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Title: [Exogenous agmatine inhibits lipopolysaccharide-induced activation and dysfunction of human umbilical vein endothelial cells].
Author: Yin SQ, Zhu JY, Luo L, Yang X, Liang HP, Luo Y.
Journal: Nan Fang Yi Ke Da Xue Xue Bao; 2018 Jun 20; 38(6):652-660. PubMed ID: 29997086.
Abstract:
OBJECTIVE: To investigate whether exogenous agmatine inhibits lipopolysaccharide (LPS)-induced activation and dysfunction of human umbilical vein endothelial cells (HUVECs) by modulating nuclear factor-κB (NF-κB) and MAPK signal pathways and the production of reactive oxygen species (ROS). METHODS: Cultured HUVECs were treated with agmatine at the optimized concentration of 1.0 mmolγL, LPS (10 µgγmL), and LPS + agmatine, with or without pretreatment with the inhibitors of NF-κB (PDTC), p38 (SB203580), and ERK (PD98059) for 1 h. The levels of soluble vascular cell adhesion molecule 1 (VCAM-1), soluble intercellular adhesion molecule 1 (sICAM-1), soluble E-selectin and monocyte chemoattractant protein 1 (MCP-1) in the supernatant were determined using ELISA, and their mRNA expressions, along with heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO-1), were assessed using real-time PCR. ROS production in the cells was determined using 2, 7-dichlorofluoresce in diacetate (DCFH-DA) as the fluorescence probe. The protein expressions of VCAM-1, ICAM-1, p65, phospho-p65 (p-p65), IκBα, p-IκBα, ERK, p-ERK, p38, p-p38, JNK, and p-JNK were detected using Western blotting. RESULTS: LPS stimulation for 6 and 24 h significantly increased the levels of sVCAM-1, sICAM-1, sE-selectin and MCP-1 in the supernatant, intracellular ROS production, and the mRNA expressions of these molecules (P<0.05). Intervention with 1 mmolγL agmatine, similar with pretreatment with p38, ERK and NF-κB inhibitors, obviously inhibited such effects of LPS in HUVECs (P<0.05). Agmatine significantly up-regulated the mRNA expression of HO-1 (P<0.05), inhibited LPS-induced phosphorylation of p38, ERK, nuclear p65 and cytoplasmic IκBα, and up-regulated the protein expression of cytoplasmic IκBα. CONCLUSION: Agmatine inhibits LPS-induced activation and dysfunction of HUVECs by modulating NF-κB and MAPK signal pathways to down-regulate the expressions of adhesion molecules and chemokines and by up-regulating the expression of HO-1 to reduce ROS production. 目的: 研究胍丁胺对脂多糖诱导的人脐静脉内皮细胞活化及损伤的影响，并探究其与NF-κB、MAPK通路及活性氧（ROS）的产生是否相关。方法: 体外培养人脐静脉内皮细胞（HUVECs）。MTT法检测0~4 mmol/L胍丁胺作用24 h对HUVECs细胞活力的影响。HUVECs细胞机分为：①空白对照组；②脂多糖（10 μg/mL）组；③胍丁胺干预组：脂多糖（10 μg/mL）+胍丁胺（0.125、0.5、1 mmol/L）。ELISA法检测24 h细胞上清中可溶性细胞间粘附分子-1（sICAM-1）、可溶性血管间粘附分子-1（sVCAM-1）、可溶性E-选择素（sE-selectin）及单核细胞趋化因子-1（MCP-1）水平，确定胍丁胺最适干预浓度（1 mmol/L）。后续实验中HUVECs细胞随机分为：对照组、脂多糖（10 μg/mL）组、胍丁胺（1 mmol/L）组及脂多糖（10 μg/mL）+胍丁胺（1 mmol/L）共同组，作用1、6、24 h；抑制剂组即在脂多糖刺激前1 h，用10 μmol/L NF-κB（PDTC）、ERK1/2（PD98059）及p38（SB203580）抑制剂进行预处理。qRT-PCR法检测ICAM-1、VCAM-1、E-selectin、MCP-1、血红素氧合酶（HO-1）、醌氧化还原酶（NQO1）mRNA表达水平；DCFH-DA作为荧光探针检测细胞内活性氧（ROS）水平；Western blot法检测细胞VCAM-1、ICAM-1、p65、磷酸化- p65（p-p65）、IκBα、p-IκBα、ERK、p-ERK、p38、p-p38、JNK、p-JNK蛋白表达水平。结果: （1）HUVECs经10 μg/mL脂多糖刺激24 h后，上清sVCAM-1、sICAM-1、sE-选择素、MCP-1含量明显升高（P < 0.05），细胞内ROS明显增加（P < 0.05）；刺激6 h后各因子mRNA水平升高（P < 0.05）；（2）胍丁胺（1 mmol/L）干预后上述指标显著下调（P < 0.05），这与p38、ERK及NF-κB信号通路抑制剂预处理细胞后的抑制效应相似；（3）胍丁胺干预组6 h HO-1 mRNA表达较脂多糖组明显增加（P < 0.05），而NQO-1无明显变化；（4）胍丁胺明显抑制脂多糖刺激引起的细胞内p38、ERK、核内p65（Ser536）与胞浆IκBα磷酸化，并上调胞浆IκBα蛋白水平。结论: 胍丁胺可能通过抑制NF-κB、MAPK通路的激活下调粘附分子及趋化因子水平，并增强HO-1表达以减少活性氧产生对抗脂多糖诱导的人脐静脉内皮细胞活化及损伤。

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