Department of Neurology, Guizhou Provincial People's Hospital, Guiyang 550002, China.
Burrell College of Osteopathic Medicine, Las Cruces, NM 88003, USA.
Oxid Med Cell Longev. 2020 Dec 22;2020:8864323. doi: 10.1155/2020/8864323. eCollection 2020.
The focus of the present study is to evaluate the effects of Angiotensin II (Ang II) on neuronal apoptosis after HIE and the potential underlying mechanisms.
Primary neonatal rat cortical neurons were used to study the oxygen-glucose deprivation (OGD) cell model. The expressions of Ang II, AT1R, GSK-3, p-GSK-3, mTOR, p-mTOR, Bax, Bcl-2, and cleaved caspase-3 were detected via western blot. IF and flow cytometry were used to evaluate neuronal apoptosis. Hypoxic-ischemic encephalopathy (HIE) was established to evaluate the therapeutic effects of Ang II in vivo. Cerebral infarction areas were detected by 2,3,5-Triphenyltetrazolium chloride staining. The righting and geotaxis reflexes were also recorded. In addition, Fluoro-Jade C staining and TUNEL staining were performed to evaluate neuronal degeneration and apoptosis.
Ang II significantly increased the rate of neuronal apoptosis, upregulated the expression of cleaved caspase-3, and downregulated Bcl-2/Bax ratio after OGD insult. For vivo assay, the expressions of endogenous Ang II and AT1R gradually increased and peaked at 24 h after HIE. Ang II increased NeuN-positive AT1R cell expression. In addition, Ang II increased the area of cerebral infarction, promoted neuronal degeneration and apoptosis, aggravated neurological deficits on righting and geotaxis reflexes, and was accompanied by increased expressions of phosphorylated GSK-3 and mTOR. The application of valsartan (Ang II inhibitor) or SB216763 (GSK-3 inhibitor) reversed these phenomena triggered by Ang II following HIE.
Ang II increased neuronal apoptosis through the AT1R/GSK-3/mTOR signaling pathway after experimental HIE both in vitro and in vivo, and Ang II may serve as a novel therapeutic target to ameliorate brain injury after HIE.
本研究旨在探讨血管紧张素 II（Ang II）在缺氧缺血性脑病（HIE）后神经元凋亡中的作用及其潜在机制。
采用原代培养的新生大鼠皮质神经元氧葡萄糖剥夺（OGD）细胞模型，通过 Western blot 检测 Ang II、AT1R、GSK-3、p-GSK-3、mTOR、p-mTOR、Bax、Bcl-2 和 cleaved caspase-3 的表达；免疫荧光和流式细胞术检测神经元凋亡；建立 HIE 动物模型，评估 Ang II 的体内治疗效果。通过 2,3,5-三苯基四氮唑氯化物（TTC）染色检测脑梗死面积；翻正反射和趋地反射实验记录大鼠的翻正和趋地反射；Fluoro-Jade C 和 TUNEL 染色评估神经元变性和凋亡。
OGD 损伤后，Ang II 显著增加神经元凋亡率，上调 cleaved caspase-3 的表达，下调 Bcl-2/Bax 比值。在体内实验中，HIE 后内源性 Ang II 和 AT1R 的表达逐渐增加，24 h 时达到峰值。Ang II 增加了 NeuN 阳性的 AT1R 细胞表达。此外，Ang II 增加了脑梗死面积，促进了神经元变性和凋亡，加重了翻正和趋地反射的神经功能缺损，同时伴随着磷酸化 GSK-3 和 mTOR 的表达增加。应用缬沙坦（Ang II 抑制剂）或 SB216763（GSK-3 抑制剂）可逆转 Ang II 对 HIE 后上述作用。
Ang II 通过 AT1R/GSK-3/mTOR 信号通路增加实验性 HIE 后神经元凋亡，Ang II 可能成为改善 HIE 后脑损伤的新治疗靶点。
