Grupo de Neurofarmacología, Departamento de Ciencias Médicas, Facultad de Medicina, Universidad de Castilla-La Mancha, Albacete, Spain.
Br J Pharmacol. 2009 Oct;158(4):1121-30. doi: 10.1111/j.1476-5381.2009.00388.x. Epub 2009 Sep 25.
The proteasome inhibitor model of Parkinson's disease (PD) appears to reproduce many of the important behavioural, imaging, pathological and biochemical features of the human disease. However, the mechanisms involved in the lactacystin-induced, mitochondria-mediated apoptotic pathway remain poorly defined.
We have used lactacystin as a specific inhibitor of the 20S proteasome in the dopaminergic neuroblastoma cell line SH-SY5Y. We over-expressed a green fluorescent protein (GFP)-Bax fusion protein in these cells to study localization of Bax. Free radical scavengers were used to assess the role of reactive oxygen species (ROS) in these pathways.
Lactacystin triggered a concentration-dependent increase in cell death mediated by the mitochondrial apoptotic pathway, and induced a change in mitochondrial membrane permeability accompanied by cytochrome c release. The participation of Bax protein was more critical than the formation of the permeability transition pore in mitochondria. GFP-Bax over-expression demonstrated Bax redistribution from the cytosol to mitochondria after the addition of lactacystin. ROS, but not p38 mitogen-activated protein kinase, participated in lactacystin-induced mitochondrial Bax translocation. Lactacystin disrupted the intracellular redox state by increasing ROS production and depleting endogenous antioxidant systems such as glutathione (GSH). Pharmacological depletion of GSH, using L-buthionine sulphoxide, potentiated lactacystin-induced cell death. Lactacystin sensitized neuroblastoma cells to oxidative damage, induced by subtoxic concentrations of 6-hydroxydopamine.
The lactacystin-induced, mitochondrial-mediated apoptotic pathway involved interactions between ROS, GSH and Bax. Lactacystin could constitute a potential factor in the development of sporadic PD.
蛋白酶体抑制剂帕金森病（PD）模型似乎再现了许多人类疾病的重要行为、成像、病理和生化特征。然而，乳酰基亮氨酸所诱导的、与线粒体相关的凋亡途径中涉及的机制仍未得到很好的定义。
我们使用乳酰基亮氨酸作为多巴胺能神经母细胞瘤细胞系 SH-SY5Y 中 20S 蛋白酶体的特异性抑制剂。我们在这些细胞中过表达绿色荧光蛋白（GFP）-Bax 融合蛋白，以研究 Bax 的定位。自由基清除剂用于评估这些途径中活性氧（ROS）的作用。
乳酰基亮氨酸触发了依赖于线粒体凋亡途径的细胞死亡的浓度依赖性增加，并诱导了线粒体膜通透性的变化，伴随着细胞色素 c 的释放。Bax 蛋白的参与比线粒体通透性转换孔的形成更为关键。GFP-Bax 的过表达表明，在加入乳酰基亮氨酸后，Bax 从细胞质重新分布到线粒体。ROS 而不是 p38 丝裂原活化蛋白激酶参与了乳酰基亮氨酸诱导的线粒体 Bax 易位。乳酰基亮氨酸通过增加 ROS 产生和耗尽内源性抗氧化系统（如谷胱甘肽（GSH））来破坏细胞内氧化还原状态。使用 L-丁硫氨酸亚砜对 GSH 进行药理学耗竭，增强了乳酰基亮氨酸诱导的细胞死亡。乳酰基亮氨酸使神经母细胞瘤细胞对 6-羟多巴胺的亚毒性浓度诱导的氧化损伤敏感。
乳酰基亮氨酸诱导的、与线粒体相关的凋亡途径涉及 ROS、GSH 和 Bax 之间的相互作用。乳酰基亮氨酸可能是散发性 PD 发展的潜在因素。
