Nox4在脓毒症小鼠内皮细胞铁死亡中的表达  

Expression of Nox4 in Iron Death of Endothelial Cells in Septic Mice

作　　者：刘锋 刘楠 刘茜 杨梅[1] 马大珍 朱国际[1] LIU Feng;LIU Nan;LIU Qian;YANG Mei;MA Da-zhen;ZHU Guo-ji(Children's Hospital of Soochow University,Suzhou,Jiangsu,215003,China)

机构地区：[1]苏州大学附属儿童医院,江苏苏州215003

出　　处：《中国血液流变学杂志》2022年第4期501-505,603,共6页Chinese Journal of Hemorheology

基　　金：苏州市科技局民生科技-医疗卫生应用基础研究(SYS2020162)。

摘　　要：目的通过盲肠结扎穿孔(cecal ligation and puncture,CLP)法建立小鼠脓毒症模型,验证脓毒症小鼠相关内皮细胞存在铁死亡及探讨Nox4在铁死亡中的作用。方法用CLP复制C57BL/6小鼠的脓毒症模型。将小鼠随机分成3组:假手术(Sham)组、CLP组和CLP+铁死亡抑制剂ferrostatin-1(Fer-1)组,每组6只。Fer-1组在CLP术前1 h经腹腔注射Fer-1(10 mg/kg),分别提取血管内皮细胞,检测CLP术后12 h各组小鼠脂质过氧化水平如丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)浓度;利用DCFH-DA测定细胞内活性氧(reactive oxygen species,ROS)水平,荧光探针技术测定细胞内Fe^(2+)水平,利用ELISA检测血管内皮VEGF、ET-1、vWF表达情况;利用Western Blot检测各组细胞GPX4、Nox4的表达情况。结果与Sham组相比,CLP组术后12 h脂质过氧化物蓄积,MDA明显升高,ROS和Fe^(2+)大量蓄积,GSH明显下降;血管内皮受损,VEGF、ET-1、vWF表达明显升高;GPX4表达明显降低,Nox4表达显著升高,证明CLP可导致铁超载和铁死亡。给予Fer-1可明显降低CLP组小鼠内皮细胞内脂质过氧化物含量,减轻铁超载,缓解上述CLP小鼠的铁代谢障碍,降低细胞内ROS和Fe^(2+)水平及VEGF、ET-1、vWF表达,恢复血管内皮细胞GPX4表达以及降低Nox4表达。证明抑制铁死亡后可减轻血管内皮受损程度。结论脓毒症可引起血管内皮细胞铁死亡,导致血管屏障功能受损,Nox4作为NADPH氧化酶亚型的一种,在氧化应激及铁死亡中发挥促进作用,抑制铁死亡可降低Nox4表达,减轻血管内皮细胞损伤。Objective To establish sepsis model in mice by cecal ligation and puncture(CLP)method,verify the existence of iron death in endothelial cells related to sepsis in mice,and explore the role of Nox4 in iron death.Methods Sepsis model of C57BL/6 mice was established by CLP.The mice were randomly divided into three groups:sham operation(Sham)group,CLP group and CLP+iron death inhibitor ferrostatin-1(Fer-1)group,with 6 mice in each group.In Fer-1 group,Fer-1(10 mg/kg)was injected intraperitoneally one hour before CLP,and vascular endothelial cells were extracted respectively.The lipid peroxidation levels,such as malondialdehyde(MDA)and glutathione(GSH)concentrations,were measured 12 hours after CLP.DCFH-DA was used to measure the level of reactive oxygen species(ROS)in cells,fluorescent probe technology was used to measure the level of Fe^(2+)in cells,and ELISA was used to detect the expression of VEGF,ET-1,vWF in vascular endothelium.Western Blot was used to detect the expression of GPX4 and Nox4 in each group.Results Compared with sham group,in CLP group,lipid peroxide accumulated at 12 h,MDA increased significantly,ROS and Fe^(2+)accumulated significantly,and GSH decreased significantly.VEGF,ET-1,vWF expression increased significantly with endothelial damage.The expression of GPX4 was significantly decreased and the expression of Nox4 was significantly increased,which proved that CLP could cause iron overload and iron death.The administration of Fer-1 can significantly reduce the content of lipid peroxide in the endothelial cells of CLP group mice,reduce iron overload,alleviate the iron metabolism disorder of the CLP mice,reduce the level of ROS and Fe^(2+)in the cells and the expression of VEGF,ET-1,vWF,restore the expression of GPX4 in vascular endothelial cells and reduce the expression of Nox4.It is proved that inhibition of iron death can reduce the damage of vascular endothelium.Conclusion Sepsis can cause iron death of vascular endothelial cells,leading to impairment of vascular barrier function.As one of NA

关 键 词：脓毒症 内皮细胞 铁死亡 NOX4 

分 类 号：R631[医药卫生—外科学]