解剖学报

解剖学报 ›› 2014, Vol. 45 ›› Issue (6): 755-760.doi: 10.3969/j.issn.0529-1356.2014.06.005

• 神经生物学 • 上一篇    下一篇

细胞外信号调节激酶/miR-133b在甲基苯丙胺致PC12细胞神经毒性的作用及机制

刘海莉1 朱德晓1 吴金涛1 岳庆伟1 王辉1 李泽岩1 李贵宝1 刘增训2 张静1 孙晋浩1*   

  1. 1.山东大学医学院人体解剖学与组织学胚胎学研究所，济南 250012; 2.山东省精神卫生中心精神科，济南 250014
  • 收稿日期:2014-06-03 修回日期:2014-07-24 出版日期:2012-12-06 发布日期:2014-12-06
  • 通讯作者: 孙晋浩 E-mail:sunjinhao@gmail.com
  • 基金资助:

    国家自然基金;教育部博士点基金;山东省科技发展计划;山东省科学基金ZR2012HM026;山东省科学基金

Role and mechanism of extracellular signal-regulated kinase/miR-133b pathway in methamphetamine-induced neurotoxicity in PC12 cells

LIU Hai-li1 ZHU De-xiao1 WU Jin-tao1 YUE Qing-wei1 WANG Hui1 LI Ze-yan1 LI Gui-bao1 LIU Zeng-xun2 ZHANG Jing1 SUN Jin-hao 1*   

  1. 1. Department of Anatomy and Embryology, School of Medicine, Shandong University, Ji’nan 250012, China; 2. Health Center of Shandong Province, the Spiritual Science, Ji’nan 250014, China
  • Received:2014-06-03 Revised:2014-07-24 Online:2012-12-06 Published:2014-12-06
  • Contact: SUN Jin-hao E-mail:sunjinhao@gmail.com
  • Supported by:

    National Science Foundation

摘要：

目的 探讨甲基苯丙胺(MA)致神经细胞毒性过程中细胞外信号调节激酶(ERK)和miR-133b的表达变化及调控机制。方法 用MA建立PC12神经细胞损伤模型，采用四甲基偶氮唑盐(MTT)检测细胞活性及镜下形态观察确定MA最佳损伤浓度；应用流式细胞术检测细胞内活性氧（ROS）水平；通过 Western blotting技术测定总ERK1/2和磷酸化ERK1/2 (p-ERK1/2)的表达变化；并应用实时定量聚合酶链反应（Real-time PCR）测定miR-133b的表达变化。为进一步分析ERK/miR133b分子通路的作用关系，经U0126特异阻断ERK通路，检测miR-133b的表达变化。 结果 给予不同浓度的MA，均可导致PC12细胞损伤，其中800μmol/L MA处理后，大部分胞体变圆，神经突起退缩，神经网络消失。MTT结果显示细胞活性明显下降。进一步的细胞毒性机制分析显示，MA处理后，细胞内ROS水平升高，p-ERK表达增高，miR-133b表达降低；并且给予ERK通路抑制剂U0126（10μmol/L）后，miR-133b表达升高，细胞活性增强，胞内ROS水平降低，镜下细胞损伤改善。 结论 MA可通过上调ERK磷酸化抑制miR-133b表达，介导神经元毒性损伤。

关键词: 甲基苯丙胺, miR-133b, 细胞外信号调节激酶, 磷酸化, 神经毒性, PC12细胞, 免疫印迹法, 实时定量聚合酶链反应

Abstract:

Objective To investigate the expression of phosphorylated extracellular signal-regulated kinase (p-ERK) and miR-133b and their regulation mechanism in methamphetamine (MA)-induced neurotoxicity. Methods PC12 cells, used as models for neuronal cellsin vitro, were treated with MA with different concentrations, and the morphological changes were observed using an inverted microscope. MTT assay was used to observe the cell viability and determine the optimal MA concentration for cellular damage. Reactive oxygen species (ROS) level was measured with flow cytometry (FCM) by staining hepatocytes with DCFH-DA. Western blotting was used to detect the changes in the expressions of ERK1/2 and activated p-ERK1/2, and realtime quantitative PCR (Real-time PCR) was performed for miR-133b. To further analyze the ERK/miR-133b molecular pathway, U0126 was added to inhibit ERK phosphorylation to detect the changes of miR-133b. Results PC12 cells were damaged under the MA treatment of all concentrations. However, when exposed to 800μmol/L MA, the morphological changes of the cells were most significant, resulting in rounded cell bodies with dendrite disruptions and disappearance of cell reticular formations. Also, cell viability decreased significantly as shown by MTT, with increased levels of ROS and p-ERK and decreased expression of miR-133b. Applying U0126(10μmol/L), which inhibits ERK phosphorylation, before inducing cell damage with MA resulted in cells with increased expression of miR-133b, decreased level of ROS, increased cell viability, and thus weaker damage compared to the MA-induce-only group.
Conclusion MA can induce cytotoxicity in the PC12 cells by up-regulating ERK phosphorylation and down-regulating miR-133b expression. U0126 can regulate the down-regulation of miR-133b by inhibiting ERK phosphorylation. Therefore, it is possible that the down-regulation of miR-133b in MA-induced neural toxicity is achieved by ERK phosphorylation.

Key words: Methamphetamine, miR-133b, Extracellular signal-regulated kinase, Phosphorylation, Neurotoxicity, PC12 cell, Western blotting, Real-time PCR