Imamura Keiko, Takeshima Takao, Kashiwaya Yoshihiro, Nakaso Kazuhiro, Nakashima Kenji
Department of Neurology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, Yonago, Japan.
J Neurosci Res. 2006 Nov 1;84(6):1376-84. doi: 10.1002/jnr.21021.
It has been postulated that the pathogenesis of Parkinson's disease (PD) is associated with mitochondrial dysfunction. Rotenone, an inhibitor of mitochondrial complex I, provides models of PD both in vivo and in vitro. We investigated the neuroprotective effect of D-beta-hydroxybutyrate (bHB), a ketone body, against rotenone toxicity by using SH-SY5Y dopaminergic neuroblastoma cells. SH-SY5Y cells, differentiated by all-trans-retinoic acid, were exposed to rotenone at concentrations ranging from 0 to 1,000 nM. We evaluated cellular oxidation reduction by the alamarBlue assay, viability by lactate dehydrogenase (LDH) assay, and survival/death ratio by live/dead assays. Exposure to rotenone for 48 hr oxidized cells and decreased their viability and survival rate in a concentration-dependent manner. Pretreatment of cells with 8 mM bHB provided significant protection to SH-SY5Y cells. Whereas rotenone caused the loss of mitochondrial membrane potential, released cytochrome c into the cytosol, and reduced cytochrome c content in mitochondria, addition of bHB blocked this toxic effect. bHB also attenuated the rotenone-induced activation of caspase-9 and caspase-3. Administration of 0-10 mM 3-nitropropionic acid, a complex II inhibitor, also decreased the reducing power of SH-SY5Y cells measured by alamarBlue assay. Pretreatment with 8 mM bHB attenuated the decrease of alamarBlue fluorescence. These data demonstrated that bHB had a neuroprotective effect that supported the mitochondrial respiration system by reversing the inhibition of complex I or II. Ketone bodies, the alternative energy source in the mammalian brain, appear to have therapeutic potential in PD.
据推测，帕金森病（PD）的发病机制与线粒体功能障碍有关。鱼藤酮是线粒体复合物I的抑制剂，可在体内和体外提供PD模型。我们使用SH-SY5Y多巴胺能神经母细胞瘤细胞研究了酮体D-β-羟基丁酸酯（bHB）对鱼藤酮毒性的神经保护作用。用全反式维甲酸分化的SH-SY5Y细胞暴露于浓度范围为0至1000 nM的鱼藤酮中。我们通过alamarBlue测定法评估细胞氧化还原，通过乳酸脱氢酶（LDH）测定法评估细胞活力，并通过活/死测定法评估存活/死亡比率。暴露于鱼藤酮48小时会使细胞氧化，并以浓度依赖性方式降低其活力和存活率。用8 mM bHB预处理细胞可对SH-SY5Y细胞提供显著保护。鱼藤酮导致线粒体膜电位丧失，细胞色素c释放到细胞质中，并降低线粒体中的细胞色素c含量，而添加bHB可阻断这种毒性作用。bHB还减弱了鱼藤酮诱导的caspase-9和caspase-3的激活。给予0-10 mM的复合物II抑制剂3-硝基丙酸也会降低通过alamarBlue测定法测得的SH-SY5Y细胞的还原能力。用8 mM bHB预处理可减弱alamarBlue荧光的降低。这些数据表明，bHB具有神经保护作用，可通过逆转复合物I或II的抑制来支持线粒体呼吸系统。酮体作为哺乳动物大脑中的替代能源，似乎在PD中具有治疗潜力。
