锌对体外海马神经细胞MEK/ERK信号通路的调控作用研究

STUDY ON MODULATION OF THE MEK/ERK SIGNALING PATHWAY OF THE HIPPOCAMPAL NEURONS BY ZINC IN VITRO

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 explore the effects of zinc on MEK/ERK signaling pathway in the hippocampal neurons. Methods The primary cultured hippocampal neurons were isolated from fetal mice and divided into control group （no treatment）, N,N,N＇,N＇tetrakis （2-pyridylmethyl） ethylenediamine （TPEN） group （hippocampal neurons were exposed to 2pmol/L TPEN for 24h）, TPEN＋Sgmol/L Zn group （hippocampal neurons were exposed to 2 μmol/L TPEN plus 5μmol/L ZnSO4 for 24h）. pMEK, pERK, Total-MEK, Total-ERK protein levels were examined by Western blot assay, and intracellular [Ca2＋]i, reactive oxygen species （ROS） generation were measured by fluorescence spectrophotometry. Results （1） LDH activity in TPEN-treated neurons was markedly increased compared with that in control group （P〈0.05）, while 5μmol/L Zn supplement was able to reduce LDH activity significantly （P〈0.05）. （2） The expression of pMEK and pERK proteins were significantly inhibited in TPEN-treated neurons compared with those in control group （P〈0.05）, while addition of zinc reversed TPEN-induced reduction of these proteins （P〈0.05）. （3） Compared with the control group, the concentration of [Ca2＋]i and the level of ROS were significantly increased in TPEN-treated neurons （P〈0.05）. Addition of 5μmol/L Zn could significantly inhibit these increases （P〈0.05）. Conclusion Abnormal changes of MEK/ERK signaling pathway may be the important mechanisms for hippocampal neuron damage induced by zinc deficiency, which is associated with the changes of intracellular [Ca2＋]i and ROS.