干扰素基因刺激因子对脓毒症状态下小鼠树突状细胞内酰基辅酶A合成酶长链家族成员4介导铁死亡的影响  

Effects of stimulator of interferon gene on ferroptosis mediated by acyl-CoA synthetase long-chain family member 4 in mouse dendritic cells under sepsis

作　　者：吴梦瑶 贺鹏翼 段昱 郑丽玉 姚人骐 周岐原 陈钰[1] 董宁[1] 吴瑶 姚咏明[1] Wu Mengyao;He Pengyi;Duan Yu;Zheng Liyu;Yao Renqi;Zhou Qiyuan;Chen Yu;Dong Ning;Wu Yao;Yao Yongming(Medical Innovation Research Division and the Fourth Medical Center of PLA General Hospital,Beijing 100853,China;Department of General Surgery,the First Medical Center of PLA General Hospital,Beijing 100853,China)

机构地区：[1]解放军总医院医学创新研究部、第四医学中心,北京100853 [2]解放军总医院第一医学中心普通外科,北京100853

出　　处：《中华烧伤与创面修复杂志》2024年第10期920-929,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);解放军总医院青年自主创新科学基金扶持项目(22QNFC017)。

摘　　要：目的探讨干扰素基因刺激因子(STING)对脓毒症状态下小鼠树突状细胞(DC)内酰基辅酶A合成酶长链家族成员4(ACSL4)介导铁死亡的影响,为改善由创面感染等因素引发的脓毒症免疫应答失调提供依据。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,按随机数字表法(分组方法下同)分为内毒素/脂多糖(LPS)刺激0 h(不刺激)组、LPS刺激6 h组、LPS刺激12 h组、LPS刺激18 h组和LPS刺激24 h组,用1μg/mL LPS(浓度下同)培养相应时间后,采用蛋白质印迹法检测细胞中磷酸化STING(p-STING)、STING和ACSL4的蛋白表达;将成功转染含STING基因小干扰RNA(下称siSTING)慢病毒的DC2.4分为siSTING+磷酸盐缓冲液(PBS)组、siSTING+LPS组,将成功转染空载慢病毒的DC2.4分为空载体+PBS组、空载体+LPS组,给予PBS或LPS刺激并培养24 h,同前检测细胞中p-STING、STING和ACSL4的蛋白表达,采用脂质过氧化检测试剂盒观察细胞脂质过氧化程度,采用流式细胞术检测细胞凋亡率。以上细胞实验中样本数均为3。将80只6~8周龄雄性C57BL/6J小鼠分为假手术+生理盐水组、盲肠结扎穿孔(CLP)+生理盐水组、假手术+C-176组、CLP+C-176组,每组20只。对2个C-176组小鼠先经腹腔注射C-176、2个生理盐水组小鼠先经腹腔注射等量生理盐水,1 h后对2个假手术组小鼠行假手术、对2个CLP组小鼠行CLP术构建脓毒症模型。术后24 h,将每组10只小鼠处死后提取脾脏DC,同前行蛋白表达、脂质过氧化程度、凋亡率检测(样本数均为3);另行苏木精-伊红染色观察小鼠心、肝、肺、肾病理组织损伤。观察各组剩余10只小鼠术后7 d内存活情况。结果LPS刺激24 h组DC2.4中p-STING、STING、ACSL4的蛋白表达及p-STING/STING比值均明显高于LPS刺激0 h组(P<0.05)。培养24 h后,siSTING+LPS组和空载体+PBS组DC2.4中p-STING、STING和ACSL4的蛋白表达均明显低于空载体+LPS组(P<0.05);siSTING+LPS组和空载体+PBS组Objective To investigate the effects of stimulator of interferon gene(STING)on ferroptosis mediated by acyl-CoA synthetase long-chain family member 4(ACSL4)in mouse dendritic cells(DCs)under sepsis,providing a basis for improving the dysregulation of immune response in sepsis caused by factors such as wound infection.MethodsThis study was an experimental research.The mouse DC line DC2.4 in the logarithmic growth phase(with passages 3-10)were divided into lipopolysaccharide(LPS)stimulation 0 h(unstimulated)group,LPS stimulation 6 h group,LPS stimulation 12 h group,LPS stimulation 18 h group,and LPS stimulation 24 h group according to the random number table(the same grouping method below),which were cultured with 1μg/mL LPS(the same concentration below)for the corresponding time.The protein expressions of phosphorylated STING(p-STING),STING,and ACSL4 in cells were determined by Western blotting.DC2.4 successfully transfected with lentivirus containing STING gene small interfering RNA(hereinafter referred to as siSTING)were divided into siSTING+phosphate buffer solution(PBS)group and siSTING+LPS group.DC2.4 successfully transfected with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group.After being stimulated with PBS or LPS and cultured for 24 hours,the protein expressions of p-STING,STING,and ACSL4 in cells were determined as above.Cell lipid peroxidation degrees were observed using the lipid peroxidation assay kit,and cell apoptosis rates were detected using flow cytometry.The sample numbers in the above cell experiments were all 3.Eighty male C57BL/6J mice aged 6 to 8 weeks were divided into sham surgery+normal saline(NS)group,cecal ligation and puncture(CLP)+NS group,sham surgery+C-176 group,and CLP+C-176 group,with 20 mice in each group.Mice in the two C-176 groups were intraperitoneally injected with C-176,while mice in the two NS groups were intraperitoneally injected with an equivalent volume of NS.One hour later,sham surgery was performed on the mice in the two sham surg

关 键 词：脓毒症 免疫调节 细胞死亡 树突细胞 铁死亡 干扰素基因刺激因子 酰基辅酶A合成酶长链家族成员4 

分 类 号：R459.7[医药卫生—急诊医学]