切换至 "中华医学电子期刊资源库"

中华肺部疾病杂志(电子版) ›› 2025, Vol. 18 ›› Issue (01) : 8 -14. doi: 10.3877/cma.j.issn.1674-6902.2025.01.002

论著

IR-780 调节糖酵解抑制特发性肺纤维化的机制分析
顾佩玉1, 曹磊1,, 刘澄英1, 曹励强1, 李杰1, 王晓雯1   
  1. 1. 214400 江阴,南通大学附属江阴医院呼吸与危重症医学科
  • 收稿日期:2024-11-23 出版日期:2025-02-25
  • 通信作者: 曹磊
  • 基金资助:
    南通大学临床医学专项项目(2022JQ001)

Mechanism analysis of IR-780 inhibits idiopathic pulmonary fibrosis by regulating glycolytic process

Peiyu Gu1, Lei Cao1,, Chengying Liu1, Liqiang Cao1, Jie Li1, Xiaowen Wang1   

  1. 1. Department of Pulmonary and Critical Care Medicine, Jiangyin Hospital Affiliated to Nantong University, Jiang Ying, 214400, China
  • Received:2024-11-23 Published:2025-02-25
  • Corresponding author: Lei Cao
引用本文:

顾佩玉, 曹磊, 刘澄英, 曹励强, 李杰, 王晓雯. IR-780 调节糖酵解抑制特发性肺纤维化的机制分析[J/OL]. 中华肺部疾病杂志(电子版), 2025, 18(01): 8-14.

Peiyu Gu, Lei Cao, Chengying Liu, Liqiang Cao, Jie Li, Xiaowen Wang. Mechanism analysis of IR-780 inhibits idiopathic pulmonary fibrosis by regulating glycolytic process[J/OL]. Chinese Journal of Lung Diseases(Electronic Edition), 2025, 18(01): 8-14.

目的

分析IR-780 在特发性肺纤维化(idiopathic pulmonary fibrosis,IPF)中的作用及与糖酵解关系。

方法

采用人类Ⅱ型肺泡上皮细胞(A549)、人胚肺成纤维细胞(human fetal lung fibroblasts,HELF)构建转化生长因子-β(transforming growth factor-β,TGF-β)诱导纤维化模型,分为对照组、TGF-β组、2-脱氧-D-葡萄糖(2-deoxy-D-glucose,2-DG)组和IR-780 组,对照组用Dulbecco 改良Eagle 培养基(Dulbecco′s modified Eagle medium,DMEM)处理,TGF-β 组用TGF-β(10 μg/ml)处理,2-DG 组用TGF-β(10 μg/ml)+2-DG(5 mmol/L)处理、IR-780 组用TGF-β(10 μg/ml)+IR-780(1 μmol/L)处理。 采用免疫荧光检测肺纤维化标志物表达水平,采用逆转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)和Western blot 方法检测纤维化标志物和糖酵解标志物表达水平。

