Center for Animal Sciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda, Punjab, 151 001, India.
Center for Biochemistry and Microbial Sciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda, Punjab, India.
Metab Brain Dis. 2017 Dec;32(6):2045-2061. doi: 10.1007/s11011-017-0093-2. Epub 2017 Aug 31.
Amyloid beta (Aβ) peptide deposition is the primary cause of neurodegeneration in Alzheimer's disease (AD) pathogenesis. Several reports point towards the role of pesticides in the AD pathogenesis, especially organophosphate pesticides (OPPs). Monocrotophos (MCP) and Chlorpyrifos (CP) are the most widely used OPPs. In this study, the role of MCP and CP in augmenting the Aβ-induced oxidative stress associated with the neurodegeneration in AD has been assessed in human neuroblastoma IMR-32 and SH-SY5Y cell lines. From the cell survival assay, it was observed that MCP and CP reduced cell survival both dose- and time-dependently. Nitro blue tetrazolium (NBT) based assay for determination of intracellular reactive oxygen species (ROS) demonstrated that Aβ(25-35), MCP or CP produce significant oxidative stress alone or synergistically in IMR-32 and SH-SY5Y cells, while pretreatment of curcumin reduced ROS levels significantly in all treatment combinations. In this study, we also demonstrate that treatment of Aβ(25-35) and MCP upregulated inducible nitric oxide synthase (iNOS/NOS2) whereas, no change was observed in neuronal nitric oxide synthase (nNOS/NOS1), but down-regulation of the nuclear factor erythroid 2-related factor 2 (Nrf2) level was observed. While curcumin pretreatment resulted in upregulation of iNOS and Nrf2 proteins. Also, the expression of key DNA repair enzymes APE1, DNA polymerase beta (Pol β), and PARP1 were found to be downregulated upon treatment with MCP, Aβ(25-35) and their combinations at 24 h and 48 h time points. In this study, pretreatment of curcumin to the SH-SY5Y cells enhanced the expression of DNA repair enzymes APE1, pol β, and PARP1 enzymes to counter the oxidative DNA base damage via base excision repair (BER) pathway, and also activated the antioxidant element (ARE) via Nrf2 upregulation. Furthermore, the immunofluorescent confocal imaging studies in SH-SY5Y and IMR-32 cells treated with Aβ(25-35) and MCP-mediated oxidative stress and their combinations at different time periods suggesting for cross-talk between the two proteins APE1 and Nrf2. The APE1's association with Nrf2 might be associated with the redox function of APE1 that might be directly regulating the ARE-mediated neuronal survival mechanisms.
淀粉样β肽（Aβ）沉积是阿尔茨海默病（AD）发病机制中神经退行性变的主要原因。有几项报告指出，农药在 AD 发病机制中起作用，特别是有机磷农药（OPPs）。久效磷（MCP）和毒死蜱（CP）是使用最广泛的 OPPs。在这项研究中，评估了 MCP 和 CP 在增强与 AD 神经退行性变相关的 Aβ诱导的氧化应激中的作用，该作用在人神经母细胞瘤 IMR-32 和 SH-SY5Y 细胞系中进行。从细胞存活测定中可以看出，MCP 和 CP 均以剂量和时间依赖性方式降低细胞存活率。用于测定细胞内活性氧（ROS）的硝基蓝四唑（NBT）测定表明，Aβ（25-35）、MCP 或 CP 单独或协同产生显著的氧化应激，而姜黄素预处理可使所有治疗组合中的 ROS 水平显著降低。在这项研究中，我们还证明，Aβ（25-35）和 MCP 的治疗可上调诱导型一氧化氮合酶（iNOS/NOS2），而神经元型一氧化氮合酶（nNOS/NOS1）没有变化，但核因子红细胞 2 相关因子 2（Nrf2）水平下调。姜黄素预处理可导致 iNOS 和 Nrf2 蛋白上调。此外，还发现，在用 MCP、Aβ（25-35）及其组合处理 24 小时和 48 小时时，关键 DNA 修复酶 APE1、DNA 聚合酶β（Polβ）和 PARP1 的表达均下调。在这项研究中，用姜黄素预处理 SH-SY5Y 细胞可增强 DNA 修复酶 APE1、polβ和 PARP1 酶的表达，以通过碱基切除修复（BER）途径对抗氧化 DNA 碱基损伤，并通过 Nrf2 上调激活抗氧化元件（ARE）。此外，用 Aβ（25-35）和 MCP 介导的氧化应激以及不同时间段的两种蛋白质 APE1 和 Nrf2 之间的相互作用对 SH-SY5Y 和 IMR-32 细胞进行免疫荧光共聚焦成像研究表明。APE1 与 Nrf2 的关联可能与 APE1 的氧化还原功能有关，该功能可能直接调节 ARE 介导的神经元存活机制。
