摘要
目的探讨脓毒症肠道损伤中是否存在铁死亡,并通过核因子E2相关因子2/谷胱甘肽过氧化物酶4(Nrf2/GPX4)通路的激发和抑制来验证脓毒症肠道损伤中的铁死亡与肠道炎症、屏障功能的关联作用。方法将48只体质量220~250 g的SPF级雄性SD大鼠按随机数字表法分为假手术组(Sham组)、脓毒症组(CLP组)、脓毒症+铁螯合剂去铁胺(DFO)组(CLP+DFO组)、脓毒症+铁死亡诱导剂Erastin组(CLP+Erastin组),每组12只。采用盲肠结扎穿孔术(CLP)制备脓毒症模型;Sham组仅行开关腹操作。CLP+DFO组在制模完成后皮下注射DFO 20 mg/kg;CLP+Erastin组则腹腔注射Erastin 20 mg/kg。各组均于术后皮下注射50 mg/kg生理盐水进行液体复苏,观察大鼠术后24 h存活状况。制模后24 h,各组取6只大鼠,先活体取小肠组织用于透射电镜下观察平滑肌细胞中线粒体形态及活性氧(ROS)测定,之后行腹主动脉取血并处死;剩余6只大鼠先完成腹主动脉取血再处死,取小肠组织,分别用蛋白质免疫印迹试验(Western blotting)检测肠道损伤指标紧密连接蛋白-1(Claudin-1)和铁死亡相关蛋白GPX4、Nrf2的表达;采用苏木素-伊红(HE)染色观察小肠组织病理学改变并完成Chiu评分;采用酶联免疫吸附试验(ELISA)检测血清中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL-1β、IL-6)水平;并测定血清铁离子(Fe 3+)、丙二醛(MDA)、D-乳酸脱氢酶(D-LDH)含量。结果①与Sham组相比,CLP组和CLP+Erastin组大鼠术后24 h存活率明显下降(66.7%、50.0%比100%,均P<0.05),而CLP+DFO组差异无统计学意义(83.3%比100%,P=0.25)。②Western blotting结果显示,与Sham组相比,CLP组、CLP+DFO组、CLP+Erastin组小肠组织GPX4、Claudin-1表达明显下降,Nrf2表达明显升高(GPX4/β-actin:0.56±0.02、1.03±0.01、0.32±0.01比1.57±0.01,Claudin-1/β-actin:0.60±0.04、0.96±0.07、0.41±0.01比1.40±0.01,Nrf2/β-actin:0.88±0.02、0.72±0.01、1.14±0.01比0.43±0.02,均P<0.05)。与CLP组相比,CLP
ObjectiveTo investigate whether ferroptosis exists in sepsis induced intestinal injury,and to verify the association between ferroptosis in sepsis induced intestinal injury and intestinal inflammation and barrier function by stimulating and inhibiting the nuclear factor E2-related factor 2/glutathione peroxidase 4(Nrf2/GPX4)pathway.MethodsForty-eight SPF grade male Sprague-Darvley(SD)rats with a body weight of 220-250 g were divided into sham operation group(Sham group),sepsis group(CLP group),sepsis+iron chelating agent deferoxamine(DFO)group(CLP+DFO group)and sepsis+ferroptosis inducer Erastin group(CLP+Erastin group)using a random number table method,with 12 rats in each group.The sepsis model was established by cecal ligation and puncture(CLP).The Sham group was only performed with abdominal opening and closing operations.After modeling,the CLP+DFO group received subcutaneous injection of 20 mg/kg of DFO,the CLP+Erastin group was intraperitoneally injected with 20 mg/kg of Erastin.Each group received subcutaneous injection of 50 mg/kg physiological saline for fluid resuscitation after surgery,and the survival status of the rats was observed 24 hours after surgery.At 24 hours after model establishment,6 rats in each group were selected.First,live small intestine tissue was taken for observation of mitochondrial morphology in smooth muscle cells under transmission electron microscopy and determination of reactive oxygen species(ROS).Then,blood was collected from the abdominal aorta and euthanized.The remaining 6 rats were sacrificed after completing blood collection from the abdominal aorta,and then small intestine tissue was taken.Western blotting was used to detect the expression of intestinal injury markers such as Claudin-1 and ferroptosis related proteins GPX4 and Nrf2.Observe the pathological changes of small intestine tissue using hematoxylin-eosin(HE)staining and complete Chiu score;Detection of tumor necrosis factor-α(TNF-α),interleukins(IL-1β,IL-6)levels in serum using enzyme-linked immunosorbent a
作者
马涛
黄玮玮
李志华
王毅
高晓明
于湘友
Ma Tao;Huang Weiwei;Li Zhihua;Wang Yi;Gao Xiaoming;Yu Xiangyou(Intensive Care Medicine Center,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,Xinjiang Uygur Autonomous Region,China;Key Laboratory of Medical Animal Model Research in Xinjiang,Urumqi 830054,Xinjiang Uygur Autonomous Region,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2023年第11期1188-1194,共7页
Chinese Critical Care Medicine
基金
国家自然科学基金(82160360)
新疆维吾尔自治区科技支疆项目计划(2021E02064)
关键词
脓毒症
铁死亡
肠道损伤
线粒体形态
Sepsis
Ferroptosis
Intestinal injury
Mitochondrial morphology
