红细胞储存损伤释放的游离血红素通过内质网应激诱导NETosis加重创伤-失血性休克小鼠模型炎症反应的作用机制研究*

doi:10.3969/j.issn.1671-2587.2025.05.002

临床输血与检验 ›› 2025, Vol. 27 ›› Issue (5): 585-593.DOI: 10.3969/j.issn.1671-2587.2025.05.002

红细胞储存损伤释放的游离血红素通过内质网应激诱导NETosis加重创伤-失血性休克小鼠模型炎症反应的作用机制研究^*

冯子阳, 黄韦华, 唐河山, 查占山

海军军医大学第一附属医院输血科,上海 200433

收稿日期:2025-09-02 修回日期:2025-09-12 出版日期:2025-10-20 发布日期:2025-10-11
通讯作者: 查占山,主要从事血液免疫研究工作,（E-mail）chazhanshan@163.com。
作者简介:冯子阳,主要从事临床输血治疗工作,（E-mail）ziyangw@126.com。
基金资助:
^*本项目受国家自然科学基金（No.82200257）、海军军医大学第一附属医院固海计划项目（No.GH-145-37）资助

Study on the Mechanism by Which NETosis Exacerbates Inflammation in a Traumatic-hemorrhagic Shock Mouse Model due to Free Heme from Red Blood Cell Storage Injury Via Endoplasmic Reticulum Stress

FENG Ziyang, HUANG Weihua, TANG Heshan, ZHA Zhanshan

Department of Transfusion Medicine, The Fist Affiliated Hospital of Naval Medical University, Shanghai 200433

Received:2025-09-02 Revised:2025-09-12 Online:2025-10-20 Published:2025-10-11

摘要/Abstract

摘要： 目的探讨红细胞储存损伤释放的游离血红素在创伤-失血性休克小鼠模型中,通过内质网应激（ERS）诱导中性粒细胞胞外诱捕网形成（NETosis）加重炎症反应的机制,进一步评估ERS抑制对NETosis及相关信号通路蛋白表达的干预效果。方法本研究采用创伤-失血性休克小鼠模型,实验分组包括假手术组、新鲜红细胞悬液复苏组、储存损伤红细胞复苏组以及游离血红素+ERS抑制组,每组10只。对各组体温、血压和病理组织进行评估,并检测外周血中中性粒细胞弹性蛋白酶（NE）阳性细胞和髓过氧化物酶（MPO）阳性细胞的比例。蛋白免疫印迹法（Western Blot）用于检测蛋白激酶R样内质网激酶（PERK）、真核翻译起始因子2α（eIF2α）和激活转录因子4（ATF4）蛋白表达水平。采用酶联免疫吸附试验（ELISA）检测血浆游离血红素、MPO-DNA复合物和炎症因子白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）浓度,采用PicoGreen荧光染料法测定细胞外DNA水平。结果与假手术组相比,储存损伤红细胞复苏组小鼠体温和血压显著降低（P<0.01）,ERS相关蛋白PERK、eIF2α和ATF4的表达水平显著增高（P均<0.01）,血清中IL-1β、TNF-α和IL-6等炎症因子浓度及血浆游离血红素水平升高（P均<0.01）,外周血中NE和MPO阳性细胞比例显著增高（P<0.01）,MPO-DNA复合物和细胞外DNA水平显著升高（P<0.01）。与储存损伤红细胞复苏组相比,新鲜红细胞悬液复苏组和游离血红素+ERS抑制组小鼠体温和血压明显升高（P<0.05）,ERS相关蛋白PERK、eIF2α和ATF4的表达水平显著降低（P均<0.01）,血清中IL-1β、TNF-α和IL-6等炎症因子浓度及血浆游离血红素水平降低（P均<0.01）,外周血中NE和MPO阳性细胞比例显著降低（P<0.01）,MPO-DNA复合物和细胞外DNA水平显著下降（P<0.01）。与新鲜红细胞悬液复苏组相比,游离血红素+ERS抑制组小鼠体温和血压、ERS相关蛋白PERK、eIF2α和ATF4的表达水平、血清中IL-1β、TNF-α和IL-6等炎症因子浓度及血浆游离血红素水平、外周血中NE和MPO阳性细胞比例、MPO-DNA复合物和细胞外DNA水平均无显著性差异。病理显示储存损伤红细胞复苏组器官炎症和细胞凋亡显著,而其他组较轻微。结论红细胞储存损伤释放的游离血红素在创伤-失血性休克过程中发挥关键作用,可通过诱导ERS显著加剧NETosis和炎症反应。ERS干预能够有效阻断游离血红素的这一效应,减轻NETosis、降低炎症因子IL-1β、TNF-α和IL-6浓度及游离血红素水平,并抑制PERK/eIF2α/ATF4信号通路的激活。这为靶向游离血红素-ERS-NETosis轴治疗创伤-失血性休克中的炎性损伤提供了重要的实验依据和潜在新策略。

