内质网应激反应介导反复高热惊厥大鼠海马神经元的凋亡

Endoplasmic Reticulum Stress Mediated Neuronal Apoptosis in Hippocampus of Rats with Recurrent Febrile Seizures

目的观察反复高热惊厥(FS)大鼠海马神经元凋亡,及内质网分子伴侣葡萄糖调节蛋白(GRP)78和内质网促凋亡因子半胱氨酸蛋白酶-12(Caspase-12)表达的改变,探讨内质网应激反应在热性惊厥后脑损伤中的作用。方法SD大鼠80只,雌雄各半,随机分为对照组和反复FS组,每组40只。采用反复热水浴诱导大鼠FS,隔日诱导1次,共10次。应用原位细胞凋亡检测法(TUNEL)检测凋亡细胞,免疫组织化学、免疫蛋白印记(Westernblot)和荧光实时定量聚合酶链反应(RT-PCR)方法检测反复FS后3、6、12、24、48h和对照组大鼠海马内GRP78及Caspase-12mRNA和蛋白的表达。结果反复FS后随着时间的延长,反复FS组大鼠TUNEL阳性神经元数目明显增加,于惊厥后24h达高峰。Hoechst染色可见其凋亡细胞的核浓缩和核碎裂。Westernblot显示反复FS组大鼠3、6、12h海马内GRP78蛋白的表达较对照组明显增高,表达高峰在FS后6h。GRP78mRNA的表达时相改变和高峰出现时间与蛋白表达类似。免疫组织化学显示其GRP78蛋白广泛分布于海马内各个区,与对照组分布相同。FS后12、24和48hCaspase-12活性片段的表达较对照组明显增多,24h达峰值。12、24、48hCaspase-12mRNA的表达与对照组的比较有显著性差异,24h达高峰。免疫荧光双标记显示Caspase-12活性片段阳性细胞大多为神经元,并与TUNEL染色共定位,表现为核浓缩和核碎裂。结论Caspase-12介导的内质网应激凋亡途径参与了反复FS导致的神经元凋亡过程。

Objective To clarify the involvement of endoplasmic reticulum stress （ERS） response in neuronal apoptosis in the hippocampus of rats with recurrent febrile seizure （FS）. Methods Rats aged 21 d （ n = 80） were randomly divided into 2 groups ： control group and FS group,40 rats in each group. In a recurrent FS rat model, the neuronal apoptosis in the hippocampus was detected by terminal deoxynucleotidyl transferase -mediated deoxyuridine triphosphate -biotin nick end - labeling （TUNEL） method. And the expression patterns of Caspase - 12 and glucose - regulated protein 78 （ GRP78 ） were detected by Western blot, real - time PCR, and immunohistochemistry. Double fluorescent staining of Caspase - 12 and TUNEL method was performed to clarify the neuronal apoptosis involvement of Caspase - 12. And double fluorescent staining of Caspase -12 and neuronal nuclei （NeuN） was applied to identify the Caspase- 12 positive cells as neu-rons. Results Apoptotic cells in the hippocampus increased remarkably at 12, 24 and 48 h after seizures compared with control rats. In FS group, chromatin condensation and breakdown of the nucleus in neurons in hippocampus were also easily observed by Hoechst staining. Meanwhile, the ERS was induced by recurrent FS, as witness by up - regulated both GRP78 mRNA and protein expression. Western blot revealed that the expression of GRP78 protein in the hippocampus was elevated at 3, 6 and 12 h, with the maximum elevation noted at 6 h, after recurrent FS. The GRP78 transcript expression detected by real - time PCR was the same as GRP78 protein. Immunohistochemical analysis showed that GRP78 positive cells were distributed ubiquitously in the hippocampus after recurrent FS. Activated Caspase - 12 protein increased at 12, 24 and 48 h in FS group. And the Caspase - 12 mRNA expression was elevated at 12, 24 and 48 h after recurrent FS. Immunoreactivity for Caspase - 12 was enhanced in the hippecampus at the same time points, and co - location of Caspase - 12 immunoreactivity and DNA fragmentation detected by the TUNEL method. Double fluorescent staining showed that most of Caspase - 12 positive cells were neurons. Conclusion The endoplasmic retieulum stress response mediated by Caspase - 12 seems to be involved in the neuronal apoptosis caused by recurrent FS.