摘要:
Objective To investigate the expression of miR-133b in the brain of methamphetamine(MA) dependent rats and its regulatory effects on neuronal toxic injury.MethodsThrough continuous intraperitoneal injection to rats with MA(10 mg/kg),the conditioned place preference(CPP) rats model was established,and the expression of miR-133b in the cerebral cortex of model rats was detected by real-time fluorescence quantitative PCR(RT-PCR).PC12 cells were cultured in vitro and treated with MA(800 μmol/L),and then miR-133b expression in cultured neurons was detected.miR-133b mimics and inhibitor were transfected to PC12 cells respectively to observe the effect of miR-133b on the mitochondrial membrane potential(MMP) of cultured neurons.ResultsAfter continuous intraperitoneal injection with MA for 14 days,the residence time of rats in the box with medicine((620.20±44.80)s) was significantly longer compared with the control group((341.80±25.12)s,P<0.01),which showed that MA dependent rats model was successfully established.The RT-PCR detection results showed that the expression of miR-133b in the cerebral cortex of model rats(0.36±0.05) significantly decreased compared with the control group(0.99±0.08,P<0.01).In the in vitro model,most of the neuronal cell bodies became round and the neuorites were withdrawn after MA treatment.Compared with the control group(1.00±0.02),the RT-PCR detection results showed that the expression of miR-133b in MA group(0.74±0.05) decreased(P<0.05).The JC-1 detection results showed that the MMP of the MA group(109.85±7.03) decreased significantly contrast to the control group(36.49±3.89,P<0.01),the MMP of the miR-133b mimics group(58.97±6.56) increased significantly contrast to the mimics control group(135.46±15.04,P<0.01) and the MMP of the miR-133b inhibitor group(162.84±14.15) decreased contrast to the inhibitor control group(139.81±12.26,P<0.05).ConclusionsThe expression of miR-133b in the cerebral cortex of MA dependent rats and in vitro neuron model treated with MA are significantly downregulated.By regulating the expression of miR-133b,the MMP damage of cultured neurons treated with MA is changed,indicating that miR-133b is not only involved in the nerve injury induced by MA,but also possiblely as a molecular target for intervention.%目的探讨miR-133b在甲基苯丙胺(methamphetamine,MA)依赖大鼠脑内表达情况及其对神经元毒性损伤的调控作用.方法通过腹腔内连续注射MA(10 mg/kg),建立条件性位置偏爱(conditioned place preference,CPP)大鼠模型,应用实时荧光定量PCR(Real-time PCR ,RT-PCR)技术检测模型鼠脑皮层miR-133b的表达变化.体外培养PC12细胞并给予800 μmol/L MA处理,RT-PCR检测miR-133b在培养神经细胞中的表达,并分别转染miR-133b模拟物和抑制物,观察miR-133b干扰后MA对培养神经细胞线粒体膜电位(mitochondrial membrane potential,MMP)的影响.结果连续14 d腹腔内注射MA后,大鼠在伴药箱停留时间[(620.20±44.80)s]明显长于对照组[(341.80±25.12)s,P<0.01],表明成功建立了MA依赖鼠模型.RT-PCR检测发现MA模型鼠脑皮层miR-133b表达(0.36±0.05)较对照组(0.99±0.08)明显降低(P<0.01).在体外神经元模型中,PC12细胞经MA处理后,可见大部分神经细胞胞体变圆,神经突起退缩,RT-PCR结果显示miR-133b表达(0.74±0.05)较对照组(1.00±0.02)降低(P<0.05).JC-1检测显示MA组 [(109.85±7.03)个] MMP较对照组[(36.49±3.89)个]明显下降(P<0.01),miR-133b模拟物组[(58.97±6.56)个]MMP较模拟物对照组[(135.46±15.04)个]明显增加(P<0.01),miR-133b抑制物组[(162.84±14.15)个]MMP较抑制物对照组[(139.81±12.26)个]下降(P<0.05).结论miR-133b在MA依赖大鼠脑皮层组织和MA损伤体外神经元模型中表达均明显下调,通过调控miR-133b的表达,能够改变MA处理培养神经细胞的MMP损伤,表明miR-133b不仅参与了MA导致的神经损伤,还有望成为一个干预的分子靶点.