红藻氨酸诱导内质网应激模型的途径

Kainic acid-induced endoplasmic reticulum stress model

背景:前期研究表明,海马内注射红藻氨酸海可诱发兴奋性红藻氨酸受体KA1亚受体在海马神经元的表达明显上调,内质网应激标志物磷酸化真核翻译起始因子2α表达增加并伴随细胞死亡。目的:探讨红藻氨酸海马内注射后内质网应激发生的机制。方法:取昆明小鼠32只,将0.15 nmol红藻氨酸注入海马CA1区域,注射时间为60 s。分别于红藻氨酸注射后第1,2,3,4,5,6,8,12小时灌注取脑,灌注取脑前进行Bederson体征评分,然后行全脑切片FJB染色分析与免疫荧光双标记观察。结果与结论:1红藻氨酸注射后第3,4,5,6,8小时,Bederson体征评分表明中枢神经功能出现明显损伤,FJB染色示小鼠海马内神经元死亡明显;注射后第1,2,12小时,Bederson体征评分中枢神经功能未见明显损伤,FJB染色小鼠海马神经元死亡结果不明显。2根据FJB结果,取第3,8小时的脑片做免疫组化。海马内注射红藻氨酸后导致海马神经元中KA1和磷酸化真核翻译起始因子2α在相同的时间点表达明显上调,将KA1与磷酸化真核翻译起始因子2α图片结果进行叠加处理,两者完全重合,表明KA1的表达和内质网应激发生在同一个神经细胞内。结果表明红藻氨酸首先诱导了兴奋性膜上受体KA1表达的上调,其KA1的表达上调可能引起细胞内质网功能紊乱,导致内质网应激反应,并进一步促进了神经细胞的死亡。

BACKGROUND： Previous studies have shown that kainic acid injected into hippocampus can significantly upregulate the expression of excitatory KA1 subunit of the kainate receptor in the hippocampus, and endoplasmic reticulum stress markers, phosphorylation of the alpha subunit of eukaryotic initiation factor 2, accompanied by cell death. OBJECTIVE： To explore the mechanism of endoplasmic reticulum stress after kainic acid is injected into the hippocampus.METHODS： 0.15 nmol kainic acid was injected into the hippocampal CA1 region of 32 adult male Kunming mice, the injection time was 60 seconds. At different time points（1, 2, 3, 4, 5, 6, 8 and 12 hours） after kainic acid was injected, the Bederson score analysis was performed, and then the brain was harvested after cerebral perfusion. FJB staining of brain sections and immunofluorescence double labeled observation were also performed. RESULTS AND CONCLUSION：（1） At 3, 4, 5, 6, 8 hours after kainic acid injection, Bederson score showed severe injury of central nervous system function, and FJB staining showed the increased of cell death in the hippocampus（P 〈 0.05）; At 1, 2, 12 hours after injection, Bederson score showed no obvious injury of central nervous system function, and FJB staining showed unobvious cell death in the hippocampus（P 〉 0.05）.（2） According to the results of FJB staining, the brain sections were selected at 3, 8 hours for immunohistochemistry. The expressionlevels of KA1 receptors and endoplasmic reticulum stress marker P-eIF2α were up-regulated at the same time after kainic acid was injected into hippocampus. Two single-staining KA1 and P-eIF2α immunofluorescence images were synthesized into one over-lapped double-stained image, and two images overlapped, indicating that the up-regulated expression of KA1 and endoplasmic reticulum stress occurred in the same nerve cells. Kainic acid first up-regulated the excitatory receptor KA1 expression, which may cause cell endoplasmic reticulum dysfunction and result in the endoplasmic reticulum stress response, further promoting neuronal cell death.