N-乙酰转移酶10对博来霉素诱导肺上皮细胞氧化应激损伤的影响

doi:10.3969/j.issn.1006-7795.2026.01.015

摘要/Abstract

摘要： 目的本研究旨在探究N-乙酰转移酶10（N-acetyltransferase 10, NAT10）在肺纤维化（pulmonary fibrosis, PF）中的作用及其与氧化应激的关联。方法通过整合基因表达综合数据库（Gene Expression Omnibus, GEO）中的多个公共数据集，包括特发性肺纤维化（idiopathic pulmonary fibrosis, IPF）患者肺组织数据集GSE110147、博来霉素（Bleomycin, BLM）诱导小鼠模型数据集GSE282477以及IPF患者单细胞转录组数据集GSE128033，系统解析NAT10在PF中的mRNA表达水平。气管滴注BLM建立小鼠肺纤维化模型，免疫荧光染色检测小鼠肺组织中NAT10的表达情况。采用BLM刺激人永生化支气管上皮细胞（bronchial epithelium transformed with Ad12-SV40 2B, BEAS-2B）构建体外纤维化模型，通过逆转录定量聚合酶链反应（reverse transcription-quantitative polymerase chain reaction, RT-qPCR）检测纤维化标志物及NAT10的mRNA表达变化，并通过酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）检测炎症因子的分泌水平变化。为探究NAT10功能，在BEAS-2B细胞中干扰NAT10表达后给予BLM刺激，采用流式细胞术检测细胞内活性氧（reactive oxygen species, ROS）水平变化，采用ELISA检测细胞上清中白细胞介素6（interleukin 6, IL-6）、转化生长因子-β1（transforming growth factor-β1, TGF-β1）浓度、超氧化物歧化酶（superoxide dismutase, SOD）活性及脂质过氧化产物丙二醛（malondialdehyde, MDA）含量。结果基于公共转录组分析，IPF患者（GSE110147）及BLM诱导小鼠（GSE282477）肺组织中NAT10的mRNA表达水平分别上调至对照组的1.05倍与1.38倍（P均<0.05）。IPF单细胞测序数据（GSE128033）分析进一步显示，与内皮细胞、巨噬细胞等其他肺内细胞类型相比，细气道club细胞和其他上皮细胞的NAT10表达上调最为显著（P 均<0.05）。在动物水平，免疫荧光检测证实BLM诱导的肺纤维化小鼠肺组织NAT10蛋白表达约为对照组的1.94倍（P<0.05）。在人支气管上皮细胞BEAS-2B中，BLM刺激48 h成功诱发了纤维化细胞表型。在此模型中，NAT10的mRNA表达上调至0 h组的约1.36倍（P<0.05）。与对照组相比，BLM刺激BEAS-2B细胞上清中ROS水平、MDA含量及TGF-β1、IL-6浓度均显著上升，而SOD活性则显著下降，差异有统计学意义（P 均<0.05）。功能实验结果表明，与BLM刺激组相比，敲低NAT10可显著提升SOD活性，并降低ROS水平、MDA含量及TGF-β1、IL-6浓度，差异有统计学意义（P均<0.05）。结论 NAT10是肺纤维化中调控氧化应激损伤的关键因子，其表达水平在病变组织与细胞模型中均显著升高，提示可能与疾病严重程度相关。本研究结果显示，在细胞水平靶向干预NAT10可有效缓解BLM诱导的氧化应激及纤维化反应,后续动物实验将有助于进一步明确其治疗潜力。

