中性粒细胞胞外陷阱促进气道损伤后纤维化进展的机制研究

doi:10.3969/j.issn.1674-8115.2026.09.007

上海交通大学学报（医学版） ›› 2026, Vol. 46 ›› Issue (9): 1222-1232.doi: 10.3969/j.issn.1674-8115.2026.09.007

• 论著 · 基础研究 • 上一篇

中性粒细胞胞外陷阱促进气道损伤后纤维化进展的机制研究

黄丹青¹, 尹玉婷¹^,², 徐莉¹^,³, 郭述良¹^,³()

^1.重庆医科大学附属第一医院呼吸与危重症医学科，重庆 400016
^2.重庆市沙坪坝区人民医院感染性疾病科，重庆 400030
^3.呼吸慢病与共病重庆市高校重点实验室，重庆 400016

收稿日期:2025-11-05 接受日期:2026-02-24 出版日期:2026-09-14 发布日期:2026-09-14
通讯作者: 郭述良，主任医师，博士；电子信箱：GUOSL999@sina.com。
基金资助:
重庆市科卫联合医学科研项目(2022MSXM082)

Mechanism of neutrophil extracellular traps in promoting post-injury airway fibrosis

Huang Danqing¹, Yin Yuting¹^,², Xu Li¹^,³, Guo Shuliang¹^,³()

^1.Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
^2.Department of Infectious Diseases, Shapingba District People's Hospital of Chongqing, Chongqing 400030, China
^3.Chongqing Key Laboratory of Chronic Respiratory Diseases and Comorbidities, Higher Education Institution, Chongqing 400016, China

Received:2025-11-05 Accepted:2026-02-24 Online:2026-09-14 Published:2026-09-14
Contact: Guo Shuliang, E-mail: GUOSL999@sina.com.
Supported by:
Science-Health Joint Medical Scientific Research Project of Chongqing(2022MSXM082)

摘要/Abstract

摘要：

目的·探讨中性粒细胞胞外陷阱（neutrophil extracellular traps，NETs）在气道损伤后异常修复中的作用及相关机制。方法·纳入2024年4—10月重庆医科大学附属第一医院因肉芽组织或瘢痕致气道狭窄患者及健康志愿者，收集外周血样本；同时收集部分患者的气道肉芽组织及癌旁正常气道组织。采用酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）、免疫荧光染色及试剂盒检测血清及组织中NETs标志物，包括髓过氧化物酶（myeloperoxidase，MPO）、瓜氨酸化组蛋白H3（citrullinated histone H3，CitH3）、细胞外游离双链DNA（double-stranded DNA，dsDNA）。NETs刺激气道成纤维细胞后，采用实时荧光定量PCR（real-time quantitative PCR，qPCR）、Western blotting、免疫荧光染色检测α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）和Ⅰ型胶原蛋白（collagen Ⅰ，COL1），采用Cell Counting Kit-8（CCK-8）法和Transwell细胞迁移实验评估其增殖和迁移能力；用小干扰RNA（small interfering RNA，siRNA）敲低气道成纤维细胞Toll样受体4（toll-like receptor 4，Tlr4）基因，观察上述指标及SMAD家族成员2（SMAD family member 2，SMAD2）磷酸化水平变化。构建大鼠气道刮擦模型，并另设二苯基氯化碘盐（diphenyleneiodonium chloride，DPI）预处理组和对照组；ELISA检测血清MPO和白细胞介素-8（interleukin-8，IL-8），qPCR和Western blotting检测气道组织α-SMA和COL1表达，Masson染色观察气道组织结构，免疫荧光染色观察CitH3的定位和表达情况。结果·① 气道狭窄患者外周血NETs标志物高于健康对照人群，肉芽组织中CitH3增多。② NETs刺激成纤维细胞后α-SMA、COL1表达水平升高，增殖和迁移能力增强，SMAD2磷酸化水平上调，脱氧核糖核酸酶Ⅰ（deoxyribonuclease Ⅰ，DNaseⅠ）可抑制NETs的作用。③ 敲低Tlr4后，NETs的作用被抑制。④ 大鼠气道刮擦模型中，血清MPO、IL-8水平较对照组显著升高，气道结构损伤和胶原沉积严重，气道组织中α-SMA、COL1、CitH3表达水平升高；DPI预处理可一定程度上抑制上述变化。结论·NETs可能经TLR4/SMAD2通路激活气道损伤后的成纤维细胞，促进细胞迁移、增殖及细胞外基质沉积，参与气道纤维化。

