麦芽酚铝通过GSK-3β调控CRMP2致小鼠海马神经元突起损伤的作用研究  

Effect of aluminum-maltolate on primary hippocampal neuron neurite damage by regulating CRMP2 through GSK-3β in mice

作　　者：张慧芳[1] 韩颖超 蔡小亚 向长鑫 牛侨[1] ZHANG Huifang;HAN Yingchao;CAI Xiaoya;XIANG Changxin;NIU Qiao(Department of Occupational Health,School of Public Health,Shanxi Medical University,Taiyuan,Shanxi 030001,China)

机构地区：[1]山西医科大学公共卫生学院劳动卫生教研室,山西太原030001

出　　处：《环境与职业医学》2021年第11期1207-1213,共7页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金项目(81703202,81872599)。

摘　　要：[背景]铝可对神经突触结构和功能造成不可逆的损伤,其机制可能与糖原合成酶-3β(GSK-3β)/脑衰反应调节蛋白2(CRMP2)调控的神经元突起损伤有关。[目的]探讨麦芽酚铝[Al(mal)_(3)]对小鼠海马原代神经元突起的影响,揭示GSK-3β-CRMP2在其中的作用。[方法]取出生24 h以内的新生ICR小鼠海马提取神经元进行原代培养。细胞培养至第5天,应用激光共聚焦检测神经元纯度。细胞培养至第7天,加入慢病毒载体介导的mNeonGreen基因对神经元进行转染。细胞培养第10天,选择生长状态良好带有mNeonGreen荧光的神经元进行Al(mal)_(3)染毒,实验分组为空白对照组、麦芽酚组(120μmol·L^(−1))以及10、20、40μmol·L^(−1)Al组。后续实验选择20μmol·L^(−1) Al建立神经元突起损伤模型并进行干预,实验分组为空白对照组、二甲基亚砜(DMSO)组、Al组(20μmol·L^(−1))、SB组(GSK-3β抑制剂,1μmol·L^(−1))、SB(1μmol·L^(−1))+Al(20μmol·L^(−1))􀀁组。采用CCK-8法检测神经元细胞活力。在Al(mal)_(3)或SB处理海马原代神经元0、48 h􀀁时,用高内涵成像分析仪对小鼠海马原代神经元突起数及长度进行扫描和分析。采用蛋白印迹法检测小鼠海马原代神经元GSK-3β、CRMP2蛋白表达及磷酸化水平。[结果]免疫荧光结果显示原代神经元纯度大于90%。经Al(mal)_(3)染毒48 h后,与空白对照组相比,10、20和40μmol·L–1Al组细胞存活率下降(P<0.05),而麦芽酚组细胞存活率无变化。经SB处理48h后,DMSO组、SB组细胞存活率与空白对照组之间的差异无统计学意义;SB+Al联合处理组神经元的细胞存活率高于Al组(P<0.05)。空白对照组神经元平均突起数量、平均突起长度的48h/0h值分别是90.13%±11.70%、113.24%±8.34%。Al组神经元平均突起数量、神经元平均突起长度的48h/0h值分别是56.47%±16.36%、62.06%±6.75%,均低于空白组(P<0.05)。SB组神经元平均突起数量的48 h/0 h值(99.0[Background]Aluminum can induce irreversible structural and synaptic functional damage,and the associated mechanism may be related to the neurite damage regulated by glycogen synthase kinase-3β(GSK-3β)/collapsin response mediator protein 2(CRMP2).[Objective]This experiment is conducted to investigate the effect of aluminum-maltolate[Al(mal)_(3)]on primary hippocampal neuron neurites in mice,and reveal the role of GSK-3β-CRMP2 in this process.[Methods]The hippocampus of newborn ICR mice(≤24 h old)was used for primary neuronal cultures.On the 5th day in vitro(DIV5),neuron purity detection were performed by confocal laser scanning microscopy.On DIV7,the neurons were transfected with lentiviral vector-mediated mNeonGreen.On DIV10,the neurons with mNeonGreen fluorescence in good growth state were treated with Al(mal)_(3).The stage I experimental groups were blank control group,maltol group,10μmol·L^(−1) Al group,20μmol·L^(−1) Al group,and 40μmol·L^(−1) Al group.Then 20μmol·L^(−1) Al was used to establish a model of neurite injury and for the intervention.The stage II experimental groups were blank control group,dimethyl sulfoxide(DMSO)group,Al(20μmol·L^(−1))group,SB(GSK-3βinhibitor,1μmol·L^(−1)),and SB(1μmol·L^(−1))+Al(20μmol·L^(−1))group.CCK-8 method was used to detect the viability of neurons.The primary hippocampal neurons of mice were scanned with high content analysis system at 0 h and 48 h after Al or SB treatment,and the density and length of neurites were analyzed.Western blotting was used to detect the expression and phosphorylation levels of CRMP2 and GSK-3β in primary hippocampal neurons of mice.[Results]The immunofluorescence results showed that the purity of primary neurons was more than 90%.Compared with the blank control group in stage I,the cell viability rates of the 10,20,and 40μmol·L^(−1) Al groups were decreased after 48h of Al(mal)_(3) treatment(P<0.05),while the cell viability rate of the maltol group had no significant change.There was no significant di

关 键 词：铝 神经元 突起 糖原合成酶-3β 脑衰反应调节蛋白2 

分 类 号：R114[医药卫生—卫生毒理学]