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TLR4/NF-κB信号通路激活对大鼠海马神经元淀粉样β蛋白沉积的影响

吴丹, 张弦, 赵敏, 周爱玲

南通大学病理生理学教研室；南通大学附属医院神经内科；南通大学附属医院传染科；南通市肿瘤医院病理科，南通 226001

摘要

本文旨在探讨海马神经元Toll样受体4 (Toll-like receptor 4, TLR4)/核转录因子κB (nuclear factor κB, NF-κB)信号通路激活后对淀粉样β蛋白(β-amyloid, Aβ)沉积的影响。采用体外培养7 d的新生大鼠海马神经元，细胞免疫荧光双标法鉴定海马神经元的纯度。TLR4配体脂多糖(lipopolysaccharide, LPS)、TLR4抑制剂CLI-095及NF-κB抑制剂PDTC分别预处理海马神经元，激活或阻断TLR4/NF-κB信号通路。酶联免疫吸附法测定海马神经元培养上清中肿瘤坏死因子α (tumor necrosis factor-α, TNF-α)、白介素-1β (interleukin-1β, IL-1β)及Aβ1–42的水平；实时荧光定量PCR (real-time quantitative PCR, RT-qPCR)法检测海马神经元TNF-α、IL-1β、去整合金属蛋白酶10 (a disintegrin and metalloproteinase domain-containing protein 10, ADAM10)、β-分泌酶(β-site APP cleaving enzyme 1, BACE-1)、早老素-1 (Presenilin-1, PS-1)、β-淀粉样前体蛋白(β-amyloid precursor protein, β-APP) mRNA的表达；Western blot法检测海马神经元ADAM10、BACE-1、PS-1和β-APP蛋白的表达。RT-qPCR、Western blot法检测不同浓度Aβ1–42刺激海马神经元后TLR4 mRNA、蛋白的表达。结果显示，新生大鼠海马神经元无血清培养7 d后，其纯度可达95%以上。与正常对照组相比，LPS激活TLR4/NF-κB信号通路后，TNF-α、IL-1β、BACE-1、PS-1、β-APP、Aβ1–42的表达增加，相反，ADAM10的表达下降，而CLI-095或PDTC预处理则可以逆转这些改变。同时，给予不同浓度的Aβ1–42刺激海马神经元后，TLR4的表达明显上升。这表明海马神经元表面的TLR4/NF-κB信号通路被激活后，可以导致炎症因子水平的升高，进一步引起ADAM10水平的下降和BACE-1、PS-1水平的升高，最终导致β-APP、Aβ的沉积；而同时Aβ又能正反馈地引起海马神经元自身受体TLR4的升高，形成一个恶性循环。

关键词： 海马神经元; Toll样受体4; NF-κB; Aβ; 阿尔茨海默病; 炎症

分类号：R742

The role of the TLR4/NF-κB signaling pathway in Aβ accumulation in primary hippocampal neurons

WU Dan, ZHANG Xian, ZHAO Min, ZHOU Ai-Ling

Department of Pathophysiology, Medical School of Nantong University, Nantong 226001, China； Department of Neurology, Affiliated Hospital of Nantong University, Nantong 226001, China； Department of Infectious Disease, Affiliated Hospital of Nantong University, Nantong 226001, China； Department of Pathology, Nantong Tumour Hospital, Nantong 226001, China

Abstract

The present study aimed to investigate the role of the Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) signaling pathway in the accumulation of amyloid β protein (Aβ) in primary hippocampal neurons of rats. The purity of these cultured neurons was determined by using immunofluorescence techniques. Lipopolysaccharide (LPS, a TLR4 ligand) or CLI-095 (a TLR4 inhibitor) was used to activate or inhibit TLR4 signaling, respectively. Pyrrolidine dithiocarbamate (PDTC), on the other hand, was used to inhibit NF-κB, a downstream effector of the TLR4 signaling pathway. The contents of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and Aβ1–42 in the supernatant were assessed by enzyme-linked immunosorbent assay (ELISA). The mRNA levels of TNF-α, IL-1β, a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), β-site APP cleaving enzyme 1 (BACE-1), Presenilin-1 (PS-1), and β-amyloid precursor protein (β-APP) were examined by real-time quantitative PCR (RT-qPCR). The protein levels of ADAM10, BACE-1, PS-1 and β-APP were examined by Western blotting. Meanwhile, the levels of TLR4 mRNA and protein in hippocampal neurons were tested by RT-qPCR and Western blotting, respectively, after stimulation with Aβ1–42 at different concentrations. We observed that the purity of cultured hippocampal neurons after being cultured for 7 days was above 95%. Compared with untreated neurons, LPS-treated neurons showed higher expression levels of TNF-α, IL-1β, BACE-1, PS-1, β-APP, and Aβ1–42, but a lower expression level of ADAM10. These effects were reversed upon pre-treatment with CLI-095 or PDTC. Furthermore, TLR4 expression was upregulated in the presence of Aβ1–42. Taken together, these results provide evidence that elevation in the level of inflammatory cytokines accompanies the activation of TLR4 signaling, and that the consequent downregulation of ADAM10 and upregulation of BACE-1/PS-1 are likely responsible for the accumulation of β-APP and Aβ, which in turn increases TLR4 level to create a positive feedback loop that may constitute the basis for the progression of Alzheimer’s disease.

Key words: Hippocampal neurons; Toll-like receptor 4; NF-κB; Aβ; Alzheimer’s disease; neuroinflammation

收稿日期：2014-10-31　　录用日期：2015-04-07

通讯作者：周爱玲　　E-mail: ALZ@ntu.edu.cn

引用本文：

吴丹, 张弦, 赵敏, 周爱玲. TLR4/NF-κB信号通路激活对大鼠海马神经元淀粉样β蛋白沉积的影响[J]. 生理学报 2015; 67 (3): 319-328.

WU Dan, ZHANG Xian, ZHAO Min, ZHOU Ai-Ling. The role of the TLR4/NF-κB signaling pathway in Aβ accumulation in primary hippocampal neurons. Acta Physiol Sin 2015; 67 (3): 319-328 (in Chinese with English abstract).