葡萄糖浓度的增加改变了体外无毛细血管畸形肢端成纤维细胞的 TGF-β1 和 NFκB 信号通路。

IF 3 2区医学 Q1 OPHTHALMOLOGY

Experimental eye research Pub Date : 2024-11-20 DOI:10.1016/j.exer.2024.110163

Zhen Li , Tanja Stachon , Sabrina Häcker , Fabian N. Fries , Ning Chai , Berthold Seitz , Lei Shi , Shao-Lun Hsu , Shuailin Li , Shanhe Liu , Maryam Amini , Shweta Suiwal , Nóra Szentmáry

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引用次数: 0

摘要

目的：确定葡萄糖浓度升高对体外原代健康人肢端成纤维细胞（LFCs）和先天性无毛细血管畸形人肢端成纤维细胞（AN-LFCs）基因表达的影响。分离 LFCs（n=8）和 AN-LFCs（n=8），分别在含血清的 DMEM（包括正常葡萄糖（17.5 mM）或高浓度葡萄糖（70 mM））中培养 48 小时或 72 小时。转化生长因子β1（TGF-β1）、α-平滑肌肌动蛋白（ACTA）2A1、SMAD 2/3、核因子卡巴B（NFκB）、诱导型一氧化氮合酶（iNOS）等缺氧标志物的 mRNA 和蛋白表达、利用 qPCR 和 Western 印迹分析了缺氧诱导因子 1-α（HIF-1ɑ）、氧化应激标志物如核因子红细胞 2 相关因子 2（Nrf2）和过氧化氢酶（CAT）。在 70 mM 葡萄糖浓度的培养基中培养 48 h，TGF-β1 mRNA 的表达明显降低（p=0.001，p=0.002）。

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Increased glucose concentration modifies TGF-β1 and NFκB signaling pathways in aniridia limbal fibroblasts, in vitro

To determine the impact of increased glucose concentration on gene expression of primary healthy human limbal fibroblasts (LFCs) and congenital aniridia human limbal fibroblasts (AN-LFCs), in vitro.

LFCs (n = 8) and AN-LFCs (n = 8) were isolated and cultured in serum containing DMEM, including either normal glucose (17.5 mM) or increased glucose (70 mM) concentration for 48h or 72h, respectively. mRNA and protein expression of transforming growth factor beta 1 (TGF-β1), alpha-smooth muscle actin (ACTA)2A1, SMAD 2/3, hypoxia markers such as nuclear factor kappa B (NFκB), inducible nitric oxide synthase (iNOS), hypoxia-inducible factor 1-alpha (HIF-1ɑ), oxidative stress markers such as nuclear factor erythroid 2-related factor 2 (Nrf2) and Catalase (CAT) were analyzed using qPCR and Western blot.

In 70 mM glucose concentration medium for 48 h, TGF-β1 mRNA expression was significantly lower (p = 0.001, p < 0.001), Nrf2 (p = 0.001, p = 0.001) and CAT (p = 0.001, p = 0.001) mRNA expression was significantly higher in LFCs and AN-LFCs, than using 17.5 mM glucose concentration medium. In addition, in 70 mM glucose concentration medium for 48 h, SMAD 2, SMAD 3, NFκB, HIF-1ɑ mRNA expression was significantly lower in AN-LFCs, than in 17.5 mM glucose concentration medium (p = 0.003, p = 0.002, p = 0.008, p = 0.020). At this time-point in 70 mM glucose concentration medium, at protein level, TGF-β1, SMAD2/3 and NFκB were significantly lower in AN-LFCs, than in 17.5 mM glucose concentration medium (p = 0.041, p = 0.002, p = 0.012).

In 70 mM glucose concentration medium for 72h, TGF-β1 was significantly higher (p < 0.001, p < 0.001) and Nrf2 (p = 0.001, p = 0.001) and CAT (p < 0.001, p < 0.001) mRNA were significantly lower in LFCs and AN-LFCs, than in 17.5 mM glucose concentration medium. At this time-point, in 70 mM glucose concentration medium, NFκB mRNA was significantly higher (p < 0.001) in LFCs, than in 17.5 mM glucose concentration DMEM medium. In 70 mM glucose concentration medium for 72 h, TGF-β1 and NFκB protein were significantly lower in AN-LFCs, than in 17.5 mM glucose concentration medium (p < 0.001, p < 0.001).

Our study confirmed that high glucose concentration has an impact on TGF-β1 and NFκB signaling both in AN-LFCs and LFCs. These findings highlight that prolonged exposure to high glucose levels may contribute to cellular stress and dysfunction in LFCs and AN-LFCs.

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来源期刊

Experimental eye research 医学-眼科学

CiteScore

6.80

自引率

5.90%

发文量

323

审稿时长

66 days

期刊介绍： The primary goal of Experimental Eye Research is to publish original research papers on all aspects of experimental biology of the eye and ocular tissues that seek to define the mechanisms of normal function and/or disease. Studies of ocular tissues that encompass the disciplines of cell biology, developmental biology, genetics, molecular biology, physiology, biochemistry, biophysics, immunology or microbiology are most welcomed. Manuscripts that are purely clinical or in a surgical area of ophthalmology are not appropriate for submission to Experimental Eye Research and if received will be returned without review.