Zhou You, You Hui, Zhang Aijie, Jiang Xingliang, Pu Zheyan, Xu Guoqiang, Zhao Mingcai
Department of Medical Laboratory, Central Hospital of Suining, Suining, Sichuan 629100, P.R. China.
Department of Ophthalmology, Central Hospital of Suining, Suining, Sichuan 629100, P.R. China.
Exp Ther Med. 2020 Aug;20(2):1682-1692. doi: 10.3892/etm.2020.8812. Epub 2020 May 28.
LipoxinA4 (LXA4) is a well-known key mediator of endogenous anti-inflammation and of the resolution of inflammation. Considerable oxidative stress occurs during inflammation due to the generation of reactive oxidative species (ROS). Moreover, high levels of uric acid (UA) contribute to endothelial cell dysfunction, which can promote disease-related morbidity, and NADPH oxidase-derived ROS are crucial regulatory factors in these responses. However, LXA4 also has the potential to reduce oxidative stress. The aim of the present study was to examine whether LXA4 could suppress UA-induced oxidative stress in human umbilical vein endothelial cells (HUVECs) and to investigate its mechanisms of action in vitro. HUVECs were incubated with or without LXA4, followed by the addition of UA. ROS levels were then measured using 2,7-dichlorodihydrofluorescein diacetate and lucigenin-enhanced chemiluminescence was used to evaluate NADPH oxidase activity. p47phox or p22phox small interfering (si)RNA were transfected into HUVECs and protein levels of p47phox were detected using western blot analysis. LXA4 significantly inhibited UA-induced generation of ROS to the same extent as the NADPH oxidase inhibitor, diphenyleneiodonium chloride. Notably, transfection of p47phox siRNA attenuated the generation of ROS and the activation of NADPH oxidase. Cells transfected with p22phox siRNA demonstrated a significant reduction in the expression of p47phox on the membrane. Further experiments demonstrated that LXA4 interfered with the transfer of p47phox from the cytoplasm to the cell membrane. These findings suggested that LXA4 inhibited the release of NADPH oxidase derived ROS in HUVECs stimulated by UA. A potential mechanism of action underlying this effect could be LXA4-mediated suppression of NADPH oxidase activity, leading to inhibition of p47phox translocation from the cytoplasm to the cell membrane.
脂氧素A4（LXA4）是一种众所周知的内源性抗炎和炎症消退的关键介质。由于活性氧化物质（ROS）的产生，炎症期间会发生相当程度的氧化应激。此外，高水平的尿酸（UA）会导致内皮细胞功能障碍，这可能会促进疾病相关的发病率，而NADPH氧化酶衍生的ROS是这些反应中的关键调节因子。然而，LXA4也有降低氧化应激的潜力。本研究的目的是检测LXA4是否能抑制UA诱导的人脐静脉内皮细胞（HUVECs）中的氧化应激，并在体外研究其作用机制。将HUVECs与LXA4一起或不与LXA4一起孵育，然后加入UA。然后使用2,7-二氯二氢荧光素二乙酸酯测量ROS水平，并使用光泽精增强化学发光来评估NADPH氧化酶活性。将p47phox或p22phox小干扰（si）RNA转染到HUVECs中，并使用蛋白质印迹分析检测p47phox的蛋白质水平。LXA4与NADPH氧化酶抑制剂二苯碘鎓氯化物一样，在相同程度上显著抑制UA诱导的ROS生成。值得注意的是，p47phox siRNA转染减弱了ROS的生成和NADPH氧化酶的激活。用p22phox siRNA转染的细胞显示膜上p47phox的表达显著降低。进一步的实验表明，LXA4干扰了p47phox从细胞质向细胞膜的转移。这些发现表明，LXA4抑制了UA刺激的HUVECs中NADPH氧化酶衍生的ROS的释放。这种效应潜在的作用机制可能是LXA4介导的NADPH氧化酶活性的抑制，导致p47phox从细胞质向细胞膜的易位受到抑制。
