Zeng Xianwei, Wang Tony, Jiang Liangliang, Ma Guozhao, Tan Shuhui, Li Jialong, Gao Jianxin, Liu Kejing, Zhang Yong
Department of Neurosurgery, Affiliated Hospital of Weifang Medical University, Shandong, China.
Neurol Res. 2013 Jun;35(5):529-36. doi: 10.1179/1743132813Y.0000000202. Epub 2013 Apr 16.
Activation of mitochondrial (MitoKATP) channels was found to protect against anoxic and chemical stress in brain. This present study sought to investigate the ability of diazoxide and cyclosporin A to antagonize cytotoxicity induced by beta-amyloid peptide (A-beta1-42) in cultured rat primary basal forebrain cholinergic neurons.
Cytotoxicity was induced by A-beta1-42 (2 μM) in the presence of either diazoxide (500 μM), a selective opener of the mitochondrial ATP-sensitive potassium channel (MitoKATP), or cyclosporin A (20 μM), an inhibitor of the mitochondrial permeability transition pore (MTP), or the combination of both the reagents. We determined cell morphology and cell viability using MTT assay and expression levels of anti-apoptotic protein (Bcl-2), pro-apoptotic proteins (Bax, cytochrome C, caspase-3 and cleaved caspase-3) using Western blotting at 24 hours and 72 hours.
Cell viability decreased markedly after exposure to A-beta1-42 for 72 hours with a decrease in the expression of Bcl-2 protein and cytochrome C and an increase in the caspase-3 and cleaved caspase-3 levels. Both diazoxide and cyclosporin A exerted significant protective effects on cell viability by ameliorating the decrease in Bcl-2 and the increase in cytochrome c and caspase-3 activity induced by A-beta1-42. The combination of both the reagents had a greater protective effect than either one alone.
The present research demonstrates that activation of MitoKATP channels independently or in combination with inhibitors of the MTP can elicit a protective effect against primary cholinergic neuron cytotoxicity induced by A-beta1-42. These findings suggest new mitochondrial targets for the development of therapeutic agents against A-beta-induced cytotoxicity.
已发现线粒体（MitoKATP）通道的激活可保护大脑免受缺氧和化学应激。本研究旨在探讨二氮嗪和环孢素A拮抗β-淀粉样肽（A-β1-42）诱导的培养大鼠原代基底前脑胆碱能神经元细胞毒性的能力。
在存在二氮嗪（500μM，一种线粒体ATP敏感性钾通道（MitoKATP）的选择性开放剂）、环孢素A（20μM，一种线粒体通透性转换孔（MTP）的抑制剂）或两种试剂组合的情况下，用A-β1-42（2μM）诱导细胞毒性。我们在24小时和72小时时使用MTT法测定细胞形态和细胞活力，并使用蛋白质印迹法测定抗凋亡蛋白（Bcl-2）、促凋亡蛋白（Bax、细胞色素C、半胱天冬酶-3和裂解的半胱天冬酶-3）的表达水平。
暴露于A-β1-42 72小时后，细胞活力显著下降，Bcl-2蛋白和细胞色素C的表达降低，半胱天冬酶-3和裂解的半胱天冬酶-3水平升高。二氮嗪和环孢素A均通过改善A-β1-42诱导的Bcl-2降低和细胞色素c及半胱天冬酶-3活性增加，对细胞活力发挥显著的保护作用。两种试剂的组合比单独使用任何一种具有更大的保护作用。
本研究表明，独立激活MitoKATP通道或与MTP抑制剂联合使用可对A-β1-42诱导的原代胆碱能神经元细胞毒性产生保护作用。这些发现为开发抗A-β诱导的细胞毒性治疗药物提出了新的线粒体靶点。
