Abstract: Objective To investigate the mechanism by which free heme released from stored red blood cells exacerbates the inflammatory response through endoplasmic reticulum stress (ERS)-induced neutrophil extracellular trap formation (NETosis) in a mouse model of trauma-hemorrhagic shock, and further to assess the intervention effect of ERS inhibition on NETosis and the expression of related signaling pathway proteins. Methods This study employed a trauma-hemorrhagic shock mouse model with experimental groups including sham operation group, fresh red blood cell suspension resuscitation group, storage-damaged red blood cell resuscitation group, and free heme+ERS inhibition group, with 10 mice in each group. Body temperature, blood pressure, and pathological tissues were evaluated in each group, and the proportions of neutrophil elastase (NE) positive cells and myeloperoxidase (MPO) positive cells in peripheral blood were detected. Western Blot was used to detect the protein expression levels of protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), and activating transcription factor 4 (ATF4). Enzyme-linked immunosorbent assay (ELISA) was used to detect plasma free heme, MPO-DNA complexes, and inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) concentrations. PicoGreen fluorescent dye method was used to determine extracellular DNA levels. Results Compared with the sham surgery group, mice in the stored damaged red blood cell resuscitation group showed significantly decreased body temperature and blood pressure (P<0.01), significantly increased expression levels of ERS-related proteins PERK, eIF2α and ATF4 (all P<0.01), elevated concentrations of inflammatory factors IL-1β, TNF-α and IL-6 in serum and increased plasma free heme levels (all P<0.01), significantly increased proportions of NE and MPO positive cells in peripheral blood (P<0.01), and significantly elevated MPO-DNA complex and extracellular DNA levels (P<0.01). Compared with the stored damaged red blood cell resuscitation group, mice in the fresh red blood cell suspension resuscitation group and the free heme+ERS inhibition group showed significantly increased body temperature and blood pressure (P<0.05), significantly decreased expression levels of ERS-related proteins PERK, eIF2α and ATF4 (all P<0.01), reduced concentrations of inflammatory factors IL-1β, TNF-α and IL-6 in serum and decreased plasma free heme levels (all P<0.01), significantly reduced proportions of NE and MPO positive cells in peripheral blood (P<0.01), and significantly decreased MPO-DNA complex and extracellular DNA levels (P<0.01). Compared with the fresh red blood cell suspension resuscitation group, there were no significant differences in body temperature and blood pressure, expression levels of ERS-related proteins PERK, eIF2α and ATF4, concentrations of inflammatory factors IL-1β, TNF-α and IL-6 in serum and plasma free heme levels, proportions of NE and MPO positive cells in peripheral blood, and MPO-DNA complex and extracellular DNA levels in the free heme+ERS inhibition group mice. Pathological examination showed that organ inflammation and cell apoptosis were significant in the stored damaged red blood cell resuscitation group, while they were relatively mild in other groups. Conclusion Free heme released from red blood cell storage lesions plays a crucial role in trauma-hemorrhagic shock by significantly exacerbating NETosis and inflammatory responses through the induction of ERS. ERS intervention can effectively block this effect of free heme, reducing NETosis, decreasing levels of inflammatory factors IL-1β, TNF-α, and IL-6, as well as free heme levels, and inhibiting the activation of the PERK/eIF2α/ATF4 signaling pathway. This provides important experimental evidence and potential new strategies for targeting the free heme-ERS-NETosis axis in the treatment of inflammatory injury in trauma-hemorrhagic shock.

Key words: Endoplasmic reticulum stress, Free heme, NETosis, Trauma-hemorrhagic shock, ERS inhibition