关键词: 内质网应激, 游离血红素, NETosis, 创伤-失血性休克, ERS抑制

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

中图分类号:

R457.1⁺3

冯子阳, 黄韦华, 唐河山, 查占山. 红细胞储存损伤释放的游离血红素通过内质网应激诱导NETosis加重创伤-失血性休克小鼠模型炎症反应的作用机制研究^*[J]. 临床输血与检验, 2025, 27(5): 585-593.

FENG Ziyang, HUANG Weihua, TANG Heshan, ZHA Zhanshan. Study on the Mechanism by Which NETosis Exacerbates Inflammation in a Traumatic-hemorrhagic Shock Mouse Model due to Free Heme from Red Blood Cell Storage Injury Via Endoplasmic Reticulum Stress[J]. JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE, 2025, 27(5): 585-593.

参考文献

[1] 邓慧敏,徐凤程,李梅婷,等. 孕产妇住院期间不同红细胞输注量的血液保护措施应用及对产后结局的影响[J]. 中国输血杂志,2025,38(5):691-698.
[2] CHAUDHARY R, KATHARIA R.Oxidative injury as contributory factor for red cells storage lesion during twenty eight days of storage[J]. Blood Transfus, 2012, 10(1):59-62.
[3] 吴晓双,安宁,陈要臻,等. 游离血红素直接致肝细胞损伤的研究[J]. 临床输血与检验,2023,25(4):444-449.
[4] CHEN X,CUBILLOS-RUIZ J R. Endoplasmic reticulum stress signals in the tumour and its microenvironment[J]. Nat Rev Cancer,2021,21(2):71-88.
[5] PU L,YI F,YU W J,et al.Endoplasmic reticulum stress mediates environmental particle-induced inflammatory response in bronchial epithelium[J]. J Immunotoxicol, 2023,20(1):2229428.
[6] LIU B C,DENG Y X,DUAN Z H,et al.Neutrophil extracellular traps promote intestinal barrier dysfunction by regulating macrophage polarization during trauma/hemorrhagic shock via the TGF-β signaling pathway[J]. Cell Signal,2024,113:110941.
[7] ZHANG H,WANG Y,QU M D,et al.Neutrophil, neutrophil extracellular traps and endothelial cell dysfunction in sepsis[J]. Clin Transl Med,2023,13(1): e1170.
[8] CHU C N,WANG X Y,YANG C,et al.Neutrophil extracellular traps drive intestinal microvascular endothelial ferroptosis by impairing Fundc1-dependent mitophagy[J]. Redox Biol,2023,67:102906.
[9] CHOI Y,LEE E G,JEONG J H,et al.4-Phenylbutyric acid, a potent endoplasmic reticulum stress inhibitor, attenuates the severity of collagen-induced arthritis in mice via inhibition of proliferation and inflammatory responses of synovial fibroblasts[J]. Kaohsiung J Med Sci,2021,37(7): 604-615.
[10] 郭雨阳. 血红素通过内质网应激诱导主动脉瓣瓣膜间质细胞成骨表型转变机制研究[D]. 广州:南方医科大学, 2021.
[11] KOLB P S,AYAUB E A,ZHOU W,et al.The therapeutic effects of 4-phenylbutyric acid in maintaining proteostasis[J]. Int J Biochem Cell Biol,2015,61:45-52.
[12] XIAO C T,GIACCA A,LEWIS G F.Sodium phenylbutyrate, a drug with known capacity to reduce endoplasmic reticulum stress,partially alleviates lipid-induced insulin resistance and beta-cell dysfunction in humans[J]. Diabetes, 2011,60(3):918-924.
[13] 卞晓华,闫雁,董士民. 碳酸氢钠林格液对创伤失血性休克炎症因子及血栓弹力图的影响[J]. 实用休克杂志(中英文),2021,5(5):277-282.
[14] SIERRA F D A,MELZAK K A,JANETZKO K,et al. Flow morphometry to assess the red blood cell storage lesion[J]. Cytometry A,2017,91(9):874-882.
[15] SHAH A M,ZAMORA R,KORFF S,et al.Inferring tissue-specific,TLR4-dependent type 17 immune interactions in experimental trauma/hemorrhagic shock and resuscitation using computational modeling[J]. Front Immunol,2022,13:908618.
[16] ESSER P R,HUBER M,MARTIN S F.Endoplasmic reticulum stress and the inflammatory response in allergic contact dermatitis[J]. Eur J Immunol,2023,53(7): e2249984.
[17] LUÍS A,HACKL M,JAFARMADAR M,et al. Circulating miRNAs associated with ER stress and organ damage in a preclinical model of trauma hemorrhagic shock[J]. Front Med,2020,7:568096.
[18] MI L,MIN X B,SHI M M,et al.Neutrophil extracellular traps aggravate neuronal endoplasmic reticulum stress and apoptosis via TLR9 after traumatic brain injury[J]. Cell Death Dis,2023,14(6):374.
[19] SUN S L,DUAN Z H,WANG X Y,et al.Neutrophil extracellular traps impair intestinal barrier functions in sepsis by regulating TLR9-mediated endoplasmic reticulum stress pathway[J]. Cell Death Dis,2021,12(6): 606.
[20] CUI Y,HONG S B,XIA Y H,et al.Melatonin engineering M2 macrophage-derived exosomes mediate endoplasmic reticulum stress and immune reprogramming for periodontitis therapy[J]. Adv Sci,2023,10(27):e2302029.
[21] MANDULA J K,CHANG S,MOHAMED E,et al. Ablation of the endoplasmic reticulum stress kinase PERK induces paraptosis and typeⅠ interferon to promote anti-tumor T cell responses[J]. Cancer Cell, 2022,40(10):1145-1160.e9.
[22] LIU A T,CHEN Z W,LI X X,et al.C5a-C5aR1 induces endoplasmic reticulum stress to accelerate vascular calcification via PERK-eIF2α-ATF4-CREB3L1 pathway[J]. Cardiovasc Res,2023,119(15):2563-2578.
[23] CHEN Y P,CHEN Z M,CHENG W N,et al.The role of the GRP78/PERK/ATF4 pathway in the ability of Gua Lou Gui Zhi decoction to attenuate apoptosis by inhibiting endoplasmic reticulum stress after ischemia-reperfusion injury[J]. Front Biosci,2022,27(10):296.