关键词: 气道损伤, 中性粒细胞胞外陷阱, 成纤维细胞, 纤维化

Abstract:

Objective ·To investigate the role of neutrophil extracellular traps (NETs) in aberrant repair following airway injury and the underlying mechanisms. Methods ·Patients with airway stenosis caused by granulation tissue or scarring and healthy volunteers were enrolled from The First Affiliated Hospital of Chongqing Medical University between April and October 2024. Peripheral blood, airway granulation tissue, and paracancerous normal airway tissue were collected. The levels of NETs markers—myeloperoxidase (MPO), citrullinated histone H3 (CitH3), and extracellular free double-stranded DNA (dsDNA)—in serum and tissues were detected using enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining, and a dsDNA quantification kit. After stimulation of airway fibroblasts with NETs, the expression levels of α-smooth muscle actin (α-SMA) and collagen Ⅰ (COL1) were examined by real-time quantitative PCR (qPCR), Western blotting, and immunofluorescence staining. Cell proliferation and migration were evaluated using the Cell Counting Kit-8 (CCK-8) assay and Transwell migration assay. The Toll-like receptor 4 (Tlr4) gene in rat airway fibroblasts was knocked down using small interfering RNA (siRNA), and the above indices, as well as phosphorylation levels of SMAD family member 2 (SMAD2), were assessed. A rat airway scraping injury model was established. A diphenyleneiodonium chloride (DPI) pretreatment group and a control group were additionally included. Serum MPO and interleukin-8 (IL-8) levels were measured by ELISA. The expression levels of α-SMA and COL1 in airway tissue were detected by qPCR and Western blotting. Airway tissue structure and the localization and expression of CitH3 were observed using Masson staining and immunofluorescence staining, respectively. Results ·① Patients with airway stenosis exhibited higher levels of serum NETs markers than healthy controls, and increased CitH3 was observed in the granulation tissue. ② After stimulation with NETs, fibroblasts showed elevated expression of α-SMA and COL1, enhanced proliferation and migration abilities, and upregulated SMAD2 phosphorylation; these effects were inhibited by deoxyribonuclease Ⅰ (DNaseⅠ). ③ Knockdown of Tlr4 suppressed the effects induced by NETs. ④ In the rat airway scraping injury model, serum MPO and IL-8 levels were significantly elevated, airway structure was damaged with severe collagen deposition, and the expression levels of α-SMA, COL1, and CitH3 in the airway tissue were increased compared with those in the control group; DPI pretreatment partially attenuated these changes. Conclusion ·NETs may activate fibroblasts via the TLR4/SMAD2 pathway following airway injury, promoting cell migration, proliferation, and extracellular matrix deposition, thereby contributing to airway fibrosis.

Key words: airway injury, neutrophil extracellular traps (NETs), fibroblast, fibrosis

中图分类号:

R562.1⁺2

黄丹青, 尹玉婷, 徐莉, 郭述良. 中性粒细胞胞外陷阱促进气道损伤后纤维化进展的机制研究[J]. 上海交通大学学报（医学版）, 2026, 46(9): 1222-1232.

Huang Danqing, Yin Yuting, Xu Li, Guo Shuliang. Mechanism of neutrophil extracellular traps in promoting post-injury airway fibrosis[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026,(9): 1222-1232.

