GPX3抑制ROS介导的NET形成减轻肾移植缺血-再灌注损伤

GPX3 attenuates ischemia-reperfusion injury in kidney transplantation by inhibiting ROS-mediated NET formation

摘要

目的 探讨谷胱甘肽过氧化物酶3（GPX3）在肾移植缺血-再灌注损伤中的作用和潜在机制。方法 将C57BL/6小鼠按照干预条件分为4组，分别为假手术组（Sham组）、肾移植组（KT组）、肾移植+GPX3过表达组（KT+GPX3组）和肾移植+GPX3过表达+活性氧簇（ROS）激动剂组（KT+GPX3+CADD522组）。模型建立24 h后，取下腔静脉血检测血尿素氮（BUN）和血清肌酐（Scr）。取左侧肾脏组织，免疫荧光染色检测ROS和NADPH氧化酶2（NOX2），酶联免疫吸附试验检测粒细胞明胶酶相关脂质运载蛋白（NGAL）、肾损伤分子1（KIM-1）、肿瘤坏死因子（TNF）-α和白细胞介素（IL）-1β，蛋白质印迹法检测髓过氧化物酶（MPO）和瓜氨酸化组蛋白H3（CitH3）。结果 与Sham组比较，KT组GPX3表达水平降低；与KT组相比，KT+GPX3组GPX3表达水平升高；与KT+GPX3组比较，KT+GPX3+CADD522组GPX3表达水平降低（均为 P<0.05）。与Sham组比较，KT组Scr、BUN、NGAL和KIM-1表达水平升高；与KT组比较，KT+GPX3组Scr、BUN、NGAL和KIM-1表达水平下降；与KT+GPX3组比较，KT+GPX3+CADD522组Scr、BUN、NGAL和KIM-1表达水平升高（均为 P<0.05）。与Sham组比较，KT组NOX2和ROS表达水平升高；与KT组相比，KT+GPX3组NOX2和ROS表达水平降低；与KT+GPX3组比较，KT+GPX3+CADD522组NOX2和ROS表达水平升高（均为 P<0.05）。与Sham组比较，KT组MPO和CitH3表达水平升高；与KT组比较，KT+GPX3组MPO和CitH3表达水平降低；与KT+GPX3组比较，KT+GPX3+CADD522组MPO和CitH3表达水平升高（均为 P<0.05）。与Sham组比较，KT组TNF-ɑ和IL-1β表达水平升高；与KT组比较，KT+GPX3组TNF-ɑ和IL-1β表达水平降低；与KT+GPX3组比较，KT+GPX3+CADD522组TNF-ɑ和IL-1β表达水平升高（均为 P<0.05）。结论 GPX3可能通过抑制ROS介导的中性粒细胞胞外诱捕网（NET）形成，减轻肾脏组织中的炎症反应，进而缓解肾移植IRI。

Abstract

Objective To investigate the role and potential mechanism of glutathione peroxidase 3 (GPX3) in ischemia-reperfusion injury (IRI) during kidney transplantation. Methods C57BL/6 mice were divided into four groups according to different interventions: sham operation group (Sham group), kidney transplantation group (KT group), kidney transplantation + GPX3 overexpression group (KT+GPX3 group) and kidney transplantation + GPX3 overexpression + reactive oxygen species (ROS) agonist group (KT+GPX3+CADD522 group). Twenty-four hours after model establishment, inferior vena cava blood was collected to detect blood urea nitrogen (BUN) and serum creatinine (Scr). Left renal tissues were harvested for immunofluorescence staining to detect ROS and NADPH oxidase 2 (NOX2). Enzyme-linked immunosorbent assay (ELISA) was performed to measure neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), tumor necrosis factor (TNF)-α and interleukin (IL)-1β. Western blotting was used to detect myeloperoxidase (MPO) and citrullinated histone H3 (CitH3). Results Compared with the Sham group, the expression level of GPX3 was decreased in the KT group; compared with the KT group, GPX3 expression was increased in the KT+GPX3 group; compared with the KT+GPX3 group, GPX3 expression was reduced in the KT+GPX3+CADD522 group (all P<0.05). Compared with the Sham group, the levels of Scr, BUN, NGAL and KIM-1 were elevated in the KT group; compared with the KT group, the above indicators were decreased in the KT+GPX3 group; compared with the KT+GPX3 group, the levels of Scr, BUN, NGAL and KIM-1 were increased in the KT+GPX3+CADD522 group (all P<0.05). Compared with the Sham group, the expression levels of NOX2 and ROS were increased in the KT group; compared with the KT group, NOX2 and ROS levels were decreased in the KT+GPX3 group; compared with the KT+GPX3 group, NOX2 and ROS levels were upregulated in the KT+GPX3+CADD522 group (all P<0.05). Compared with the Sham group, the expression levels of MPO and CitH3 were increased in the KT group; compared with the KT group, MPO and CitH3 levels were decreased in the KT+GPX3 group; compared with the KT+GPX3 group, MPO and CitH3 levels were increased in the KT+GPX3+CADD522 group (all P<0.05). Compared with the Sham group, the expression levels of TNF-α and IL-1β were increased in the KT group; compared with the KT group, TNF-α and IL-1β levels were decreased in the KT+GPX3 group; compared with the KT+GPX3 group, TNF-α and IL-1β levels were increased in the KT+GPX3+CADD522 group (all P<0.05). Conclusions GPX3 may alleviate renal tissue inflammation by inhibiting ROS-mediated neutrophil extracellular trap (NET) formation, thereby attenuating IRI in renal transplantation.

关键词

肾移植 / 谷胱甘肽过氧化物酶3 / 缺血-再灌注损伤 / 中性粒细胞胞外诱捕网 / NADPH氧化酶2 / 活性氧簇 / 髓过氧化物酶 / 瓜氨酸化组蛋白H3

Key words

Kidney transplantation / Glutathione peroxidase 3 / Ischemia-reperfusion injury / Neutrophil extracellular trap / NADPH oxidase 2 / Reactive oxygen species / Myeloperoxidase / Citrullinated histone H3

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基金资助

贵州省科技厅基础研究计划青年引导项目(黔科合基础QN（2025）407)

贵州省人民医院人才基金项目(院人才项目（2025）-1)

贵州省卫生健康委科学技术基金项目(gzwkj2026-297)

上海交通大学医学院附属上海儿童医学中心贵州医院院级科研项目基金资助(YG2025-GJ01001)