College of Medicine, Hunan Normal University, Changsha, Hunan 410013, P.R. China.
Center for Molecular Medicine, Xiangya Hospital, Collaborative Innovation Center for Cancer Medicine, Central South University, Changsha, Hunan 410078, P.R. China.
Int J Mol Med. 2018 May;41(5):2619-2628. doi: 10.3892/ijmm.2018.3511. Epub 2018 Feb 23.
7‑difluoromethoxy‑5,4'‑dimethoxy‑genistein (DFMG) is a novel active chemical entity, which modulates the function and signal transduction of endothelial cells and macrophages (MPs), and is essential in the prevention of atherosclerosis. In the present study, the activity and molecular mechanism of DFMG on MPs was investigated using a Transwell assay to construct a non‑contact co‑culture model. Human umbilical vein endothelial cells (HUVE‑12), which were incubated with lysophosphatidylcholine (LPC), were seeded in the upper chambers, whereas PMA‑induced MPs were grown in the lower chambers. The generation of reactive oxygen species (ROS) and the release of lactate dehydrogenase (LDH) were measured using the corresponding assay kits. The proliferation and migration were assessed using 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide and wound healing assays, respectively. Foam cell formation was examined using oil red O staining and a total cholesterol assay. The protein expression levels of Toll‑like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)‑κB p65 were detected by western immunoblotting. The secretion of interleukin (IL)‑1β was examined using an enzyme‑linked immunosorbent assay. It was found that LPC significantly increased the generation of ROS and the release of LDH in HUVE‑12 cells. The LPC‑injured HUVE‑12 cells activated MPs under co‑culture conditions and this process was inhibited by DFMG treatment. LPC upregulated the expression levels of TLR4, MyD88 and NF‑κB p65, and the secretion of IL‑1β in the supernatant of the co‑cultured HUVE‑12 cells and MPs. These effects were reversed by the application of DFMG. Furthermore, CLI‑095 and IL‑1Ra suppressed the activation of MPs that was induced by co‑culture with injured HUVE‑12 cells. These effects were further enhanced by co‑treatment with DFMG, and DFMG exhibited synergistic effects with a TLR4‑specific inhibitor. Take together, these findings revealed that DFMG attenuated the activation of MP induced by co‑culture with LPC‑injured HUVE‑12 cells. This process was mediated via inhibition of the TLR4/MyD88/NF‑κB signaling pathway in HUVE‑12 cells.
7-二氟甲氧基-5,4'-二甲氧基染料木黄酮（DFMG）是一种新型的活性化学实体，可调节内皮细胞和巨噬细胞（MPs）的功能和信号转导，是预防动脉粥样硬化的关键。在本研究中，采用 Transwell 测定法构建非接触共培养模型，研究了 DFMG 对 MPs 的活性和分子机制。孵育溶血磷脂酰胆碱（LPC）的人脐静脉内皮细胞（HUVE-12）接种在上室，而 PMA 诱导的 MPs 则在下室中生长。使用相应的测定试剂盒测定活性氧（ROS）的产生和乳酸脱氢酶（LDH）的释放。使用 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐和划痕愈合测定法分别评估增殖和迁移。使用油红 O 染色和总胆固醇测定法检查泡沫细胞形成。通过 Western 免疫印迹法检测 Toll 样受体 4（TLR4）、髓样分化因子 88（MyD88）和核因子（NF）-κB p65 的蛋白表达水平。使用酶联免疫吸附测定法检测白细胞介素（IL）-1β的分泌。结果发现，LPC 可显著增加 HUVE-12 细胞中 ROS 的产生和 LDH 的释放。在共培养条件下，LPC 损伤的 HUVE-12 细胞激活 MPs，DFMG 处理可抑制该过程。LPC 上调共培养 HUVE-12 细胞和 MPs 上清液中 TLR4、MyD88 和 NF-κB p65 的表达水平以及 IL-1β的分泌。DFMG 的应用可逆转这些作用。此外，CLI-095 和 IL-1Ra 抑制了与受损 HUVE-12 细胞共培养诱导的 MPs 激活。与 DFMG 共同处理进一步增强了这些作用，DFMG 与 TLR4 特异性抑制剂表现出协同作用。综上所述，这些发现表明，DFMG 可减轻与 LPC 损伤的 HUVE-12 细胞共培养诱导的 MPs 激活。该过程通过抑制 HUVE-12 细胞中的 TLR4/MyD88/NF-κB 信号通路介导。
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