Dectin-1受体介导肺炎克雷伯菌诱导的ARDS炎症反应及其病理特征  

Dectin-1 receptor mediates ARDS inflammation induced by K.pneumoniae and its pathological features

作　　者：王丽辉[1] 施安[1] 唐佳佳 施志强[1] 李卉[1] 吴卉[1] 艾田逸 朱铭力[1] 余跃天[1,2,3,4] WANG Lihui;SHI An;TANG Jiajia;SHI Zhiqiang;LI Hui;WU Hui;AI Tianyi;ZHU Mingli;YU Yuetian(Department of Critical Care Medicine,Renji Hospital,Shanghai JiaoTong University School of Medicine,Shanghai 200127;Key Laboratory of Multiple Organ Failure of Ministry of Education,Zhejiang University,Hangzhou 310027;Key Laboratory of Intelligent Pharmacy and Individualized Therapy of Huzhou City,Huzhou Zhejiang 313100;Health Commission Key Laboratory of Diagnosis and Treatment ofAcute Respiratory Distress Syndrome of Guangxi,Nanning 530021,China)

机构地区：[1]上海交通大学医学院附属仁济医院重症医学科,上海200127 [2]浙江大学多脏器衰竭预警与干预教育部重点实验室,杭州310027 [3]湖州市智能药学与个体化治疗重点实验室,浙江湖州313100 [4]广西急性呼吸窘迫综合征诊治研究重点实验室,南宁530021

出　　处：《临床与病理杂志》2023年第12期2133-2143,共11页Journal of Clinical and Pathological Research

基　　金：多脏器衰竭预警与干预教育部重点实验室开放课题资助项目(2023KF07);湖州市智能药学与个体化治疗重点实验室开放基金(HZKF-20240101);广西急性呼吸窘迫综合征诊治研究重点实验室开放基金(ZZH2020013-3)。

摘　　要：目的:免疫反应在急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)的病理生理机制中发挥重要作用,而Dectin-1受体介导多个炎症反应通路。本研究旨在探讨Dectin-1受体介导肺炎克雷伯菌诱导的ARDS炎症反应特点及其与病理特征的相关性。方法:提取C57BL/6野生型或Dectin-1^(−/−)小鼠腹腔巨噬细胞,采用酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法检测不同感染复数(multiplicity of infection,MOI)的肺炎克雷伯菌或不同浓度的脂多糖(lipopolysaccharide,LPS)刺激下巨噬细胞中白细胞介素(interleukin,IL)-6、IL-1β和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的表达水平;采用蛋白质印迹法检测巨噬细胞相关炎症通路蛋白质胞外信号调节激酶(extracellular signal-regulated kinase,ERK)、p38蛋白、c-Jun氨基端激酶(c-Jun N-terminal kinase,JNK)的表达水平。取健康野生型小鼠或Dectin-1^(−/−)小鼠随机分为ARDS模型小鼠和对照小鼠,小鼠采用肺炎克雷伯菌经气道感染建立ARDS模型,对照小鼠采用与肺炎克雷伯菌等体积的PBS经气道感染。观察小鼠7 d生存率;采用ELISA法检测各组小鼠3、8、24、48 h血浆IL-6表达水平;采用HE染色法比较小鼠肺组织损伤情况;计算并比较小鼠肺组织载菌量的差异。结果:给予肺炎克雷伯菌刺激后,Dectin-1^(−/−)小鼠巨噬细胞中IL-6、IL-1β和TNF-α的表达水平均高于野生型小鼠巨噬细胞(P<0.05);给予巨噬细胞LPS刺激后,Dectin-1^(−/−)小鼠巨噬细胞表达IL-6明显高于野生型小鼠巨噬细胞(P<0.05)。蛋白质印迹法结果显示:Dectin-1^(−/−)小鼠巨噬细胞在肺炎克雷伯菌刺激45、60 min时ERK表达水平均高于野生型小鼠巨噬细胞(均P<0.05);刺激30、60 min时,Dectin-1^(−/−)小鼠巨噬细胞表达JNK、p38水平均高于野生型小鼠巨噬细胞(均P<0.05)。ARDS建模7 d内,Dectin-1^(−/−)ARDS模型小鼠生存率低于野生型ARDS模型小�Objective:Immune response plays an important role in the pathophysiological mechanisms of acute respiratory distress syndrome(ARDS),and the Dectin-1 receptor mediates multiple inflammatory response pathways.This study aims to explore the characteristics of the Dectin-1 receptor in the inflammatory response induced by K.pneumoniae-induced ARDS and its correlation with pathological features.Methods:Peritoneal macrophages from C57BL/6 wild-type or Dectin-1^(−/−)mice were extracted.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression levels of interleukin(IL)-6,IL-1βand tumor necrosis factor-α(TNF-α)in macrophages stimulated with different multiplicities of infection(MOI)of K.pneumoniae or different concentration of lipopolysaccharide(LPS).Western blotting was performed to detect the expression of extracellular signal-regulated kinase(ERK),p38 protein,and c-Jun N-terminal kinase(JNK).Healthy wild-type mice or Dectin-1^(−/−)mice were randomly divided into an ARDS group and a control group.The ARDS model mice were established by intratracheal infection with K.pneumoniae,while the control mice were intratracheally infected with PBS of equal volume.The 7-day survival rate of mice in 4 groups was observed.ELISA was used to detect the difference of IL-6 expression level in plasma at 3,8,24,and 48 h.HE staining was used to compare the injury of lung tissue in mice.The difference of bacterial load in lung tissue of mice was calculated and compared.Results:The expression levels of IL-6,IL-1β,and TNF-αin macrophages from Dectin-1^(−/−)mice were higher than those from wild-type mice after stimulation with K.pneumoniae(P<0.05).After LPS stimulation,the expression of IL-6 in macrophages from Dectin-1^(−/−)mice was significantly higher than that from wild-type mice(P<0.05).Western blotting results showed that the expression levels of ERK in macrophages from Dectin-1^(−/−)mice were higher than those from wild-type mice at 45 and 60 min after K.pneumoniae stimulation(both P<0.05).After 3

关 键 词：Dectin-1受体 肺炎克雷伯菌 急性呼吸窘迫综合征 炎症反应 

分 类 号：R563.8[医药卫生—呼吸系统]