The endoplasmic reticulum stress inhibitor salubrinal inhibits the activation of autophagy and neuroprotection induced by brain ischemic preconditioning
The endoplasmic reticulum stress inhibitor salubrinal inhibits the activation of autophagy and neuroprotection induced by brain ischemic preconditioning
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内质网应激抑制剂salubrinal抑制脑缺血预处理诱导的自噬激活和神经保护
DOI：
10.1038/aps.2013.34
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发表时间：
2013-05-01
影响因子：
8.2
通讯作者：
Sheng, Rui
中科院分区：
文献类型：
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作者：
Gao, Bo;Zhang, Xiang-yang;Sheng, Rui
目的：探讨内质网（ER）应激是否参与了缺血预处理（IPC）诱导的神经保护和自噬激活的脑保护作用。方法：采用SD大鼠右侧大脑中动脉阻断10 min诱导局灶性脑IPC，24 h后永久阻断，诱导永久性局灶性缺血（PFI）。IPC开始前10 min脑室注射ER应激抑制剂salubrinal（SAL）。在缺血损伤后检查脑体积和运动行为缺陷。免疫印迹法检测LC 3、p62、HSP 70、葡萄糖调节蛋白78（GRP 78）、p-eIF 2 α和caspase-12在同侧皮质的蛋白水平。结果：SAL（150 pmol）预处理可明显抑制IPC的神经保护作用，PFI后死亡率、梗死体积和运动功能障碍明显增加。在分子水平上，SAL（150 pmol）预处理可显著增加PFI后p-eIF 2 α水平，降低GRP 78水平，表明SAL可有效抑制皮层内质网应激。此外，SAL预处理阻断了IPC诱导的皮层LC 3-II上调和p62下调，从而抑制了自噬的激活。此外，SAL阻断了HSP 70的上调，但显着增加裂解caspase-12的水平，从而促进ER应激依赖的细胞凋亡信号在cortex.Conclusion：ER应激诱导的自噬可能有助于脑缺血precondition.IntroductionThe应用程序的神经保护作用的亚阈值缺血损伤的一个器官可能会激活某些细胞通路，有助于减少随后严重缺血发作所造成的损害量。这种现象被称为缺血预处理（IPC）。研究IPC可以提供深入了解内源性保护机制，可以利用治疗1，2，3，4。内质网（ER）是合成分泌蛋白或膜蛋白的细胞器。已知引起ER应激的四个主要因素：a）葡萄糖/营养缺乏，B）蛋白质糖基化的抑制，c）二硫键形成的破坏和d）钙消耗5。ER应激触发了一条进化上保守的信号通路，涉及RNA调节的蛋白激酶样ER激酶（PERK）和细胞真核起始因子2（eIF 2 α）激酶，使eIF 2的α亚基磷酸化，导致蛋白质合成减弱。具有核酸内切酶活性的PERK易位到细胞核并诱导几种ER分子伴侣如GRP 78的表达，其防止蛋白质-蛋白质聚集并有助于蛋白质重折叠。然而，过度或延长的ER应激可能通过CHOP（C/EBP同源蛋白，生长停滞和DNA损伤诱导基因153，GADD 153）和半胱天冬酶-12的激活导致ER依赖性细胞凋亡6，7。有证据表明，轻度ER应激，其特征在于GRP 78的上调，参与预处理8，9，10，11，但ER应激促进预处理的神经保护作用的途径仍有待阐明。
Aim:To investigate whether endoplasmic reticulum (ER) stress participates in the neuroprotective effects of ischemic preconditioning (IPC)-induced neuroprotection and autophagy activation in rat brains.Methods:The right middle cerebral artery in SD rats was occluded for 10 min to induce focal cerebral IPC, and was occluded permanently 24 h later to induce permanent focal ischemia (PFI). ER stress inhibitor salubrinal (SAL) was injected via intracerebral ventricle infusion 10 min before the onset of IPC. Infarct volume and motor behavior deficits were examined after the ischemic insult. The protein levels of LC3, p62, HSP70, glucose-regulated protein 78 (GRP 78), p-eIF2α and caspase-12 in the ipsilateral cortex were analyzed using immunoblotting. LC3 expression pattern in the sections of ipsilateral cortex was observed with immunofluorescence.Results:Pretreatment with SAL (150 pmol) abolished the neuroprotective effects of IPC, as evidenced by the significant increases in mortality, infarct volume and motor deficits after PFI. At the molecular levels, pretreatment with SAL (150 pmol) significantly increased p-eIF2α level, and decreased GRP78 level after PFI, suggesting that SAL effectively inhibited ER stress in the cortex. Furthermore, the pretreatment with SAL blocked the IPC-induced upregulation of LC3-II and downregulation of p62 in the cortex, thus inhibiting the activation of autophagy. Moreover, SAL blocked the upregulation of HSP70, but significantly increased the cleaved caspase-12 level, thus promoting ER stress-dependent apoptotic signaling in the cortex.Conclusion:ER stress-induced autophagy might contribute to the neuroprotective effect of brain ischemic preconditioning.IntroductionThe application of a sub-threshold ischemic insult to an organ may activate certain cellular pathways that help to reduce the amount of damage caused by subsequent severe ischemic episodes. This phenomenon is known as ischemic preconditioning (IPC). Studying IPC may provide insight into endogenous protective mechanisms that could be exploited therapeutically 1, 2, 3, 4. The endoplasmic reticulum (ER) is an organelle in which secretory or membrane proteins are synthesized. Four main factors are known to cause ER stress: a) glucose/nutrient deficiencies, b) the inhibition of protein glycosylation, c) the disruption of disulfide bond formation and d) calcium depletion 5. ER stress triggers an evolutionarily conserved signaling pathway involving RNA-regulated protein kinase-like ER kinase (PERK) and cell eukaryotic initiation factor 2 (eIF2α) kinase that phosphorylate the α subunit of eIF2, leading to the attenuation of protein synthesis. PERK, which has an endonuclease activity, translocates to the nucleus and induces the expression of several ER chaperones such as GRP78, which prevents protein-protein aggregation and helps to refold the proteins. However, excessive or prolonged ER stress may lead to ER-dependent apoptosis through the activation of CHOP (C/EBP homologous protein, growth arrest and DNA damage inducible gene 153, GADD153) and caspase-12 6, 7. Evidence has shown that mild ER stress, characterized by the upregulation of GRP78, is involved in preconditioning 8, 9, 10, 11, but the pathway through which ER stress promotes the neuroprotective effects of preconditioning remains to be elucidated.