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

doi:10.3969/j.issn.0529-1356.2014.06.005

Abstract

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

LIU Hai-li ZHU De-xiao WU Jin-tao YUE Qing-wei WANG Hui LI Ze-yan LI Gui-bao LIU Zeng-xun ZHANG Jing SUN Jin-hao*. Role and mechanism of extracellular signal-regulated kinase/miR-133b pathway in methamphetamine-induced neurotoxicity in PC12 cells[J]. AAS, 2014, 45(6): 755-760.

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