减少TUNEL阳性细胞,增加存活神经元。发作后KA注射同侧海马CA3区神经元caspase-9免疫反应性增强,Smac／DIABLO和XIAP弥散于整个神经元内。对侧海马未检测到TUNEL阳性细胞及Smac／DIABLO和XIAP蛋白的上述变化。以上结果提示,癫痫发作可诱导Smac／DIABLO蛋白从线粒体向细胞浆的移位、XIAP亚细胞分布改变和caspase-9的激活,Smac／DIABLO、XIAP和caspase-9可能参与了癫痫神经元损伤的病理生理机制,caspase-9可能是潜在的治疗靶点。

The expression of Smac and XIAP in rat hippocampus following limbic seizure induced by kainic acid injection into amygdaloid nucleus

To determine whether Smac/DIABLO (second mitochondrial activator of caspases/direct inhibitor of apoptosis protein-binding protein of low isoelectric point PI]) and XIAP (X-chromosome-linked inhibitor of apoptosis protein) serve to regulate neuronal apoptosis following seizures, we investigated seizure-induced changes in caspase-9, Smac/DIABLO and XIAP protein expression and the in vivo effect of caspase-9 inhibition. Animals received unilateral intra-amygdaloid injection of kainic acid (0.5 microg) to induce seizures for 1 h. The seizures were then terminated by diazepam (30 mg/kg). Animals were killed 0, 2, 4, 8, 24 or 72 h following diazepam administration. The apoptotic and surviving neurons in hippocampus were observed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and cresyl violet staining, the expression of Smac/DIABLO, XIAP and caspase-9 was detected with immunofluorescence and western blot. The results showed that the levels of XIAP and the 46-kDa proenzyme form of caspase-9 were unaffected by the seizures. The expression of Smac increased at 2 h and the 37-kD cleaved fragment of caspase-9 was detected at 4 h, TUNEL-positive neurons appeared at 8 h and reached maximal at 24 h following seizure cessation within the ipsilateral (the same side as the intra-amygdaloid injection of kainic acid) CA3 subfield of the hippocampus. Intracerebroventricular infusion of caspase-9 inhibitor z-LEHD-fluoromethyl ketone (z-LEHD-fmk) significantly decreased TUNEL-positive neurons and increased the number of surviving cells. Caspase-9 immunoreactivity increased and Smac/DIABLO, XIAP immunoreactivity became extensive within the ipsilateral CA3 neurons. TUNEL-positive neurons and the alterations of the expression of Smac/DIABLO and XIAP within the ipsilateral CA3 were not detected within the contralateral hippocampus. These results suggest that seizures lead the translocation of Smac/DIABLO into the cytosol, the activation of caspase-9 and the change of subcellular locoalization of XIAP. These changes may play a role in the brain damage induced by seizures. Caspase-9 is possibly a potential therapeutic target in the treatment of brain injury associated with seizures.