缺锌致海马神经细胞损伤的表观遗传机制初探

A PRIMARY STUDY ON EPIGENETIC MECHANISM OF THE INJURY INDUCED BY ZINC DEFICIENCY IN HIPPOCAMPAL NEURONS

目的观察锌缺乏致原代培养的大鼠海马神经细胞损伤中DNA甲基化及组蛋白去乙酰化相关酶的变化,对缺锌致海马神经细胞损伤的表观遗传机制进行初探。方法将原代培养的大鼠海马神经细胞随机分为对照(control);缺锌[四吡啶甲基乙二胺,N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine,TPEN];补锌5和50μmol/L(TPEN+5μmol/L Zn SO_4,TPEN+50μmol/L Zn SO_4)4组,除对照组不加任何处理外,其余3组分别在培养液中加入2μmol/L TPEN,补锌组则对应加入5和50μmol/L Zn SO_4,作用24h。MTT法检测细胞存活率;免疫酶学法检测HDAC活性;RT-PCR法检测DNA甲基转移酶(DNMT3a、DNMT3b)及组蛋白去乙酰化酶HDACs(HDAC1、HDAC2、HDAC3)的m RNA表达水平。结果与对照组比较,(1)缺锌组海马神经存活率明显降低(P<0.05);5μmol/L补锌和50μmol/L补锌均可显著抑制TPEN诱导的细胞存活率降低(P<0.05)。(2)缺锌组海马神经细胞内DNMT1 m RNA的表达明显上调,DNMT3a m RNA的表达明显下调(P<0.05),而5μmol/L补锌组或50μmol/L补锌组均能够显著抑制TPEN诱导的DNMT1 m RNA和DNMT3a m RNA表达异常(P<0.05)。与对照组比较,缺锌组海马神经细胞内DNMT3b m RNA的表达无明显变化(P>0.05),与对照组比较,50μmol/L补锌后,DNMT3b m RNA表达明显上调(P<0.05)。(3)缺锌组海马神经细胞内HDAC活性及HDAC2的m RNA表达明显升高(P<0.05),HDAC1及HDAC3 m RNA的表达无明显变化(P>0.05);补锌能显著抑制TPEN诱导的HDAC活性及HDAC2 m RNA表达异常(P<0.05)。结论细胞内缺锌诱导海马神经细胞损伤,造成DNA甲基化和组蛋白去乙酰化修饰的改变。

Objective To explore the effects of zinc deficiency on DNA methylation and histone acetylation in hippocampal neurons and provide scientific basis for the potential epigenetic mechanisms of hippocampal damage induced by zinc deficiency. Methods The cultured primary hippocampal neurons were divided into control group （no treatment factor）, TPEN group （hippocampal neurons exposed to 2μmol/L TPEN for 24h）, TPEN ＋5μmol/L Zn group （hippocampal neurons exposed to 2μmol/L TPEN plus 5 μmol/L ZnSO4 for 24h）, and TPEN＋50μmol/L Zn group （hippocampal neurons exposed to 2 μmol/L TPEN plus 50μmol/L ZnSO4 for 24h）. Cell viability was detected by a MTT. HDAC activity in the nucleus of hippocampal neurons was detected by a colorimetric method, and the expression of DNMTs and HDACs mRNA by RT-PCR. Results （1） the viability in TPEN-treated neurons decreased significantly （P〈0.05）. Zn at 5 μmol/L or 50 μmol/L significantly inhibited the drop of survival rate of hippocampal neurons exposed to by TPEN （P〈0.05）. （2） Treatment with TPEN enhanced DNMT1 mRNA expression and inhibited DNMT3a mRNA expression （P〈0.05）, but no significant effect on the mRNA expression of DNMT3b was observed （P〉0.05）. Zn at 5μmol/L or 50 μmol/L significantly inhibited the abnormal changes of mRNA expression of DNMTI, DNMT3a and DNMT3b induced by TPEN. （3） HDAC activity was increased significantly after treated with TPEN （P〈0.05）. Zn at 5 μmol/L or 50μmol/L significantly inhibited the activation of HDAC stimulated by TPEN, The mRNA expression of HDAC2 increased significantly in TPEN-treated neurons （P〈0.05）, whereas no significant changes were found in the mRNA expression of HDACl and HDAC3 （P〉0.05）. The addition of 5μmol/L Zn or 50 μmol/L Zn completely reversed the increase ofHDAC2 mRNA induced by TPEN （P〈0.05）. Conclusion Intracellular zinc dificiency may cause damage to hippocampal neurons, which is related to the changes of DNA methylation and histone acetylation.