脱嘌呤/脱嘧啶脱氧核糖核酸内切酶1对模拟脓毒症状态下小鼠树突状细胞铁死亡的作用  

Role of apurinic/apyrimidinic endodeoxyribonuclease 1 in ferroptosis of mouse dendritic cells under simulated sepsis

作　　者：周岐原 李京宴 姚咏明[2] 田英平[1] Zhou Qiyuan;Li Jingyan;Yao Yongming;Tian Yingping(Emergency Department,the Second Hospital of Hebei Medical University,Shijiazhuang 050000,China)

机构地区：[1]河北医科大学第二医院急诊医学科,石家庄050000 [2]解放军总医院医学创新研究部、第四医学中心,北京100853

出　　处：《中华烧伤与创面修复杂志》2024年第10期930-939,共10页Chinese Journal of Burns And Wounds

基　　金：国家自然科学基金重点项目(82130062,82241062);国家自然科学基金青年科学基金项目(82302412);北京市自然科学基金(7244296)。

摘　　要：目的探讨脱嘌呤/脱嘧啶脱氧核糖核酸内切酶1(APE1)对模拟脓毒症状态下小鼠树突状细胞(DC)铁死亡的作用,为改善创面感染等引起的脓毒症免疫抑制提供依据。方法该研究为实验研究。取第3~10代对数生长期的小鼠DC系DC2.4进行研究(样本数均为3),采用1μg/mL内毒素/脂多糖(LPS,浓度下同)处理DC 0(未处理)、6、12、24、48、72 h构建脓毒症模型,采用蛋白质印迹法检测细胞中APE1及抗铁死亡蛋白谷胱甘肽过氧化物酶4(GPX4)、溶质载体家族7成员11(SLC7A11)蛋白表达,采用流式细胞术检测细胞中活性氧水平,采用活细胞成像技术检测细胞脂质过氧化水平;将成功转染含APE1基因短发夹RNA序列慢病毒的DC分为敲减APE1+磷酸盐缓冲液(PBS)组、敲减APE1+LPS组,将成功转染空载慢病毒的DC分为空载体+PBS组、空载体+LPS组,给予PBS或LPS刺激并培养24 h后同前行相应检测;将成功转染含APE1基因过表达RNA序列慢病毒的DC分为过表达APE1+PBS组、过表达APE1+LPS组,将成功转染空载慢病毒的DC分为空载体+PBS组、空载体+LPS组,给予PBS或LPS刺激并培养24 h后同前行相应检测。将88只6~8周龄雄性C57BL/6J小鼠按随机数字表法分为玉米油+假伤组、玉米油+盲肠结扎穿孔(CLP)组、抑制剂+假伤组、抑制剂+CLP组,每组22只。对2个抑制剂组小鼠按照40 mg/kg每日灌胃1 mg/mL APE1抑制剂E3330,对2个玉米油组小鼠每日灌胃等量玉米油,2周后对2个CLP组小鼠行CLP术构建脓毒症模型,对2个假伤组小鼠行假手术。从4组小鼠中各选取16只,观察术后7 d内存活情况;术后24 h采用CD11c阳选磁珠提取4组分别剩余的6只小鼠脾脏DC同前行相应检测(样本数均为3)。结果与LPS处理0 h比较,LPS处理6 h时细胞中APE1蛋白表达显著升高(P<0.05),LPS处理24、48、72 h时细胞中APE1与GPX4蛋白表达及LPS处理24 h时细胞中SLC7A11蛋白表达均显著降低(P<0.05),LPS处理24、48、72 h时细胞中活性Objective To investigate the role of apurinic/apyrimidinic endodeoxyribonuclease 1(APE1)in ferroptosis of mouse dendritic cells(DCs)under simulated sepsis,providing evidence for improving immunosuppression in sepsis caused by wound infection.MethodsThis study was an experimental research.The mouse DC line DC2.4 in the logarithmic growth phase(with passages 3-10)was used for the experiments(with each sample size of 3).The sepsis model was established by treating DCs with 1μg/mL lipopolysaccharide(LPS,same concentration throughout)for 0(untreated),6,12,24,48,and 72 h.Western blotting was used to detect the protein expressions of APE1 and anti-ferroptosis proteins glutathione peroxidase 4(GPX4)and solute carrier family 7 member 11(SLC7A11)in cells,flow cytometry was employed to detect reactive oxygen species(ROS)levels in cells,and live-cell imaging technology was used to measure cell lipid peroxidation levels.DCs successfully transfected with lentivirus containing APE1 short hairpin RNA sequence were divided into APE1-knockdown+phosphate buffer solution(PBS)group and APE1-knockdown+LPS group.DCs successfully transfected with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group.After stimulation with PBS or LPS and 24 h of culture,corresponding assays were conducted as above.DCs transfected with lentivirus containing APE1 overexpression RNA sequence were divided into APE1-overexpression+PBS group and APE1-overexpression+LPS group.DCs transfected with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group.After stimulation with PBS or LPS and 24 h of culture,corresponding assays were conducted as above.A total of 88 male C57BL/6J mice aged 6-8 weeks were divided into corn oil+sham injury group,corn oil+cecal ligation and puncture(CLP)group,inhibitor+sham injury group,and inhibitor+CLP group(n=22)according to the random number table.Mice in the two inhibitor groups were gavaged with APE1 inhibitor E3330(1 mg/mL in concentration)at 40 mg/kg per day,while mi

关 键 词：脓毒症 免疫 树突细胞 氧化性应激 铁死亡 脱嘌呤/脱嘧啶脱氧核糖核酸内切酶1 

分 类 号：R459.7[医药卫生—急诊医学]