图/表 7

表1 人及大鼠qPCR目的基因引物序列

Tab 1 Primer sequences of target genes for qPCR in humans and rats

Gene	Species	Sequence (5′→3′)
ACTB	Human	F: TTCCTTCCTGGGCATGGAGTCC R: TGGCGTACAGGTCTTTGCGG
Actb	Rat	F: CCTTTGCCGATCCGCCG R: GATATCATCATCCATGGTGAGCTGG
COL1A1	Human	F: CCCCGAGGCTCTGAAGGT R: GCAATACCAGGAGCACCATTG
Col1a1	Rat	F: CTTTGTGGACCTCCGGCTC R: AGCTGACTTCAGGGATGTCTTC
ACTA2	Human	F: CCCCGGGAGCCAGTCT R: GCTTCACAGGATTCCCGTCT
Acta2	Rat	F: CAGCTATGTGGGGGACGAAG R: AGTGGTGCCAGATCTTTTCCA

图1 NETs标志物在临床样本中的表达与定位Note： A. Detection of dsDNA levels in serum samples from control subjects (n=17) and patients with airway stenosis (n=18) using the PicoGreen dsDNA assay kit. B. Detection of MPO levels in serum samples from control subjects (n=17) and patients with airway stenosis (n=18) by ELISA. C. Immunofluorescence staining for α‑SMA and CitH3 in normal airway tissue and granulation tissue (×100). ①P<0.001.

Fig 1 Expression and localization of NETs markers in clinical samples

图2 气道成纤维细胞经NETs刺激后α‑SMA和COL1的表达变化Note： A. qPCR analysis of Acta2 and Col1a1 expression in different groups of RF. B. Western blotting analysis of α‑SMA and COL1 expression in different groups of RF and HF. C. Immunofluorescence staining of α‑SMA and COL1 in different groups of RF (×500). ①P=0.001; ②P=0.025; ③P<0.001; ④P=0.011.

Fig 2 Changes in α‑SMA and COL1 expression in airway fibroblasts after NETs stimulation

图3 气道成纤维细胞经NETs刺激后的功能变化Note： A. Migration ability of RF in different groups assessed by Transwell assay (×40). B. Proliferation of RF under different concentrations of NETs detected by CCK‑8 assay. C. Proliferation of RF and HF in different groups detected by CCK‑8 assay. D. IL‑8 levels in the supernatant of HF in different groups detected by ELISA. ①P<0.001; ②P=0.002; ③P=0.015.

Fig 3 Functional changes in airway fibroblasts after stimulation by NETs

图4 NETs激活气道成纤维细胞的分子通路Note： A. TLR4 expression in rat airway tissues detected by Western blotting. B. TLR4 expression in RF detected by Western blotting. C. Phosphorylation levels of SMAD2 in RF detected by Western blotting. D. Knockdown of Tlr4 in RF by siTlr4. E. Phosphorylation levels of SMAD2 in RF after Tlr4 knockdown followed by NETs stimulation detected by Western blotting. F. Expression of α-SMA and COL1 in RF after Tlr4 knockdown followed by NETs stimulation detected by Western blotting.

Fig 4 Molecular pathway of NETs activating airway fibroblasts

图5 大鼠气道刮擦模型中血清NETs标志物表达情况及其与成纤维细胞活化的关系Note： A. Serum MPO levels in the 3 groups of rats measured by ELISA. B. Serum IL‑8 levels in the 3 groups of rats measured by ELISA. C. mRNA levels of Acta2 and Col1a1 in rat airway tissues from the 3 groups detected by qPCR. D. Protein levels of α‑SMA and COL1 in rat airway tissues from the 3 groups detected by Western blotting. E. Masson′s trichrome staining of rat airway tissue sections from the 3 groups (×30). ①P<0.001; ②P=0.002.

Fig 5 Expression of serum NETs markers and their relationship with fibroblast activation in the rat airway injury model

图6 大鼠气道刮擦模型中气道组织NETs标志物表达情况及其与成纤维细胞活化的关系Note： Immunofluorescence staining of α‑SMA and CitH3 in rat airway tissue sections from the 3 groups (upper panel: ×20; lower panel: ×200).

Fig 6 Expression of NETs markers in airway tissues and their relationship with fibroblast activation in the rat airway injury model

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中性粒细胞胞外陷阱促进气道损伤后纤维化进展的机制研究

Mechanism of neutrophil extracellular traps in promoting post-injury airway fibrosis

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