Hou Yujun, Wang Wen, Ye Jiangnan, Sun Luqiang, Zhou Siyuan, Zheng Qianhua, Shi Yunzhou, Chen Ying, Yao Junpeng, Wang Lu, Yan Xiangyun, Wan Renhong, Chen Shuai, Li Ying
Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
Department of Acupuncture and Moxibustion, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, China.
Sci Rep. 2025 Apr 9;15(1):12109. doi: 10.1038/s41598-025-95880-4.
The homeostasis of gastric mucosa is extremely delicate. Neutrophils, the most abundant immune cells in human circulation, are regarded crutial in the regulation of gastric mucosal immune response. Non-steroidal anti-inflammatory drugs (NSAIDs) induced gastric injury is the second major reason for gastric ulcers. The relations between neutrophils and Indomethacin-induced gastric injury are not fully understood. A mouse model of gastric injury was established using Indomethacin, followed by proteomic analysis (raw data are available via ProteomeXchange with identifier PXD058482). GO functional annotations and KEGG pathway enrichment analysis were conducted on significant differential proteins. The formation of neutrophil extracellular traps (NETs) was observed using ELISA and immunofluorescence. TEM, Western blot and Real-time PCR were applied to observe programmed death of gastric epithelial cells (GECs), and ELISA was conducted to measure levels of TNF-α and IL-1β in the gastric tissue. Deoxyribonuclease 1 (DNase 1), a NETs inhibitor, was administered intraperitoneally to inhibit NETs formation. In vitro, neutrophils were isolated from peripheral blood of mice and co-cultured with mouse GECs cell line, different dosage of Indomethacin were added to the culture dish, the levels of inflammatory factors, formation of NETs and GECs programmed death were assessed in vitro. Poly morphonuclear neutrophils (PMN) were extracted from mouse peripheral blood and single-cell RNA-sequencing (scRNA-seq) was further applied (raw data are available via Genome Sequence Archive with identifier CRA020950) to explore the intracellular mechanism of NETs formation. ELISA and immunofluorescence were performed to validate expression of IL-17 signaling pathway. After Indomethacin gavage, obvious gastric injury was observed. Proteomic analysis indicated that NETs formation played a crucial role in Indomethacin-induced gastric injury. Compared to control group, Indomethacin treatment resulted in NETs formation, elevated levels of TNF-α and IL-1β and GECs programmed death. Inhibition of NETs significantly reduced inflammatory factor levels and mitigated gastric injury caused by indomethacin. In vitro, 200 µL, 400 µL and 600 µL of Indomethacin caused excessive NETs formation in neutrophils. Besides, Indomethacin-induced NETs formation led to GECs programmed death in vitro. scRNA-seq revealed that neutrophils enrichment in the peripheral blood of Indomethacin-induced gastric injury and IL-17 signaling might be the key intracellular of NETs formation. Expressions of neutrophil IL-17R and concentration of IL-17 were significantly higher in model group. NETs formation is pivotal in Indomethacin-induced gastric injury, contributing to programmed cell death of GECs and inflammation; IL-17 signaling might be the key intracellular mechanism of NETs formation.
胃黏膜的内环境稳定极其微妙。中性粒细胞是人体循环中最丰富的免疫细胞，被认为在胃黏膜免疫反应的调节中至关重要。非甾体抗炎药（NSAIDs）诱导的胃损伤是胃溃疡的第二大主要原因。中性粒细胞与吲哚美辛诱导的胃损伤之间的关系尚未完全明确。使用吲哚美辛建立胃损伤小鼠模型，随后进行蛋白质组学分析（原始数据可通过ProteomeXchange获得，标识符为PXD058482）。对显著差异蛋白进行基因本体（GO）功能注释和京都基因与基因组百科全书（KEGG）通路富集分析。使用酶联免疫吸附测定（ELISA）和免疫荧光观察中性粒细胞胞外陷阱（NETs）的形成。应用透射电子显微镜（TEM）、蛋白质免疫印迹法（Western blot）和实时聚合酶链反应（Real-time PCR）观察胃上皮细胞（GECs）的程序性死亡，并通过ELISA测定胃组织中肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）的水平。腹腔注射脱氧核糖核酸酶1（DNase 1），一种NETs抑制剂，以抑制NETs的形成。在体外，从小鼠外周血中分离中性粒细胞，并与小鼠GECs细胞系共培养，向培养皿中加入不同剂量的吲哚美辛，在体外评估炎症因子水平、NETs的形成和GECs的程序性死亡。从小鼠外周血中提取多形核中性粒细胞（PMN），并进一步应用单细胞RNA测序（scRNA-seq）（原始数据可通过基因组序列档案获得，标识符为CRA020950）来探索NETs形成的细胞内机制。进行ELISA和免疫荧光以验证白细胞介素-17信号通路的表达。吲哚美辛灌胃后，观察到明显的胃损伤。蛋白质组学分析表明，NETs的形成在吲哚美辛诱导的胃损伤中起关键作用。与对照组相比，吲哚美辛处理导致NETs形成、TNF-α和IL-1β水平升高以及GECs程序性死亡。抑制NETs可显著降低炎症因子水平，并减轻吲哚美辛引起的胃损伤。在体外，200 μL、400 μL和600 μL的吲哚美辛导致中性粒细胞中过度的NETs形成。此外，吲哚美辛诱导的NETs形成在体外导致GECs程序性死亡。scRNA-seq显示，吲哚美辛诱导的胃损伤外周血中中性粒细胞富集以及白细胞介素-17信号可能是NETs形成的关键细胞内机制。模型组中性粒细胞白细胞介素-17受体（IL-17R）的表达和白细胞介素-17的浓度显著更高。NETs的形成在吲哚美辛诱导的胃损伤中起关键作用，导致GECs程序性细胞死亡和炎症；白细胞介素-17信号可能是NETs形成的关键细胞内机制。
