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目的通过应用N-甲基-D-天门冬氨酸(NMDA)受体非竞争性拮抗剂MK801探讨兴奋性氨基酸神经递质在乐果诱导的新生大鼠皮层神经元凋亡中发挥的作用。方法在纯化的新生鼠皮层神经元中,加入终浓度为100μmol/L的乐果,并用50和100μmol/L NMDA受体非竞争性拮抗剂MK801对100μmol/L乐果染毒组进行干预。染毒后48小时收获细胞,TUNEL染色检测神经元凋亡情况;HPLC-FLD方法测定细胞内兴奋性氨基酸递质含量,RT-PCR检测NMDA受体NR2B亚基mRNA表达的变化,荧光探针DCFH-DA试剂盒检测细胞内活性氧水平。结果在纯化培养的皮层神经元中,100μmol/L乐果染毒48h后,与对照组相比,Tunel染色强度为对照组1.40倍(P<0.01);兴奋性氨基酸的含量均上升(P<0.01);细胞内ROS水平逐渐增高为对照组的2.47倍(P<0.01)。对100μmol/L乐果染毒组给予50和100μmol/L MK801干预后,高剂量干预组凋亡减少为干预前的79.6%(P<0.01),EAA含量下降(P<0.01);细胞内ROS水平下降为干预前的88.9%和74.8%(P<0.01),但仍远高于对照组细胞内活性氧水平(P<0.01);NR2B mRNA表达上升为干预前的1.59和2.22倍(P<0.01),显著高于对照组水平(P<0.01)。结论兴奋性氨基酸递质和细胞内活性氧共同参与乐果诱导的神经元凋亡。NMDA受体阻断剂MK801不仅能减少活性氧的生成并能降低神经元兴奋性氨基酸含量,从而减少乐果诱导的神经元凋亡。
OBJECTIVE: To investigate the role of excitatory amino acid neurotransmitters in the neuroprotection induced by dimethoate in neonatal rat cortical neurons by using N-methyl-D-aspartate (NMDA) receptor non-competitive antagonist MK801. Methods Purified neonatal rat cortical neurons were treated with 100 μmol / L dimethoate and 100 μmol / L dimethoate with 50 and 100 μmol / L NMDA receptor noncompetitive antagonist MK801. The cells were harvested at 48 hours after exposure. TUNEL staining was used to detect neuronal apoptosis. The content of intracellular excitatory amino acid neurotransmitters was determined by HPLC-FLD. The expression of NMDA receptor NR2B subunit mRNA was detected by RT-PCR. Fluorescence probe DCFH -DA kit to detect intracellular reactive oxygen species level. Results In purified cortical neurons, the Tunel staining intensity was 1.40 times higher than that of the control group (P <0.01) after 48 h exposure to 100 μmol / L dimethoate, while the content of excitatory amino acids increased (P < 0.01). The intracellular ROS level increased 2.47 folds (P <0.01) in control group. After intervention with 50 and 100 μmol / L MK801, the apoptosis of the high dose group was reduced to 79.6% (P <0.01) and the content of EAA was decreased (P <0.01). The intracellular ROS (P <0.01), but still much higher than that in the control group (P <0.01), while the level of NR2B mRNA increased to 1.59 and 2.22 times before intervention (P < 0.01), which was significantly higher than that of the control group (P <0.01). Conclusion Excitatory amino acid transmitter and intracellular reactive oxygen are involved in the apoptosis of neurons induced by dimethoate. NMDA receptor antagonist MK801 can not only reduce the generation of reactive oxygen species (ROS) but also reduce the excitatory amino acid content of neurons, thereby reducing the apoptosis of neurons induced by dimethoate.