红细胞储存损伤释放的游离血红素通过内质网应激诱导NETosis加重创伤-失血性休克小鼠模型炎症反应的作用机制研究^*

Study on the Mechanism by Which NETosis Exacerbates Inflammation in a Traumatic-hemorrhagic Shock Mouse Model due to Free Heme from Red Blood Cell Storage Injury Via Endoplasmic Reticulum Stress

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 6

编辑推荐

Metrics

本文评价

关于本刊

作者中心

在线期刊

访问统计

联系我们

[1]	乔剑, 刁春红, 于洋, 张娜, 常梦远. 吴茱萸碱通过调节HMGB1/TLR4/NF-κB信号通路减轻大鼠输血相关性急性肺损伤^*[J]. 临床输血与检验, 2023, 25(5): 599-606.
[2]	刘丽娟, 杜肖刚, 王丽荣, 武洪琳. 输血前预防性用药对输血不良反应的影响[J]. 临床输血与检验, 2017, 19(6): 559-561.
[3]	张志敏, 李碧娟. 输血相关性循环超负荷[J]. 临床输血与检验, 2017, 19(6): 650-653.
[4]	贾波. 2015年济源市非传染性输血不良反应联动处置与分析^*[J]. 临床输血与检验, 2017, 19(4): 332-335.
[5]	安宁,张秋会,夏爱军,胡兴斌,尹文,安群星. 西安地区女性献血者HLA抗体筛查分析[J]. 临床输血与检验, 2016, 18(6): 568-570.
[6]	罗圆圆, 张晓娟, 马春娅, 付丽辉, 王泽珊, 于洋. 单采血小板细菌污染致输血不良反应的病例统计分析[J]. 临床输血与检验, 2025, 27(1): 59-63.