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

作     者：Guo-Qian He Wen-Ming Xu Hui-Juan Liao Chuan Jiang Chang-Qing Li Wei Zhang 

作者机构：Department of PediatricsKey Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University)Ministry of EducationWest China Second University HospitalSichuan UniversityChengduSichuan ProvinceChina Joint Laboratory of Reproductive MedicineKey Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University)Ministry of EducationWest China Second University HospitalSichuan UniversityChengduSichuan ProvinceChina Department of NeurologyThe Second Affiliated Hospital of Chongqing Medical UniversityChongqingChina Department of Medical OncologySichuan Cancer Hospital & InstituteSichuan Cancer CenterCancer Hospital Affiliated to School of MedicineUniversity of Electronic Science and Technology of ChinaChengduSichuan ProvinceChina 

出 版 物：《Neural Regeneration Research》 (中国神经再生研究（英文版）)

年 卷 期：2019年第14卷第11期

页      面：1977-1985页

核心收录：

学科分类：1002[医学-临床医学] 1001[医学-基础医学(可授医学、理学学位)] 1010[医学-医学技术（可授医学、理学学位）] 100104[医学-病理学与病理生理学] 100215[医学-康复医学与理疗学] 10[医学] 

基　　金：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) 

主　　题：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 

摘      要：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 p38 MAPK 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 p38 MAPK and of apoptosis-related proteins(p53, Gadd45 a, 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 Gadd45 a 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 p38 MAPK signaling pathways as well as the expression of apoptosis-related genes to provide neuroprotection during ische