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

doi:10.19982/j.issn.1000-6621.20240275

摘要/Abstract

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、Fe²⁺、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)%]、Fe²⁺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Fe²⁺[(11.70±0.91)μmol/mg vs. (17.92±1.22)μmol/mg]和脂质过氧化[(19.17±1.75)% vs. (53.28±3.01)%]水平均明显降低(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),细胞中GPX4蛋白相对表达水平(0.57±0.04 vs. 0.20±0.02)和GSH水平[(8.15±0.47)μmol/mg vs. (4.69±0.22)μmol/mg]则明显升高(t=-15.044,P<0.001;t=-9.316,P<0.001)。此外,BCG感染GSK2606414处理后的巨噬细胞,其24h和48h的菌载量[(1.72±0.15)×10⁵CFU和(1.48±0.12)×10⁵CFU]较BCG组[(3.51±0.28)×10⁵CFU和(2.94±0.21)×10⁵CFU]明显降低(t=17.576,P<0.001;t=15.225,P<0.001)。与BCG组(3.24±0.14)比较,BCG+NAC组细胞中的相对ROS水平(1.59±0.11)明显降低(t=20.215,P<0.001)。BCG感染NAC处理后的巨噬细胞,其24h和48h的菌载量[(0.91±0.12)×10⁵CFU和(0.80±0.13)×10⁵CFU]较BCG组[(2.18±0.18)×10⁵CFU和(2.37±0.21)×10⁵CFU]明显降低(t=16.672,P<0.001;t=20.630,P<0.001)。结论: BCG感染诱导的巨噬细胞铁死亡与ROS/PERK信号的激活有关。

关键词: 分枝杆菌, 结核, 巨噬细胞, 铁死亡, 活性氧

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) μmol/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)×10⁵CFU and (1.48±0.12)×10⁵CFU) compared with those in the BCG group ((3.51±0.28)×10⁵CFU and (2.94±0.21)×10⁵CFU; 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)×10⁵CFU and (0.80±0.13)×10⁵CFU) compared to those of the BCG group ((2.18±0.18)×10⁵CFU and (2.37±0.21)×10⁵CFU; 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.

Key words: Mycobacterium tuberculosis, Macrophages, Ferroptosis, Reactive oxygen species

中图分类号:

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. doi: 10.19982/j.issn.1000-6621.20240275

Chen Feifei, Zheng Yongzhi, Wu Sufang, Kang Qian, Jin Chunyang. Regulating effect of reactive oxygen species/protein kinase RNA-like endoplasmic reticulum kinase signaling axis on BCG-induced ferroptosis in mouse macrophages[J]. Chinese Journal of Antituberculosis, 2024, 46(12): 1448-1458. doi: 10.19982/j.issn.1000-6621.20240275

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