基于对脑型肌酸激酶的抑制作用研究食源铝致神经元能量代谢障碍的作用机制

Aluminum (Al) and its compounds are an important type of chemical contaminants in food. It has been well known as a neurotoxicant. Al could interfere with the energy supply and lead to apoptosis in neuron. Nevertheless, the mechanisms underlying this toxicological process, especially Al-induced upstream mechanism, are still largely elusive. In previous research, A novel post-electrophoresis staining procedure relied on the reaction of Al3+ with 8-hydroxyquinoline has been performed to achieve visualization of the high Al-affinity protein. Brain type creatine kinase (BCK), which plays a vital role in providing energy supply cushion in neuron, has been identified to be the protein with high Al3+ affinity. Hence, this project attempts to study of Al3+-induced the inhibition kinetics of BCK and the underlying repression mechanism in vitro by using methods such as fluorescent spectroscopy, molecular docking simulation and so on. Then, the affinity interactions of BCK with Al3+ and the time-effect relationship are analyzed in primary neuronal cells by immunochemical methods. Then, the mitochondrial function and expression of apoptosis-regulated proteins have been tested to investigate how Al3+ induce neurotoxicity. Based on this study, the expected results would provide theoretic evidence for food safety risk assessment of Al exposure.

铝（Al）及其化合物是食品中一类重要的化学污染物，铝暴露会显著干扰神经细胞的能量供应，诱导神经细胞凋亡，继而引发阿尔茨海默症等困扰人类健康的疾病。然而，Al诱导细胞凋亡的上游机制却是未知的。申请人前期实验已证实：Al3+与细胞能量供应体系中起能量缓冲作用的脑型肌酸激酶（BCK）发生了特异性亲和并抑制了BCK酶活。本项目首先以BCK为研究对象，研究Al3+对BCK酶活的抑制动力学；综合运用光谱分析及分子模拟技术，揭示Al3+对BCK蛋白构象的修饰特征，阐明二者确切的结合过程和酶活抑制机理。然后以神经元细胞为研究对象，运用细胞生物学及免疫学等技术，研究外源Al3+与BCK的胞内亲和作用及抑制BCK酶活的时效关系；并进一步探究亲和作用发生后对线粒体功能损伤及激活凋亡途径信号的影响，阐明由外源Al3+引发神经细胞能量代谢障碍的分子机制，为科学分析食源铝的危害及评估其食品安全风险提供坚实的理论基础。

铝（Al）及其化合物是食品中一类重要的化学污染物，铝暴露会显著干扰神经细胞的能量供应，诱导神经细胞凋亡，继而引发阿尔茨海默症等困扰人类健康的疾病。然而，Al诱导细胞凋亡的上游机制却是未知的。申请人前期实验已证实：Al3+与细胞能量供应体系中起能量缓冲作用的脑型肌酸激酶（BCK）发生了特异性亲和并抑制了BCK酶活。本项目首先以BCK为研究对象，研究了AlCl3和Al(mal)3对BCK催化活性的影响，结果表明AlCl3对BCK催化活性展现出较强的抑制作用，其半数抑制浓度为0.67mM，抑制类型为混合型抑制。而Al(mal)3的半数抑制浓度为3.81mM，当底物为PCr时抑制类型为双曲线非竞争性抑制、当底物为ADP时抑制类型为反竞争性抑制。两种形态的铝均会导致BCK疏水性增强，Al(mal)3对BCK内源性荧光的影响较AlCl3更为强烈。最终利用分子模拟对接研究了AlCl3和Al(mal)3与BCK的结合位点及相互作用的氨基酸残基，结果显示AlCl3位于BCK活性位点附近。在C6细胞中，3.5mM AlCl3处理后CK-BB酶活性降低了23.92%。AlCl3处理导致细胞局部ATP供应不足，从而影响F-肌动蛋白的形成和细胞形态。Al(mal)3处理后，线粒体膜电位荧光强度提高141.37%，说明细胞能量供应受损，激活了CytC/Caspase9/Caspase3凋亡通路。然后，我们发现H2O2和Al(mal)3对C6细胞有明显的联合毒性作用，表现为抗氧化酶活性降低和胞内Al增加。Al(mal)3可抑制AMPK正常的磷酸化过程，改变脑肌酸激酶的疏水性，导致BCK不能与SERCA共定位。Al(mal)3和H2O2对BCK的联合作用导致Ca2+超载引起线粒体损伤，导致能量供应障碍，最终表现为细胞凋亡。本研究的结果为科学分析食源铝的危害及评估其食品安全风险提供了理论基础。