NETs通过调节铁死亡对肾缺血再灌注损伤的 影响

The Impact of Neutrophil Extracellular Traps on Renal Ischemia-Reperfusion Injury via the Modulation of Ferroptosis

摘要: 目的：探讨中性粒细胞胞外诱捕网(NETs)在肾脏缺血再灌注损伤(IRI)中的作用及内在机制，并进一步探究其与细胞铁死亡之间的关联。方法：将18只C57BL/6J小鼠随机分为肾缺血再灌注组(I/R组，n = 6)、假手术组(Sham组，n = 6)、I/R + 脱氧核糖核酸酶I组(I/R + DNase I, n = 6)。采用无创动脉夹夹闭双侧肾蒂45分钟、恢复供血24小时的方法构建肾缺血再灌注小鼠模型；检测各组小鼠外周血肌酐(Scr)、尿素氮(BUN)水平，HE染色观察小鼠肾组织病理变化；免疫荧光双染检测各组肾组织中NETs标志物中性粒细胞髓过氧化物酶(MPO)与淋巴细胞抗原6G (LY6G)蛋白情况，免疫印迹检测小鼠肾组织中谷胱甘肽过氧化物酶4 (GPX4)、酰基辅酶A合成酶长链家族成员4 (ACSL4)、铁蛋白重链(FTH1)蛋白水平。结果：Sham组小鼠肾脏结构完整，I/R组小鼠可见明显的肾小管损伤，表现为部分肾小管上皮细胞空泡变性及胞质嗜酸性增强，肾组织内MPO与LY6G蛋白荧光强度表达增加(P < 0.01)，I/R + DNase I组小鼠肾小管上皮细胞的空泡变性程度减轻，细胞核脱落现象得到改善，肾组织内肾组织内MPO与LY6G蛋白强度表达减弱(P < 0.05)。I/R组小鼠血清Scr、BUN水平[(314.1 ±13.95) μmol/L、(137.7 ± 6.44) mmol/L]及肾组织中ACSL4蛋白水平(1.01 ± 0.12)高于Sham组[(30.48 ± 2.360) μmol/L、(23.81 ± 2.42) mmol/L、0.38 ± 0.12]，而GPX4、FTH1蛋白水平(0.67 ± 0.09、0.29 ± 0.08)低于Sham组(1.29 ± 0.01、1.19 ± 0.11)，差异均有统计学意义(P < 0.05)。I/R + DNase I组血清Scr、BUN水平[(30.48 ± 2.360) μmol/L、(23.81 ± 2.42) mmol/L]及肾组织中ACSL4蛋白水平(0.47 ± 0.13)低于I/R组，而GPX4、FTH1蛋白水平(1.12 ± 0.03、0.97 ± 0.05)高于I/R组，差异均有统计学意义(P < 0.05)。结论：IRI可诱导NETs生成并促进细胞铁死亡。抑制NETs后，可缓解IRI以及细胞铁死亡，NETs可能是通过促进细胞铁死亡参与肾脏IRI。

Abstract: Objective: This paper aims to investigate the role and underlying mechanisms of neutrophil extracellular traps (NETs) in renal ischemia-reperfusion injury (IRI) and to further explore their association with ferroptosis. Methods: Eighteen C57BL/6J male mice were randomly divided into three groups (n = 6 per group): renal ischemia-reperfusion (I/R group), sham-operated (Sham group), and I/R treated with DNase I (I/R + DNase I group). Renal IRI was induced by bilaterally clamping the renal pedicles for 45 minutes with a non-invasive arterial clip, followed by 24 hours of reperfusion. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were assessed. Hematoxylin and eosin (H&E) staining was performed to evaluate histopathological changes. Immunofluorescence co-staining for myeloid peroxidase (MPO) and lymphocyte antigen 6 complex locus G (Ly6G) was used to detect NETs, while western blotting was employed to analyze the expression levels of glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4), and ferritin heavy chain 1 (FTH1). Results: Kidneys from the Sham group maintained intact architecture. In the I/R group, significant renal tubular damage was evident, characterized by vacuolar degeneration of tubular epithelial cells and enhanced cytoplasmic eosinophilia. The co-expression intensity of MPO and Ly6G proteins within the renal tissue was elevated (P < 0.01). In the I/R + DNase I group, the severity of vacuolar degeneration in tubular epithelial cells was reduced, nuclear shedding was ameliorated, and the expression intensity of MPO and Ly6G was attenuated (P < 0.05). Compared to the Sham group (30.48 ± 2.360 µmol/L, 23.81 ± 2.42 mmol/L, 0.38 ± 0.12), the I/R group exhibited significantly higher levels of serum Scr (314.1 ± 13.95 µmol/L), BUN (137.7 ± 6.44 mmol/L), and renal ACSL4 protein (1.01 ± 0.12), while showing lower levels of GPX4 (0.67 ± 0.09) and FTH1 (0.29 ± 0.08), all statistically significant (P < 0.05). Conversely, the I/R + DNase I group demonstrated significantly lower levels of Scr (30.48 ± 2.360 µmol/L), BUN (23.81 ± 2.42 mmol/L), and renal ACSL4 (0.47 ± 0.13), but higher levels of GPX4 (1.12 ± 0.03) and FTH1 (0.97 ± 0.05) compared to the I/R group, with statistical significance (P < 0.05). Conclusion: Renal IRI induces NET formation and promotes ferroptosis. Inhibiting NETs mitigates both IRI and ferroptosis, suggesting that NETs may participate in renal IRI by potentiating ferroptosis.