Qi Zhifeng, Dong Wen, Shi Wenjuan, Wang Rongliang, Zhang Chencheng, Zhao Yongmei, Ji Xunming, Liu Ke Jian, Luo Yumin
Cerebrovascular Diseases Research Institute, Xuanwu Hospital of Capital Medical University, 45 Changchun Street, Beijing, 100053, China.
Transl Stroke Res. 2015 Jun;6(3):198-206. doi: 10.1007/s12975-015-0393-y. Epub 2015 Mar 7.
Autophagy, an important intracellular degradation pathway, has been reported to clear impaired mitochondria and reduce mitochondria-mediated injury in ischemic disease. Our study and other recent investigations have shown that AKT-dependent autophagy contributes to the neuroprotection afforded by limb remote ischemic conditioning (RIC) in experimental stroke. However, how AKT triggers RIC-based autophagy and whether RIC-afforded autophagy is beneficial for mitochondrial function after cerebral ischemia remains unclear. The disruption of the Bcl-2/Beclin1 complex has been reported to trigger autophagy formation in the condition of Bcl-2 phosphorylation at Ser70. We investigated whether Bcl-2 phosphorylation triggers RIC-based autophagy and thereby confers RIC-induced neuroprotection against mitochondrial injury, using a transient cerebral ischemic rat model. We demonstrated that rats undergoing RIC treatment 30 min after the onset of ischemia (I-30) and at reperfusion (R-0) significantly upregulated Bcl-2 phosphorylation. Immunoprecipitation revealed that RIC increased dissociation of the Bcl-2/Beclin1 complex, leading to a higher level of autophagy than in ischemia/reperfusion rats. Furthermore, AKT activation was shown to play a critical role in regulating Bcl-2-mediated autophagy, as an AKT inhibitor (LY294002, AKTi) administered 30 min prior to ischemia significantly suppressed Bcl-2 phosphorylation and Bcl-2/Beclin1 complex dissociation, thereby reducing autophagy in RIC rats. Blocking Bcl-2 phosphorylation-dependent autophagy with AKTi suppressed RIC-afforded protection on mitochondrial potential and mitochondrial-dependent cell death effector pathway. These findings indicate that Bcl-2 phosphorylation and thereby Bcl-2/Beclin1 complex disruption play a crucial role in triggering autophagy and reducing mitochondrial damage in RIC rats after cerebral ischemia and require the involvement of the AKT activation.
自噬是一种重要的细胞内降解途径，据报道它可清除受损的线粒体，并减轻缺血性疾病中线粒体介导的损伤。我们的研究以及其他近期调查表明，AKT依赖的自噬有助于肢体远程缺血预处理（RIC）在实验性中风中提供神经保护作用。然而，AKT如何触发基于RIC的自噬，以及RIC诱导的自噬在脑缺血后对线粒体功能是否有益仍不清楚。据报道，在Bcl-2的Ser70位点发生磷酸化的情况下，Bcl-2/Beclin1复合物的破坏会触发自噬形成。我们使用短暂性脑缺血大鼠模型，研究了Bcl-2磷酸化是否触发基于RIC的自噬，从而赋予RIC诱导的针对线粒体损伤的神经保护作用。我们证明，在缺血发作后30分钟（I-30）和再灌注时（R-0）接受RIC治疗的大鼠，其Bcl-2磷酸化显著上调。免疫沉淀显示，RIC增加了Bcl-2/Beclin1复合物的解离，导致自噬水平高于缺血/再灌注大鼠。此外，AKT激活在调节Bcl-2介导的自噬中起关键作用，因为在缺血前30分钟给予AKT抑制剂（LY294002，AKTi）可显著抑制Bcl-2磷酸化和Bcl-2/Beclin1复合物的解离，从而减少RIC大鼠的自噬。用AKTi阻断Bcl-2磷酸化依赖性自噬可抑制RIC对线粒体电位和线粒体依赖性细胞死亡效应途径的保护作用。这些发现表明，Bcl-2磷酸化以及由此导致的Bcl-2/Beclin1复合物破坏在触发自噬和减少脑缺血后RIC大鼠的线粒体损伤中起关键作用，并且需要AKT激活的参与。
