缺血后处理通过激活大鼠 T 淋巴细胞可激活丝氨酸/苏氨酸激酶/蛋白激酶 B 通路减轻脑缺血再灌注损伤。
Ischemic postconditioning relieves cerebral ischemia and reperfusion injury through activating T-LAK cell-originated protein kinase/protein kinase B pathway in rats.
机构信息
From the Cerebrovascular Diseases Research Institute (H.Z., R.W., Z.T., F.Y., Z.G., X.L., X.J., Y.L.) and Department of Neurology (L.G.), Xuanwu Hospital of Capital Medical University, Beijing, China; Cerebralvascular Diseases Research Laboratory, Beijing Geriatric Medical Research Center, Beijing, China (H.Z., R.W., Z.T., F.Y., Z.G., X.L., X.J., Y.L.); and Key Laboratory of Neurodegenerative Diseases of Ministry of Education and Beijing Key Laboratory of Translational Medicine for Cerebrovascular Diseases, Beijing, China (H.Z., R.W., Z.T., F.Y., Z.G., X.L., X.J., Y.L.).
出版信息
Stroke. 2014 Aug;45(8):2417-24. doi: 10.1161/STROKEAHA.114.006135. Epub 2014 Jul 10.
BACKGROUND AND PURPOSE
Ischemic postconditioning (IPostC) protects against ischemic brain injury. To date, no study has examined the role of T-LAK-cell-originated protein kinase (TOPK) in IPostC-afforded neuroprotection. We explored the molecular mechanism related with TOPK in antioxidant effect of IPostC against ischemia/reperfusion.
METHODS
Focal ischemia was induced in rats by transient middle cerebral artery occlusion. Reactive oxygen species production in the peri-infarct cortex was detected using dihydroethidium. Malondialdehyde, as a marker of lipid peroxidation, and 3-nitrotyrosine, as a marker of protein oxidation, were detected by ELISA. The expression or location of antioxidant proteins and signal molecules TOPK, phosphatase, and tensin homolog, and Akt was analyzed by Western blotting and immunofluorescence.
RESULTS
Our results revealed that IPostC relieved transient middle cerebral artery occlusion-induced oxidative damage by reducing reactive oxygen species, malondialdehyde, and 3-nitrotyrosine accumulation in the peri-infarct cortex and raised levels of antioxidants perioxiredoxin-1, peroxiredoxin-2, and thioredoxin-1. In addition, IPostC increased p-AKT and p-TOPK levels, which colocalized in neural cells. In vitro TOPK knockdown by small interfering RNA decreased the levels of antioxidants peroxiredoxin-1, thioredoxin, and manganese superoxide dismutase activity in PC12 cells. In vivo intracerebroventricular injection of TOPK small interfering RNA reversed IPostC-induced neuroprotection by increasing infarct volume and nitric oxide content and reducing manganese superoxide dismutase activity. Moreover, IPostC-evoked Akt activation was blocked by TOPK small interfering RNA in vivo, but the decreased phosphorylated phosphatase and tensin homolog level in ischemia/reperfusion was not influenced by IPostC or by TOPK small interfering RNA treatment.
CONCLUSIONS
Our results suggest that the antioxidative effects of TOPK/Akt might contribute to the neuroprotection of IPostC treatment against transient middle cerebral artery occlusion.
背景与目的
缺血后处理（IPostC）可保护脑免受缺血性损伤。迄今为止，尚无研究探讨 T-LAK 细胞源性蛋白激酶（TOPK）在 IPostC 提供的神经保护中的作用。我们探讨了 TOPK 在 IPostC 对抗缺血/再灌注的抗氧化作用中的相关分子机制。
方法
通过短暂性大脑中动脉闭塞诱导大鼠局灶性脑缺血。使用二氢乙啶检测梗死周边皮质中的活性氧（ROS）产生。通过 ELISA 检测丙二醛（作为脂质过氧化的标志物）和 3-硝基酪氨酸（作为蛋白质氧化的标志物）。通过 Western 印迹和免疫荧光分析抗氧化蛋白和信号分子 TOPK、磷酸酶和张力蛋白同源物（PTEN）以及 Akt 的表达或定位。
结果
我们的结果表明，IPostC 通过减少梗死周边皮质中 ROS、丙二醛和 3-硝基酪氨酸的积累，减轻了短暂性大脑中动脉闭塞引起的氧化损伤，并提高了过氧化物还原酶-1、过氧化物还原酶-2 和硫氧还蛋白-1 的水平。此外，IPostC 增加了 p-AKT 和 p-TOPK 的水平，这些蛋白在神经细胞中发生共定位。通过小干扰 RNA 敲低 PC12 细胞中的 TOPK，降低了过氧化物还原酶-1、硫氧还蛋白和锰超氧化物歧化酶活性等抗氧化剂的水平。体内脑室注射 TOPK 小干扰 RNA 增加了梗死体积和一氧化氮含量，降低了锰超氧化物歧化酶活性，从而逆转了 IPostC 诱导的神经保护作用。此外，体内 TOPK 小干扰 RNA 阻断了 IPostC 诱导的 Akt 激活，但缺血/再灌注引起的磷酸化 PTEN 水平降低不受 IPostC 或 TOPK 小干扰 RNA 处理的影响。
结论
我们的结果表明，TOPK/Akt 的抗氧化作用可能有助于 IPostC 治疗短暂性大脑中动脉闭塞的神经保护作用。
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