Lin Yinyao, Zhan Zexin, Hu Mengyan, Li Haiyan, Zhang Bingjun, Wu Ruizhen, Tan Sha, Shan Yilong, Lu Zhengqi, Qin Bing
Department of Neurology, Mental and Neurological Disease Research Center, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, People's Republic of China.
Department of Rehabilitation Medicine, Mental and Neurological Disease Research Center, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, People's Republic of China.
J Neurochem. 2023 Jan;164(2):172-192. doi: 10.1111/jnc.15721. Epub 2022 Nov 20.
Acute ischemic stroke (AIS) induces cerebral endothelial cell death resulting in the breakdown of the blood-brain barrier (BBB). Endothelial cell autophagy acts as a protective mechanism against cell death. Autophagy is activated in the very early stages of ischemic stroke and declines after prolonged ischemia. Previous studies have shown that Rubicon can inhibit autophagy. The current study aimed to investigate whether continuous long-term ischemia can inhibit autophagy in endothelial cells after ischemic stroke by regulating the function of Rubicon and its underlying mechanism. Wild-type male C57BL/6J mice were subjected to transient middle cerebral artery occlusion (tMCAO). ROCK1, ROCK2, and NOX2 inhibitors were injected into male mice 1 h before the onset of tMCAO. Disease severity and BBB permeability were evaluated. bEnd.3 cells were cultured in vitro and subjected to oxygen-glucose deprivation (OGD). bEnd.3 cells were pretreated with or without ROCK1, ROCK2, or NOX2 inhibitors overnight and then subjected to OGD. Cell viability and permeability were also evaluated. The expression of Rubicon, ROCK1, and autophagy-related proteins were analyzed. Increased BBB permeability was correlated with Rubicon expression in tMCAO mice and Rubicon was upregulated in endothelial cells subjected to OGD. Autophagy was inhibited in endothelial cells after long-term OGD treatment and knockdown of Rubicon expression restored autophagy and viability in endothelial cells subjected to 6-h OGD. ROCK1 inhibition decreased the interaction between Beclin1 and Rubicon and restored cell viability and autophagy suppressed by 6-h OGD treatment in endothelial cells. Additionally, ROCK1 inhibition suppressed Rubicon, attenuated BBB disruption, and brain injury induced by prolonged ischemia in 6-h tMCAO mice. Prolonged ischemia induced the death of brain endothelial cells and the breakdown of the BBB, thus aggravating brain injury by increasing the interaction of ROCK1 and Rubicon with Beclin1 while inhibiting canonical autophagy. Inhibition of ROCK1 signaling in endothelial cells could be a promising therapeutic strategy to prolong the therapeutic time window in AIS.
急性缺血性卒中（AIS）可导致脑内皮细胞死亡，进而引起血脑屏障（BBB）破坏。内皮细胞自噬作为一种对抗细胞死亡的保护机制。自噬在缺血性卒中的极早期被激活，并在长时间缺血后下降。先前的研究表明，Rubicon可抑制自噬。本研究旨在探讨持续长期缺血是否可通过调节Rubicon的功能及其潜在机制来抑制缺血性卒中后内皮细胞的自噬。对野生型雄性C57BL/6J小鼠进行短暂性大脑中动脉闭塞（tMCAO）。在tMCAO发作前1小时向雄性小鼠注射ROCK1、ROCK2和NOX2抑制剂。评估疾病严重程度和血脑屏障通透性。体外培养bEnd.3细胞并使其经历氧葡萄糖剥夺（OGD）。bEnd.3细胞用或不用ROCK1、ROCK2或NOX2抑制剂预处理过夜，然后进行OGD处理。还评估细胞活力和通透性。分析Rubicon、ROCK1和自噬相关蛋白的表达。血脑屏障通透性增加与tMCAO小鼠中Rubicon表达相关，且在经历OGD的内皮细胞中Rubicon上调。长期OGD处理后内皮细胞中的自噬受到抑制，敲低Rubicon表达可恢复经历6小时OGD的内皮细胞中的自噬和活力。抑制ROCK1可减少Beclin1与Rubicon之间的相互作用，并恢复经历6小时OGD处理的内皮细胞中被抑制的细胞活力和自噬。此外，抑制ROCK1可抑制Rubicon，减轻6小时tMCAO小鼠中长时间缺血诱导的血脑屏障破坏和脑损伤。长时间缺血诱导脑内皮细胞死亡和血脑屏障破坏，从而通过增加ROCK1和Rubicon与Beclin1的相互作用同时抑制经典自噬来加重脑损伤。抑制内皮细胞中的ROCK1信号可能是延长AIS治疗时间窗的一种有前景的治疗策略。
