硫酸铜染毒对小鼠海马神经细胞突触可塑性相关蛋白表达的影响

Effects of copper sulfate on expressions of synaptic plasticity-related proteins in mice hippocampal nerve cells

[背景]铜稳态失衡与多种神经退行性疾病密切相关,而铜暴露对学习记忆损害作用的具体机制尚不明确。突触可塑性是学习记忆的细胞生物学基础,突触可塑性相关蛋白可反映突触生长、发育、连接的过程。[目的]探讨硫酸铜染毒对小鼠海马神经元(HT22细胞)突触可塑性相关蛋白表达的影响,为研究铜暴露对学习记忆的作用机制提供科学依据。[方法]以HT22细胞为靶细胞,同时设立CuSO4染毒组(CuSO4浓度分别为10、20、40、80μmol·L^-1)和对照组(予以同等体积完全培养基处理)。采用CCK-8法检测染毒24、36、48 h后HT22细胞增殖活性。染毒48 h后,采用Western blotting法检测突触可塑性相关蛋白突触泡膜素(SYP)、突触后致密蛋白-95(PSD-95)、脑源性神经营养因子(BDNF)及酪氨酸激酶B(TrkB)的蛋白表达水平。[结果]染毒24、36、48 h后,各组间细胞增殖活性的差异均有统计学意义(F=27.99、13.80、29.15,均P <0.01)。染毒48 h后,10、20、40、80μmol·L^-1硫酸铜组HT22细胞增殖活性分别为对照组的89.26%、88.72%、81.50%、67.42%,均P <0.05。染毒48 h后,与对照组相比,20、40、80μmol·L^-1染毒组HT22细胞SYP蛋白相对表达量分别下调15.93%、29.55%、42.69%(均P <0.05);40、80μmol·L^-1染毒组HT22细胞PSD-95蛋白相对表达量分别下调41.24%、47.19%(均P <0.05);20、40、80μmol·L^-1染毒组BDNF蛋白相对表达量分别下调23.17%、24.49%、58.99%(均P <0.05);各组间HT22细胞TrkB蛋白相对表达量差异有统计学意义(P <0.05),与对照组相比,80μmol·L^-1染毒组TrkB蛋白相对表达量下调38.03%(P <0.05)。[结论]硫酸铜染毒可降低海马神经细胞增殖活性,可能与突触可塑性相关蛋白的表达下调有关。

[Background] The imbalance of copper homeostasis is closely related to a variety of neurodegenerative diseases, but the specific mechanism of copper-induced learning and memory impairment is still unclear. Synaptic plasticity is the cell biological basis of learning and memory, and synaptic plasticity proteins reflect the processes of synaptic growth, development, and connectivity.[Objective] This in vitro experiment is designed to investigate the effects of copper sulfate exposure on the expressions of synaptic plasticity-related proteins of hippocampal neurons(HT22 cells), and provide a scientific basis for studying the mechanism of copper-induced learning and memory impairment.[Methods] HT22 cells were divided into one control group(equal volume of complete medium) and four CuSO4 exposure groups(CuSO4 concentrations were 10, 20, 40, and 80 μmol·L^-1, respectively). The proliferation activity of HT22 cells was detected by CCK-8 method after CuSO4 exposure for 24, 36, and 48 h. The expressions of synaptic plasticity related proteins including synaptophysin (SYP), post-synaptic dense protein-95(PSD-95), brain-derived neurotrophic factor(BDNF), and tyrosine kinase B(Trk B) were detected by Western blotting after CuSO4 exposure for 48 h.[Results] After 24, 36, and 48 h of designed treatment, the cell proliferation activities among groups were statistically different(F=27.99, 13.80, and 29.15, all P < 0.01);after 48 h of treatment, the proliferation activities of HT22 cells in the 10, 20, 40, and 80 μmol·L^-1 CuSO4 groups were 89.26%, 88.72%, 81.50%, and 67.42%, respectively, and were lower than that in the control group(all P < 0.05). After 48 h of treatment, compared with the control group, the SYP relative protein expression levels of HT22 cells in the 20, 40, and 80 μmol·L^-1 CuSO4 groups were down-regulated by 15.93%, 29.55%, and 42.69%, respectively(all P < 0.05);the PSD-95 relative protein expression levels in the 40 and 80 μmol·L^-1 CuSO4 groups were down-regulated by 41.24% and 47.19%, respectively(both P < 0.05);the BDNF relative protein expression levels in the 20, 40, and 80 μmol·L^-1 CuSO4 groups were down-regulated by 23.17%, 24.49%, and 58.99%, respectively(all P < 0.05);there was a statistically significant difference in Trk B relative protein expression levels among the groups(P < 0.05), and the Trk B relative protein expression level of HT22 cells in the 80 μmol·L^-1 CuSO4 group was down-regulated by 38.03% compared with the control group(P < 0.05).[Conclusion] The exposure to copper sulfate decreases the proliferation activity of hippocampal nerve cells, which may be related to the down-regulation of synaptic plasticity-related proteins.