Evidence of phosphorylation of Akt and neuronal survival after transient focal cerebral ischemia in mice
Evidence of phosphorylation of Akt and neuronal survival after transient focal cerebral ischemia in mice
复制标题
DOI：
10.1097/00004647-200112000-00009
复制
发表时间：
2001-12-01
影响因子：
6.3
通讯作者：
Chan, PH
中科院分区：
文献类型：
--
作者：
Noshita, N;Lewén, A;Chan, PH
丝氨酸-苏氨酸激酶 Akt 在多种细胞系统中通过丝氨酸 473 磷酸化来防止细胞凋亡。磷酸化后，激活的 Akt 会使其他凋亡因子失活，例如 Bad 或 caspase-9，从而抑制细胞死亡。本研究检测了小鼠短暂局灶性脑缺血后，Akt 473 丝氨酸的磷酸化和 DNA 片段化，该小鼠通过腔内阻断大脑中动脉而遭受 60 分钟的局灶性脑缺血。通过免疫组织化学和蛋白质印迹分析对磷酸化 Akt 进行分析。 DNA 片段化通过末端脱氧核苷酸转移酶介导的尿苷 5'-三磷酸-生物素缺口末端标记 (TUNEL) 进行评估。免疫组化结果显示，再灌注4小时大脑中动脉区皮层磷酸化Akt表达较对照组明显升高，而24小时则下降。 Western blot 分析显示，局灶性脑缺血 4 小时后，皮质中的磷酸化 Akt 显着增加，而缺血核心区的磷酸化 Akt 减少。磷酸 Akt 和 TUNEL 双重染色显示磷酸 Akt 和 TUNEL 阳性染色的不同细胞分布。 LY294002（一种磷脂酰肌醇 3-激酶抑制剂）可在局灶性脑缺血后阻止 Akt 的磷酸化，从而促进随后的 DNA 断裂。这些结果表明，Akt 的磷酸化可能参与决定短暂性局灶性脑缺血后细胞的存活或死亡。
The serine-threonine kinase, Akt, prevents apoptosis by phosphorylation at serine-473 in several cell systems. After phosphorylation, activated Akt inactivates other apoptogenic factors, such as Bad or caspase-9, thereby inhibiting cell death. The present study examined phosphorylation of Akt at serine-473 and DNA fragmentation after transient focal cerebral ischemia in mice subjected to 60 minutes of focal cerebral ischemia by intraluminal blockade of the middle cerebral artery. Phospho-Akt was analyzed by immunohistochemistry and Western blot analysis. The DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL). Immunohistochemistry showed the expression of phospho-Akt was markedly increased in the middle cerebral artery territory cortex at 4 hours of reperfusion compared with the control, Whereas it was decreased by 24 hours. Western blot analysis showed a significant increase of phospho-Akt 4 hours after focal cerebral ischemia in the cortex, whereas phospho-Akt was decreased in the ischemic core. Double staining With phospho-Akt and TUNEL showed different cellular distributions of phospho-Akt and TUNEL-positive staining. Phosphorylation of Akt was prevented after focal cerebral ischemia by LY294002, a phosphatidylinositol 3-kinase inhibitor, which facilitated subsequent DNA fragmentation. These results suggest that phosphorylation of Akt may be involved in determining cell survival or cell death after transient focal cerebral ischemia.