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目的探讨锌对海马神经细胞MEK/ERK通路的调控机制。方法将原代培养乳鼠海马神经细胞分为3组。(1)对照组(Control组):不加任何处理因素。(2)四吡啶甲基乙二胺[N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine,TPEN]干预组(TPEN组):2μmol/L TPEN处理海马神经细胞24h,以诱导海马神经细胞缺锌。(3)TPEN+5μmol/L Zn组:同时加入终浓度为2μmol/L TPEN及5μmol/L的Zn SO4。采用Western-Blot法检测pMEK,perk,Total-MEK、Total-ERK蛋白表达;免疫荧光法检测细胞内[Ca~(2+)]_i浓度;分子探针DCFH-DA检测细胞内活性氧(ROS)含量。结果 (1)与对照组比较,TPEN组海马神经细胞上清液中LDH活性明显升高(P<0.05);加入5μmol/L Zn后LDH活性显著降低(P<0.05)。(2)与对照组比较,TPEN组pMEK及pERK蛋白表达均明显下降(P<0.05);5μmol/L Zn可明显抑制TPEN诱导的海马神经细胞pMEK及pERK蛋白表达的降低(P<0.05)。(3)与对照组比较,TPEN组细胞内[Ca~(2+)]_i浓度及ROS水平明显升高(P<0.05);5μmol/L Zn能明显抑制细胞内[Ca~(2+)]_i及ROS水平的升高(P<0.05)。结论缺锌可下调海马神经细胞MEK/ERK信号通路,其作用机制可能与细胞内[Ca~(2+)]_i及ROS的调控有关。
Objective To investigate the regulatory mechanism of zinc on MEK / ERK pathway in hippocampal neurons. Methods Primary cultured neonatal rat hippocampal neurons were divided into 3 groups. (1) Control group: without any treatment factors. (2) The TPEN treatment group was treated with TPEN at a concentration of 2 μmol / L for 24 h, and induced by tetrakis (triphenylmethylene) ethylenediamine (TPN) Hippocampal nerve cells lack of zinc. (3) TPEN + 5μmol / L Zn group: 2μmol / L TPEN and 5μmol / L ZnSO4 were added at the same time. The protein expression of pMEK, perk, Total-MEK and Total-ERK was detected by Western-Blot. The [Ca 2+] i concentration in cells was detected by immunofluorescence. )content. Results (1) Compared with the control group, LDH activity in the hippocampal neuron supernatant of TPEN group was significantly increased (P <0.05); LDH activity was significantly decreased (P <0.05) after addition of 5μmol / L Zn. (2) Compared with the control group, the expression of pMEK and pERK protein in TPEN group was significantly decreased (P <0.05); 5μmol / L Zn significantly inhibited the decrease of pMEK and pERK protein expression in TPEN-induced hippocampal neurons (P <0.05). (3) Compared with the control group, [Ca ~ (2 +)] i concentration and ROS level in TPEN group were significantly increased (P <0.05); 5μmol / L Zn significantly inhibited intracellular [ ] _i and ROS levels (P <0.05). Conclusion Zinc deficiency can down-regulate the MEK / ERK signal pathway in hippocampal neurons, and its mechanism may be related to the regulation of intracellular [Ca 2+] i and ROS.