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