Protease Omi cleaving Hax-1 protein contributes to OGD/R-induced mitochondrial damage in neuroblastoma N2a cells and cerebral injury in MCAO mice
Protease Omi cleaving Hax-1 protein contributes to OGD/R-induced mitochondrial damage in neuroblastoma N2a cells and cerebral injury in MCAO mice
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蛋白酶 Omi 裂解 Hax-1 蛋白导致 OGD/R 诱导的神经母细胞瘤 N2a 细胞线粒体损伤和 MCAO 小鼠脑损伤
DOI：
10.1038/aps.2015.50
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发表时间：
2015-08
影响因子：
8.2
通讯作者：
Wang Guang-hui
中科院分区：
文献类型：
--
作者：
Wu Jia-yuan;Li Mei;Cao Li-juan;Sun Mei-ling;Chen Dong;Ren Hai-gang;Xia Qin;Tao Zhou-teng;Qin Zheng-hong;Hu Qing-song;Wang Guang-hui
目的：在局灶性脑缺血后的半暗带中，Omi蛋白酶的增加与Hs 1相关蛋白X-1（Hax-1）的减少有关，Hax-1是一种属于Bcl-2家族的蛋白。本研究旨在探讨Omi蛋白酶对Hax-1在脑缺血/再灌注损伤中的调控机制。方法：采用小鼠神经母细胞瘤N2 a细胞进行氧糖剥夺再给氧（OGD/R），MTT法检测细胞活力。小鼠大脑中动脉闭塞（MCAO）2 h后再灌注，TTC染色测定脑梗死体积。采用免疫印迹和免疫荧光法检测Omi和Hax-1的表达。结果：MCAO小鼠脑组织中Omi蛋白表达明显增加，而Hax-1蛋白表达明显减少。在OGD/R处理的N2 a细胞中观察到类似的变化，但Hax-1的mRNA水平没有变化。此外，在OGD/R处理的N2 a细胞中，敲低Omi显著增加Hax-1蛋白水平。免疫荧光检测显示Omi和Hax-1共定位于N2 a细胞线粒体。OGD/R可引起N2 a细胞线粒体损伤和凋亡，UCF-101（10 μmol/L）抑制Omi蛋白酶活性或过表达Hax-1可恢复线粒体膜电位，减轻细胞凋亡。结论：Omi蛋白酶通过切割Hax-1损伤线粒体功能，诱导OGD/R处理的N2 a细胞凋亡，导致MCAO小鼠I/R损伤。
Aim:In the penumbra after focal cerebral ischemia, an increase of protease Omi is linked to a decrease of Hs1-associated protein X-1 (Hax-1), a protein belonging to the Bcl-2 family. In this study we investigated the mechanisms underlying the regulation of Hax-1 by protease Omi in cerebral ischemia/reperfusion (I/R) injury.Methods:Mouse neuroblastoma N2a cells were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R); cell viability was assessed with MTT assay. Mice underwent 2-h middle cerebral artery occlusion (MCAO) and reperfusion, and the infarct volume was determined with TTC staining. The expression of Omi and Hax-1 was detected using immunoblot and immunofluorescence assays. The mitochondrial membrane potential was measured using TMRM staining.Results:In the brains of MCAO mice, the protein level of Omi was significantly increased, while the protein level of Hax-1 was decreased. Similar changes were observed in OGD/R-treated N2a cells, but the mRNA level of Hax-1 was not changed. Furthermore, in OGD/R-treated N2a cells, knockdown of Omi significantly increased Hax-1 protein level. Immunofluorescence assay showed that Omi and Hax-1 were co-localized in mitochondria of N2a cells. OGD/R caused marked mitochondrial damage and apoptosis in N2a cells, while inhibition of Omi protease activity with UCF-101 (10 μmol/L) or overexpression of Hax-1 could restore the mitochondrial membrane potential and attenuate cell apoptosis. Moreover, pretreatment of MCAO mice with UCF-101 (7.15 mg/kg, ip) could restore Hax-1 expression, inhibit caspase activation, and significantly reduce the infarct volume.Conclusion:Protease Omi impairs mitochondrial function by cleaving Hax-1, which induces apoptosis in OGD/R-treated N2a cells and causes I/R injury in MCAO mice.
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DOI：
10.1111/j.1747-4949.2012.00838.x
发表时间：
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期刊：
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10.1002/ar.20910
发表时间：
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