Department of Cardiology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China; The Key Laboratory of Myocardial Ischemia, Chinese Ministry of Education, Harbin, Heilongjiang Province, China.
Department of Cardiology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu Province, China.
Mol Immunol. 2018 Sep;101:221-228. doi: 10.1016/j.molimm.2018.05.028. Epub 2018 Jul 11.
Erythropoietin (EPO), which is clinically used for renal anaemia, reportedly exerts beneficial pleiotropic effects in atherosclerosis. This aim of this study was to investigate the effects of EPO on macrophage inflammation and polarization under hyperglycaemic conditions and to identify the effects of EPO-treated macrophage supernatants (SNs) on endothelial cell (EC) function.
Peritoneal macrophages (pMΦs) were isolated from normal, diabetic or EPO-injected mice. Pro-inflammatory factors were detected by qRT-PCR and ELISA, and macrophage phenotype markers were evaluated by flow cytometry. High glucose culture was used to mimic the hyperglycaemic microenvironment of diabetes mellitus (DM) in vitro. After exposure to various doses of stimuli, macrophage inflammation and phenotype were detected via ELISA, qRT-PCR and flow cytometry. The underlying mechanism was investigated through western blotting. To examine the communication between macrophages and ECs, ECs were cultured with the SN of macrophages treated with different stimuli, and the tube formation ability of ECs was detected using Matrigel. The VEGF, ICAM-1 and VCAM-1 protein expression levels were determined by western blotting, and the nitric oxide (NO) and endothelin-1 (ET-1) expression levels were measured with a nitric oxide indicator and by ELISA, respectively.
EPO treatment increased the M2 macrophage population and decreased the number of M1 macrophages. EPO decreased the secretion of pro-inflammatory factors, including TNF-α, iNOS and IL-6. The JAK2/STAT3 signalling pathway was also identified as being involved in the M1 macrophage transition. The SN of macrophages treated with EPO (SN-EPO) presented increased NO and ET-1 levels and decreased ICAM-1 and VCAM-1 levels. Tube formation assays revealed that the SN-EPO promoted the ability of ECs to form capillary-like structures in vitro. In contrast, AZD1480, a JAK2 inhibitor, abolished this SN-EPO effect.
EPO treatment alleviated the inflammatory reaction in DM mice and inhibited M1 polarization through the JAK2/STAT3 pathway. Moreover, EPO treatment promoted the tube formation ability of ECs in a VEGF-dependent manner and decreased the production of adhesion molecules, a vasodilator and a vasoconstrictor.
促红细胞生成素（EPO）临床上用于治疗肾性贫血，据报道其在动脉粥样硬化中具有有益的多效作用。本研究旨在探讨 EPO 在高血糖条件下对巨噬细胞炎症和极化的影响，并确定 EPO 处理的巨噬细胞上清液（SN）对内皮细胞（EC）功能的影响。
从正常、糖尿病或 EPO 注射的小鼠中分离出腹腔巨噬细胞（pMΦ）。通过 qRT-PCR 和 ELISA 检测促炎因子，通过流式细胞术评估巨噬细胞表型标志物。体外高糖培养模拟糖尿病（DM）的高血糖微环境。用不同剂量的刺激物处理后，通过 ELISA、qRT-PCR 和流式细胞术检测巨噬细胞炎症和表型。通过 Western blot 研究潜在机制。为了研究巨噬细胞和 EC 之间的通讯，将不同刺激物处理的巨噬细胞的 SN 与 EC 共培养，并用 Matrigel 检测 EC 的管形成能力。通过 Western blot 测定 VEGF、ICAM-1 和 VCAM-1 蛋白表达水平，通过一氧化氮指示剂和 ELISA 分别测定一氧化氮（NO）和内皮素-1（ET-1）表达水平。
EPO 处理增加了 M2 巨噬细胞群体，减少了 M1 巨噬细胞的数量。EPO 减少了 TNF-α、iNOS 和 IL-6 等促炎因子的分泌。还确定 JAK2/STAT3 信号通路参与了 M1 巨噬细胞的转化。用 EPO 处理的巨噬细胞的 SN（SN-EPO）表现出增加的 NO 和 ET-1 水平，降低的 ICAM-1 和 VCAM-1 水平。管形成实验表明，SN-EPO 促进了 EC 体外形成毛细血管样结构的能力。相反，JAK2 抑制剂 AZD1480 消除了这种 SN-EPO 效应。
EPO 治疗通过 JAK2/STAT3 通路减轻 DM 小鼠的炎症反应并抑制 M1 极化。此外，EPO 处理以 VEGF 依赖的方式促进 EC 的管形成能力，并减少粘附分子、血管扩张剂和血管收缩剂的产生。