关键词: N-乙酰转移酶10, 肺纤维化, 氧化应激, BEAS-2B细胞, 博来霉素, 基因敲低, 活性氧

Abstract: Objective To investigate the role of N-acetyltransferase 10 (NAT10) in pulmonary fibrosis (PF) and its association with oxidative stress. Methods Public transcriptome datasets (GEO: GSE110147, GSE282477, GSE128033) were integrated to analyze NAT10 mRNA levels in pulmonary fibrosis (PF). A murine PF model was established by intratracheal Bleomycin (BLM) administration. The expression of NAT10 protein in lung tissue was assessed by immunofluorescence. Human bronchial epithelial cell line BEAS-2B was stimulated by BLM to establish an in vitro fibrosis model. The mRNA expression of fibrotic markers and NAT10 were analyzed by using reverse transcription polymerase chain reaction (RT-qPCR), while the protein levels of inflammatory factors were evaluated by enzyme-linked immunosorbent assay (ELISA). To study the function of NAT10, BEAS-2B cells with NAT10 inhibition were treated with BLM. Intracellular reactive oxygen species (ROS) levels were analyzed by flow cytometry. The concentrations of interleukin 6 (IL-6), transforming growth factor-β1 (TGF-β1), superoxide dismutase (SOD) activity, and the lipid peroxidation product malondialdehyde (MDA) content were measured via ELISA. Results Based on public transcriptome analysis, NAT10 mRNA expression was elevated to 1.05- and 1.38-fold of controls in lung tissues from idiopathic pulmonary fibrosis (IPF) patients (GSE110147) and BLM-induced fibrotic mice (GSE282477), respectively (both P<0.05). Single-cell RNA sequencing of IPF lungs (GSE128033) indicated that bronchiolar club and other epithelial cells exhibited the most pronounced NAT10 upregulation compared to other pulmonary cell types (all P<0.05). Immunofluorescence quantification confirmed a 1.94-fold increase in NAT10 protein in BLM-induced fibrotic murine lungs versus model controls (P<0.05). In BEAS-2B cells, BLM stimulation for 48 hours induced fibrotic changes and increased NAT10 mRNA to 1.36-fold of baseline (P<0.05). This was accompanied by elevated supernatant levels of ROS, MDA content, TGF-β1 and IL-6 levels, alongside reduced SOD activity (all P<0.05). NAT10 knockdown rescued these alterations, increasing SOD activity and decreasing ROS levels, MDA content, TGF-β1, and IL-6 levels compared to the BLM-stimulated group (all P<0.05). Conclusion NAT10 is a critical regulator of oxidative stress injury in pulmonary fibrosis. Its expression is significantly elevated in fibrotic tissues and cellular models, suggesting a possible correlation with disease severity. At the cellular level, targeting NAT10 effectively mitigates BLM-induced oxidative stress and fibrotic responses. Further animal studies will help clarify its therapeutic potential.

Key words: N-acetyltransferase 10, pulmonary fibrosis, oxidative stress, BEAS-2B cells, bleomycin, gene knockdown, reactive oxygen species

中图分类号:

R563
R392.5

王妍然, 许钰铃, 宋楠. N-乙酰转移酶10对博来霉素诱导肺上皮细胞氧化应激损伤的影响[J]. 首都医科大学学报, 2026, 47(1): 115-125.

Wang Yanran, Xu Yuling, Song Nan. Effect of N-acetyltransferase 10 on bleomycin-induced oxidative stress in lung epithelial cells[J]. Journal of Capital Medical University, 2026, 47(1): 115-125.

