Kaur Satinder, Sohnen Peri, Kumar Simran, Vohra Mehak, Swamynathan Sudha, Swamynathan Shivalingappa
Department of Ophthalmology, Morsani College of Medicine, University of South Florida, United States.
Invest Ophthalmol Vis Sci. 2025 Mar 3;66(3):30. doi: 10.1167/iovs.66.3.30.
Previously, we reported that the secreted Ly6/uPAR-related protein-1 (SLURP1), abundantly expressed by the corneal epithelium (CE) and secreted into the tear fluid, suppresses NF-κB signaling in healthy corneas and is downregulated in response to a variety of stressors, allowing helpful inflammation to progress. Here we investigate whether SLURP1 manifests its broad protective effects by promoting corneal redox homeostasis.
Oxidative stress was induced in the wild-type (WT) and Slurp1-null (Slurp1X-/-) mouse corneas using 1350 J/m2 UV-B, and in human corneal limbal epithelial (HCLE) and SLURP1-overexpressing HCLE-SLURP1 cells with 100 J/m2 UV-B, 0.4 µg/mL mitomycin-C, or 0-100 µM H2O2. We evaluated their (i) redox status (GSH:GSSG ratio) using O-phthalaldehyde; (ii) reactive oxygen species (ROS) accumulation using 2',7'-dichlorodihydrofluorescein diacetate; (iii) antioxidants GPX4, CAT, and SOD2 expression by qRTPCR; (iv) lipid peroxidation by staining for 4-hydroxynonenol, malondialdehyde, and BODIPY-C11; and (v) DNA damage and NF-κB activation by immunostaining for γH2AX, 8-OHdG, NF-κB, and IκB.
Slurp1 was significantly downregulated in the UV-B-irradiated WT corneas. Oxidatively stressed HCLE-SLURP1 cells displayed relatively less ROS accumulation, lipid peroxidation, DNA damage and NF-κB activation, and a higher GSH/GSSG ratio and antioxidant gene expression than the similarly treated control HCLE cells. UV-B-irradiated Slurp1X-/- corneas displayed relatively more ROS accumulation, DNA damage and less GPX4 expression than the similarly treated WT corneas.
Collectively, these results elucidate that SLURP1 serves as an insult-agnostic immunomodulator that upregulates antioxidants and suppresses ROS accumulation to promote redox homeostasis in corneal epithelial cells and protect them from diverse genotoxic stressors.
此前，我们报道分泌型Ly6/uPAR相关蛋白-1（SLURP1）由角膜上皮（CE）大量表达并分泌到泪液中，在健康角膜中抑制NF-κB信号传导，并且在多种应激源作用下表达下调，从而使有益的炎症得以进展。在此，我们研究SLURP1是否通过促进角膜氧化还原稳态发挥其广泛的保护作用。
使用1350 J/m2的UV-B在野生型（WT）和Slurp1基因敲除（Slurp1X-/-）小鼠角膜中诱导氧化应激，使用100 J/m2的UV-B、0.4 μg/mL丝裂霉素-C或0-100 μM H2O2在人角膜缘上皮（HCLE）细胞和过表达SLURP1的HCLE-SLURP1细胞中诱导氧化应激。我们使用邻苯二甲醛评估它们的（i）氧化还原状态（谷胱甘肽：氧化型谷胱甘肽比值）；（ii）使用2',7'-二氯二氢荧光素二乙酸酯评估活性氧（ROS）积累；（iii）通过qRTPCR评估抗氧化剂GPX4、CAT和SOD2的表达；（iv）通过对4-羟基壬烯醛、丙二醛和BODIPY-C11进行染色评估脂质过氧化；以及（v）通过对γH2AX、8-羟基脱氧鸟苷、NF-κB和IκB进行免疫染色评估DNA损伤和NF-κB激活。
在UV-B照射的WT角膜中，Slurp1显著下调。与同样处理的对照HCLE细胞相比，氧化应激的HCLE-SLURP1细胞表现出相对较少的ROS积累、脂质过氧化、DNA损伤和NF-κB激活，以及更高的谷胱甘肽/氧化型谷胱甘肽比值和抗氧化基因表达。与同样处理的WT角膜相比，UV-B照射的Slurp1X-/-角膜表现出相对更多的ROS积累、DNA损伤和更少的GPX4表达。
总体而言，这些结果表明SLURP1作为一种与损伤无关的免疫调节剂，上调抗氧化剂并抑制ROS积累，以促进角膜上皮细胞的氧化还原稳态，并保护它们免受多种遗传毒性应激源的影响。
