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Toll样受体4通过Akt/FoxO3a/Bim信号通路参与海马神经元凋亡

徐玲, 周爱玲, 赵敏

南通大学医学院病理生理学系；南通大学附属医院神经内科，南通 226001

摘要

为了探讨海马神经元中是否有Toll样受体4 (Toll-like receptor 4, TLR4)介导的Akt/FoxO3a/Bim信号通路，及该通路在海马神经元凋亡中的作用和机制，本实验运用脂多糖(lipopolysaccharide, LPS)作用于原代培养的大鼠海马神经元，利用不同的工具药，以减弱或加强TLR4/Akt/FoxO3a/Bim通路的作用。应用CCK-8法检测细胞活力，Western blot法检测神经元p-Akt (Ser473)、Akt、p-FoxO3a (Thr32)、FoxO3a、Bim、活化的Caspase-3蛋白的表达变化，real-time PCR法检测海马神经元Bim的mRNA表达变化，免疫荧光法观察FoxO3a核易位情况，流式细胞术检测细胞凋亡率。结果显示：LPS作用于海马神经元不同时间后，各组细胞活力均下降(P < 0.05)，且具有一定的时间依赖性；LPS作用后p-Akt (Ser473)、p-FoxO3a (Thr32)表达减少，FoxO3a易位进入胞核，而促凋亡蛋白Bim及活化的Caspase-3表达增加，且海马神经元的凋亡率也增加(P < 0.05)；PI3K特异性抑制剂LY294002预处理后p-Akt (Ser473)、p-FoxO3a (Thr32)表达较LPS组降低，而促凋亡蛋白Bim及活化的Caspase-3表达升高，细胞凋亡率也明显升高(P < 0.05)；TLR4抗体预处理后p-Akt (Ser473)、p-FoxO3a (Thr32)较LPS组增多，FoxO3a易位进入胞核的活动减弱，而促凋亡蛋白Bim、活化的Caspase-3及细胞的凋亡率均减少(P < 0.05)。以上结果表明，海马神经元中有TLR4介导的Akt/FoxO3a/Bim通路；神经元可通过该通路引起自身的凋亡。

关键词： 海马神经元; Toll样受体4; PI3K/Akt; FoxO3a; Bim; 凋亡

分类号：R3

[Involvement of Toll-like receptor 4 in apoptosis of hippocampal neurons through Akt/FoxO3a/Bim signaling pathways.] [Article in Chinese]

XU Ling, ZHOU Ai-Ling, ZHAO Min

Department of Pathophysiology, Medical College of Nantong University； Department of Neurology, Affiliated Hospital of Nantong University, Nantong 226001, China

Abstract

The present study was to investigate whether Toll-like receptor 4 (TLR4)-mediated Akt/FoxO3a/Bim signaling pathway participated in lipopolysaccharide (LPS)-induced apoptosis in hippocampal neurons. The primarily cultured rat hippocampal neurons were treated with LPS, TLR4 antibody+LPS, and LY294002+LPS, respectively. Cell vitality was assayed by CCK-8. Expressions of p-Akt, Akt, p-FoxO3a, FoxO3a, Bim and active-Caspase-3 of each group were detected by Western blot analysis; the mRNA expression of Bim was detected by real-time quantitative PCR; FoxO3a nuclear translocation was detected by fluorescence microscope. The rate of cell apoptosis was assayed by flow cytometry. The results showed that cell vitality of hippocampal neurons decreased after being treated with LPS in a time-dependent way. Compared with the control group, the expressions of p-Akt and p-FoxO3a decreased significantly, FoxO3a translocated into the nucleus, meanwhile, the expression of Bim and active-Caspase-3, and the apoptotic ratio of hippocampal neurons increased in LPS treated neurons. Pretreatment with TLR4 antibody significantly blocked, while PI3K antagonist LY294002 further strengthened these changes induced by LPS. In conclusion, the present study suggests that Akt/FoxO3a/Bim signaling pathways mediated by TLR4 participate in the apoptotic processes of primarily cultured hippocampal neurons treated with LPS, and the activation of TLR4 causes neuronal apoptosis.

Key words: Hippocampal neurons; Toll-like receptor 4; PI3K/Akt; FoxO3a; Bim; apoptosis

收稿日期：2013-11-12　　录用日期：2014-02-20

通讯作者：周爱玲　　E-mail: alz@ntu.edu.cn

引用本文：

徐玲, 周爱玲, 赵敏. Toll样受体4通过Akt/FoxO3a/Bim信号通路参与海马神经元凋亡[J]. 生理学报 2014; 66 (3): 315-322.

XU Ling, ZHOU Ai-Ling, ZHAO Min. [Involvement of Toll-like receptor 4 in apoptosis of hippocampal neurons through Akt/FoxO3a/Bim signaling pathways.] [Article in Chinese]. Acta Physiol Sin 2014; 66 (3): 315-322 (in Chinese with English abstract).