中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (5): 841-849.doi: 10.4103/1673-5374.249232

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

ESE1表达与脑缺血再灌注损伤后海马神经元的凋亡相关

  

  • 出版日期:2019-05-15 发布日期:2019-05-15
  • 基金资助:

    国家自然科学基金资助(81400963)，江苏省333号项目(BRA 2015187)；江苏省医学青年人才基金(QNRC 2016327，QNRC 2016328，QNRC 2016326)

ESE1 expression correlates with neuronal apoptosis in the hippocampus after cerebral ischemia/reperfusion injury

Hai-Long Yu 1, 2, 3, 4 , Liang-Zhu Wang 5 , Ling-Ling Zhang 1, 2 , Bei-Lei Chen 1, 2 , Hui-Juan Zhang 1, 2 , Yu-Ping Li 1, 2 , Guo-Dong Xiao 6 , Ying-Zhu Chen 1, 2   

  1. 1 Clinical Medical College of Yangzhou University, Yangzhou, Jiangsu Province, China
    2 Department of Neurology, Northern Jiangsu People’s Hospital, Yangzhou, Jiangsu Province, China
    3 Institute of Neuroscience, Northern Jiangsu People’s Hospital, Yangzhou, Jiangsu Province, China
    4 Drum Tower Hospital, Medical School of Nanjing University, Nanjing, Jiangsu Province, China
    5 Dalian Medical University, Dalian, Liaoning Province, China
    6 Department of Neurology, Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China
    Funding: This study was supported by the National Natural Science Foundation of China, No. 81400963 (to
  • Online:2019-05-15 Published:2019-05-15
  • Contact: Guo-Dong Xiao, PhD, yarrowshaw@hotmail.com; Ying-Zhu Chen, PhD, yzchendr@163.com.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 81400963 (to HLY); the 333 Project of Jiangsu Province of China, No. BRA2015187 (to YZC); the Medical Youth Talent Foundation of Jiangsu Province of China, No. QNRC2016327 (to HLY), QNRC2016328 (to BLC), and QNRC2016326 (to YPL).

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摘要：

ESE1是ETS转录因子家族的成员，在许多组织中广泛表达，且在炎症反应中起多种作用。以往研究表明ESE1在神经炎症过程中可促进神经元凋亡，但其在脑缺血过程中的表达及对神经元凋亡的影响仍然未知，对此，作者予以体内外实验加以论证。(1)实验探讨了ESE1在脑缺血损伤中的作用：成年SD大鼠利用改良的四血管闭塞法建立全脑缺血再灌注模型，于建模后6，12，24，48，72 h取缺血后海马组织进行实验。Western blot法检测缺血后海马组织ESE1的表达、与调控细胞凋亡有关的NF-κB亚单位p-p65和细胞凋亡相关因子active caspase3的表达，免疫组织化学染色检测海马组织中ESE1的表达和分布，采用免疫荧光双染法观察缺血后海马组织中ESE1的细胞分布及其与active caspase3表达的关系；(2)体外试验：将氯化钴(CoCl2)加入扬州大学细胞资源中心的大鼠源PC12细胞的培养基构建细胞的化学性缺氧模型。Western blot检测PC12细胞凋亡过程中ESE1和p-p65的表达变化。通过siRNA干扰ESE1表达，并通过MTT法检测干扰6 h后缺氧的细胞活力变化，Western blot检测细胞active caspase3和p-p65蛋白的表达；(3)Western blot和免疫组织化学结果显示：建模后大鼠海马组织中ESE1、p-p65和active caspase3的蛋白表达及免疫反应显著增高，免疫荧光双染发现ESE1在全脑缺血再灌注24 h后主要与海马神经元存在共定位，并且与active caspase3共表达；(4)体外实验结果显示，经CoCl2 刺激后，PC12细胞中ESE1，p-p65和active caspase3的蛋白表达显著增加。siRNA干扰ESE1表达后增加了缺氧PC12细胞活力，其中siESE1#3作用最强，同时也下调缺氧后的p-p65和active caspase-3蛋白表达；(5)上述数据提示：脑缺血再灌注后海马组织中ESE1蛋白表达增加，并且可能通过激活NF-κB通路而促进神经元凋亡。

orcid: 0000-0001-9448-3127 (Guo-Dong Xiao)
           0000-0002-2110-236X (Ying-Zhu Chen)

关键词: ESE1, 上皮特异性ETS-1, 脑缺血/再灌注, 海马, 神经元, 凋亡, NF-κB, 炎症, caspase 3, p-p65, 神经保护, 卒中, 神经再生

Abstract:

Epithelial-specific ETS-1 (ESE1), a member of the ETS transcription factor family, is widely expressed in multiple tissues and performs various functions in inflammation. During neuroinflammation, ESE1 promotes neuronal apoptosis; however, the expression and biolog¬ical functions of ESE1 remain unclear after cerebral ischemia/reperfusion. We performed in vivo and in vitro experiments to explore the role of ESE1 in cerebral ischemic injury. A modified four vessel occlusion method was used in adult Sprague-Dawley rats. At 6, 12, 24, 48, and 72 hours after model induction, the hippocampus was collected for analysis. Western blot assays and immunohistochemistry showed that the expression of ESE1, phosphorylated p65 and active caspase-3 was significantly up-regulated after ischemia. Double immunofluo-rescence staining indicated that ESE1 and NeuN were mostly co-located in the hippocampus after ischemia. Furthermore, ESE1 was also co-expressed with active caspase-3. PC12 cells were stimulated with cobalt chloride (CoCl2) to establish a chemical hypoxia model. After ESE1 knockdown by siRNA for 6 hours, cell viability was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assays. The levels of ESE1, phosphorylated p65 and active caspase-3 were also remarkably increased in PC12 cells after CoCl2 stimulation. After ESE1 knockdown, PC12 cell viability was increased after hypoxia. siRNA knockdown of ESE1 decreased the level of p-p65 and active caspase-3 after CoCl2 stimulation. These data reveal that ESE1 levels are elevated in the hippocampus after cerebral ischemia/reperfusion injury. This may play a role in neuronal apoptosis via activation of the nuclear factor-κB pathway.

Key words: stroke, epithelial specific ETS-1, cerebral ischemia/reperfusion, nuclear factor-κB, inflammation, caspase-3, neuroprotective effects, neural regeneration, cobalt chloride (CoCl2), siRNA transfection