活性氧/蛋白激酶RNA样内质网激酶信号轴对卡介苗诱导RAW264.7小鼠巨噬细胞铁死亡的调控作用  

Regulating effect of reactive oxygen species/protein kinase RNA-like endoplasmic reticulum kinase signaling axis on BCG-induced ferroptosis in mouse macrophages

作　　者：陈飞飞[1] 郑永智[2] 吴素芳 康乾 晋春阳 Chen Feifei;Zheng Yongzhi;Wu Sufang;Kang Qian;Jin Chunyang(Department of Tuberculosis,Henan Provincial Chest Hospital,Zhengzhou 450000,China;Department of Cartilage,Henan Provincial Hospital of Traditional Chinese Medicine,Zhengzhou 450000,China)

机构地区：[1]河南省胸科医院结核病科,郑州450000 [2]河南省中医院关节科,郑州450000

出　　处：《中国防痨杂志》2024年第12期1448-1458,共11页Chinese Journal of Antituberculosis

基　　金：河南省教育厅项目(23A360023)。

摘　　要：目的:探讨活性氧(reactive oxygen species,ROS)/蛋白激酶RNA样内质网激酶(protein kinase RNA-like endoplasmic reticulum kinase,PERK)信号通路对卡介苗(BCG)感染引起的RAW264.7小鼠巨噬细胞铁死亡的调控作用。方法:采用铁死亡螯合剂去铁胺(deferoxamine,DFO)研究铁死亡在BCG感染诱导细胞死亡中的作用,将RAW264.7细胞分为对照组、BCG组、DFO组、BCG+DFO组;采用PERK抑制剂GSK2606414研究PERK信号通路在BCG感染诱导细胞铁死亡中的作用,将RAW264.7细胞分为对照组、BCG组、GSK2606414组、BCG+GSK2606414组;采用ROS清除剂N-乙酰半胱氨酸(N-acetylcysteine,NAC)观察ROS在调控PERK中的作用,将RAW264.7细胞分为对照组、BCG组、NAC组和BCG+NAC组。通过比色法检测细胞乳酸脱氢酶(lactate dehydrogenase,LDH)释放率、Fe^(2+)、谷胱甘肽(glutathione,GSH)及脂质过氧化水平;通过蛋白免疫印迹法检测谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、磷酸化PERK(p-PERK)、活化转录因子4(activating transcription factor 4,ATF4)及C/EBP同源蛋白(C/EBP-homologous protein,CHOP)表达水平;通过流式细胞术检测ROS含量;通过免疫荧光法检测p-PERK蛋白水平;通过菌落形成单位(colony-forming unit,CFU)实验计算BCG在细胞内的存活。结果:与BCG组比较,BCG+DFO组细胞存活率[(84.72±3.62)%vs.(58.41±2.73)%]、GSH水平[(8.85±0.54)μmol/mg vs.(4.81±0.36)μmol/mg]和GPX4蛋白相对表达水平(0.82±0.06 vs.0.33±0.03)升高(t=-4.263,P=0.004;t=-10.116,P<0.001;t=-10.519,P<0.001),LDH释放率[(15.70±3.18)%vs.(56.24±4.98)%]、Fe^(2+)含量[(8.15±0.64)μmol/mg vs.(18.68±1.27)μmol/mg]和脂质过氧化水平[(22.18±2.24)%vs.(58.13±4.47)%]均明显降低(t=35.982,P<0.001;t=20.203,P<0.001;t=32.528,P<0.001)。与BCG组比较,BCG+GSK2606414组细胞中p-PERK蛋白相对表达水平(0.23±0.02 vs.0.69±0.04)、ATF4蛋白相对表达水平(0.39±0.02 vs.0.91±0.06)和CHOP蛋白相对表达水平(0.24±0.03 vs.0.61±0.04),以及Fe^(2+)[(11.70±0.91)�Objective:To investigate the role of reactive oxygen species(ROS)/protein kinase RNA-like endoplasmic reticulum kinase(PERK)signaling pathway in the regulation of ferroptosis in RAW264.7 macrophages induced by BCG infection.Methods:The ferroptosis inhibitor deferoxamine(DFO)was used to investigate the role of ferroptosis in BCG infection-induced apoptosis by dividing RAW264.7 cells into control,BCG,DFO,and BCG+DFO groups;the PERK inhibitor GSK2606414 was used to investigate the role of PERK signaling pathway in BCG infection-induced cell ferroptosis by dividing RAW264.7 cells into control,BCG,GSK2606414,and BCG+GSK2606414 groups.The ROS scavenger N-acetylcysteine(NAC)was used to investigate the role of ROS in the regulation of PERK by dividing RAW264.7 cells into control,BCG,NAC and BCG+NAC groups.Colorimetric assay was used to detect cellular lactate dehydrogenase(LDH)release rate,Fe^(2+),glutathione(GSH)and lipid peroxidation concentration.Immunoblotting was used to detect expression of glutathione peroxidase 4(GPX4),phosphorylated PERK(p-PERK),activating transcription factor 4(ATF4),and C/EBP-homologous protein(CHOP);flow cytometry was used to detect ROS concentration.Immunofluorescence was used to detect p-PERK protein concentration.Colony-forming unit(CFU)assay was used to calculate the survival of BCG in cells.Results:Compared with the BCG group,the BCG+DFO group had elevated cell survival rate((84.72±3.62)%vs.(58.41±2.73)%,t=-4.263,P=0.004),GSH level((8.85±0.54)μmol/mg vs.(4.81±0.36)μmol/mg,t=-10.116,P<0.001),and relative GPX4 protein expression level(0.82±0.06 vs.0.33±0.03,t=-10.519,P<0.001);LDH release rate((15.70±3.18)%vs.(56.24±4.98)%),Fe^(2+)((8.15±0.64)μmol/mg vs.(18.68±1.27)μmol/mg)and lipid peroxidation level((22.18±2.24)%vs.(58.13±4.47)%)were significantly reduced(t=35.982,P<0.001;t=20.203,P<0.001;t=32.528,P<0.001).Compared with the BCG group,the BCG+GSK2606414 group had p-PERK(0.23±0.02 vs.0.69±0.04),ATF4(0.39±0.02 vs.0.91±0.06),CHOP protein(0.24±0.03 vs.0.61±0.04),Fe^(2+)((

关 键 词：分枝杆菌 结核 巨噬细胞 铁死亡 活性氧 

分 类 号：R34[医药卫生—基础医学]