Epidermal Growth Factor Receptor Mediates Neuronal Apoptosis After Subarachnoid Hemorrhage in Mice
Epidermal Growth Factor Receptor Mediates Neuronal Apoptosis After Subarachnoid Hemorrhage in Mice
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DOI：
10.1161/strokeaha.122.041977
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发表时间：
2023-05
期刊：
影响因子：
8.3
通讯作者：
Fumi Nakano;Hideki Kanamaru;Fumihiro Kawakita;Lei Liu;Y. Nakatsuka;Hirofumi Nishikawa;Takeshi Okada
中科院分区：
文献类型：
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作者：
Fumi Nakano;Hideki Kanamaru;Fumihiro Kawakita;Lei Liu;Y. Nakatsuka;Hirofumi Nishikawa;Takeshi Okada
背景：早期脑损伤包括神经元凋亡是蛛网膜下腔出血（SAH）后神经功能恶化的主要原因。本研究旨在探讨表皮生长因子受体（EGFR）/核因子-κB（NFκB）诱导激酶（NIK）/NFκB（p65和p50）通路是否参与小鼠SAH后神经元凋亡。方法：C57 BL/6成年雄性小鼠286只，行血管内穿孔SAH模型或假手术，排除轻度SAH小鼠86只。在实验1中，在建模后30分钟时，脑室内施用媒介物或EGFR抑制剂（632.0 ng AG 1478）。在24或72小时，在神经学评分后，检测脑含水量，用末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）和神经元标记物抗微管相关蛋白-2抗体进行双重免疫标记，使用全组织裂解物或左侧皮质的核蛋白提取物进行蛋白质印迹，以及对裂解的半胱天冬酶-3、磷酸化（p-）EGFR、NIK、p-NFκB p65、检测NFκB p105/50。在实验2中，在假手术或SAH建模后，脑室内施用AG 1478+媒介物或AG 1478 + 4.0ng EGF。观察24 h后取脑组织行TUNEL染色和免疫组化。结果如下：SAH组神经功能评分下降（P<0.01，Mann-Whitney U检验），TUNEL和切割的caspase-3阳性神经元增多（P<0.01，ANOVA），脑含水量增加（P<0.01，Mann-Whitney U检验），SAH-AG 1478组改善了这些观察结果。Western blotting结果显示SAH后p-EGFR、p-p65、p50和核NIK的表达水平升高（P<0.05，ANOVA），AG 1478处理后表达水平降低。免疫组化显示这些分子定位于变性神经元。EGF给药导致神经功能恶化，TUNEL阳性神经元增加，EGFR、NIK和NFκB活化。结论：在SAH后皮质变性神经元中观察到激活的EGFR、核NIK和NFκB表达，并通过给予AG 1478降低，与TUNEL和裂解的caspase-3阳性神经元的抑制相关。EGFR/NIK/NFκB通路参与了SAH后神经元凋亡的发生。
Background: Early brain injury including neuronal apoptosis is a main contributor to neurological deterioration after subarachnoid hemorrhage (SAH). This study was aimed to investigate whether EGFR (epidermal growth factor receptor)/NFκB (nuclear factor-kappa B) inducing kinase (NIK)/NFκB (p65 and p50) pathway is involved in the neuronal apoptosis after SAH in mice. Methods: C57BL/6 adult male mice underwent endovascular perforation SAH modeling or sham-operation (n=286), and 86 mild SAH mice were excluded. In experiment 1, vehicle or an EGFR inhibitor (632.0 ng AG1478) was administered intraventricularly at 30 minutes postmodeling. At 24 or 72 hours, after neurological score was tested, brain water content, double immunolabeling with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and a neuronal marker antimicrotubule-associated protein-2 antibody, Western blotting using whole tissue lysate or nuclear protein extraction of the left cortex, and immunohistochemistry for cleaved caspase-3, phosphorylated (p-) EGFR, NIK, p-NFκB p65, and NFκB p105/50 were evaluated. In experiment 2, after sham or SAH modeling, AG1478+vehicle or AG1478+4.0 ng EGF was administered intraventricularly. The brain was used for TUNEL staining and immunohistochemistry after 24-hour observation. Results: SAH group showed deteriorated neurological score (P<0.01, Mann-Whitney U test), more TUNEL- and cleaved caspase-3-positive neurons (P<0.01, ANOVA), and higher brain water content (P<0.01, Mann-Whitney U test), and these observations were improved in SAH-AG1478 group. Western blotting showed that expression levels of p-EGFR, p-p65, p50, and nuclear-NIK were increased after SAH (P<0.05, ANOVA), and decreased by AG1478 administration. Immunohistochemistry revealed these molecules localized in degenerating neurons. EGF administration resulted in neurological deterioration, increased TUNEL-positive neurons, and activation of EGFR, NIK, and NFκB. Conclusions: Activated EGFR, nuclear-NIK, and NFκB expressions were observed in cortical degenerating neurons after SAH, and were decreased by administration of AG1478, associated with suppression of TUNEL- and cleaved caspase-3-positive neurons. EGFR/NIK/NFκB pathway is suggested to be involved in neuronal apoptosis after SAH in mice.