miR-133b在甲基苯丙胺依赖大鼠脑内表达及对神经元毒性损伤的调控作用

The expression of miR-133b in the brain of methamphetamine dependent rats and its regulation on neuronal toxic injury

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（10mg／kg），建立条件性位置偏爱（conditionedplacepreference，CPP）大鼠模型，应用实时荧光定量PCR（Real—timePCR，RT—PCR）技术检测模型鼠脑皮层miR-133b的表达变化。体外培养PCI2细胞并给予800μmol／LMA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（mitochondfialmembranepotential，MMP）的影响。结果连续14d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，PCI2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-个干预的分子靶点。

Objective To investigate the expression of miR-133h in the brain of methamphetamine （MA） dependent rats and its regulatory effects on neuronal toxic injury. Methods Through continuous in- traperitoneal 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 in- hibitor were transfected to PC12 cells respectively to observe the effect of miR-133b on the mitochondrial membrane potential（MMP） of cultured neurons. Results After continuous intraperitoneal injection with MAfor 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group （0.99± 0.08 ,P〈0.01 ）.In the in vitro model, most of the neuronal cell bodies became round and the neuorites were withdrawn after MA treatment.Compared with the control group （ 1.00± 0.02 ） , the RT-PCR detection results showed that the expression of miR-133b in MA group（0.74±0.05） deereased（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 miPt-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 ）. Conclusions The 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.