结果

免疫荧光检测显示,TGF-β 组较对照组A549、HFLF 细胞CollagenⅠ和α-SMA 表达上升(P <0.05)。 RT-PCR 和Western blot 显示,TGF-β 组A549 及HFLF 细胞mRNA CollagenⅠ(3.17±0.06)、(1.74±0.03),α-SMA(2.26±0.08)、(1.65±0.00),GLUT1(2.11±0.16)、(1.57±0.08),PKM2(2.66±0.19)、(1.51±0.06),HK2(2.84±0.10)、(1.75±0.12)和蛋白表达CollagenⅠ(4.71±0.07)、(1.76±0.02),α-SMA(4.11±0.04)、(1.57±0.03),GLUT1 (4.06±0.10)、(1.51±0.04),PKM2 (4.78±0.15)、(1.35±0.01),HK2 (4.07±0.07)、(1.69±0.06)高于对照组A549 及HFLF 细胞mRNA CollagenⅠ(1.00±0.05)、(0.57±0.05),α-SMA(1.00±0.05)、(0.71±0.01),GLUT1(1.00±0.06)、(0.72±0.04),PKM2(1.00±0.04)、(0.64±0.01),HK2(1.00±0.04)、(0.67±0.01)和蛋白表达CollagenⅠ(1.00±0.02)、(0.21±0.01),α-SMA(1.00±0.02)、(0.23±0.02),GLUT1(1.00±0.01)、(0.20±0.00),PKM2 (1.00±0.06)、(0.22±0.01),HK2 (1.00±0.04)、(0.29±0.02)(P<0.05)。2-DG 组、IR-780 组A549、HFLF 细胞CollagenⅠ、α-SMA 蛋白及mRNA 表达低于TGF-β 组(P<0.05)。 2-DG 组与IR-780 组A549 及HFLF 细胞CollagenⅠ、α-SMA 表达差异无统计学意义(P>0.05)。 TGF-β 组A549 和HFLF 细胞糖酵解相关蛋白GLUT1、PKM2、HK2 表达升高,2-DG 组、IR-780 组糖酵解相关蛋白GLUT1、PKM2、HK2 表达低于TGF-β 组(P<0.05)。 2-DG 组与IR-780 组糖酵解相关蛋白表达差异无统计学意义(P>0.05)。

结论

IR-780 抑制TGF-β 诱导A549、HFLF 细胞纤维化和糖酵解过程,作用机制与2-DG 相似,IR-780 可通过调节糖酵解代谢过程抑制肺纤维化。

Objective

To analyze the role of IR-780 in idiopathic pulmonary fibrosis (IPF) and its relationship with glycolysis.

Methods

Transforming growth factor-β (TGF-β) -induced fibrosis model was constructed using human type Ⅱalveolar epithelial cells (A549) and human embryonic lung fibroblasts(HELF).They were divided into control group,TGF-β group,2-deoxy-D-glucose (2-DG) group and IR-780 group.The control group was treated with DMEM,and the TGF-β group was treated with TGF-β(10μg/ml).2-DG group use TGF-beta (10 mu g/ml) +2 DG (5 tendency/L) processing,IR-780 group with TGF-beta (10 mu g/ml) +IR-780 mu (1 mol/L).Immunofluorescence was used to detect the expression of pulmonary fibrosis markers,and RT-PCR and Western blot were used to detect the expression of fibrosis markers and glycolysis markers.

Results

Immunofluorescence detection showed that the expressions of Collagen I and α-SMA in TGF-β group were increased compared with those in control group (P<0.05).RT-PCR and Western blot showed that mRNA Collagen I (3.17±0.06) and (1.74±0.03) of A549 and HFLF cells in TGF-β group,α-SMA (2.26±0.08) and (1.65±0.00),GLUT1 (2.11±0.16),(1.57±0.08),PKM2 (2.66±0.19),(1.51±0.06),HK2 (2.84±0.10),(1.75±0.12) and Collagen protein expression Ⅰ(4.71±0.07),(1.76±0.02) and alpha SMA (4.11 ±0.04),(1.57±0.03),GLUT1 (4.06±0.10),(1.51±0.04),PKM2 (4.78±0.15),(1.35±0.01),HK2 (4.07±0.07),(1.69±0.06) were higher than those of A549 and HFLF cells mRNA CollagenⅠ(1.00±0.05),(0.57±0.05),α-SMA (1.00±0.05),(0.71±0.01),GLUT1 (1.00±0.06),(0.72±0.04),PKM2 (1.00±0.04),(0.64±0.01),HK2 (1 ±0.04),(0.67±0.01) and protein expression CollagenⅠ(1.00±0.02),(0.21±0.01),α-SMA(1.00±0.02),(0.23±0.02),GLUT1 (1.00± 0.01),(0.20±0.00),PKM2 (1.00±0.06),(0.22±0.01),HK2 (1.00±0.04),(0.29±0.02) respectively(P<0.05).The protein and mRNA expressions of CollagenⅠand α-SMA in A549 and HFLF cells of 2-DG and IR-780 groups were lower than those of TGF-β group (P<0.05).There were no significant differences in the expressions of Collagen I and α-SMA in A549 and HFLF cells between 2-DG group and IR-780 group (P>0.05).The expressions of GLUT1,PKM2,HK2 in A549 and HFLF cells of TGF-β group were increased,and the expressions of GLUT1,PKM2,HK2 in 2-DG and IR-780 groups were lower than those in TGF-β group (P<0.05).There was no significant difference in glycolytic protein expression between 2-DG group and IR-780 group (P >0.05).

