Silencing Huwe1 reduces apoptosis of cortical neurons exposed to oxygen-glucose deprivation and reperfusion
Silencing Huwe1 reduces apoptosis of cortical neurons exposed to oxygen-glucose deprivation and reperfusion
复制标题
沉默Huwe1可减少暴露于氧糖剥夺和再灌注的皮质神经元的凋亡
DOI：
10.4103/1673-5374.259620
复制
发表时间：
2019-11-01
影响因子：
6.1
通讯作者：
Zhang, Wei
中科院分区：
文献类型：
--
作者：
He, Guo-Qian;Xu, Wen-Ming;Zhang, Wei
HECT、乌巴和WWE domain containing 1（Huwe1）是一种参与泛素-蛋白酶体系统的E3泛素连接酶，在脑组织中广泛表达。Huwe1参与多种底物的周转，包括p53，Mcl-1，Cdc6和N-myc，从而在细胞凋亡和神经发生中发挥关键作用。然而，Huwe1在脑缺血再灌注损伤中的作用尚不清楚。因此，在本研究中，我们研究了Huwe1在体外缺血和再灌注损伤模型中的作用。在体外3天，用对照或shRNA-Huwe1慢病毒载体转导原代皮层神经元以沉默Huwe1的表达。在体外培养第7天，将细胞暴露于氧-葡萄糖剥夺3小时并再灌注24小时。为了研究c-Jun N-末端激酶（JNK）/p38通路的作用，在体外用JNK抑制剂（SP600125）或p38MAPK抑制剂（SB 203508）预处理皮层神经元30分钟，7天，随后缺血和再灌注。TUNEL法检测神经细胞凋亡。Western blot检测JNK、p38MAPK和凋亡相关蛋白（p53、Gadd45a、切割型caspase-3、Bax和Bcl-2）的蛋白表达水平。对裂解的caspase-3进行免疫荧光标记。我们观察到缺血和再灌注后神经元凋亡和Huwe1表达显著增加。shRNA-Huwe1慢病毒载体治疗显著降低了Huwe1水平，并显著减少了缺血和再灌注后TUNEL阳性细胞的数量。沉默载体还下调促凋亡蛋白Bax和裂解的半胱天冬酶-3，并上调抗凋亡蛋白Gadd45a和Bcl-2。沉默Huwe1还显著降低了p-JNK水平并增加了p-p38水平。我们的研究结果表明，下调Huwe1影响JNK和p38 MAPK信号通路以及凋亡相关基因的表达，在缺血和再灌注期间提供神经保护。所有动物实验和程序均于2018年1月获得中国四川大学动物伦理委员会批准（批准号2018013）。
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).