张慧芳, 韩颖超, 蔡小亚, 向长鑫, 牛侨. 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[J]. 环境与职业医学, 2021, 38(11): 1207-1213. DOI: 10.13213/j.cnki.jeom.2021.21085
引用本文: 张慧芳, 韩颖超, 蔡小亚, 向长鑫, 牛侨. 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[J]. 环境与职业医学, 2021, 38(11): 1207-1213. DOI: 10.13213/j.cnki.jeom.2021.21085
ZHANG Huifang, HAN Yingchao, CAI Xiaoya, XIANG Changxin, NIU Qiao. Effect of aluminum-maltolate on primary hippocampal neuron neurite damage by regulating CRMP2 through GSK-3β in mice[J]. Journal of Environmental and Occupational Medicine, 2021, 38(11): 1207-1213. DOI: 10.13213/j.cnki.jeom.2021.21085
Citation: ZHANG Huifang, HAN Yingchao, CAI Xiaoya, XIANG Changxin, NIU Qiao. Effect of aluminum-maltolate on primary hippocampal neuron neurite damage by regulating CRMP2 through GSK-3β in mice[J]. Journal of Environmental and Occupational Medicine, 2021, 38(11): 1207-1213. DOI: 10.13213/j.cnki.jeom.2021.21085

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

  • 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(120 µmol·L−1)以及10、20、40 µmol·L−1Al组。后续实验选择20 µmol·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(20 µmol·L−1)、SB组(GSK-3β抑制剂,1 µmol·L−1)、SB(1 µmol·L−1)+Al(20 µmol·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40 µmol·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(99.03%±21.83%)与空白对照组相比差异无统计学意义,但神经元平均突起长度的48h/0h值(128.72%±15.39%)高于空白对照组(P<0.05)。SB+Al联合处理组神经元平均突起数量和平均突起长度的48h/0h值分别是72.60%±10.89%、93.84%±14.65%,高于Al组(P<0.05)。蛋白印迹结果显示:各组间GSK- 3β蛋白水平差异无统计学意义;与空白对照组(1.00±0.18)相比,Al组神经元p-GSK-3β蛋白水平(0.45±0.05)降低,SB组p-GSK-3β蛋白水平(1.32±0.23)升高;SB+Al联合处理组p-GSK-3β蛋白水平(0.80±0.05)高于Al组(P<0.05)。与对照组(1.00±0.07)相比,Al组CRMP2蛋白水平(0.66±0.11)降低(P<0.05),SB组CRMP2蛋白水平(1.01±0.017)无变化。与对照组(1.00±0.13)相比,Al组p-CRMP2蛋白水平(1.50±2.18)增加,SB组p-CRMP2蛋白水平(0.62±0.09)降低(P<0.05);SB+Al联合处理组p-CRMP2蛋白水平(1.28±0.24)低于Al组(P<0.05)。

    结论

    铝可能通过激活GSK-3β,增加CRMP2蛋白磷酸化水平,损伤神经元突起生长。

     

    Abstract: 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, 10 µmol·L−1 Al group, 20 µmol·L−1 Al group, and 40 µmol·L−1 Al group. Then 20 µmol·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 (20 µmol·L−1) group, SB (GSK-3β inhibitor, 1 µmol·L−1), and SB (1 µmol·L−1)+Al (20 µmol·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 40 µmol·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 difference in cell viability rate among the DMSO group, the SB group, and the control group after 48h of SB treatment, and the viability rate of neurons in the SB+Al group was higher than that in the Al group (P<0.05) in stage II. The 48 h/0 h ratios of average number and length of neurites in the control group were 90.13%±11.70% and 113.24%±8.34%, respectively. The 48 h/0 h ratios in the Al group were 56.47%±16.36% and 62.06%±6.75%, respectively, which were lower than those in the control group (P<0.05). The 48 h/0 h ratios of average number of neurites in the SB group (99.03%±21.83%) was not significantly different from that in the control group, but the 48 h/0 h ratio of average length of neurites in the SB group (128.72%±15.39%) was higher than that in the control group (P<0.05). The 48 h/0 h ratios of average number (72.59%±10.89%) and length of neurites (93.84%±14.65%) in the SB+Al group were significantly increased compared with those in the Al group (P<0.05). Western blotting results showed that: There was no significant difference in GSK-3β protein level among all groups; compared with the control group (1.00±0.18), the protein level of p-GSK-3β in the Al group (0.45±0.05) was significantly decreased, and that in the SB group (1.32±0.23) was significantly increased; the protein level of p-GSK-3β in the SB+Al group (0.80±0.05) was significantly higher than that in the Al group (P<0.05). Compared with the control group (1.00±0.07), the CRMP2 protein level in the Al group (0.66±0.11) was significantly decreased (P<0.05), while that in the SB group (1.01±0.02) was not significantly changed. Compared with the control group (1.00±0.13), the p-CRMP2 protein level in the Al group (1.50±2.18) was significantly increased, and that in the SB group (0.62±0.09) was significantly decreased (P<0.05); the protein level of p-CRMP2 in the SB+Al group (1.28±0.24) was lower than that in the Al group (P<0.05).

    Conclusion

    Aluminum may activate GSK-3β, increase CRMP2 phosphorylation level, and damage neurite growth.

     

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