Conclusion

IR-780 inhibits fibrosis and glycolysis of A549 and HFLF cells induced by TGF-β.The mechanism of action is similar to that of 2-DG.IR-780 can inhibit pulmonary fibrosis by regulating glycolysis metabolism.

表1 Real-timePCR 引物序列
图1 免疫荧光检测TGF-β 处理后A549、HFLF 细胞Collagen Ⅰ
图2 免疫荧光检测TGF-β 处理后A549、HFLF 细胞α-SMA 表达
图3 免疫荧光检测TGF-β 处理后HFLF 细胞Collagen Ⅰ表达
图4 免疫荧光检测TGF-β 处理后HFLF 细胞α-SMA 表达
表2 IR-780、2-DG 处理TGF-β 诱导细胞中纤维化相关蛋白及mRNA 表达水平比较(±s
表3 IR-780、2-DG 处理TGF-β 诱导细胞中糖酵解相关蛋白及mRNA 表达比较(±s
1
Richeldi L,Collard HR,Jones MG.Idiopathic pulmonary fibrosis[J].Lancet,2017,389(10082): 1941-1952.
2
Hewlett JC,Kropski JA,Blackwell TS.Idiopathic pulmonary fibrosis:Epithelial-mesenchymal interactions and emerging therapeutic targets[J].J Int Soc Matrix Biol,2018,71-72: 112-127.
3
Zhang E,Luo S,Tan X,et al.Mechanistic study of IR-780 dye as a potential tumor targeting and drug delivery agent [ J ].Biomaterials,2014,35(2): 771-778.
4
Meng X,Yu Z,Xu W,et al.Control of fibrosis and hypertrophic scar formation via glycolysis regulation with IR780[J].Burns &trauma,2022,10: tkac015.
5
Li J,Shen C,Qiu H,et al.Intravesical IR-780 instillation prevents radiation cystitis by protecting urothelial integrity[J].Neurourol Urodynam,2023,42(1): 40-48.
6
Luo M,Chen L,Zheng J,et al.Mitigation of radiation-induced pulmonary fibrosis by small-molecule dye IR-780[J].Free Rad Biol Med,2021,164: 417-428.
7
Kang YP,Lee SB,Lee JM,et al.Metabolic profiling regarding pathogenesis of idiopathic pulmonary fibrosis[J].J Proteome Res,2016,15(5): 1717-1724.
8
Xie N,Tan Z,Banerjee S,et al.Glycolytic reprogramming in myofibroblast differentiation and lung fibrosis[J].Am J Respir Crit Care Med,2015,192(12): 1462-1474.
9
Lemons JM,Feng XJ,Bennett BD,et al.Quiescent fibroblasts exhibit high metabolic activity[J].PLoS biology,2010,8(10):e1000514.
10
Kottmann RM,Kulkarni AA,Smolnycki KA,et al.Lactic acid is elevated in idiopathic pulmonary fibrosis and induces myofibroblast differentiation via pH-dependent activation of transforming growth factor-beta[J].Am J Respir Crit Care Med,2012,186(8): 740-751.
11
Goodwin J,Choi H,Hsieh MH,et al.Targeting hypoxia-inducible factor-1alpha/pyruvate dehydrogenase kinase 1 axis by dichloroacetate suppresses bleomycin-induced pulmonary fibrosis[J].Am J Respir Cell Molecul Biol,2018,58(2): 216-231.
12
Lederer DJ,Martinez FJ.Idiopathic pulmonary fibrosis[J].New Engl J Med,2018,379(8): 797-798.
