小鼠脂肪肝缺血再灌注损伤中铁死亡的作用及机制研究  

Role and mechanism of ferroptosis mediated ischemia reperfusion injury in steatosis liver

作　　者：夏煜寒 顾劲扬 邓鉴 王健东[1] Xia Yuhan;Gu Jingyang;Deng Jian;Wang Jiandong(Department of General Surgery,Xinhua Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200092,China;Liver Transplantation Center,Wuhan Union Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430022;Department of Anesthesia,Jiangsu Provincial Hospital of Traditional Chinese Medicine,Nanjing University of Chinese Medicine,Nanjing 210029,China)

机构地区：[1]上海交通大学医学院附属新华医院普外科,上海200092 [2]华中科技大学同济医学院附属协和医院肝移植中心,武汉430022 [3]南京中医药大学附属江苏省中医院麻醉科,南京210029

出　　处：《中华器官移植杂志》2023年第8期487-495,共9页Chinese Journal of Organ Transplantation

基　　金：国家自然科学基金(82130020)。

摘　　要：目的研究脂肪肝缺血再灌注损伤(ischemia reperfusion injury,IRI)中发生铁死亡的现象与机制,阐明靶向抑制铁死亡可减轻脂肪肝IRI的机制。方法先按照随机数字法取小鼠20只按照喂养方式不同分为2组:正常饮食(Normal chow diet,NCD)组(n=10)和高脂饮食(high fat diet,HFD)组(n=10),经实验室检测相关指标和油红染色验证,脂肪肝建模成功。然后另取两组小鼠分别构建假手术组(sham组,每组n=10)和缺血再灌注组(IRI组,每组n=10),免疫印迹法检测NCD-sham组(n=10)、NCD-IRI组(n=10)、HFD-sham组(n=10)和HFD-IRI组(n=10)肝组织谷胱甘肽过氧化物酶4(Gpx4)的表达水平,电子显微镜观察各组小鼠肝细胞线粒体形态等,综合判断小鼠肝脏IRI中是否存在铁死亡的发生。另取NCD小鼠和HFD小鼠按照不同手术模型分为6组:NCD-sham组(n=10)、NCD-IRI组(n=10)、NCD缺血再灌注铁死亡组(NCD-IRI-fer-1,n=10),HFD-sham组(n=10)、HFD-IRI组(n=10)、HFD缺血再灌注铁死亡组(HFD-IRI-fer-1,n=10),检测各组血清ALT、AST含量以及HE染色检测肝损伤程度;免疫组织化学染色及酶联免疫吸附试验(ELISA)检测炎症因子水平及炎性细胞浸润水平。同样,分别提取NCD、HFD小鼠原代肝细胞,根据对提取细胞的处理不同将其分为8组:NCD-sham组、NCD-Erastin组、NCD-Erastin-fer-1组、NCD-二甲基亚砜(DMSO)组,HFD-sham组、HFD-Erastin组、HFD-Erastin-fer-1组和HFD-DMSO组。通过C11-BODIPY(581/591)荧光染色检测铁死亡的发生并分析比较各组指标的数据。结果相较于NCD-IRI组,HFD-IRI组Gpx4蛋白表达水平低,血清ALT、AST较NCD-IRI组高(均P<0.01),HFD-IRI组4-羟基壬烯酸(4-HNE)的免疫组织化学染色(IHC)为阳性,线粒体特异性损伤更加明显,炎症浸润强;相较于IRI组,IRI-fer-1组血清ALT、AST水平低,炎症细胞浸润少,炎症因子分泌少(均P<0.01)。结论小鼠脂肪肝缺血再灌注(ischemia reperfusion,IR)过程中发生的高水平铁死亡及剧烈炎症反应是脂�Objective To explore the phenotype and mechanism of ferroptosis in steatosis liver ischemia reperfusion injury(IRI)and elucidate the mechanism of targeted inhibition of ferroptosis down-regulating steatosis liver IRI.Methods First,20 mice are divided into 2 group according to the different feeding mode:Normal chow diet(NCD group,n=10)and high fat diet(HFD group,n=10).The fatty liver constructs are successful as verified by laboratory tests for relevant indicators and oil red staining.NCD and HFD mice were randomized into two groups sham and IRI.The expression of glutathione peroxidase 4(Gpx4)in liver tissues of each group is detected by Western blot.Morphological phenotypes of mitochondria in liver tissue are observed by transmission electron microscope.Accordingly,it was determined whether ferroptosis occurred in mouse liver IRIs.NCD(n=30)and HFD(n=30)are further randomized into 3 groups:sham(n=10),IRI(n=10)and inhibitor(IRI-fer-1 group,n=10).The serum contents of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)are detected by ALT/AST kits.Hematoxylin-eosin(HE)stain is employed for detecting the degree of liver injury;immunohistochemistry(IHC)and enzyme-linked immunosorbent assay(ELISA)for detecting the levels of inflammatory factors and inflammatory cell infiltration.Similarly,primary hepatocytes from NCD/HFD mice are extracted and the occurrence of ferroptosis is verified by detecting C11-BODIPY(581/591)by fluorescent stain after hypoxia-reoxygenation(H/R).Results As compared with NCD-IRI group,Gpx4 protein expression declined obviously in HFD-IRI group while serum ALT/AST level spiked markedly(P<0.01).IHC staining of 4-HNE is positive,mitochondrial specific damage is more pronounced and inflammatory infiltration became enhanced.As compared with IRI group,serum level of ALT/AST dropped obviously and infiltration of inflammatory cells and secretion of inflammatory factors are blunted markedly in IRI-fer-1 group(P<0.01).Conclusions A high degree of ferroptosis and severe inflammatory response dur

关 键 词：脂肪肝 缺血再灌注损伤 铁死亡 

分 类 号：R575.5[医药卫生—消化系统]