14,15-EET减轻脂多糖诱导的小鼠急性肺损伤的作用及其机制  

Effect and mechanism of 14,15-EET attenuates lipopolysaccharide-induced acute lung injury in mice

作　　者：于悦 杨霭琳 何馨[1] 于刚刚[1] 王浩彦[1] 吴艳军[1] YU Yue;YANG Ai-lin;HE Xin(Department of Respiratory Medicine,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China)

机构地区：[1]首都医科大学附属北京友谊医院呼吸内科,北京100050

出　　处：《临床和实验医学杂志》2024年第2期113-117,共5页Journal of Clinical and Experimental Medicine

基　　金：国家自然科学基金(编号:82000042,81870029);北京市自然科学基金(编号:7204247);首都医科大学附属北京友谊医院院启动课题控烟专项(编号:yyqdktzx2020-3);北京市临床重点专科建设项目(编号:2020-2022)。

摘　　要：目的探讨14,15-环氧二十碳三烯酸(14,15-EET)减轻脂多糖(LPS)诱导小鼠急性肺损伤(ALI)的作用及其机制。方法将32只6~8周的C57BL/6J雄性小鼠按照随机数字表法分为4组:对照组、LPS组、LPS+铁死亡抑制剂(Fer-1)组以及LPS+14,15-EET组,每组各8只。对照组小鼠气管内滴注无菌磷酸盐缓冲液(PBS)50μL,另外3组小鼠气管内分别滴注0.2 mg/mL LPS 50μL。气管内滴注LPS刺激6 h后,LPS+Fer-1组尾静脉注射Fer-1(0.8 mg/kg),LPS+14,15-EET组尾静脉注射14,15-EET(100 nmol/L),分别作用16 h。造模成功后取小鼠肺组织及支气管肺泡灌洗液(BALF),苏木精-伊红染色观察各组小鼠肺组织病理学变化,测量肺湿/干(W/D)比重;记录各组小鼠BALF中总蛋白浓度及总细胞数量;采用实时荧光定量聚合酶链式反应(RT-qPCR)检测小鼠肺组织白细胞介素(IL)-1β和单核细胞趋化蛋白-1(MCP-1)mRNA表达;采用冰冻切片活性氧簇(ROS)染色观察肺组织中ROS的生成;分别使用试剂盒检测肺组织丙二醛水平及铁含量;采用蛋白质印迹法检测铁死亡相关分子谷胱甘肽过氧化物酶4(GPX4)、环氧化物酶2(COX2)的表达水平。结果与对照组相比,LPS组肺损伤评分及W/D值均明显升高,差异均有统计学意义(P<0.05);与LPS组相比,LPS+Fer-1组和LPS+14,15-EET组肺损伤评分及W/D值均明显下降,差异均有统计学意义(P<0.05)。与对照组相比,LPS组BALF中总蛋白浓度、细胞数量明显升高,肺组织中IL-1β和MCP-1 mRNA表达水平均明显升高,差异均有统计学意义(P<0.05);与LPS组相比,LPS+Fer-1组和LPS+14,15-EET组BALF蛋白、细胞的数量及肺组织中IL-1β和MCP-1表达均明显下降,差异均有统计学意义(P<0.05)。与对照组相比,LPS组ROS红色荧光信号明显增强,丙二醛、铁离子、COX2水平均显著升高,GPX4水平下降,差异均有统计学意义(P<0.05);与LPS组相比,LPS+Fer-1组和LPS+14,15-EET组肺组织中ROS红色荧光信号明显减弱,丙二醛、铁离子、COX2�Objective To investigate the effect of 14,15-epoxyeicosatrienoic acid(14,15-EET)in attenuating lipopolysaccharide(LPS)-induced acute lung injury(ALI)in mice and its mechanism.Methods Thirty-two male C57BL/6J mice aged 6-8 weeks were divided into four groups according to the random number table method:control group,LPS group,LPS+ferroptosis inhibi ferrostatin-1(Fer-1)group,and LPS+14,15-EET group,each group with 8 mice.The sterile phosphate buffer solution(PBS)50μL was used for intratracheal instillation in control group and 0.2 mg/mL LPS 50μL were used for intratracheal instillation in another 3 groups,respectively.After 6 hours of intratracheal stimulation with LPS,Fer-1(0.8 mg/kg)was injected into the tail vein of the LPS+Fer-1 group,and 14,15-EET(100 nmol/L)was injected into the tail vein of the LPS+14,15-EET group for 16 hours,respectively.After successful modeling,lung tissues and bronchoalveolar lavage fluid(BALF)were collected from each group.The pathological changes in lung tissue of each group of mice were observed using hematoxylin-eosin staining,and the wet/dry(W/D)density of the lungs was measured.The total protein concentration and total cell count in BALF of each group of mice were recorded.The expression levels of interleukin(IL)-1βand monocyte chemotactic protein-1(MCP-1)mRNA in mouse lung tissue were detected using real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).The generation of reactive oxygen species(ROS)in lung tissue was observed by ROS staining in frozen sections.The levels of malondialdehyde and iron content in lung tissue were detected using reagent kits,respectively.The expression levels of iron death related molecules glutathione peroxidase 4(GPX4)and epoxidase 2(COX2)were detected using Western blotting.Results Compared with the control group,the lung injury score and W/D value in the LPS group were significantly increased,and the differences were statistically significant(P<0.05);compared with the LPS group,the lung injury score and W/D value in the LPS+Fer-1

关 键 词：小鼠 脂多糖类 14 15-环氧二十碳三烯酸 急性肺损伤 铁死亡 

分 类 号：R563[医药卫生—呼吸系统]