D-AP5对氟砷联合染毒原代培养海马神经元突触相关蛋白表达的影响
Effect of D-AP5 on expression of synapse-associated proteins in hippocampal neurons treated by fluoride and arsenic co-exposure
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摘要:
目的 探讨谷氨酸NMDA受体拮抗剂D-2-氨基-5-磷酰基戊酸(D-AP5) 对氟砷联合染毒后原代海马神经元突触相关蛋白表达的影响。
方法 将体外培养的海马神经元分为4组, 空白对照组、氟砷联合染毒组(0.4 μg/mL氟化钠+0.1 μg/mL亚砷酸钠)、D-AP5组(25 μmol/L D-AP5) 和D-AP5+氟砷联合染毒组(0.4 μg/mL氟化钠+0.1 μg/mL亚砷酸钠+25 μmol/L D-AP5)。各组海马神经元置于37℃、含有体积分数5%CO2培养箱中培养24 h。把培养好的各组海马神经元用CCK8检测活性, 用蛋白免疫印迹法检测海马神经元中NR2B、SYN及GAP43突触相关蛋白的表达水平。
结果 与空白对照组(100.00±0.00)%比较, 氟砷联合染毒组(75.81±6.27)%、D-AP5+氟砷联合染毒组(81.58±5.20)%的细胞存活率均明显降低(P < 0.05);与氟砷联合染毒组(75.81±6.27)%比较, D-AP5组(98.78±3.53)%、D-AP5+氟砷联合染毒组(81.58±5.20)%海马神经元存活率均明显升高(P < 0.05)。氟砷联合染毒组(0.425±0.06)、D-AP5组(0.469±0.03)、D-AP5+氟砷联合染毒组(0.354±0.04) 的NR2B相对表达量较空白对照组(0.572±0.05) 均有所下降(P < 0.05), D-AP5+氟砷联合染毒组(0.354±0.04) 受体蛋白NR2B表达较氟砷联合染毒组(0.425±0.06) 降低(P < 0.05);氟砷联合染毒组与D-AP5+氟砷联合染毒组的SYN和GAP43相对表达量均较空白对照组低(P < 0.05)。而D-AP5组与D-AP5+氟砷联合染毒组的SYN和GAP43表达较氟砷联合染毒组均升高(P < 0.05)。
结论 氟砷联合染毒能够下调突触相关蛋白SYN及GAP-43表达水平, 谷氨酸NMDA受体拮抗剂D-AP5可以抑制氟砷联合染毒对海马神经元突触相关蛋白表达的影响。
Abstract:Objective To test the effect of glutamate NMDA receptor antagonist D-2-amino-5-phosphonopentanoate (D-AP5) on the expression of synapse-associated proteins in primary cultured hippocampal neurons after fluoride and arsenic co-exposure.
Methods In vitro cultured hippocampal neurons were divided into four groups, including control group, fluoride-arsenic coexposure group (0.4 μg/mL sodium fluoride+0.1 μg/mL sodium arsenite), D-AP5 group (25 μmol/L D-AP5), and D-AP5+ fluoridearsenic co-exposure group (0.4 μg/mL sodium fluoride+0.1 μg/mL sodium arsenite+25 μmol/L D-AP5). Hippocampal neurons in each group were cultured in 5%CO2 incubator at 37℃ for 24 h. Cell viability was detected by CCK8, and the expression levels of GAP43, NR2B, and SYN proteins in hippocampal neurons were detected by Western blot.
Results Compared with the control group (100.00±0.00)%, the cell viability rates of hippocampal neurons in the fluoridearsenic co-exposure group (75.81±6.27)% and the D-AP5+ fluoride-arsenic co-exposure group (81.58±5.20)% were significantly decreased (P < 0.05). Compared with the fluoride-arsenic co-exposure group (75.81±6.27)%, the cell viability rates of hippocampal neurons in the D-AP5 group (98.78±3.53)% and the D-AP5+ fluoride-arsenic co-exposure group (81.58±5.20)% were significantly increased (P < 0.05). Compared with the control group (0.572±0.05), the protein expression levels of NR2B in the fluoride-arsenic co-exposure group (0.425±0.06), D-AP5 group (0.469±0.03), and D-AP5+ fluoride-arsenic co-exposure group (0.354±0.04) were significantly decreased (P < 0.05). The protein expression level of NR2B in the D-AP5+ fluoride-arsenic co-exposure group (0.354±0.04) were significantly lower than that of the fluoride-arsenic co-exposure group (0.425±0.06). The protein expression levels of SYN and GAP43 in the fluoride-arsenic co-exposure group and the D-AP5+ fluoride-arsenic co-exposure group were also significantly decreased (P < 0.05). The protein expression levels of SYN and GAP43 in the D-AP5 group and the D-AP5+ fluoride-arsenic co-exposure group were higher than those of the fluoride-arsenic co-exposure group (P < 0.05).
Conclusion Fluoride-arsenic co-exposure could decrease the levels of synapse-associated proteins SYN and GAP-43. Glutamate NMDA receptor antagonist D-AP5 could inhibit the effect of fluoride and arsenic co-exposure on the expression of synapse-related proteins in hippocampal neurons.
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