【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。更多还原

【Abstract】 Objective To investigate the expression of phosphorylated extracellular signal-regulated kinase( pERK) and miR-133 b 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-133 b. To further analyze the ERK / miR-133 b molecular pathway,U0126 was added to inhibit ERK phosphorylation to detect the changes of miR-133 b. 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 shownby MTT,with increased levels of ROS and p-ERK and decreased expression of miR-133 b. Applying U0126( 10μmol / L),which inhibits ERK phosphorylation,before inducing cell damage with MA resulted in cells with increased expression of miR-133 b,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 downregulating miR-133 b expression. U0126 can regulate the down-regulation of miR-133 b by inhibiting ERK phosphorylation.Therefore,it is possible that the down-regulation of miR-133 b in MA-induced neural toxicity is achieved by ERK phosphorylation.更多还原