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内皮Adora2b活化可减轻脂多糖诱导的肺微血管内皮炎症 被引量:4

Role of adenosine A2b receptors in pulmonary microvascular endothelial inflammation induced by lipopolysaccharide
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摘要 目的探讨内皮低亲和力A2b腺苷受体(Adora2b)对脂多糖(LPS)诱导肺微血管内皮炎症的调控作用及机制.方法原代培养大鼠肺微血管内皮细胞(PMVEC),血清饥饿24 h后,采用Adora2b特异性激动剂BAY60-6583(0.1、1、10μmol/L)或抑制剂PSB1115(1μmol/L)预处理1 h,然后再加入LPS(100μg/L);同时设立空白对照组、LPS组、BAY60-6583单独处理组和PSB1115单独处理组.各组细胞培养24 h后,采用膜联蛋白V/碘化丙啶(Annexin V/PI)双染色法检测早期细胞凋亡率,用酶联免疫吸附试验(ELISA)测定细胞上清液中炎性因子含量,用实时荧光定量聚合酶链反应(RT-qPCR)测定趋化因子和黏附分子的mRNA表达;分离提取大鼠静脉血中性粒细胞(PMN),观察体外PMN黏附迁移情况,用异硫氰酸荧光素-白蛋白(FITC-albumin)法检测PMN黏附迁移后PMVEC单层通透性,用荧光探针DCFH-DA检测细胞内氧化应激水平.结果与空白对照组相比,LPS组早期细胞凋亡率明显升高,上清液中早期炎性因子白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)含量明显增加,趋化因子如CXC基序趋化因子配体1(CXCL-1)、CXC基序趋化因子配体3(CXCL-3)、单核细胞趋化蛋白1(MCP-1)以及黏附分子如E-选择素、细胞间黏附分子-1(ICAM-1)、血管细胞黏附分子-1(VCAM-1)的mRNA表达均明显升高,黏附迁移的PMN增多,PMVEC单层通透性升高,细胞内活性氧水平升高.与LPS组比较,BAY60-6583预处理能剂量依赖性地降低早期细胞凋亡率,减少PMN迁移,降低PMVEC单层通透性,0.1μmol/L时差异即有统计学意义〔凋亡率:(21.12±2.12)%比(27.66±3.57)%,PMN迁移细胞数(个/HP):260.60±18.24比290.20±16.48,通透系数(Pd,×10-6 cm/s):28.28±2.04比32.55±2.13,均P<0.05〕,并能剂量依赖性减少早期促炎因子分泌,降低趋化因子和黏附分子的mRNA表达,1μmol/L时差异均有统计学意义〔IL-1β(ng/L):475.75±63.15比755.25±67.42,TNF-α(ng/L):560.25±69.96比818.75±60.92,CXCL-1 mRNA(2-ΔΔCt):3.57±0.28比5.27±0.69,CXCL-3 mRNA(2-ΔΔCt):4.56±0.48比7.32±0.54,MCP-1 mRNA(2-ΔΔCt):2.21±0.31比3.35±0.21,E-选择素mRNA(2-ΔΔCt):4.64±0.09比7.28±0.73,ICAM-1 mRNA(2-ΔΔCt):4.14±0.30比5.89±0.25,VCAM-1 mRNA(2-ΔΔCt):2.23±0.19比2.92±0.33,均P<0.05〕,10μmol/L BAY60-6583预处理还能降低细胞内氧化应激水平〔活性氧(荧光强度):629.05±33.10比781.45±64.59,P<0.05〕;而PSB1115预处理能进一步增加LPS诱导的细胞凋亡〔凋亡率:(34.36±4.57)%比(27.66±3.57)%〕,上调炎性因子和趋化因子、黏附分子的表达〔IL-1β(ng/L):889.00±63.11比755.25±67.42,TNF-α(ng/L):939.00±43.44比818.75±60.92,CXCL-1 mRNA(2-ΔΔCt):6.66±0.65比5.27±0.69,CXCL-3 mRNA(2-ΔΔCt):10.42±0.51比7.32±0.54,MCP-1 mRNA(2-ΔΔCt):4.85±0.34比3.35±0.21,E-选择素mRNA(2-ΔΔCt):8.42±0.47比7.28±0.73,ICAM-1 mRNA(2-ΔΔCt):7.46±0.72比5.89±0.25,VCAM-1 mRNA(2-ΔΔCt):4.35±0.26比2.92±0.33〕,并能增加PMN黏附迁移(个/HP:348.40±22.68比290.20±16.48)及PMVEC单层通透性〔Pd(×10-6 cm/s):39.65±2.69比32.55±2.13〕,加重氧化应激损伤〔活性氧(荧光强度):847.04±29.26比781.45±64.59〕,差异均有统计学意义(均P<0.05).结论内皮Adora2b活化可通过减少早期炎性因子释放、下调细胞趋化和黏附分子表达、减少PMN黏附迁移、降低氧化应激等机制减轻LPS诱导的肺微血管内皮炎症. Objective To explore the role of the low-affinity A2b adenosine receptors(Adora2b)in pulmonary microvascular endothelial inflammation induced by lipopolysaccharide and its mechanism.Methods Rat pulmonary microvascular endothelial cells(PMVECs)were isolated and cultured in vitro.After serum deprivation for 24 hours,cells were pretreated with Adora2b specific agonist BAY60-6583(0.1,1,10μmol/L)or Adora2b specific antagonist PSB1115(1μmol/L)for 1 hour,respectively,and then challenged with LPS(100μg/L).Cells without treatment were served as the control group,and those treated with LPS,BAY60-6583 or PSB1115 alone were served as single challenge groups.After incubation with specific drugs for 24 hours,the apoptosis of PMVECs was analyzed by flow cytometry using Annexin V/propidium iodide(PI)technique.The levels of early inflammatory factors in cultured medium were measured using enzyme linked immunosorbent assay(ELISA).The mRNA expressions of chemotactic factors and adhesion molecules were determined by real-time quantitative-polymerase chain reaction(RT-qPCR).Polymorph nuclear neutrophils(PMNs)from venous blood of healthy rats were isolated,and PMN migration through PMVECs monolayer under stimulation of drugs was observed in transwell inserts.The monolayer permeability of PMVECs after adhesion of PMNs was determined by fluorescein isothiocyanate(FITC)-albumin assay.Oxidative stress