脂多糖诱导内皮细胞O-GlcNAc修饰参与炎症信号通路  

O-linked N-acetylglucosamine modification induced by lipopolysaccharide is involved in inflammatory signaling pathway in endothelial cells

作　　者：陈赫赫[1] 石燕华 应佳云 董卓亚[1] 王艳[1] 郑耀 阮培森 Chen Hehe;Shi Yanhua;Ying Jiayun;Dong Zhuoya;Wang Yan;Zheng Yao;Ruan Peisen(Department of Pediatric Critical Care Medicine,Ningbo Women and Children's Hospital,Ningbo 315000,Zhejiang,China;Department of Critical Care Medicine,Children's Hospital of Fudan University,Shanghai 200023,China)

机构地区：[1]宁波市妇女儿童医院儿童重症医学科,浙江宁波315000 [2]复旦大学附属儿科医院重症医学科,上海200023

出　　处：《中华危重病急救医学》2023年第2期164-169,共6页Chinese Critical Care Medicine

基　　金：浙江省医药卫生科技计划项目(2020KY283);浙江省宁波市科技计划项目(2019A21002);浙江省宁波市医学重点学科建设计划项目(2022-B17)。

摘　　要：目的探讨脂多糖(LPS)诱导的O-连接的N-乙酰葡萄糖胺(O-GlcNAc)修饰是否参与内皮细胞炎症信号通路。方法体外培养人脐静脉内皮细胞(HUVEC),取对数生长期细胞用于实验,分为空白对照组、LPS组(2000 mg/L的LPS)、O-GlcNAc转移酶(OGT)过表达(OGT-OE)+LPS组(转染OGT-OE质粒+2000 mg/L的LPS)、蛋白激酶C(PKC)抑制剂+LPS组(10μmol/L的Go 6983+2000 mg/L的LPS)、Rho蛋白家族RhoA抑制剂+LPS组(40μmol/L盐酸罗红+2000 mg/L的LPS)、磷脂酰肌醇3激酶(PI3K)抑制剂+LPS组(1μmol/L的SL-2052+2000 mg/L的LPS)、丝氨酸/苏氨酸蛋白激酶(Akt)抑制剂+LPS组(10μmol/L的PP2+2000 mg/L的LPS)和小干扰RNA(siRNA)作用的Akt(si-AKT)+LPS组(si-Akt+2000 mg/L的LPS)。各组细胞经LPS处理24 h后,采用实时荧光定量反转录-聚合酶链反应(RT-qPCR)检测炎症细胞因子〔白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、细胞间黏附分子-1(ICAM-1)和血管细胞黏附分子-1(VCAM-1)〕的转录水平;采用蛋白质免疫印迹试验(Western blotting)测定OGT、O-GlcNAc、Akt、细胞外信号调节激酶(ERK)、p38丝裂素活化蛋白激酶(p38MAPK)、核转录因子-κB p65(NF-κB p65)和信号转导及转录激活因子3(STAT3)的蛋白表达或磷酸化水平。结果与空白对照组相比,LPS组细胞中OGT表达及O-GlcNAc修饰水平均降低,并伴随ERK、p38MAPK和STAT3的磷酸化水平升高,且炎症因子的转录水平亦显著升高〔IL-6 mRNA(2^(-ΔΔCt)):4.71±0.60比1.03±0.29,TNF-αmRNA(2^(-ΔΔCt)):1.89±0.11比1.04±0.35,ICAM-1 mRNA(2^(-ΔΔCt)):2.06±0.18比1.02±0.21,VCAM-1 mRNA(2^(-ΔΔCt)):2.94±0.57比1.01±0.17,均P<0.05〕,说明LPS会导致O-GlcNAc修饰水平降低、炎症信号通路激活及炎症因子表达升高。与LPS组相比,OGT-OE+LPS组细胞ERK、p38MAPK、NF-κB p65和STAT3磷酸化水平下降,伴随炎症因子表达显著下降〔IL-6 mRNA(2^(-ΔΔCt)):0.12±0.01比0.90±0.17,TNF-αmRNA(2^(-ΔΔCt)):0.31±0.01比0.91±0.14,ICAM-1 mRNA(2^(-ΔΔCt)):0.64±0.02比1.13±Objective To explore whether the lipopolysaccharide(LPS)-induced modification of O-linked N-acetylglucosamine(O-GlcNAc)is involved in the inflammatory signaling pathway of endothelial cells.Methods Human umbilical vein endothelial cells(HUVEC)were cultured in vitro,and cells in logarithmic growth phase were used for experiments.Cells were divided into blank control group,LPS group(2000 mg/L LPS),O-GlcNAc transferase(OGT)overexpression(OGT-OE)+LPS group(plasmid transfection OGT+2000 mg/L LPS),protein kinase C(PKC)inhibitor+LPS group(10μmol/L Go 6983+2000 mg/L LPS),RhoA inhibitor+LPS group(40μmol/L Rhoin hydrochloride+2000 mg/L LPS),phosphatidylinositol-3-kinase(PI3K)inhibitor+LPS group(1μmol/L SL-2052+2000 mg/L LPS),serine/threonine kinase(Akt)inhibitor+LPS group(10μmol/L PP2+2000 mg/L LPS)and small interfering RNA(siRNA)treated Akt(si-AKT)+LPS group(si-Akt+2000 mg/L LPS).After 24 hours of LPS treatment,real-time fluorescence quantitative reverse transcription-polymerase chain reaction(RT-qPCR)was used to detect the transcription levels of inflammatory cytokines[interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),intercellular adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule-1(VCAM-1)].The protein expression or phosphorylation of OGT,O-GlcNAc,Akt,extracellular signal-regulated kinase(ERK),p38 mitogen-activated protein kinase(p38MAPK),nuclear factor-κB p65(NF-κB p65),and signal transducer and activator of transcription 3(STAT3)were determined by Western blotting.Results Compared with the blank control group,the expression of OGT and the modification of O-GlcNAc in the LPS group were decreased,while the expressions of phosphorylated ERK,p38MAPK,and STAT3 were increased,and the transcript levels of inflammatory cytokines were also significantly increased[IL-6 mRNA(2^(-ΔΔCt)):4.71±0.60 vs.1.03±0.29,TNF-αmRNA(2^(-ΔΔCt)):1.89±0.11 vs.1.04±0.35,ICAM-1 mRNA(2^(-ΔΔCt)):2.06±0.18 vs.1.02±0.21,VCAM-1 mRNA(2^(-ΔΔCt)):2.94±0.57 vs.1.01±0.17,all P<0.05],indicating that LPS could decrease O-

关 键 词：O-GLCNAC 内皮细胞 信号通路 细胞因子 

分 类 号：R364.5[医药卫生—病理学]