13
Diamantopoulos A,Wright E,Vlahopoulou K,et al.The burden of illness of idiopathic pulmonary fibrosis: a comprehensive evidence review[J].Pharmaco Economics,2018,36(7): 779-807.
14
Raghu G,Rochwerg B,Zhang Y,et al.An official ATS/ERS/JRS/ALAT clinical practice guideline: Treatment of idiopathic pulmonary fibrosis.An update of the 2011 clinical practice guideline[J].Am J Respir Crit Care Med,2015,192(2): e3-19.
15
Fridlender ZG,Cohen PY,Golan O,et al.Telomerase activity in bleomycin-induced epithelial cell apoptosis and lung fibrosis[J].European Respir J,2007,30(2): 205-213.
16
Conte E,Gili E,Fagone E,et al.Effect of pirfenidone on proliferation,TGF-beta-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts [ J].European J Pharmaceut Sci,2014,58: 13-19.
17
Katzen J,Beers MF.Contributions of alveolar epithelial cell quality control to pulmonary fibrosis[J].J Clin Invest,2020,130(10):5088-5099.
18
Bargagli E,Refini RM,d′Alessandro M,et al.Metabolic dysregulation in idiopathic pulmonary fibrosis[J].Int J Molecul Sci,2020,21(16): doi:10.3390/ijms21165663.
19
Michaeloudes C,Bhavsar PK,Mumby S,et al.Role of metabolic reprogramming in pulmonary innate immunity and its impact on lung diseases[J].J Innate Immun,2020,12(1): 31-46.
20
Liu X,Zhang L,Zhang W.Metabolic reprogramming:A novel metabolic model for pulmonary hypertension[J].Front Cardiovascul Med,2022,9: 957524.
21
Chung KP,Hsu CL,Fan LC,et al.Mitofusins regulate lipid metabolism to mediate the development of lung fibrosis[J].Nature Communicat,2019,10(1): 3390.
22
Chu SG,Villalba JA,Liang X,et al.Palmitic acid-rich high-fat diet exacerbates experimental pulmonary fibrosis by modulating endoplasmic reticulum stress[J].Am J Respirat Cell Molecul Biol,2019,61(6): 737-746.
23
Koudelka A,Cechova V,Rojas M,et al.Fatty acid nitroalkene reversal of established lung fibrosis[J].Redox Biol,2022,50:102226.
24
Shi X,Chen Y,Liu Q,et al.LDLR dysfunction induces LDL accumulation and promotes pulmonary fibrosis[J].Clin Translat Med,2022,12(1): e711.
25
Wang L,Yuan H,Li W,et al.ACSS3 regulates the metabolic homeostasis of epithelial cells and alleviates pulmonary fibrosis[J].Biochim Biophys Acta Molecul Basis Dis,2024,1870 (2):166960.
26
Selvarajah B,Azuelos I,Plate M,et al.mTORC1 amplifies the ATF4-dependent de novo serine-glycine pathway to supply glycine during TGF-beta(1)-induced collagen biosynthesis[J].Sci Signal,2019,12(582): doi:10.1126/scisignal.aav3048.
