有机磷暴露引起的AChE与Aβ时程变化和AD的关联性研究

Organophosphorus compound (OP) is the most widely used pesticide in our country and worldwide. OP exposure in large population could induce many health issues, which is a major public health problem. Epidemiological studies have shown that OP exposure is closely associated with the risk of developing Alzheimer's disease (AD). However, the mechanism underlying its pathological role in AD remains elusive. Thus, it is crucial to elucidate the potential mechanism. In our previous study, we found live mice treated with OP have reduced acetylcholinesterase (AChE) activity, but after a short recovery period, their AChE activity rebounds to levels that exceed pretreatment levels following inhibition. AChE activity and neuronal apoptosis are both induced in mouse brain; cells that were positive in the apoptosis TUNEL assay, stained heavily for AChE activity. In cultured SH-SY5Y cells treated with OP, AChE activity and apoptosis are both induced, and AChE inhibitor could attenuate OP-induced apoptosis. Mice exposed to low dose OP for a long time showed elevated β-amyloid protein (Aβ) level and Aβ plaque in the brain. Taken the important role of AChE in apoptosis and the interaction with Aβ, in this proposal, we will consolidate the role of OP in the development of pathology of AD in APP/PS1 mice model. To further elucidate the mechanism underlying OP induced AD pathology, we will perform experiment to determine the signaling pathway leading to the increased AChE activity in different cell models. We will also investigate the spatial-temporal relationship between Aβ production and AChE activity enhanced by OP and their effect on apoptosis. Collectively, our proposed research will broadly impact the field of neurotoxicity of OP by introducing a new paradigm on the role in the pathogenesis of AD and provide some hints for therapeutic interest in the treatment of this disease.

急慢性有机磷（OP）暴露的神经系统危害是重大公共卫生问题，但致病机制还不清楚。我们前期研究发现：OP暴露引起小鼠大脑和体外培养神经细胞的乙酰胆碱酯酶（AChE）活性异常升高并诱导细胞凋亡，AChE抑制剂处理可减弱凋亡，表明AChE介导OP诱导的细胞凋亡；OP长期暴露可导致小鼠大脑中β淀粉样蛋白（Aβ）斑块增多并损伤神经元。文献报道Aβ诱导神经元凋亡是导致AD的主要原因之一，AChE和Aβ存在互作关系，但两者在OP诱导细胞凋亡过程中的具体机制，存在何种cross-talk，目前知之甚少。因此本项目拟采用APP/PS1小鼠建立AD动物模型，分析OP暴露促进AD发生发展；并在多种细胞模型分析OP暴露对AChE和Aβ表达的影响，探索两者相互作用诱导神经细胞凋亡的分子机制。项目预期明确OP暴露引起的AChE与Aβ时程变化及促进AD发展作用，研究成果将为OP致AD提供证据并为AD防治研究提供理论基础。

有机磷（OP）暴露的神经系统危害是重大公共卫生问题。我们发现OP暴露引起小鼠大脑和体外培养神经细胞AChE 活性异常增加并诱导细胞凋亡。本课题通过建立细胞模型分析OP暴露影响AChE和A-beta表达、活性，探索AChE和A-beta相互作用致神经细胞凋亡的分子机制。本研究发现，高于400µmol/L的CPO可以诱导N2a APP细胞死亡（P <0.05），低于300µmol/L的CPO对N2a APP细胞的凋亡率无明显影响。同时我们还发现；N2a APP细胞分别以不同CPO浓度处理，用乙酰胆碱酯酶检测试剂盒检测AChE活性，用ELISA试剂盒检测Aβ1-42表达。结果表明，接触CPO可以显着提高AChE活性至200µmol/L以上（P <0.05）。同时，我们选择400µmol/L作为实验浓度，CPO暴露后Aβ1-42浓度可以显着增加（P <0.05）。表明CPO暴露可以诱导AChE活性增加和Aβ1-42表达增加。在CPO暴露后，通过流式细胞术检测有无多奈哌齐时的细胞凋亡率。 使用多奈哌齐预处理后发现，AChE活性可能会降低至多奈哌齐20µmol/L以上（P <0.05）。然后以20μmol/ L的多奈哌齐浓度作为实验浓度检测细胞凋亡。结果发现多奈哌齐预处理后凋亡细胞死亡可能减少。在之后的实验中，我们研究了P-Akt在CPO诱导AChE活性增加中的作用。 首先利用P-Akt抑制剂LY，P-Akt的抑制剂不仅显着降低了P-Akt的表达，而且有效降低了CPO诱导的凋亡细胞比例。 然后，在LY294002预处理之后，测量AChE活性和AChE蛋白表达。 结果表明，LY294002预处理后，AChE活性可能显着降低，AChE蛋白表达可能显着降低。我们将N2aAPP细胞用20μm的AChE活性抑制剂多奈哌齐预处理2小时，然后使用400μm的CPO处理24h。结果表明多奈哌齐预处理显着降低了CPO诱导的Aβ1-42浓度增加和BACE1的表达增加。将N2a APP细胞用抑制剂BACE1酶40µm AZD3839预处理2h，结果表明，AZD3839预处理可显着降低Aβ表达和凋亡率诱导的CPO。这些数据表明，AChE增加导致活性诱导的Aβ表达增加，这取决于BACE1酶在N2a APP细胞凋亡中的增加。