Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, Chengdu, Sichuan 610041, P.R. China.
Department of Medical Oncology, Sichuan Cancer Hospital and Institute, Sichuan Cancer Center, Cancer Hospital Affiliated to School of Medicine, Chengdu, Sichuan 610041, P.R. China.
Mol Med Rep. 2020 Dec;22(6):5083-5094. doi: 10.3892/mmr.2020.11611. Epub 2020 Oct 19.
Autophagy and the ubiquitin proteasome system (UPS) are two major protein degradation pathways involved in brain ischemia. Autophagy can compensate for UPS impairment‑induced cellular dysfunction. HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1 (Huwe1), an E3 ubiquitin ligase, serves critical roles in nervous system plasticity, regeneration and disease. However, the role of Huwe1 in autophagy in brain ischemia/reperfusion (I/R) injury remains unknown. The aim of the present study was to investigate the crosstalk between autophagy and the UPS in brain ischemia. The present study established an oxygen‑glucose deprivation and reperfusion (OGD/R) model in rat primary cortex neurons in vitro. Lentiviral interference was used to silence the expression of Huwe1. An autophagy promoter (rapamycin), an autophagy inhibitor (wortmannin) and a JNK pathway inhibitor (SP600125) were also used in the current study. Cellular autophagy‑related proteins, including Beclin‑1, autophagy related (ATG) 7, ATG5, ATG3 and microtubule associated protein 1 light chain 3 α, and apoptosis‑related proteins, such as P53, cleaved caspase 3, Bax and Bcl2, were detected via western blotting and immunocytochemistry. Neuronal apoptosis was evaluated using a TUNEL assay. The results demonstrated that silencing Huwe1 increased the expression levels of autophagy‑related proteins at 24 h after OGD/R. Treatment with a JNK inhibitor or cotreatment with Huwe1 shRNA significantly increased autophagy. Rapamycin increased apoptosis under OGD/R conditions. However, treatment with Huwe1 shRNA decreased the number of TUNEL‑positive cells at 24 h after OGD/R. Cotreatment with Huwe1 shRNA and wortmannin alleviated neuronal apoptosis under OGD/R conditions compared with cotreatment with DMSO. Collectively, the present results suggested that silencing Huwe1 was accompanied by a compensatory induction of autophagy under OGD/R conditions. Furthermore, the JNK pathway may be a key mediator of the interaction between Huwe1 and autophagy in response to UPS impairment.
自噬和泛素蛋白酶体系统（UPS）是参与脑缺血的两种主要蛋白质降解途径。自噬可以补偿 UPS 损伤引起的细胞功能障碍。HECT、UBA 和 WWE 结构域包含 E3 泛素蛋白连接酶 1（Huwe1）是一种 E3 泛素连接酶，在神经系统的可塑性、再生和疾病中发挥着关键作用。然而，Huwe1 在脑缺血/再灌注（I/R）损伤中的自噬作用尚不清楚。本研究旨在探讨自噬与脑缺血中 UPS 之间的相互作用。本研究在体外建立了大鼠原代皮质神经元的氧葡萄糖剥夺再灌注（OGD/R）模型。慢病毒干扰用于沉默 Huwe1 的表达。本研究还使用了自噬促进剂（雷帕霉素）、自噬抑制剂（wortmannin）和 JNK 通路抑制剂（SP600125）。通过 Western blot 和免疫细胞化学检测细胞自噬相关蛋白，包括 Beclin-1、自噬相关（ATG）7、ATG5、ATG3 和微管相关蛋白 1 轻链 3α，以及凋亡相关蛋白，如 P53、裂解的 caspase-3、Bax 和 Bcl2。用 TUNEL 测定法评估神经元凋亡。结果表明，OGD/R 后 24 小时沉默 Huwe1 增加了自噬相关蛋白的表达水平。JNK 抑制剂处理或 Huwe1 shRNA 共处理显著增加了自噬。雷帕霉素增加了 OGD/R 条件下的细胞凋亡。然而，OGD/R 后 24 小时 Huwe1 shRNA 处理减少了 TUNEL 阳性细胞的数量。与 DMSO 共处理相比，Huwe1 shRNA 和 wortmannin 共处理减轻了 OGD/R 条件下的神经元凋亡。综上所述，本研究结果表明，在 OGD/R 条件下沉默 Huwe1 伴随着自噬的代偿性诱导。此外，JNK 通路可能是 UPS 损伤后 Huwe1 与自噬相互作用的关键介导者。
