CCAAT／增强子结合蛋白介导的内质网应激参与癫痫脑损伤的机制

Mechanism of CCAAT/enhancer-binding protein-homologous protein mediated endoplasmic reticulum stress involving in the epileptic brain damage

目的在建立戊四氮癫痫持续状态（SE）模型的基础上观察大鼠海马神经元的凋亡，内质网分子伴侣葡萄糖调节蛋白78（GRP78）和内质网应激（ERS）特异性促凋亡因子即CCAAT／增强子结合蛋白（CHOP）在癫痫脑损伤（EBD）大鼠中的表达，探讨GRP78及CHOP在EBD中的作用，以及2-脱氧葡萄糖（2-DG）通过激活一定程度的ERS发挥脑保护作用的可能机制。方法SD大鼠120只，雌雄各半，随机分为正常对照组、SE组和sE＋DG组，每组40只。采用腹腔注射戊四氮诱导大鼠SE。应用原位细胞凋亡检测法检测凋亡细胞，Westernblot和Real—timePCR方法检测SE发作后3、6、12、24、48h大鼠海马内GRP78及CHOPmRNA和蛋白的表达。结果SE发作后12、24、48h神经细胞凋亡增多，SE＋DG组凋亡细胞数在同一时间点较sE组明显减少；Westernblot和Real—timePCR发现GRP78蛋白和mRNA的表达在SE发作后3、6、12h均高于正常对照组，6h达峰值；SE＋DG组GRP78mRNA和蛋白的表达在早期（3h）较sE组明显增高，6h和12h明显减少；在SE发作后6h开始增多，12、24、48hCHOP蛋白和mRNA的表达高于正常对照组，24h达高峰；SE＋DG组在同一时间点均较SE明显减少。正常对照组各个时间点GRP78和CHOP蛋白和基因的表达均无差异。结论SE发作后早期可能通过GRP78表达升高启动ERS以保护细胞功能；后期CHOP被激活，参与EBD发生过程；2-DG通过激活一定程度的ERS，保护脑细胞。内质网CHOP凋亡通路可能是EBD的又一机制。

Objective To explore the neuronal apoptosis, endoplasmic reticulum stress （ERS）-related factors glucose-regulated protein 78 （GRP78） and CCAAT/enhaneer-binding protein-homologous protein（CHOP） expressions in hippocampus and possible mechanism of brain protection by 2-deoxy-D-glucose（2-DG） on their expressions in epi- leptic brain damage rats induced by pentetrazole. Methods Rats aged 21 d （ n = 120） were randomly divided into 3 groups： control group,status epileptieus （SE） group and SE ＋ DG group,with 40 rats in each group. On a SE rat mo- del, the neuronal apoptosis in the hippoeampus was detected by terminal deoxynueleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling method. And the expression of GRP78 and CHOP were detected by Western blot and Real-time PCR. Results Apoptotic cells in the hippocampus were increased remarkably at 12 h, 24 h, and 48 h after the seizures, as compared with those of control rats. In SE group, chromatin condensation and break- down of the nucleus in neurons in hippocampus were also easily observed by Hoechst staining. Meanwhile, the ERS was induced by SE, as witness by up-regulating GRP78 expression at both protein and mRNA level. The expression of GRP78 protein in the hippocampus was elevated at 3 h,6 h and 12 h, with the maximum elevation noted at 6 h after SE. The GRP78 transcript expression was increased at 3 h,6 h,and 12 h after SE. DG treatment enhanced the changes of GRP78 protein and transcript at 3 h, and abrogated the increase of GRP78 at 6 h and 12 h, as compared with those in SE group. CHOP activation occulted at 12 h,24 h and 48 h in SE group. And the CHOP-mRNA expression was the same as the CHOP protein expression. The expression of CHOP protein and mRNA was decreased at 12 h,24 h,and 48 h after using DG. Conclusion The endoplasmic reticulum stress response mediated by CHOP seems to be involved in the neuronal apoptosis caused by status epilepticus.