参考文献

［1］Chapman H A. Disorders of lung matrix remodeling［J］. J Clin Invest, 2004, 113(2): 148－157.
［2］Phan T H G, Paliogiannis P, Nasrallah G K, et al. Emerging cellular and molecular determinants of idiopathic pulmonary fibrosis［J］. Cell Mol Life Sci, 2021, 78(5): 2031－2057.
［3］Chambers R C, Mercer P F. Mechanisms of alveolar epithelial injury, repair, and fibrosis［J］. Ann Am Thorac Soc, 2015, 12(Suppl 1): S16-S20.
［4］Song L C, Li K, Chen H Y, et al. Cell cross-talk in alveolar microenvironment: from lung injury to fibrosis［J］. Am J Respir Cell Mol Biol, 2024, 71(1): 30－42.
［5］Henderson N C, Rieder F, Wynn T A. Fibrosis: from mechanisms to medicines［J］. Nature, 2020, 587(7835): 555－566.
［6］Pott H, Sykes D L, Charriot J, et al. Breathing barriers: bridging lung health, research, and awareness［J］. Lancet Respir Med, 2025, 13(8): 665－667.
［7］Spagnolo P, Guler S A, Chaudhuri N, et al. Global epidemiology and burden of interstitial lung disease［J］. Lancet Respir Med, 2025, 13(8): 739－755.
［8］Liu T J, De Los Santos F G, Phan S H. The bleomycin model of pulmonary fibrosis［J］. Methods Mol Biol, 2017, 1627: 27－42.
［9］El-Baz L M, Shoukry N M, Hafez H S, et al. Experimental mouse model of bleomycin-induced pulmonary fibrosis［J］. Int J Cancer Biomed Res, 2020, 4(3): 1－8.
［10］Burger R M, Peisach J, Horwitz S B. Activated bleomycin. A transient complex of drug, iron, and oxygen that degrades DNA［J］. J Biol Chem, 1981, 256(22): 11636－11644.
［11］Ayilya B L, Balde A, Ramya M, et al. Insights on the mechanism of bleomycin to induce lung injury and associated in vivo models: a review［J］. Int Immunopharmacol, 2023, 121: 110493.
［12］Teixeira K C, Soares F S, Rocha L G C, et al. Attenuation of bleomycin-induced lung injury and oxidative stress by N-acetylcysteine plus deferoxamine［J］. Pulm Pharmacol Ther, 2008, 21(2): 309－316.
［13］Liu X J, Chen Z H. The pathophysiological role of mitochondrial oxidative stress in lung diseases［J］. J Transl Med, 2017, 15(1): 207.
［14］Rogers L K, Cismowski M J. Oxidative stress in the lung-the essential paradox［J］. Curr Opin Toxicol, 2018, 7: 37－43.
［15］Della Latta V, Cecchettini A, Del Ry S, et al. Bleomycin in the setting of lung fibrosis induction: from biological mechanisms to counteractions［J］. Pharmacol Res, 2015, 97: 122－130.
［16］Cheresh P, Kim S J, Tulasiram S, et al. Oxidative stress and pulmonary fibrosis［J］. Biochim Biophys Acta, 2013, 1832(7): 1028－1040.
［17］Liu J T, Han X Y, Zhang T Y, et al. Reactive oxygen species (ROS) scavenging biomaterials for anti-inflammatory diseases: from mechanism to therapy［J］. J Hematol Oncol, 2023, 16(1): 116.
［18］Wang Z W, Chen S Y, Guo W X, et al. Oxidative stress promotes intervertebral disc degeneration through NAT10-mediated IKKβ N4-acetylcytidine modification［J］. Cell Signal, 2025, 134: 111918.
［19］Qu Z Z, Pang X C, Mei Z T, et al. The positive feedback loop of the NAT10/Mybbp1a/p53 axis promotes cardiomyocyte ferroptosis to exacerbate cardiac I/R injury［J］. Redox Biol, 2024, 72: 103145.
［20］Wu S S, Yin L J, Han K, et al. NAT10 accelerates pulmonary fibrosis through N4-acetylated TGFB1-initiated epithelial-to-mesenchymal transition upon ambient fine particulate matter exposure［J］. Environ Pollut, 2023, 322: 121149.
［21］Spagnolo P, Kropski J A, Jones M G, et al. Idiopathic pulmonary fibrosis: disease mechanisms and drug development［J］. Pharmacol Ther, 2021, 222:107798.
［22］Otoupalova E, Smith S, Cheng G J, et al. Oxidative stress in pulmonary fibrosis［J］. Compr Physiol, 2020, 10(2): 509－547.
［23］Veith C, Boots A W, Idris M, et al. Redox imbalance in idiopathic pulmonary fibrosis: a role for oxidant cross-talk between NADPH oxidase enzymes and mitochondria［J］. Antioxid Redox Signal, 2019, 31(14): 1092－1115.
［24］Ranneh Y, Ali F, Akim A M, et al. Crosstalk between reactive oxygen species and pro-inflammatory markers in developing various chronic diseases: a review［J］. Appl Biol Chem, 2017, 60(3): 327－338.
［25］Richter K, Konzack A, Pihlajaniemi T, et al. Redox-fibrosis: impact of TGFβ1 on ROS generators, mediators and functional consequences［J］. Redox Biol, 2015, 6: 344－352.
［26］Kinnula V L, Fattman C L, Tan R J, et al. Oxidative stress in pulmonary fibrosis: a possible role for redox modulatory therapy［J］. Am J Respir Crit Care Med, 2005, 172(4): 417－422.
［27］Makena P, Kikalova T, Prasad G L, et al. Oxidative stress and lung fibrosis: towards an adverse outcome pathway［J］. Int J Mol Sci, 2023, 24(15): 12490.

[1]	崔玮, 庞淇丹, 相韩悦, 肖猱, 姜德春, 李深, 沈光莉. 利用BIBP-H荧光探针进行脑缺血再灌注损伤检测[J]. 首都医科大学学报, 2025, 46(1): 76-82.
[2]	李亚堃, 李蔚, 杨雪琦, 师文娟, 朱玥荃, 赵咏梅. 活性氧自由基清除剂EUK-134对脑缺血再灌注损伤大鼠线粒体相关蛋白PGC-1α和VDAC1的影响[J]. 首都医科大学学报, 2024, 45(6): 1016-1022.
[3]	苟东芸, 梁加一, 郭梓煜, 闫方圆, 王帅翔, 渠硕, 高雁, 张雨佳, 马晓伟. 脑蛋白水解物通过p38 MAPK信号通路降低阿尔茨海默病小鼠的细胞凋亡、炎症及氧化应激水平[J]. 首都医科大学学报, 2024, 45(6): 1062-1070.
[4]	江玉梅, 张紫薇, 王吉天, 杨宇. 一氧化氮供体型药物对牙龈卟啉单胞菌体外抗菌活性的影响[J]. 首都医科大学学报, 2024, 45(1): 104-110.