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 (11): 1977-1985.doi: 10.4103/1673-5374.259620

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

抑制Huwe1表达可减轻皮质神经元缺氧再灌注后的神经元凋亡

  

  • 出版日期:2019-11-15 发布日期:2019-11-15
  • 基金资助:

    国家自然科学基金(81771642); 中国西部第二大学医院新芽研究基金

Silencing Huwe1 reduces apoptosis of cortical neurons exposed to oxygen-glucose deprivation and reperfusion

Guo-Qian He 1 , Wen-Ming Xu 2 , Hui-Juan Liao 2 , Chuan Jiang 2 , Chang-Qing Li 3 , Wei Zhang 4   

  1. 1 Department of Pediatrics, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu, Sichuan Province, China
    2 Joint Laboratory of Reproductive Medicine, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu, Sichuan Province, China
    3 Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
    4 Department of Medical Oncology, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, Cancer Hospital Affiliated to School of Medicine, University of Electronic Science and Technology of China, Chengdu, Sichuan Province, China
  • Online:2019-11-15 Published:2019-11-15
  • Contact: Wei Zhang, MD, zw493493@163.com.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 81771642 (to WMX); the New Bud Research Foundation of West China Second University Hospital of China (to GQH).

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摘要：

Huwe1作为泛素化系统E3泛素连接酶成员之一，在脑组织中广泛表达，已被证实可通过泛素化不同底物(如p53、Mcl-1、Cdc6、N-myc)参与调控细胞凋亡，对大脑皮质神经发生起着重要作用。然而在脑缺血再灌注病理过程中，Huwe1对细胞凋亡的作用及机制尚不清楚。为此，实验在体外原代培养大鼠皮质神经元，并进行氧糖剥夺再灌注处理(OGD/R，即OGD 3 h和再灌注24 h)。为了沉默Huwe1蛋白的表达，实验在神经元细胞体外培养第3天，采用慢病毒shRNA-Huwe1感染大鼠皮质神经元细胞，并在体外培养第7天予以氧糖剥夺再灌注24 h处理。此外，为了探讨MAPKs家族JNK/p38信号通路的作用，在上述皮质神经元细胞体外培养第7天，采用JNK通路抑制剂(SP600125)和p38通路抑制剂(SB203508)对细胞进行预处理30 min，然后再对神经元细胞进行OGD/R 24 h处理。采用TUNEL染色检测皮质神经元的凋亡情况，利用蛋白印迹技术检测JNK/p38 信号通路和凋亡通路分子(p53、Gadd45a、cleaved caspase 3、Bax、Bcl2)蛋白的表达变化，采用cleaved caspase 3 免疫荧光染色技术检测其在皮质神经元细胞的表达情况。结果显示：(1)在皮质神经元OGD/R的病理过程中，Huwe1和神经元凋亡在OGD/R 24 h表达明显增加，在给予慢病毒shRNA沉默Huwe1表达后，Huwe1在再灌注24 h表达明显降低，同时凋亡阳性神经元比例也明显降低；(2)慢病毒shRNA沉默Huwe1表达后，降低了神经元中促凋亡蛋白Bax和cleaved caspase 3表达，并增加了神经元中抑制凋亡蛋白Gadd45a和Bcl-2表达；(3)沉默Huwe1表达后，神经元中p-JNK磷酸化水平明显降低，而p-p38 磷酸化水平增加；(4)上述数据说明，在脑缺血再灌注损伤病理过程中，抑制Huwe1表达可通过调节凋亡相关JNK/p38信号通路，从而发挥抑制神经元凋亡的作用。实验于2018年1月经四川大学动物伦理委员会批准（批准号：2018013）。

orcid: 0000-0002-4552-2588(Wei Zhang)

关键词: 缺血性脑卒中, 氧糖剥夺再灌注损伤, 缺血再灌注, 皮质神经元, 泛素化系统, Huwe1, 凋亡, 治疗靶点, 细胞培养, 细胞死亡, 神经再生

Abstract:

HECT, UBA and WWE domain-containing 1 (Huwe1), an E3 ubiquitin ligase involved in the ubiquitin-proteasome system, is widely expressed in brain tissue. Huwe1 is involved in the turnover of numerous substrates, including p53, Mcl-1, Cdc6 and N-myc, thereby playing a critical role in apoptosis and neurogenesis. However, the role of Huwe1 in brain ischemia and reperfusion injury remains unclear. Therefore, in this study, we investigated the role of Huwe1 in an in vitro model of ischemia and reperfusion injury. At 3 days in vitro, primary cortical neurons were transduced with a control or shRNA-Huwe1 lentiviral vector to silence expression of Huwe1. At 7 days in vitro, the cells were exposed to oxygen-glucose deprivation for 3 hours and reperfusion for 24 hours. To examine the role of the c-Jun N-terminal kinase (JNK)/p38 pathway, cortical neurons were pretreated with a JNK inhibitor (SP600125) or a p38MAPK inhibitor (SB203508) for 30 minutes at 7 days in vitro, followed by ischemia and reperfusion. Neuronal apoptosis was assessed by TUNEL assay. Protein expression levels of JNK and p38MAPK and of apoptosis-related proteins (p53, Gadd45a, cleaved caspase-3, Bax and Bcl-2) were measured by western blot assay. Immunofluorescence labeling for cleaved caspase-3 was performed. We observed a significant increase in neuronal apoptosis and Huwe1 expression after ischemia and reperfusion. Treatment with the shRNA-Huwe1 lentiviral vector markedly decreased Huwe1 levels, and significantly decreased the number of TUNEL-positive cells after ischemia and reperfusion. The silencing vector also downregulated the pro-apoptotic proteins Bax and cleaved caspase-3, and upregulated the anti-apoptotic proteins Gadd45a and Bcl-2. Silencing Huwe1 also significantly reduced p-JNK levels and increased p-p38 levels. Our findings show that downregulating Huwe1 affects the JNK and p38MAPK signaling pathways as well as the expression of apoptosis-related genes to provide neuroprotection during ischemia and reperfusion. All animal experiments and procedures were approved by the Animal Ethics Committee of Sichuan University, China in January 2018 (approval No. 2018013).

Key words: nerve regeneration, ischemic stroke, oxygen-glucose deprivation and reperfusion, ischemia/reperfusion, cortical neuron, ubiquitin proteasome system, Huwe1, apoptosis, therapeutic targets, cell culture, cell death, neural regeneration