摘要
目的:分析在脓毒症诱导的小鼠肺上皮细胞系MLE-12凋亡和小鼠急性肺损伤(ALI)过程中铁死亡的作用,并探讨谷胱甘肽过氧化物酶4(GPX4)在这一过程的调节作用。方法:体外实验1将MLE-12细胞分为对照组、铁死亡抑制剂ferrostatin-1(Fer-1)组、脂多糖(LPS)组和LPS+Fer-1组,以考察铁死亡参与LPS诱导的MLE-12细胞凋亡;实验2将细胞分为:载体组和GPX4组,以考察GPX4对LPS刺激诱导的MLE-12细胞损伤的影响;实验3将细胞分为:si-WTAP+si-NC组和si-WTAP+si-GPX4组,以考察Wilms肿瘤1相关蛋白(WTAP)对GPX4驱动的铁死亡影响。LPS刺激MLE-12细胞建立体外模型,然后将细胞用Fer-1和转染GPX4过表达质粒、WTAP特异性siRNA(si-WTAP)、GPX4特异性siRNA(si-GPX4)进行处理。分别通过CCK-8测定细胞活力,流式细胞术分析细胞凋亡,DCFH-DA测定细胞内活性氧(ROS)水平,荧光探针测定细胞内Fe^(2+)水平和Western blot分析GPX4和WTAP表达水平。体内实验将小鼠随机分为4组:对照组、假手术组、盲肠结扎穿刺(CLP)12 h组和CLP 24 h组,每组12只。进行H&E染色以评估小鼠的肺损伤,并采用试剂盒法检测肺匀浆中的铁含量、丙二醛(MDA)和谷胱甘肽(GSH)水平。结果:与对照组相比,随着LPS暴露时间的延长,细胞中ROS水平和Fe^(2+)水平显著增加(P<0.05),铁死亡标志物GPX4的蛋白表达逐渐降低,WTAP蛋白水平以及WTAP/GPX4比例显著增加(P<0.05)。相对于LPS组,在LPS+Fer-1组中细胞活力显著增加(P<0.05),细胞凋亡百分率、ROS水平和Fe^(2+)水平显著降低(P<0.05)。与载体组相比,GPX4组细胞中GPX4表达及细胞活力显著增加(P<0.05),细胞凋亡百分比、ROS水平和Fe^(2+)水平显著降低(P<0.05)。与si-WTAP+si-NC组相比,si-WTAP+si-GPX4组MLE-12细胞中ROS水平和Fe^(2+)水平显著增加(P<0.05)。与假手术组相比,CLP 12 h组和CLP 24 h组小鼠肺组织学评分和肺匀浆中铁含量、MDA水平和WTAP蛋白表达显著升高(P<0.05),肺组织中GPX4蛋白�
AIM:To analyze the role of ferroptosis in the apoptosis of mouse lung epithelial cell line MLE-12and acute lung injury(ALI)in mice induced by sepsis,and to explore the regulatory role of glutathione peroxidase 4(GPX4)in these processes.METHODS:For in vitro experiment 1,the MLE-12 cells were divided into control group,ferroptosis inhibitor ferrostatin-1(Fer-1)group,lipopolysaccharide(LPS)group and LPS+Fer-1 group to investigate the participation of ferroptosis in LPS-induced MLE-12 cell apoptosis. For experiment 2,the cells were divided into vector group and GPX4 group to investigate the effect of GPX4 on MLE-12 cell damage induced by LPS stimulation. For experiment 3,the cells were divided into si-WTAP(Wilms tumor 1-associated protein)+si-NC group and si-WTAP+si-GPX4group to investigate the effect of WTAP on GPX4-driven ferroptosis. The MLE-12 cells were stimulated by LPS to build an in vitro model,and then the cells were treated with Fer-1 or transfected with GPX4 overexpression plasmid(GPX4),WTAP-specific siRNA(si-WTAP)and GPX4-specific siRNA(si-GPX4). Cell viability was determined by CCK-8 assay,apoptosis was analyzed by flow cytometry,intracellular reactive oxygen species(ROS)levels were determined by DCFHDA,intracellular Fe^(2+)levels were determined by fluorescent probes,and GPX4 and WTAP expression levels were analyzed by Western blot. For in vivo experiments,C57BL/6J mice were randomly divided into 4 groups:control group,sham surgery group,cecal ligation and puncture(CLP)12 h group,and CLP 24 h group,with 12 individuals in each group.HE staining was performed to assess lung damage in mice,and the iron,malondialdehyde(MDA)and glutathione(GSH)levels in lung homogenates were measured using the kit method.RESULTS:Compared with control group,GPX4,the marker of ferroptosis,was decreased gradually,and the protein level of WTAP and WTAP/GPX4 ratio were increased significantly with the prolonged LPS exposure time(P<0. 05). Compared with LPS group,cell viability was significantly increased in LPS+Fer-1 group(P<0. 0
作者
柳红英
丁璐
王卉
范桄溥
LIU Hong-ying;DING Lu;WANG Hui;FAN Guang-pu(Departemnt of Critical Care Medicine,Peking University People's Hospital,Beijing 100044,China;Departemnt of Cardiology,Peking University People's Hospital,Beijing 100044,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2022年第9期1659-1666,共8页
Chinese Journal of Pathophysiology
基金
北京市卫生健康科研基金项目(No.20200081)。
