外源性胍丁胺抑制脂多糖诱导的人脐静脉内皮细胞活化及损伤  

Exogenous agmatine inhibits lipopolysaccharide-induced activation and dysfunction of human umbilical vein endothelial cells

作　　者：殷商启 朱俊宇[2] 罗莉[2] 杨霞[3] 梁华平[2] 罗艳[1] YIN Shangqi;ZHU Junyu;LUO Li;YANG Xia;LIANG Huaping;LUO Yan(Department of Clinical Laboratory, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China;Research Institute of Surgery, Daping Hospital, Army Medical University, Chongqing 400042, China;Department of Clinical Pharmacy, West China Hospital, Sichuan University, Chengdu 610041, China)

机构地区：[1]重庆医科大学附属第一医院检验科,重庆400016 [2]陆军军医大学大坪医院野战外科研究所//创伤烧伤与复合伤国家重点实验室第一研究室,重庆400042 [3]四川大学华西医院临床药学部,四川成都610041

出　　处：《南方医科大学学报》2018年第6期652-660,共9页Journal of Southern Medical University

基　　金：国家自然科学基金(81671906);第三军医大学成果转化基金项目(2016XZH11)~~

摘　　要：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(10μg/m L)组;(3)胍丁胺干预组:脂多糖(10μg/m L)+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(s ICAM-1)、可溶性血管间粘附分子-1(s VCAM-1)、可溶性E-选择素(s E-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(10μg/m L)组、胍丁胺(1 mmol/L)组及脂多糖(10μg/m L)+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10μmol/L NF-κB(PDTC)、ERK1/2(PD98059)及p38(SB203580)抑制剂进行预处理。q RT-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)m RNA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10μg/m L脂多糖刺激24 h后,上清s VCAM-1、s ICAM-1、s E-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m RNA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 m RNA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 mmol/L, LPS（10 μg/m L）, 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（s ICAM-1）, soluble E-selectin and monocyte chemoattractant protein 1（MCP-1） in the supernatant were determined using ELISA, and their m RNA 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 s VCAM-1, s ICAM-1, s E-selectin and MCP-1 in the supernatant, intracellular ROS production, and the m RNA expressions of these molecules（P〈0.05）. Intervention with 1 mmol/L 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 m RNA 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

关 键 词：胍丁胺 NF-κB MAPK 活性氧 人脐静脉内皮细胞 脂多糖 

分 类 号：R459.7[医药卫生—急诊医学]