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  

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

作　　者：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] 

机构地区：[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

出　　处：《解剖学报》2014年第6期755-760,共6页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(81371471);教育部博士点基金资助项目(20120131110039);山东省科技发展计划资助项目(2011GSF11810);山东省自然科学基金资助项目(ZR2012HM026;ZR2010HM051)

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。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 ceils 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 ceils 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.

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 

分 类 号：R741.02[医药卫生—神经病学与精神病学]