27
Xu Q,Cheng D,Li G,et al.CircHIPK3 regulates pulmonary fibrosis by facilitating glycolysis in miR-30a-3p/FOXK2-dependent manner[J].Int J Biolog Sci,2021,17(9): 2294-2307.
28
Inoki K,Haneda M,Maeda S,et al.TGF-beta 1 stimulates glucose uptake by enhancing GLUT1 expression in mesangial cells[J].Kidney Int,1999,55(5): 1704-1712.
29
Hertenstein H,McMullen E,Weiler A,et al.Starvation-induced regulation of carbohydrate transport at the blood-brain barrier is TGF-beta-signaling dependent[J].Elife,2021,10: doi:10.7554/eLife.62503.
30
Andrianifahanana M,Hernandez DM,Yin X,et al.Profibrotic upregulation of glucose transporter 1 by TGF-beta involves activation of MEK and mammalian target of rapamycin complex 2 pathways[J].FASEB J,2016,30(11): 3733-3744.
[1] 陈天恩, 刘树安, 薛欢家, 王慧, 邱会格, 高姗姗, 陈兰新, 经慧, 武焱旻, 王凯. 尼达尼布及吡非尼酮治疗特发性肺纤维化及非特发性肺纤维化的荟萃分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(06): 861-868.
[2] 王亚岚, 倪婧, 余世庆, 陶银花, 张荣. 尼达尼布抗纤维化治疗特发性肺纤维化的耐受性和疗效预测因素分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 750-755.
[3] 周璇, 谢莉, 邹娟. 尼达尼布对特发性肺纤维化肺功能、肺纤维化程度及PDGF、PGE2、TGF-β1的影响[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(03): 368-372.
[4] 邱凌霄, 王创业, 卿斌, 刘锦程, 张鑫烨, 武文娟, 邢德冰, 郭亮, 徐智, 王斌. 基于转录组学筛选特发性肺纤维化的枢纽基因和信号通路[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(02): 212-217.
[5] 吴沛玲, 娄月妍, 张洪艳, 陈东方, 刘雪青, 赵丽芳, 薛姗, 蒋捍东. 线粒体相关基因在特发性肺纤维化中的分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(02): 178-184.
[6] 拉周措毛, 山春玲, 李国蓉, 华毛. 青海西宁地区IPF-LC的病理类型及临床特征分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(01): 25-29.
[7] 谷先勇, 徐娟. 特发性肺纤维化合并T2DM炎症水平与预后相关性分析[J/OL]. 中华肺部疾病杂志(电子版), 2023, 16(05): 721-723.
[8] 蒋梦洁, 钱治军, 徐思, 梁伟. 低强度脉冲超声波治疗冻结肩模型兔的实验研究[J/OL]. 中华肩肘外科电子杂志, 2023, 11(01): 30-34.
[9] 王静, 丁红. 益肾化湿颗粒对慢性肾衰竭大鼠肾组织转化生长因子-β1、α-平滑肌肌动蛋白表达的影响[J/OL]. 中华肾病研究电子杂志, 2024, 13(03): 161-165.
[10] 洪权. 肾脏疾病中的代谢重编程:新机制与新的治疗机会[J/OL]. 中华肾病研究电子杂志, 2024, 13(01): 60-60.
[11] 刘一, 文旖旎, 吴映辉. 过敏性紫癜患儿外周血辅助性T细胞、调节性T细胞细胞因子与肾损害的相关性分析[J/OL]. 中华肾病研究电子杂志, 2023, 12(05): 271-275.
[12] 郑鑫蓥, 张惠勇, 黄星, 邱磊, 方庆亮, 鹿振辉, 王蕾. TGF-β在放射治疗中的双重调控作用[J/OL]. 中华临床医师杂志(电子版), 2024, 18(03): 309-314.
[13] 李易飞, 李文冉, 刘欢. 乳酸脱氢酶A在乳腺癌诊疗中的研究进展[J/OL]. 中华诊断学电子杂志, 2024, 12(02): 128-132.
[14] 吕昆明, 王沙沙, 万军, 令狐恩强. 胃食管反流病与特发性肺纤维化关系的研究进展[J/OL]. 中华胃肠内镜电子杂志, 2023, 10(02): 121-124.
[15] 申超, 吴雪, 张政, 杜毓锋. 龟龄集对小鼠肺纤维化的改善作用及作用机制[J/OL]. 中华老年病研究电子杂志, 2024, 11(04): 9-15.
阅读次数
全文


摘要