核因子红细胞系2相关因子2抑制铁死亡对高氧肺损伤的影响  

The effect of nuclear factor erythroid 2 related factor 2-induced inhibition of ferroptosis on hyperoxia lung injury

作　　者：殷晓桐 罗好 施佳 楚晓云 蔡成 Yin Xiaotong;Luo Hao;Shi Jia;Chu Xiaoyun;Cai Cheng(Department of Neonatology,Shanghai Children′s Hospital,School of Medicine,Shanghai Jiao Tong University,Shanghai 200062,China)

机构地区：[1]上海交通大学医学院附属儿童医院,上海市儿童医院新生儿科,上海200062

出　　处：《中华实用儿科临床杂志》2023年第7期532-537,共6页Chinese Journal of Applied Clinical Pediatrics

摘　　要：目的观察不同条件下人肺微血管内皮细胞(HPMEC)中核因子红细胞系2相关因子2(Nrf2)、谷胱甘肽过氧化物酶4(GPX4)的表达变化, 探讨Nrf2抑制铁死亡在减轻高氧肺损伤(HLI)过程中的作用。方法采用HPMEC建立高氧暴露模型。将HPMEC按照简单随机分组分为4组:对照组、高氧组(氧体积分数950 mL/L)、铁死亡抑制剂组(氧体积分数950 mL/L, 10 μmol/L Ferrostatin)及Nrf2抑制剂组(氧体积分数950 mL/L, 10 μmol/L ML385), 分别在24 h、48 h使用细胞活性检测试剂盒测定细胞活性, 荧光活性氧(ROS)试剂盒检测各组细胞内ROS水平, 应用实时荧光定量PCR和Western blot法分别检测Nrf2、GPX4的mRNA及其蛋白表达水平。2组间比较采用两样本t检验;多组间比较采用单因素方差分析。结果 1.与对照组相比, 高氧组HPMEC细胞活性降低, 细胞内ROS含量增加, 24 h及48 h GPX4 mRNA(24 h:0.750±0.010比1.010±0.160, 48 h:0.690±0.050比1.000±0.070)和蛋白表达(24 h:0.160±0.010比0.290±0.010, 48 h:0.190±0.010比0.250±0.010)均下降, Nrf2 mRNA(24 h:1.740±0.050比1.000±0.050, 48 h:2.130±0.020比1.000±0.030)和蛋白表达(24 h:0.840±0.010比0.480±0.010, 48 h:0.840±0.010比0.550±0.030)均增加, 差异均有统计学意义(均P<0.05);2.与高氧组相比, 铁死亡抑制剂组细胞活性增加, 细胞内ROS含量减少, 24 h及48 h GPX4 mRNA(24 h:1.520±0.110, 48 h:1.880±0.050)和蛋白表达(24 h:0.290±0.010, 48 h:0.250±0.004)均增加, Nrf2 mRNA(24 h:0.780±0.040, 48 h:0.760±0.030)和蛋白表达(24 h:0.480±0.010, 48 h:0.540±0.020)均下降, 差异均有统计学意义(均P<0.05);3.与高氧组相比, Nrf2抑制剂组细胞活性进一步下降, 细胞内ROS水平进一步增加, 24 h及48 h GPX4 mRNA(24 h:0.600±0.030, 48 h:0.590±0.003)及蛋白表达(24 h:0.150±0.001, 48 h:0.180±0.001)均下降, Nrf2 mRNA表达量在24 h表达升高(3.360±0.130), 48 h表达下降(1.430±0.130), 差异均有统计学意义(均P<0.05), Nrf2蛋白表达差异无统计�Objective To observe the expression changes of nuclear factor erythroid 2 related factor 2(Nrf2)and glutathione peroxidase(GPX4)in human pulmonary microvascular endothelial cells(HPMEC)under different experimental conditions,and to explore the role of Nrf2 in inhibiting ferroptosis in the process of alleviating hyperoxic lung injury(HLI).Methods Hyperoxic model was established by hyperoxia exposure.HPMEC were treated with blank control(control group),oxygen exposure at the concentration of 950 mL/L(hyperoxia group),oxygen exposure at the concentration of 950 mL/L+10μmol/L Ferrostatin(ferroptosis inhibitor group)and oxygen exposure at the concentration of 950 mL/L+10μmol/L ML385(Nrf2 inhibitor group).Cell viability at 24 h and 48 h was tested by the Cell Counting Kit-8 assay,and reactive oxygen species(ROS)levels were detected by a commercial ROS kit.The mRNA and protein levels of Nrf2 and GPX4 were detected by real-time quantitative polymerase chain reaction and Western blot,respectively.Differences were analyzed using the Student′s t-test for a two-group comparison or one-way ANOVA test among groups.Results(1)Compared with the control group,significantly decreased viability and increased ROS levels were detected in hyperoxia group.Meanwhile,the mRNA(24 h:0.750±0.010 vs.1.010±0.160,48 h:0.690±0.050 vs.1.000±0.070)and protein levels of GPX4(24 h:0.160±0.010 vs.0.290±0.010,48 h:0.190±0.010 vs.0.250±0.010)at 24 h and 48 h were significantly downregulated,while the mRNA(24 h:1.740±0.050 vs.1.000±0.050,48 h:2.130±0.020 vs.1.000±0.030)and protein levels of Nrf2(24 h:0.840±0.010 vs.0.480±0.010,48 h:0.840±0.010 vs.0.550±0.030)at 24 h and 48 h were significantly upregulated in hyperoxia group than those of control group(all P<0.05).(2)Compared with the hyperoxia group,significantly increased viability and decreased ROS levels were detected in ferroptosis inhibitor group.Meanwhile,the mRNA(24 h:1.520±0.110,48 h:1.880±0.050)and protein levels of GPX4(24 h:0.290±0.010,48 h:0.250±0.004)at 24 h and 48 h

关 键 词：铁死亡 高氧肺损伤 人肺微血管内皮细胞 谷胱甘肽过氧化物酶4 核因子红细胞系2相关因子2 

分 类 号：R563[医药卫生—呼吸系统]