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曲古抑菌素A对脂多糖诱导的巨噬细胞炎症细胞因子、Toll样受体4表达及核因子-κB乙酰化的影响

胡晓兰^*, 张晓, 李倩, 邱水凤, 梅汝焕

浙江大学医学院生理学系，杭州 310058

摘要

本研究旨在探讨组蛋白去乙酰化酶抑制剂曲古抑菌素A (trichostatin A, TSA)对LPS/TLR4/NF-κB炎症信号通路影响的可能机制。以小鼠巨噬细胞系RAW264.7细胞为研究对象，MTT法检测TSA对巨噬细胞存活率的影响，ELISA测定细胞培养上清中TNF-α、IL-1β和IL-6的含量，Western Blot检测TLR4蛋白表达及NF-κB/p65(Lys310)的乙酰化程度，应用TransAMTM NF-κB/p65活性检测试剂盒检测NF-κB/p65 DNA结合活性。结果显示，和对照组(DMSO处理)相比，TSA (浓度大于10 ng/mL)处理的细胞存活率显著降低(P < 0.05)。LPS处理显著升高培养上清中TNF-α、IL-1β和IL-6含量，而TSA (40 ng/mL)预处理显著降低培养上清中这三种炎症细胞因子含量(P < 0.05)。LPS处理显著上调RAW264.7细胞TLR4蛋白表达，而TSA预处理可显著降低TLR4蛋白表达(P < 0.05)。LPS处理增强NF-κB/p65(Lys310)的乙酰化和NF-κB/p65 DNA结合活性，TSA预处理进一步增强NF-κB/p65(Lys310)的乙酰化，而降低NF-κB/p65 DNA结合活性(P < 0.05)。以上结果提示，组蛋白去乙酰化酶抑制剂TSA通过降低TLR4表达以及NF-κB/p65 DNA结合活性发挥其抗炎作用。

关键词： 组蛋白去乙酰化酶抑制剂; 脂多糖; Toll样受体4; 核因子-κB; 炎症因子

分类号：Q291;R392

[Effects of trichostatin A on the expressions of inflammatory cytokines and toll-like receptor 4 and the acetylation of nuclear factor-κB induced by lipopolysaccharide in macrophage.] [Article in Chinese]

HU Xiao-Lan^*, ZHANG Xiao, Li Qian, QIU Shui-Feng, MEI Ru-Huan

Department of Physiology, Zhejiang University School of Medicine, 588 Yuhangtang Road, Hangzhou 310058, China

Abstract

The present study aims to explore the possible mechanisms that trichostatin A (TSA), a histone deacetylases inhibitor (HDACi), affects the inflammatory signaling pathways of lipopolysaccharide/toll-like receptor 4/nuclear factor-κB (LPS/TLR4/NF-κB). Murine macrophage cell line RAW264.7 cells were employed. MTT assay was used to assess cell viability. The contents of TNF-α, IL-1β and IL-6 in culture supernatant were assayed by enzyme-linked immunosorbent assay (ELISA). TLR4 expression and NF-κB/p65 (Lys310) acetylation were examined by Western blotting. DNA binding activity of NF-κB/p65 was detected by using TransAM(TM) NF-κB/p65 activity assay kit. The results showed that, compared with control group, which was treated by DMSO, the cells treated with TSA (20, 40, 80 ng/mL) showed decreased percentages of cell survival (P < 0.05). The contents of TNF-α, IL-1β and IL-6 in culture supernatant were all increased by LPS (100 ng/mL), whereas reduced by 40 ng/mL TSA pretreatment (P < 0.05). TSA pretreatment inhibited LPS-induced up-regulation of TLR4 protein expression. Acetylation of NF-κB/p65(Lys310), which was already increased by LPS, was further enhanced by TSA (P < 0.05). On the contrary, LPS-increased DNA binding activity of NF-κB/p65 was decreased by pretreatment with TSA (P < 0.05). The results suggest that TSA-induced anti-inflammation may be attributed to decreases in the expression of TLR4 and DNA binding activity of NF-κB/p65.

Key words: histone deacetylases inhibitor; lipopolysaccharide; Toll-like receptor 4; nuclear factor-κB; inflammatory factor

收稿日期：2012-04-26　　录用日期：2012-06-19

通讯作者：胡晓兰　　E-mail: huxiaolan1998@yahoo.com.cn

引用本文：

胡晓兰, 张晓, 李倩, 邱水凤, 梅汝焕. 曲古抑菌素A对脂多糖诱导的巨噬细胞炎症细胞因子、Toll样受体4表达及核因子-κB乙酰化的影响[J]. 生理学报 2012; 64 (6): 651-656.

HU Xiao-Lan, ZHANG Xiao, Li Qian, QIU Shui-Feng, MEI Ru-Huan. [Effects of trichostatin A on the expressions of inflammatory cytokines and toll-like receptor 4 and the acetylation of nuclear factor-κB induced by lipopolysaccharide in macrophage.] [Article in Chinese]. Acta Physiol Sin 2012; 64 (6): 651-656 (in Chinese with English abstract).