人类虹膜睫状体成纤维细胞更易受到氧化应激的影响。

IF 3 2区 医学 Q1 OPHTHALMOLOGY Experimental eye research Pub Date : 2024-09-19 DOI:10.1016/j.exer.2024.110105
Simon Trusen , Julia Sarah Alexandra Zimmermann , Fabian Norbert Fries , Zhen Li , Ning Chai , Berthold Seitz , Shweta Suiwal , Maryam Amini , Nóra Szentmáry , Tanja Stachon
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引用次数: 0

摘要

Aniridia 相关性角膜病源于转录因子 PAX6(PAX6+/-)的单倍体缺陷。在 PAX6+/- 小鼠的角膜上皮细胞中,观察到氧化蛋白显著增加,同时氧化应激(OS)升高的补偿功能受损。在先天性无角膜症(AN)的病例中,角膜缘成纤维细胞(LFC)对氧化应激的易感性增加,其影响程度尚未确定。我们的目的是研究OS对正常和先天性无虹膜症LFC抗氧化酶表达的影响。在分离和培养原代LFCs(n = 8)和AN-LFCs(n = 8)后,用氯化钴处理细胞48小时,以化学方法诱导缺氧条件和OS。随后,通过 qPCR 检测 HIF-1α/-2α、PHD1/2、Nrf2、CAT、SOD1、PRDX6 和 GPX1 基因的表达。通过 Western 印迹对细胞裂解液中的 SOD1、PRDX6 和 GPX1 蛋白水平进行了评估。缺氧诱导导致两组成纤维细胞中 HIF-1α 基因表达减少(p≤0.008),而 PHD1 的减少仅限于 AN-LFCs (p=0.0007)。另一方面,在缺氧条件下,PHD2在AN-LFCs中的mRNA表达高于正常LFCs(p=0.013)。由于 OS 的结果,Nrf2 的 mRNA 水平(p2 treatment.
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Increased susceptibility of human limbal aniridia fibroblasts to oxidative stress
Aniridia-associated keratopathy originates from a haploinsufficiency of the transcription factor PAX6 (PAX6+/−). In the corneal epithelium of PAX6+/− mice, a significant increase in oxidized proteins was observed, accompanied by impaired compensation for elevated oxidative stress (OS). The extent to which limbal fibroblast cells (LFCs) are affected by an increased susceptibility to OS in cases of congenital aniridia (AN) has not been determined, yet. Our aim was to examine the impact of OS on antioxidant enzyme expression in normal and AN-LFCs. Following isolation and culture of primary LFCs (n = 8) and AN-LFCs (n = 8), cells were treated with cobalt chloride for 48 h to chemically induce hypoxic conditions and OS. Subsequently, HIF-1α/-2α, PHD1/2, Nrf2, CAT, SOD1, PRDX6, and GPX1 gene expression was examined by qPCR. SOD1, PRDX6, and GPX1 protein levels were assessed from the cell lysate by Western blot. The induction of hypoxia led to reduced HIF-1α gene expression in both fibroblast groups (p 0.008), while the decrease in PHD1 was limited to AN-LFCs (p = 0.0007). On the other hand, under hypoxic conditions, PHD2 showed higher mRNA expression in AN-LFCs compared to normal LFCs (p = 0.013). As a result of OS, the mRNA levels of Nrf2 (p<0.0001) and the antioxidant enzymes CAT (p = 0.005), SOD1 (p = 0.005), GPX1 (p = 0.002) decreased in AN-LFCs. This was accompanied by an increased protein expression of SOD1 (p = 0.019) and PRDX6 (p=0.0009). In the normal LFC group, the induced extent of OS had no impact on the gene (p0.151) and protein expression (p ≥ 0.629) of antioxidant enzymes, except for the GPX1 mRNA level (p = 0.027). AN-LFCs exhibit higher susceptibility to OS than normal LFCs. Therefore, in AN-LFCs, there are sustained alterations in gene and protein expression of antioxidative enzymes even after 48 h of CoCl2 treatment.
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来源期刊
Experimental eye research
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.
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