中国防痨杂志2024，Vol.46Issue(12)：1448-1458,11.DOI:10.19982/j.issn.1000-6621.20240275
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
摘要
Abstract
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,Fe2+,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)％),Fe2+((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),Fe2+((11.70±0.91)μmnol/mg vs.(17.92±1.22)μmol/mg)and lipid peroxidation((19.17±1.75)％vs.(53.28±3.01)％)all significantly reduced(t=17.177,P＜0.001;t=21.024,P＜0.001;t=19.358,P＜0.001;t=11.999,P＜0.001;t=30.292,P＜0.001,respectively),while GPX4 protein relative expression(0.57±0.04 vs.0.20±0.02)and GSH level((8.15±0.47)μmol/mg vs.(4.69±0.22)μmol/mg)were significantly elevated in cells(t=-15.044,P＜0.001;t=-9.316,P＜0.001).In addition,BCG-infected GSK2606414-treated macrophages had significantly lower bacterial loads at 24 h and 48 h((1.72±0.15)×105 CFU and(1.48±0.12)×105 CFU)compared with those in the BCG group((3.51±0.28)× 105 CFU and(2.94±0.21)X105 CFU;t=17.576,P＜0.001;t=15.225,P＜0.001).The relative level of ROS in the cells of the BCG+NAC group(1.59±0.11)was significantly lower compared to the BCG group(3.24±0.14,t=20.215,P＜0.001).The bacterial loads of BCG-infected NAC-treated macrophages were significantly lower at 24 and 48 h((0.91±0.12)×105 CFU and(0.80±0.13)×105 CFU)compared to those of the BCG group((2.18±0.18)×105 CFU and(2.37±0.21)×105 CFU;t=16.672,P＜0.001;t=20.630,P＜0.001).Conclusion:BCG infection-induced macrophage ferroptosis is associated with activation of ROS/PERK signaling.关键词
分枝杆菌,结核/巨噬细胞/铁死亡/活性氧Key words
Mycobacterium tuberculosis/Macrophages/Ferroptosis/Reactive oxygen species分类
医药卫生引用本文复制引用
陈飞飞,郑永智,吴素芳,康乾,晋春阳..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[J].中国防痨杂志,2024,46(12):1448-1458,11.基金项目
Henan Provincial Department of Education(23A360023) 河南省教育厅项目(23A360023) （23A360023）
