毒死蜱(CPF)是一种广泛使用的农药,可引起不良的神经发育和生殖作用。然而,对潜在机制的了解是有限的,特别是在下丘脑。我们研究了CPF在永生化小鼠下丘脑GnRH神经元(GT1-7)中的人相关浓度(1nM-100nM)的作用方式,用于研究下丘脑-垂体-性腺(HPG)轴破坏的选择性模型。我们首先检查了细胞活力,扩散,和凋亡/坏死。在非细胞毒性浓度下,我们评估了神经元的功能,基因表达,透射电子显微镜(TEM)和蛋白质组学概况,通过免疫荧光和免疫印迹(WB)验证结果。CPF以剂量反应降低细胞活力,但不影响细胞增殖。在100nM时,CPF抑制GnRH基因的表达和分泌;CPF以剂量依赖性方式降低神经元标记物Map2的免疫反应性。雌激素受体α和β的基因表达(Erα,Erβ),雄激素受体(Ar),CPF诱导芳香化酶和催产素受体有不同的趋势。差异表达蛋白的功能分析鉴定自噬,mTOR信号传导和中性粒细胞胞外陷阱(NETs)形成作为在所有浓度下影响的显著途径。这一发现得到了TEM分析的表型支持,显示明显的自噬和线粒体损伤,以及通过蛋白质分析证明mTOR及其直接靶标pULK1(Ser757)的剂量依赖性降低。生物信息学网络分析确定了相互作用蛋白的核心模块,包括Erα,Ar,通过WB分析证实了mTOR和Sirt1的下调。总的来说,我们的结果表明,CPF是导致GnRH神经元自噬的mTOR通路的抑制剂;还提示了Era/Ar信号的可能参与.在纳摩尔范围内的下丘脑CPF的不良反应的证据,就像人类暴露时发生的那样,增加了对这种农药在HPG轴上引起的潜在不利结果的关注。
Chlorpyrifos (CPF) is a widely used pesticide inducing adverse neurodevelopmental and reproductive effects. However, knowledge of the underlying mechanisms is limited, particularly in the
hypothalamus. We investigated the mode of action of CPF at human relevant concentrations (1 nM-100 nM) in immortalized mouse hypothalamic GnRH neurons (GT1-7), an elective model for studying disruption of the
hypothalamus-pituitary-gonads (HPG) axis. We firstly examined cell vitality, proliferation, and apoptosis/necrosis. At not-cytotoxic concentrations, we evaluated neuron functionality, gene expression, Transmission Electron Microscopy (TEM) and proteomics profiles, validating results by immunofluorescence and western blotting (WB). CPF decreased cell vitality with a dose-response but did not affect cell proliferation. At 100 nM, CPF inhibited gene expression and secretion of GnRH; in addition, CPF reduced the immunoreactivity of the neuronal marker Map2 in a dose-dependent manner. The gene expression of Estrogen Receptor α and β (Erα, Erβ), Androgen Receptor (Ar), aromatase and oxytocin receptor was induced by CPF with different trends. Functional analysis of differentially expressed proteins identified Autophagy, mTOR signaling and Neutrophil extracellular traps (NETs) formation as significant pathways affected at all concentrations. This finding was phenotypically supported by the TEM analysis, showing marked autophagy and damage of mitochondria, as well as by protein analysis demonstrating a dose-dependent decrease of mTOR and its direct target pUlk1 (Ser 757). The bioinformatics network analysis identified a core module of interacting proteins, including Erα, Ar, mTOR and Sirt1, whose down-regulation was confirmed by WB analysis. Overall, our results demonstrate that CPF is an inhibitor of the mTOR pathway leading to autophagy in GnRH neurons; a possible involvement of the Erα/Ar signaling is also suggested. The evidence for adverse effects of CPF in the
hypothalamus in the nanomolar range, as occurs in human exposure, increases concern on potential adverse outcomes induced by this pesticide on the HPG axis.