Abstract：
OBJECTIVE: Chronic cerebral hypoperfusion (CCH) can cause a series of pathophysiological processes, including neuronal autophagy and apoptosis. VEGF-A has been reported to affect angiogenesis and neurogenesis in many CNS diseases. However, its effects on neuronal autophagy and apoptosis, as well as the underlying mechanisms in CCH remain unclear.
METHODS: To address these issues, the CCH model was established by permanent bilateral common carotid artery occlusion (2VO). Rats were sacrificed at different stages of CCH. Hippocampal morphological and ultrastructural changes were detected using HE staining and electron microscopy. The immunoreactivities of microtubule-associated protein 1 light chain 3 (LC3) and phospho-cAMP response element binding protein (p-CREB) were examined by immunofluorescence staining. The neuronal apoptosis was detected via TUNEL staining. The levels of LC3-II, Beclin-1, Akt, p-Akt, CREB, p-CREB, Caspase-3, and Bad were accessed by Western blotting. Furthermore, mouse hippocampal HT22 neurons received the oxygen and glucose deprivation (OGD) treatment, VEGF-A treatment, and GSK690693 (an Akt inhibitor) treatment, respectively.
RESULTS: LC3-II protein started to increase at 3 days of CCH, peaked at 4 weeks of CCH, then decreased. CCH increased the levels of LC3-II, Caspase-3, and Bad, and decreased the levels of p-Akt, CREB, and p-CREB, which were reversed by VEGF-A treatment. VEGF-A also improved CCH-induced neuronal ultrastructural injuries and apoptosis in the hippocampus in vitro. In HT22, the anti-apoptosis and pro-phosphorylation of VEGF-A were reversed by GSK690693.
CONCLUSIONS: Present results provide a novel neuroprotective effect of VEGF-A in CCH that is related to the inhibition of neuronal autophagy and activation of the Akt/CREB signaling, suggesting a potential therapeutic strategy for ischemic brain damage.
METHODS: To address these issues, the CCH model was established by permanent bilateral common carotid artery occlusion (2VO). Rats were sacrificed at different stages of CCH. Hippocampal morphological and ultrastructural changes were detected using HE staining and electron microscopy. The immunoreactivities of microtubule-associated protein 1 light chain 3 (LC3) and phospho-cAMP response element binding protein (p-CREB) were examined by immunofluorescence staining. The neuronal apoptosis was detected via TUNEL staining. The levels of LC3-II, Beclin-1, Akt, p-Akt, CREB, p-CREB, Caspase-3, and Bad were accessed by Western blotting. Furthermore, mouse hippocampal HT22 neurons received the oxygen and glucose deprivation (OGD) treatment, VEGF-A treatment, and GSK690693 (an Akt inhibitor) treatment, respectively.
RESULTS: LC3-II protein started to increase at 3 days of CCH, peaked at 4 weeks of CCH, then decreased. CCH increased the levels of LC3-II, Caspase-3, and Bad, and decreased the levels of p-Akt, CREB, and p-CREB, which were reversed by VEGF-A treatment. VEGF-A also improved CCH-induced neuronal ultrastructural injuries and apoptosis in the hippocampus in vitro. In HT22, the anti-apoptosis and pro-phosphorylation of VEGF-A were reversed by GSK690693.
CONCLUSIONS: Present results provide a novel neuroprotective effect of VEGF-A in CCH that is related to the inhibition of neuronal autophagy and activation of the Akt/CREB signaling, suggesting a potential therapeutic strategy for ischemic brain damage.
摘要：
目的:慢性脑低灌注(CCH)可引起一系列病理生理过程,包括神经元自噬和凋亡。已经报道VEGF-A在许多CNS疾病中影响血管生成和神经发生。然而,其对神经元自噬和凋亡的影响，以及CCH的基本机制仍不清楚。
方法：为了解决这些问题，采用双侧颈总动脉永久性闭塞(2VO)建立CCH模型。在CCH的不同阶段处死大鼠。HE染色和电镜观察海马形态和超微结构变化。通过免疫荧光染色检查微管相关蛋白1轻链3（LC3）和磷酸-cAMP反应元件结合蛋白（p-CREB）的免疫反应性。通过TUNEL染色检测神经元凋亡。LC3-II的水平，Beclin-1Akt,p-Akt,CREB,p-CREB,Caspase-3和Bad通过Western印迹获得。此外，小鼠海马HT22神经元接受氧糖剥夺（OGD）治疗，VEGF-A治疗,和GSK690693（Akt抑制剂）治疗，分别。
结果：LC3-II蛋白在CCH治疗3天开始增加，在CCH的4周达到峰值，然后减少。CCH提高了LC3-II的水平，胱天蛋白酶-3，坏，降低了p-Akt的水平,CREB,和p-CREB,VEGF-A治疗可逆转。VEGF-A还可以改善CCH诱导的海马神经元超微结构损伤和凋亡。在HT22中，GSK690693逆转了VEGF-A的抗凋亡和前磷酸化。
结论：目前的结果提供了VEGF-A在CCH中的新型神经保护作用，该作用与抑制神经元自噬和激活Akt/CREB信号有关。提示缺血性脑损伤的潜在治疗策略。
方法：为了解决这些问题，采用双侧颈总动脉永久性闭塞(2VO)建立CCH模型。在CCH的不同阶段处死大鼠。HE染色和电镜观察海马形态和超微结构变化。通过免疫荧光染色检查微管相关蛋白1轻链3（LC3）和磷酸-cAMP反应元件结合蛋白（p-CREB）的免疫反应性。通过TUNEL染色检测神经元凋亡。LC3-II的水平，Beclin-1Akt,p-Akt,CREB,p-CREB,Caspase-3和Bad通过Western印迹获得。此外，小鼠海马HT22神经元接受氧糖剥夺（OGD）治疗，VEGF-A治疗,和GSK690693（Akt抑制剂）治疗，分别。
结果：LC3-II蛋白在CCH治疗3天开始增加，在CCH的4周达到峰值，然后减少。CCH提高了LC3-II的水平，胱天蛋白酶-3，坏，降低了p-Akt的水平,CREB,和p-CREB,VEGF-A治疗可逆转。VEGF-A还可以改善CCH诱导的海马神经元超微结构损伤和凋亡。在HT22中，GSK690693逆转了VEGF-A的抗凋亡和前磷酸化。
结论：目前的结果提供了VEGF-A在CCH中的新型神经保护作用，该作用与抑制神经元自噬和激活Akt/CREB信号有关。提示缺血性脑损伤的潜在治疗策略。
