成纤维细胞生长因子2对支气管肺发育不良的影响及机制分析。

IF 2.5 4区 医学 Q3 ALLERGY
Ling Lu, Ye-Dan Liu, Dong-Yun Liu, Rong Jin, Zheng-Hai Qu
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引用次数: 0

摘要

目的:肺泡上皮细胞上皮间质转化(epithelial -mesenchymal transition, EMT)是支气管肺发育不良(BPD)发生发展的重要机制。FGF-2在BPD中的作用目前尚不清楚。我们的研究目的是通过研究BPD小鼠肺组织中FGF-2的表达,进一步阐明FGF-2对肺泡上皮细胞EMT的影响,积极寻找可能的信号通路。方法:采用高氧诱导BPD模型。收集肺组织样本,采用苏木精和伊红(HE)染色测定模型效果。采用定量实时聚合酶链反应(QRT-PCR)、免疫组化检测BPD小鼠中FGF-2的表达。为了进一步研究FGF-2补充和缺乏对肺泡上皮细胞EMT的影响,我们对A549细胞进行了冷冻保存、重悬、培养和传代。转化生长因子-β1 (tgf -β1)诱导EMT。合成并筛选FGF-2小干扰RNA片段。BGJ398可抑制成纤维细胞生长因子受体1 (FGFR1)的表达。采用(3-(4,5 -二甲基噻唑-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)- 2h四氮唑)(MTS)法检测FGF-2和Infigratinib (BGJ398)对细胞增殖的影响。我们采用qRT-PCR和Western blot检测上皮细胞标志物、间充质细胞标志物和emt相关信号通路蛋白的表达。结果:成功建立的高氧小鼠模型具有BPD特征。高氧在第4天降低FGF-2,在第21天上调FGF-2,导致EMT。在体外,我们发现单独使用FGF-2可增加间充质标记物的表达,降低上皮标记物的表达,激活磷脂酰肌醇3-激酶/蛋白激酶B (PI3K/AKT)、小母抗十肢截瘫(Smad)、丝裂原活化蛋白激酶(P38)和细胞外信号调节激酶(ERK)信号通路。FGF-2不能逆转但能协同促进TGF-β1诱导的肺泡上皮细胞EMT。沉默FGF-2可增加上皮标志物E-cadherin的表达,抑制TGF-β1激活的PI3K/AKT、Smad、P38信号通路,激活ERK信号通路。FGF-2受体抑制剂BGJ398逆转TGF-β1诱导的EMT,降低FGFR1表达,抑制ERK信号通路激活。结论:FGF2与BPD小鼠EMT密切相关。不管是高水平还是低水平的FGF2都会促进A549的EMT。FGF-2受体抑制剂BGJ398通过抑制FGFR1/P-ERK信号通路逆转TGF-β1诱导的A549 EMT。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Analysis of Fibroblast Growth Factor 2 Impact and Mechanism on Broncho-Pulmonary Dysplasia.

Objective: Epithelial-mesenchymal transition (EMT) of alveolar epithelial cells is an important mechanism for the onset and development of broncho-pulmonary dysplasia (BPD).The role of FGF-2 in BPD is currently unclear. The aim of our study is to investigate the expression of FGF-2 in lung tissue of BPD mice, to further clarify the effect of FGF-2 on EMT in alveolar epithelial cells and to actively search for possible signaling pathways.

Methods: The BPD model was induced by exposure to hyperoxia. Lung tissue samples were collected and Hematoxylin and eosin (HE) staining was used to determine the modelling effect. Quantitative Real-time Polymerase Chain Reaction (QRT-PCR), immunohistochemistry were used to detect FGF-2 expression in BPD mice. To further investigate the effect of FGF-2 supplementation and deficiency on EMT in alveolar epithelial cells, A549 cells were cryopreserved, resuspended, cultured and passaged. Transforming growth factor-β1 (TGFβ1) was used to induce EMT. FGF-2 small interfering RNA fragments were synthesised and screened. Fbroblast growth factor receptor1 (FGFR1) expression was inhibited by BGJ398.  (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium) (MTS) assay was used to detect the effect of FGF-2 and Infigratinib (BGJ398) on cell proliferation. We used qRT-PCR and Western blot to detect the expression of epithelial cell markers, mesenchymal cell markers and EMT-related signaling pathway proteins.

Results: Our results showed that the successful established hyperoxia mice model were characteristic by BPD. Hyperoxia decreased FGF-2 on day 4, upregulated FGF-2 on day 21, which resulted in EMT. In vitro, we found that FGF-2 alone increased the expression of mesenchymal markers, decreased the expression of epithelial markers and activated Phosphatidylinositol 3-kinase/Protein kinase B (PI3K/AKT), Small mother against decapentaplegic (Smad), mitogen-activated protein kinase (P38) and extracellular signal-regulated kinase (ERK) signaling pathways. FGF-2 could not reverse but synergistically promote TGF-β1-induced EMT of alveolar epithelial cells. Silencing FGF-2 increased the expression of epithelial marker E-cadherin, inhibited the PI3K/AKT, Smad, and P38 signaling pathways activated by TGF-β1, but activated ERK signaling. FGF-2 receptor inhibitor BGJ398 reversed TGF-β1-induced EMT, decreased the expression of FGFR1, and inhibited ERK signaling pathway activation.

Conclusions: FGF2 was closely associated with EMT in BPD mice. Both high and low levels of FGF2 promoted EMT in A549. The FGF-2 receptor inhibitor BGJ398 reversed TGF-β1-induced EMT in A549 by inhibiting the FGFR1/P-ERK signaling pathway.

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来源期刊
CiteScore
5.60
自引率
3.60%
发文量
105
审稿时长
2 months
期刊介绍: ''International Archives of Allergy and Immunology'' provides a forum for basic and clinical research in modern molecular and cellular allergology and immunology. Appearing monthly, the journal publishes original work in the fields of allergy, immunopathology, immunogenetics, immunopharmacology, immunoendocrinology, tumor immunology, mucosal immunity, transplantation and immunology of infectious and connective tissue diseases.
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