Inhibition of autophagy blocks cathepsins-tBid-mitochondrial apoptotic signaling pathway via stabilization of lysosomal membrane in ischemic astrocytes.
Inhibition of autophagy blocks cathepsins-tBid-mitochondrial apoptotic signaling pathway via stabilization of lysosomal membrane in ischemic astrocytes.
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DOI：
10.1038/cddis.2017.34
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发表时间：
2017-02-16
影响因子：
9
通讯作者：
Zhang HL
中科院分区：
文献类型：
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作者：
Zhou XY;Luo Y;Zhu YM;Liu ZH;Kent TA;Rong JG;Li W;Qiao SG;Li M;Ni Y;Ishidoh K;Zhang HL
我们以前的研究和其他人已经证明，自噬在缺血性星形胶质细胞中被激活，并有助于星形胶质细胞死亡。然而，缺血诱导的自噬机制在很大程度上仍然未知。本研究建立了大鼠永久性大脑中动脉闭塞（pMCAO）模型和体外氧糖剥夺（OGD）模型。无论是药物治疗与3-甲基腺嘌呤（3-MA）和渥曼青霉素（WORT）或基因治疗与原代培养的星形胶质细胞和小鼠胚胎成纤维细胞（MEF）的Atg 5基因敲除的Atg 5分别抑制自噬。我们发现，药物或遗传抑制自噬逆转pMCAO或OGD诱导的LC 3-II，活性组织蛋白酶B和L，tBid，活性半胱天冬酶3和细胞色素c（Cyt-c）的增加，并抑制损伤诱导的线粒体Cyt-c在缺血皮质，损伤的星形胶质细胞和MEF细胞的减少。免疫荧光分析显示3-MA或Wort处理逆转了OGD诱导的组织蛋白酶B和L从溶酶体释放到细胞质以及星形胶质细胞中caspase-3的活化。此外，3-MA或麦芽汁处理降低OGD诱导的溶酶体膜通透性增加，并增强OGD诱导的星形胶质细胞溶酶体热休克蛋白70.1B（Hsp70.1B）上调。3-MA或Wort抑制自噬可减少大鼠梗死体积，并保护OGD诱导的星形胶质细胞损伤。非选择性半胱天冬酶抑制剂z-VAD-feptin或特异性半胱天冬酶-3抑制剂Q-DEVD-OPh也可挽救OGD诱导的星形胶质细胞损伤。总之，我们提出的数据表明，抑制自噬阻断组织蛋白酶-TBID-线粒体凋亡信号通路通过稳定溶酶体膜，可能是由于在缺血性星形胶质细胞中上调溶酶体Hsp70.1B。
Our previous study and others have demonstrated that autophagy is activated in ischemic astrocytes and contributes to astrocytic cell death. However, the mechanisms of ischemia-induced autophagy remain largely unknown. In this study, we established a rat's model of permanent middle cerebral artery occlusion (pMCAO) and an in vitro oxygen and glucose deprivation (OGD) model. Autophagy was inhibited by either pharmacological treatment with 3-methyladenine (3-MA) and wortmannin (Wort) or genetic treatment with knockdown of Atg5 in primary cultured astrocytes and knockout of Atg5 in mouse embryonic fibroblast (MEF) cells, respectively. We found that pharmacological or genetic inhibition of autophagy reversed pMCAO or OGD-induced increase in LC3-II, active cathepsin B and L, tBid, active caspase-3 and cytoplastic cytochrome c (Cyt-c), and suppressed the injury-induced reduction in mitochondrial Cyt-c in ischemic cortex, in injured astrocytes and MEF cells. Immunofluorescence analysis showed that 3-MA or Wort treatment reversed OGD-induced release of cathepsin B and L from the lysosome to the cytoplasm and activation of caspase-3 in the astrocytes. Furthermore, treatment of 3-MA or Wort decreased OGD-induced increase in lysosomal membrane permeability and enhanced OGD-induced upregulation of lysosomal heat shock protein 70.1B (Hsp70.1B) in astrocytes. Inhibition of autophagy by 3-MA or Wort reduced infarction volume in rats and protected OGD-induced astrocytic cell injury. A non-selective caspase inhibitor z-VAD-fmk or a specific caspase-3 inhibitor Q-DEVD-OPh also rescued OGD-induced astrocytic cell injury. In conclusion, our presenting data suggest that inhibition of autophagy blocks cathepsins–tBid–mitochondrial apoptotic signaling pathway via stabilization of lysosomal membranes, possibly due to upregulation of the lysosomal Hsp70.1B in ischemic astrocytes.