Department of Critical Care, McGill University Health Centre, Montréal, Québec, Canada Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, Québec, Canada.
Department of Critical Care, McGill University Health Centre, Montréal, Québec, Canada Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, Québec, Canada
Cardiovasc Res. 2015 Jun 1;106(3):465-77. doi: 10.1093/cvr/cvv120. Epub 2015 Mar 30.
Bacterial lipopolysaccharides (LPS) induce innate immune inflammatory responses in endothelial cells by activating toll-like receptor 4 (TLR4) signalling. Here, we investigate the effects of angiopoietin-1 (Ang-1) on LPS-induced TLR4 signalling and the role of the miR-146 family of micro RNAs in the effects of Ang-1 on TRL4 signalling.
Leucocyte adhesion to human umbilical vein endothelial cells (HUVECs) was detected using fluorescence microscopy. Adhesion molecule, pro-inflammatory cytokine, miR-146a, and miR-146b-5p expressions in HUVECs were quantified using real-time PCR. TLR4 signalling protein levels were measured using immunoblotting. Exposure of HUVECs to LPS for 4-6 h induces robust inflammatory responses, including enhanced leucocyte adhesion, up-regulation of adhesion molecule expression (VCAM1, ICAM1, E-SELECTIN), enhanced cytokine production (TNFα, IL1β, IL6, and IL8), and increased NFκB luciferase reporter activity. Addition of Ang-1 to the culture medium for 24 h prior to LPS exposure significantly attenuates these responses. Prolonged Ang-1 exposure significantly decreases IRAK1 and TRAF6 protein levels but has no effect on TLR4, MYD88, IRAK4, or TAK1 expressions. Ang-1 triggers significant up-regulation of miR-146b-5p levels but has no effect on miR-146a or miR-146b-3p expressions. Transfection of HUVECs with a miR-146b-5p mimic significantly attenuates LPS-induced inflammatory responses and IRAK1 and TRAF6 expressions. In HUVECs transfected with a miR-146b-5p inhibitor, Ang-1 has no effect on LPS-induced inflammatory responses or IRAK1 and TRAF6 expressions.
Ang-1 disrupts TLR4 signalling, resulting in inhibition of LPS-induced inflammatory responses in endothelial cells. This inhibition occurs through selective targeting of IRAK1 and TRAF6 proteins by miR-146b-5p.
细菌脂多糖（LPS）通过激活 Toll 样受体 4（TLR4）信号通路，诱导内皮细胞固有免疫炎症反应。在这里，我们研究了血管生成素-1（Ang-1）对 LPS 诱导的 TLR4 信号通路的影响，以及 miR-146 家族微 RNA 在 Ang-1 对 TLR4 信号通路影响中的作用。
通过荧光显微镜检测白细胞与脐静脉内皮细胞（HUVEC）的黏附。使用实时 PCR 定量检测 HUVEC 中黏附分子、促炎细胞因子、miR-146a 和 miR-146b-5p 的表达。使用免疫印迹法测量 TLR4 信号通路蛋白水平。HUVEC 暴露于 LPS 4-6 小时可引起强烈的炎症反应，包括增强白细胞黏附、上调黏附分子表达（VCAM1、ICAM1、E-SELECTIN）、增强细胞因子产生（TNFα、IL1β、IL6 和 IL8）以及增加 NFκB 荧光素酶报告基因活性。在 LPS 暴露前 24 小时将 Ang-1 添加到培养基中可显著减弱这些反应。长时间暴露于 Ang-1 可显著降低 IRAK1 和 TRAF6 蛋白水平，但对 TLR4、MYD88、IRAK4 或 TAK1 的表达无影响。Ang-1 可显著上调 miR-146b-5p 水平，但对 miR-146a 或 miR-146b-3p 的表达无影响。转染 HUVEC 的 miR-146b-5p 模拟物可显著减弱 LPS 诱导的炎症反应和 IRAK1 和 TRAF6 的表达。在转染 miR-146b-5p 抑制剂的 HUVEC 中，Ang-1 对 LPS 诱导的炎症反应或 IRAK1 和 TRAF6 的表达无影响。
Ang-1 破坏 TLR4 信号通路，从而抑制内皮细胞中 LPS 诱导的炎症反应。这种抑制作用是通过 miR-146b-5p 对 IRAK1 和 TRAF6 蛋白的选择性靶向作用实现的。
