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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 and regulation of extracellular signal-regulated kinase (ERK) and miR-133b during methamphetamine (MA) induced neurotoxicity. Methods The model of PC12 cell injury was established by MA. MTT assay was used to detect the cell viability and microscopic morphology. The optimal concentration of MA was determined. The level of reactive oxygen species (ROS) in cells was detected by flow cytometry The expression of total ERK1 / 2 and phosphorylated ERK1 / 2 (p-ERK1 / 2) was detected by Western blotting. The expression of miR-133b was detected by real-time PCR. To further analyze the role of ERK / miR133b molecular pathway, U0126 specific block ERK pathway, detect miR-133b expression changes. RESULTS: Different concentration of MA could lead to PC12 cell injury. After treated with 800 μmol / L MA, most of the somatic cells became round, neurites were retarded and neural network disappeared. MTT results showed that cell activity decreased significantly. Further cytotoxicity analysis showed that after MA treatment, intracellular ROS level increased, p-ERK expression increased, miR-133b expression decreased; and ERK pathway inhibitor U0126 (10μmol / L), the expression of miR-133b increased High, cell activity increased, intracellular ROS levels decreased, microscopic cell injury improved. Conclusion MA can upregulate the phosphorylation of ERK and downregulate the expression of miR-133b, which mediates neuronal toxicity.