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作者	单位	E-mail
倪雯雯	上海交通大学医学院基础医学院药理学教研室上海 200025	shirleynww@shsmu.edu.cn
谭小芳	上海交通大学医学院基础医学院药理学教研室上海 200025
于丰祥	上海交通大学医学院基础医学院药理学教研室上海 200025
宫　平	上海交通大学医学院基础医学院药理学教研室上海 200025
陈　功	上海交通大学医学院临床医学系14级八年制上海 200025
侯丽娜	上海交通大学医学院基础医学院药理学教研室上海 200025
李　娟	上海交通大学医学院基础医学院药理学教研室上海 200025

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中文摘要:

摘要目的：比较不同摩尔比Cu²⁺-Aβ复合物与Aβ单体诱导神经元H₂O₂释放作用的差异。方法：制备不同摩尔比（0.1-5）的Cu²⁺-Aβ复合物，通过检测硫磺素T (Thioflavin T，ThT) 荧光强度考察Cu²⁺对Aβ纤丝形成的影响。利用原代培养的大鼠海马神经元细胞，分别以不同摩尔比Cu²⁺-Aβ复合物，不同浓度Cu²⁺-Aβ复合物（摩尔比为1），以及Aβ单体和Cu²⁺处理细胞，检测培养上清中的H₂O₂含量；分离线粒体，分别检测不同浓度Cu²⁺-Aβ复合物（摩尔比为1），以及Aβ单体和Cu²⁺处理后H₂O₂的释放；观察不同摩尔比Cu²⁺-Aβ复合物，不同浓度Cu²⁺-Aβ复合物（摩尔比为1），以及Aβ单体和Cu²⁺对神经元细胞活力的影响。结果：（1）ThT 荧光试验结果表明，Cu²⁺ 与Aβ（10 μM）摩尔比为1~5范围内可明显抑制Aβ纤丝形成。（2）Cu²⁺-Aβ复合物（摩尔比为1~5；Aβ浓度为10 μM）以及摩尔比为1的Cu²⁺-Aβ复合物（Aβ浓度分别为5，10 μM）可显著诱导神经元释放H2O2；另外，摩尔比为1时，Cu²⁺-Aβ复合物还可诱导神经元线粒体内H2O2释放；上述作用均强于Aβ单体或Cu²⁺。（3）Cu²⁺-Aβ复合物（摩尔比为1~5）可显著降低神经元细胞活力，该作用强于Aβ单体或Cu²⁺ 。结论：与Aβ单体相比，Cu²⁺-Aβ复合物诱发神经元细胞及其线粒体释放H₂O₂作用更强，并诱发更为明显的神经元毒性。提示Cu2²⁺+与Aβ之间的配位结合可能增强其引发活性氧释放以及神经元毒性反应；Cu²⁺-Aβ复合物引发的活性氧可能主要来自线粒体。

英文摘要:

ABSTRACT Objective: To compare the effects of Cu²⁺-Aβ complex at different molar ratios with Aβ monomers on neuronal release of H₂O₂. Methods: Cu²⁺-Aβ complex at different molar ratios (0.1-5) were prepared. Thioflavin T (ThT)-based fluorometric assay was used to examine the effect of Cu²⁺ on Aβ fibril formation. The primary hippocampal neurons were treated with Cu²⁺-Aβ complex of different ratios, Cu²⁺-Aβ complex of different concentrations (with a molar ratio of 1), Aβ monomers and Cu²⁺, respectively. H₂O₂ content in the culture medium was measured. The mitochondria were separated and treated with different concentrations of Cu²⁺-Aβ complex (with a molar ratio of 1), Aβ monomers and Cu²⁺, respectively. H₂O₂ from mitochondria were also measured. Neuronal viability was detected using Cell Counting Kit-8 (CCK-8) after the neurons were treated with Cu²⁺-Aβ complex of different ratios, Cu²⁺-Aβ complex of different concentrations (with a molar ratio of 1), Aβ monomers and Cu²⁺. Results: (1) ThT-based fluorometric assay demonstrated that Cu²⁺ inhibited Aβ fibril formation when the molar ratio of Cu²⁺to Aβ (10 μM) was within 1~5. (2) Cu²⁺-Aβ complex (the molar ratio was within 1~5; the concentration of Aβ was 10 μM) and Cu²⁺-Aβ complex with molar ratio of 1 (the concentrations of A were 5, 10 μM) significantly induced neuron releasing of H₂O₂; In addition, when the molar ratio was 1, Cu²⁺-Aβ complex also induced neuronal mitochondria releasing of H₂O₂; All the above mentioned effects were stronger than that of Aβ monomer or Cu²⁺. (3) Cu²⁺-Aβ complex (with a molar ratios within 1~5) significantly reduced the viability of neuron cells, which was stronger than that of Aβ monomers or Cu²⁺. Conclusion: Compared with Aβ monomers, Cu²⁺-Aβ complex elicited a more robust H₂O₂ release from neuronal cells and their mitochondria. Moreover, it induced a more overt neuronal cell death. Our observations suggest that Cu²⁺-Aβ interactions may enhance the ability of Aβ to induce reactive oxygen species release and neuronal toxicity; Cu²⁺-Aβ complex-evokeded reactive oxygen species may primarily come from the mitochondria.

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