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10ng/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(40ng/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。
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 DNAbinding activity of NF-κB/p65.
出处
《生理学报》
CAS
CSCD
北大核心
2012年第6期651-656,共6页
Acta Physiologica Sinica
基金
supported by the National Natural Science Foundation of China(No.81072415 and 81173466)
