Department of Occupational Health, Ministry of Education Key Laboratory, School of Public Health, Shanxi Medical University, Taiyuan, China.
Restor Neurol Neurosci. 2013;31(5):543-55. doi: 10.3233/RNN-120304.
There are many in vivo and in vitro studies suggested the involvement of apoptosis in neurodegenerative processes. There is considerable evidence that various complex events may contribute to neural cell death. The present study focuses on the underlined neurodegenerative mechanism and the preventive effect of necrostatin-1 (Nec-1) on neural cell death induced by aluminum (Al).
Al-exposed primary cultures of newborn mice cortical cells were separately treated with 3-methylamphetamine (3-MA), benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone (zVAD-fmk), and Nec-1, the cell viability analysis was used to evaluate cell damage from apoptosis, necroptosis and autophagy. Morphology of neural cells treated with 2 mM Al, and 2 mM Al plus 60 μM Nec-1 were examined by fluorescent microscope, and the cell death rates were quantified by cytometry. For the in vivo experiments, male ICR mice were microinjected with normal saline, 2 mM Al, and 2 mM Al plus Nec-1 at the concentrations of 2 mM, 4 mM and 8 mM into the lateral cerebral ventricles. The Morris water maze task was performed in 20 days after intracerebroventricular injection, Nissl staining was used to demonstrate the loss of Nissl substance and the number of neural cells, and western blot was used to analyze the expressing of cell death and Alzheimer's disease related proteins.
The cell viabilities inhibited by Al could be enhanced by 3-MA, zVAD-fmk and Nec-1, of which Nec-1 improved the cell viability most significantly. Furthermore, the cell viability of neural cells treated with Nec-1 increased concentration-dependently, and the expressions of cell death-related proteins were decreased also in a concentration-dependent manner. The in vivo experiments indicated that administration of Nec-1 on Al-treated mice significantly improved learning and memory retention in the Morris water maze task, decreased the neural cells death and inhibited the expression of Alzheimer's disease related proteins in the mice brain.
The present study provides the first direct evidence of a connection between necroptosis and neurodegeneration, which indicates that necroptosis is involved in neurodegenerative cell death. Furthermore, Nec-1 may be useful for the prevention and treatment of neurodegenerative disorders.
有许多体内和体外研究表明细胞凋亡参与神经退行性过程。有相当多的证据表明，各种复杂的事件可能导致神经细胞死亡。本研究主要关注神经退行性机制和 Necrostatin-1（Nec-1）对铝诱导的神经细胞死亡的预防作用。
分别用 3-甲基苯丙胺（3-MA）、苯甲氧基羰基缬氨酰-丙氨酰-天冬氨酸（O-甲基）-氟甲基酮（zVAD-fmk）和 Nec-1 处理暴露于铝的新生小鼠皮质细胞原代培养物，细胞活力分析用于评估凋亡、坏死性凋亡和自噬引起的细胞损伤。通过荧光显微镜观察用 2 mM Al 和 2 mM Al 加 60 μM Nec-1 处理的神经细胞的形态，并通过细胞计数定量细胞死亡率。在体内实验中，雄性 ICR 小鼠分别向侧脑室注射生理盐水、2 mM Al 和 2 mM Al 加浓度为 2 mM、4 mM 和 8 mM 的 Nec-1。在脑室内注射后 20 天进行 Morris 水迷宫任务，Nissl 染色显示 Nissl 物质和神经细胞数量的丧失，Western blot 用于分析细胞死亡和阿尔茨海默病相关蛋白的表达。
Al 抑制的细胞活力可被 3-MA、zVAD-fmk 和 Nec-1 增强，其中 Nec-1 最显著地提高了细胞活力。此外，神经细胞用 Nec-1 处理的细胞活力呈浓度依赖性增加，细胞死亡相关蛋白的表达也呈浓度依赖性降低。体内实验表明，Nec-1 给药可显著改善 Morris 水迷宫任务中 Al 处理小鼠的学习和记忆保留，减少神经细胞死亡，并抑制小鼠大脑中阿尔茨海默病相关蛋白的表达。
本研究首次提供了坏死性凋亡与神经退行性之间联系的直接证据，表明坏死性凋亡参与神经退行性细胞死亡。此外，Nec-1 可能对预防和治疗神经退行性疾病有用。
