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)50 μL,另外3组小鼠气管内分别滴注0.2 mg/mL LPS 50 μL.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水平显著降低,GPX4水平升高,差异均有统计学意义(P<0.05).LPS+Fer-1组和LPS+14,15-EET组以上各指标比较,差异均无统计学意义(P>0.05).结论 14,15-EET可能通过抑制铁死亡减轻LPS诱导的小鼠ALI,发挥肺部保护作用.
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 divid-ed 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)50 μL was used for intratracheal in-stillation in control group and 0.2 mg/mL LPS 50 μL were used for intratracheal instillation in another 3 groups,respectively.After 6 hours of in-tratracheal 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 bron-choalveolar lavage fluid(BALF)were collected from each group.The pathological changes in lung tissue of each group of mice were observed u-sing 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)-1(3 and 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 group and the LPS+14,15-EET group were significant decreased,the differences were statistically significant(P<0.05).Compared with the control group,the total protein concentration and cell number in BALF of LPS group were significantly increased,and the expression levels of IL-1β and MCP-1 mRNA in lung tissue were significantly increased,the differences were statistically significant(P<0.05);compared with the LPS group,the total protein concentration and cell number in BALF,and the expression levels of IL-1β and MCP-1 mRNA in lung tissue of the LPS+Fer-1 group and the LPS+14,15-EET group were significant decreased,the differences were statistically significant(P<0.05).Compared with control group,the red fluorescence signal of ROS in the LPS group was sig-nificantly enhanced,and the levels of malondialdehyde,iron ions,and COX2 were significantly increased,while the level of GPX4 was decreased,the differences were statistically significant(P<0.05);compared with the LPS group,the ROS red fluorescence signal in the lung tissue of the LPS+Fer-1 group and LPS+14,15-EET group was significantly reduced,and the levels of malondialdehyde,iron ions,and COX2 were signif-icantly decreased,while the levels of GPX4 were increased,the differences were statistically significant(P<0.05).There were no statistically significant differences in the above indicators between the LPS+Fer-1 group and the LPS+14,15-EET group(P>0.05).Conclusion 14,15-EET may alleviate LPS induced ALI in mice and exert pulmonary protective effects by inhibiting ferroptosis.