The Study On MEK/ERK Signaling Pathway In The Damage Of Hippocampal Neurons Induced By Zinc Deficiency And The Mechanisms Involved

Zinc is essential trace elements with variety of biological roles, includingmaintaining normal biofilm structure and function, maintaining normal cell morphology,growth and development. The relationship between zinc nutriture and cognition isbecoming the hot of nutritional study. Based On our previous work,the changes of MEK/ERK signaling pathway and its epigenetic modification in the damage of hippocampalneurons induced by zinc deficiency were explored in this study.The research was performed from the following three parts:1. To explore the effects of zinc deficiency on the damage and apoptosis ofprimary cultured rat hippocampal neurons. The primary cultured hippocampalneurons were divided into control group(no treatment factor),TPEN group(hippocampalneurons were exposed to2μmol/L TPEN for24h), TPEN+5μmol/L Zn group(hippocampal neurons were exposed to2μmol/L TPEN plus5μmol/L ZnSO4for24h)and TPEN+50μmol/L Zn group(hippocampal neurons were exposed to2μmol/L TPENplus50μmol/L ZnSO4for24h). Cell viability was detected by MTT,cell cytotoxicitywas detected by lactate dehydrogenase (LDH) release and necrosis rate,while apoptosisrate was simultaneously observed by the flow cytometric analysis and Hoechst staining.Bcl-2,Bax and Caspase-3protein levels were examined by Western blot assays. Theresults showed that:(1) the viability in TPEN-incubated neurons decreased significantly(P<0.05).5μmol/L Zn or50μmol/L Zn can significantly inhibit the drop of survival rateof hippocampal neurons induced by TPEN (P<0.05).(2) LDH activity in TPEN-incubated neurons was markedly increased (P<0.05).5μmol/L Zn or50μmol/L Zn wereable to reduce the LDH activity significantly (P<0.05).(3) Necrosis and apoptosis of hippocampal neurons induced by TPEN were significantly increased (P<0.05). Additionof5μmol/L Zn or50μmol/L Zn almost completely prevented the increase of neuronalnecrosis and apoptosis induced by TPEN (P<0.05).(4)The protein expression of Bcl-2was significantly inhibited in TPEN-incubated neurons (P<0.05). Addition of5μmol/LZnor50μmol/LZn can significantly inhibited down-regulation of Bcl-2induced by TPEN(P<0.05).(5) The protein expression of Bax and Caspase-3were markedly increased inTPEN-incubated neurons (P<0.05). Combination5μmol/L Zn or50μmol/L Zn cansignificantly abolished the increase of Bax and Caspase-3protein expression induced byTPEN (P <0.05).2. To explore the effects of zinc deficiency on the MEK/ERK pathway inhippocampal neurons. Groups were divided as above. Raf-1,pMEK,pERK,pCREBand BDNF protein levels were examined by Western blot. NMDA receptors(NR1,NR2Aand NR2B) and BDNF mRNA level by RT-PCR,intracellular [Ca2+]iand reactiveoxygen species (ROS) generation by fluorescence spectrophotometric were detected. Theresults showed that:(1) The expression of Raf-1,pMEK and pERK protein weresignificantly inhibited in TPEN-incubated neurons (P<0.05),while addition of zincreversed TPEN-induced reduction of these proteins (P<0.05).(2) NR1and NR2B mRNAexpression level in TPEN-exposed neurons was significantly enhanced,while theexpression of NR2A mRNA was markedly decreased (P<0.05). Addition of5μmol/L Znor50μmol/L Zn can significantly reverse such changes (P<0.05).(3) Compared with thecontrol group,[Ca2+]iand the level of ROS were significantly increased in TPEN-incubated neurons (P<0.05). Combination of5μmol/L Zn or50μmol/L Zn cansignificantly inhibit the changes of intracellular [Ca2+]i and ROS level induced by TPEN(P<0.05).(4) The expression of pCREB, BDNF protein and BDNF mRNA weresignificantly inhibited in TPEN-incubated neurons (P<0.05). Combination5μmol/L Znor50μmol/L Zn can significantly promote pCREB, BDNF protein and mRNA expression(P<0.05).3. To explore the effects of the agonist and inhibitor of MEK/ERK signalingpathway on neuronal apoptosis and the expression of Bcl-2/Bax and Caspase-3protein. The primary cultured hippocampal neurons were divided into TPENgroup(hippocampal neurons were exposed to2μmol/L TPEN for24h), TPEN+U0126group(hippocampal neurons were pretreatment with10μmol/L U0126for30min,thenexposed to2μmol/L TPEN for24h) and TPEN+NGF group(hippocampal neurons werepretreatment with100ng/ml NGF for30min,then exposed to2μmol/L TPEN for24h). Apoptosis rate was observed by Hochest staining,the expression of Bcl-2,Bax andCaspase-3protein were detect by Western blot. The results showed that:(1) There were1.8-fold increase in apoptosis of neurons in TPEN+U0126group,while there were1.4-fold reduction in apoptosis of neurons in TPEN+NGF group.(2) U0126promoted theprotein expression of Caspase-3induced by TPEN,while NGF significantly inhibited theprotein expression of Caspase-3stimulated by TPEN (P<0.05). Both U0126and NGFhad no significant effect on the protein expression of Bcl-2and Bax (P>0.05).4. To explore the effects of zinc deficiency on the epigenetic modifications inhippocampal neurons. Groups were divided into control group, TPEN group (2μmol/LTPEN), TPEN+5μmol/L Zn group and TPEN+50μmol/L Zn group. Cells were treated asabove. HDAC activity in the hippocampal neurons of the nucleus was detected bycolorimetric, the expression of DNMTs and HDACs mRNA by RT-PCR. The resultsshowed that:(1) HDAC activity was increased significantly after treated with TPEN(P<0.05).5μmol/L Zn or50μmol/L Zn can significantly inhibited activation of HDACstimulated by TPEN.(2) The mRNA expression of HDAC2increased significantly inTPEN-incubated neurons (P<0.05),whereas no significant changes were found in themRNA expression of HDAC1and HDAC3(P>0.05). The addition of5μmol/L Zn or50μmol/L Zn completely reversed the increase of HDAC2,HDAC1mRNA induced byTPEN (P<0.05).(3) Treatment with TPEN enhanced DNMT1mRNA expression andinhibited DNMT3a mRNA expression (P<0.05),but no significant effect on the mRNAexpression of DNMT3b was observed (P>0.05).5. To explore the effects of the MEK/ERK signaling pathway on histonedeacetyl in hippocampal neurons. Groups were divided into TPEN group,TPEN+U0126group and TPEN+NGF group. Cells were treated as above. HDAC activity inthe hippocampal neurons of the nucleus was detected by colorimetric,the expression ofHDAC2mRNA by RT-PCR. The results showed that:(1) U0126significantly promotedactivation of HDAC induced by TPEN,whereas NGF significantly inhibited activation ofHDAC induced by TPEN (P<0.05).(2) U0126promoted TPEN-induced the expressionof HDAC2mRNA, while NGF significantly inhibited the expression of HDAC2mRNAstimulated by TPEN (P<0.05).In summary,abnormal changes of MEK/ERK signaling pathway and epigeneticmodifications may be the important mechanisms for hippocampal neuron damageinduced by zinc deficiency,suitable zinc supplementation can prevent hippocampalneurons from damage induced by zinc deficiency through the regulation of MEK/ERK signaling pathway. The results of this study provided new ideas and scientific basis forclarifying the molecular mechanisms of hippocampal damage induced zinc deficiencyand preventing cognitive dysfunction induced zinc deficiency through zinc intervention.