was detected by DCFH-DA assay.Results Compared with the control group,more cells entered into the apoptosis stage after LPS challenge.Meanwhile,the levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in cultured medium were significantly increased,as well as the mRNA expressions of chemotactic factors[C-X-C motif chemokine ligand 1(CXCL-1),CXCL-3 and monocyte chemoattractant protein-1(MCP-1)]and adhesion molecules[E-selectin,intercellular adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule-1(VCAM-1)].More PMNs migrated through PMVECs following adhesion and the monolayer permeability of PMVECs was rapidly enhanced.The oxidative stress was upregulated.Compared with LPS group,BAY60-6583 pretreatment could dose-dependently decrease the rate of apoptosis,attenuate trans-endothelial migration of PMNs and decrease the endothelial cell barrier leakage.There were significant differences even after incubation of 0.1μmol/L BAY60-6583[apoptosis rate:(21.12±2.12)%vs.(27.66±3.57)%,number of migrated PMNs/HP:260.60±18.24 vs.290.20±16.48,permeability coefficient(Pd,×10-6 cm/s):28.28±2.04 vs.32.55±2.13,all P<0.05].Meanwhile,BAY60-6583 pretreatment also downregulated the levels of early proinflammatory factors in a dose-dependent manner as well as the mRNA expressions of chemotactic factors and adhesion molecules.The statistic difference was significant while treated with 1μmol/L BAY60-6583[IL-1β(ng/L):475.75±63.15 vs.755.25±67.42,TNF-α(ng/L):560.25±69.96 vs.818.75±60.92,CXCL-1 mRNA(2-ΔΔCt):3.57±0.28 vs.5.27±0.69,CXCL-3 mRNA(2-ΔΔCt):4.56±0.48 vs.7.32±0.54,MCP-1 mRNA(2-ΔΔCt):2.21±0.31 vs.3.35±0.21,E-selectin mRNA(2-ΔΔCt):4.64±0.09 vs.7.28±0.73,ICAM-1 mRNA(2-ΔΔCt):4.14±0.30 vs.5.89±0.25,VCAM-1 mRNA(2-ΔΔCt):2.23±0.19 vs.2.92±0.33,all P<0.05].Furthermore,pretreatment of 10μmol/L BAY60-6583 could decrease the oxidative stress[reactive oxygen species(RFU):629.05±33.10 vs.781.45±64.59,P<0.05].Contrast,PSB1115 pretreatment aggravated apoptosis of PMVECs after LPS incubation[(34.36±4.57)%vs.(27.66±3.57)%],upregulated expressions of proinflammatory and chemotactic factors as well as adhesion molecules[IL-1β(ng/L):889.00±63.11 vs.755.25±67.42,TNF-α(ng/L):939.00±43.44 vs.818.75±60.92,CXCL-1 mRNA(2-ΔΔCt):6.66±0.65 vs.5.27±0.69,CXCL-3 mRNA(2-ΔΔCt):10.42±0.51 vs.7.32±0.54,MCP-1 mRNA(2-ΔΔCt):4.85±0.34 vs.3.35±0.21,E-selectin mRNA(2-ΔΔCt):8.42±0.47 vs.7.28±0.73,ICAM-1 mRNA(2-ΔΔCt):7.46±0.72 vs.5.89±0.25,VCAM-1 mRNA(2-ΔΔCt):4.35±0.26 vs.2.92±0.33],aggravated trans-endothelial migration of PMNs(cells/HP:348.40±22.68 vs.290.20±16.48),enhanced the leakage of PMVECs monolayer[Pd(×10-6 cm/s):39.65±2.69 vs.32.55±2.13]and increased oxidative stress in PMVECs[reactive oxygen species(RFU):847.04±29.26 vs.781.45±64.59],with statistically significant difference(all P<0.05).Conclusion Activation of endothelial Adora2b attenuates LPS-induced pulmonary microvascular inflammation by decreasing the release of early inflammatory factors,downregulating expressions of chemotactic factors and adhesion molecules,attenuating trans-endothelial migration of PMNs and oxidative stress in PMVECs,which suggest endothelial Adora2b is apotential anti-inflammatory target in the treatment of LPS-induced acute lung injury.
作者 郭晓夏 安友仲 Guo Xiaoxia;An Youzhong(Department of Intensive Care Unit,People's Hospital,Peking University,Beijing 100044,China)
出处 《中华危重病急救医学》 CAS CSCD 北大核心 2019年第12期1485-1490,共6页 Chinese Critical Care Medicine
基金 北京市自然科学基金(7194328)。
关键词 急性呼吸窘迫综合征 A2b腺苷受体 肺微血管内皮 炎症 Acute respiratory distress syndrome A2b adenosine receptor Pulmonary microvascular endothelium Inflammation
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