EGCG干预LPS诱导的THP-1细胞TLR4及下游通路的活化

Effect of EGCG on the expression of TLR4 and its downstream signal transduction pathway in human THP-1 monocytes

目的:探讨表没食子儿茶素-3-没食子酸酯(EGCG)对细菌脂多糖(LPS)诱导的人单核细胞株THP-1 Toll样受体4(TLR4)及下游信号转导通路的干预作用。方法:采用不同浓度的EGCG和LPS处理THP-1细胞,MTT检测细胞增殖能力;实时荧光定量RT-PCR(qRT-PCR)检测细胞TLR4和肿瘤坏死因子α(TNF-α)mRNA的表达;Western blotting检测TLR4以及下游信号分子胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)、p38的蛋白水平变化;ELISA检测细胞培养上清TNF-α蛋白含量。结果:EGCG(20 mg·L-1)能降低LPS刺激的THP-1细胞TLR4(蛋白和mRNA)的表达(P<0.05);EGCG(5,10,20 mg·L-1)能抑制TLR4下游分子ERK,JNK,p38的磷酸化,并呈现一定的浓度依赖性(P<0.05);同时,EGCG(20 mg·L-1)能干预LPS刺激的细胞TNF-α(蛋白和mRNA)的表达(P<0.05)。结论:EGCG可通过干预LPS刺激的TLR4表达及下游分子ERK,JNK,p38磷酸化,抑制细胞TNF-α表达,这可能是EGCG的抗炎机制之一。

Objective： To investigate the effect of epigallocatechin-3-gallate（ EGCG） on the expression of Toll-like receptor 4（ TLR4） and its downstream signal transduction pathway in THP-1 mononuclear cells. Methods：THP-1 cells were treated with lipopolysaccharide（ LPS） and various centrations of EGCG. Then,the proliferation ability of THP-1 cells was assessed by MTT assay,the mRNA levels of TLR4 and tumor necrosis factor-α（ TNF-α）in THP-1 cells were detected by real time quantitative PCR（ qRT-PCR）,and the levels of TLR4,extracellular signal-regulated kinase（ ERK）,c-Jun N-terminal kinase（ JNK）,p38 protein by Western blotting. The content of TNF-α protein in the culture supernatant of THP-1 cells was measured by enzymelinked immunosorbent assay（ ELISA）. Results： EGCG（ 20 mg·L- 1） significantly reduced the mRNA and protein expressions of TLR4 and TNF-α（ P 0. 05） in the LPS-stimulated THP-1 cells. EGCG（ 5,10 and 20 mg·L- 1） concentration-dependently decreased the levels of phospho-ERK,phospho-JNK and phospho-p38（ P 0. 05） in the LPS-stimulated THP-1cells. Conclusion： EGCG inhibits the expression of TNF-α by reducing the expression of TLR4,and the phosphorylation of JNK,ERK and p38,which may be one of the anti-inflammatory mechanisms of EGCG.