低剂量TCDD通过AhR调节Sirt1诱导神经元和神经前体细胞衰老的机制研究

2,3,7,8-tetrachlorodibenzo-p-dioxin（TCDD）are widespread environmental pollutants that could cause significant neurotoxicity. However, the underlying mechanism of its neurotoxic effect remains much elusive. In this study, we found exposure with low level of TCDD would trigger senescence in neural pc12 cell line. Further, we scanned the differentially-expressed genes in TCDD-induced senescent cells using gene-chip analysis, and verified the differentially expressed genes with RT-PCR and chromatin immunoprecipitation assays. In this way, an NAD-dependent deacetylase Sirt1, which plays a crucial role in regulating cellular lifespan and senescence, was identified to be a direct target of dioxins-AhR signaling. Further, we found dioxins could induce neural senescence, which was accompanied with a dramatic reduction of Sirt1 expression, indicating that the decreased expression of Sirt1 might be involved in the regulation of dioxin-induced neural senescence and the impairment of learning and memory ability of adult rats. Based on these findings, we aim to first clarify the transcriptional regulation of Sirt1 by Dioxins, and observe its effects on hippocampal neurons, senescence of neural precursor cells and learning and memory ability to analyze the Sirt 1 expression in rat hippocampal neurons. Further, we will use TCDD to induce PC12 and primary neurons, C17.2 and precursors of primary neurons, and study the expression of Sirt1 down regulated by AhR and its downstreaming signalings in the process of dioxin-induced neural senescence. Finally, we will investigate the effect of dioxin on neural senescence, Sirt1 expression and it's down signalings in animal models, and see whether overexpressing Sirt1 would block neuron senescence in brain cortex following dioxin exposure. Our project might help clarify the molecular mechanism of dioxin-induced neurotoxic effect, and thus benefit the prevention and therapy of dioxin-mediated neurotoxicity.

TCDD具有明显的神经毒性，但其作用机制尚未明确。课题组首次发现，低剂量TCDD可以诱导PC12细胞DNA损伤和衰老表型增加，利用基因芯片筛选结合实时定量和免疫印迹实验发现Sirt1表达下降，成年大鼠受低剂量TCDD暴露其海马组织Sirt1表达亦降低，ChIP实验证明TCDD通过激活AhR直接抑制sirt1启动子活性。故提出"TCDD通过AhR抑制sirt1，诱导成年大鼠神经元和神经前体细胞衰老和学习记忆力下降"的假说。课题拟先以低剂量TCDD处理大鼠，观察其对海马神经元、神经前体细胞衰老表型和学习记忆的影响，分析大鼠海马组织Sirt1表达；接着以TCDD刺激PC12与原代神经元、C17.2与原代神经前体细胞，分析AhR下调Sirt1基因表达、并进而调控其下游通路及靶基因的分子机制；最后在细胞和整体动物水平使用白黎芦醇（Sirt1激动剂）观察其能否拮抗TCDD诱导神经元衰老。本课题开展的意义在于能拓展TCDD致神经毒性的分子机制；并可为阐述环境毒物对神经退行性疾病的影响提供新的实验证据。

在体内和体外水平探讨低剂量TCDD新的神经毒性及其作用机制。体外实验主要研究包括：低剂量TCDD诱导原代神经元/PC12细胞、神经前体细胞/C17.2细胞衰老，重点探讨TCDD对神经元和神经前体细胞中衰老相关蛋白Sirt1及其下游靶基因乙酰化的改变，观察使用Sirt1激动剂白黎芦醇能否逆转TCDD致神经元和神经前体细胞过早衰老。整体动物实验主要研究包括：低剂量TCDD单次染毒对C57BL6小鼠海马齿状回区细胞衰老的影响、Sirt1及其下游靶基因乙酰化的改变、低剂量TCDD对C57BL6小鼠恐惧记忆能力的影响及白黎芦醇预防作用等。研究结果表明：低剂量TCDD能诱导PC12神经细胞和C17.2神经前体细胞过早衰老，其机制涉及TCDD能改变神经元和神经前体细胞的细胞周期先关蛋白p16/p21/p27/p53等的表达水平、增加细胞氧化应激和DNA损伤、下调抗衰老相关蛋白Sirt1表达水平及增加其下游相关靶基因乙酰化水平等，一些细胞内信号通路，如Sirt1/p53/FOXO3a，Akt-mTOR-p21, keap1/p62/Nrf2，ROS/NF-κB/IL-6等在低剂量TCDD诱导神经元或神经前体细胞过早衰老中发挥了重要作用。此外，体内与体外实验中，使用白藜芦醇均能有效拮抗低剂量TCDD上述神经毒性。本课题的开展不仅能帮助学术界更新认识低剂量TCDD新的神经毒性（致神经元和神经前体细胞过早衰老）、作用机制以及白藜芦醇的预防作用，而且有助于我们重新认识和评估环境持久性污染物对神经退行性疾病的影响和探寻有益预防措施。