Chronic hyperglycemia involves persistent high-glucose exposure and correlates with retinal degeneration. It causes various diseases, including diabetic retinopathy (DR), a major cause of adult vision loss. Most in vitro studies have investigated the damaging short-term effects of high glucose exposure on retinal pigment epithelial (RPE) cells. DR is also a severe complication of diabetes. In this study, we established a model with prolonged high-glucose exposure (15 and 75 mM exogenous glucose for two months) to mimic RPE tissue pathophysiology in patients with hyperglycemia. Prolonged high-glucose exposure attenuated glucose uptake and clonogenicity in ARPE-19 cells. It also significantly increased reactive oxygen species levels and decreased antioxidant protein (superoxide dismutase 2) levels in RPE cells, possibly causing oxidative stress and DNA damage and impairing proliferation. Western blotting showed that autophagic stress, endoplasmic reticulum stress, and genotoxic stress were induced by prolonged high-glucose exposure in RPE cells. Despite a moderate apoptotic cell population detected using the Annexin V-staining assay, the increases in the senescence-associated proteins p53 and p21 and SA-β-gal-positive cells suggest that prolonged high-glucose exposure dominantly sensitized RPE cells to premature senescence. Comprehensive next-generation sequencing suggested that upregulation of oxidative stress and DNA damage-associated pathways contributed to stress-induced premature senescence of ARPE-19 cells. Our findings elucidate the pathophysiology of hyperglycemia-associated retinal diseases and should benefit the future development of preventive drugs.

Graphical Abstract

Prolonged high-glucose exposure downregulates glucose uptake and oxidative stress by increasing reactive oxygen species (ROS) production through regulation of superoxide dismutase 2 (SOD2) expression. Autophagic stress, ER stress, and DNA damage stress (genotoxic stress) are also induced by prolonged high-glucose exposure in RPE cells. Consequently, multiple stresses induce the upregulation of the senescence-associated proteins p53 and p21. Although both apoptosis and premature senescence contribute to high glucose exposure-induced anti-proliferation of RPE cells, the present work shows that premature senescence rather than apoptosis is the dominant cause of RPE degeneration, eventually leading to the pathogenesis of DR.

中文翻译：

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长期暴露于高血糖会通过视网膜色素上皮细胞的氧化应激和自噬诱导过早衰老

慢性高血糖涉及持续的高血糖暴露，并与视网膜变性相关。它会导致多种疾病，包括糖尿病视网膜病变（DR），这是成人视力丧失的主要原因。大多数体外研究都调查了高葡萄糖暴露对视网膜色素上皮（RPE）细胞的短期破坏性影响。DR也是糖尿病的严重并发症。在这项研究中，我们建立了一个长期高葡萄糖暴露（15 和 75 mM 外源葡萄糖两个月）的模型来模拟高血糖患者的 RPE 组织病理生理学。长时间的高葡萄糖暴露会减弱 ARPE-19 细胞的葡萄糖摄取和克隆形成能力。它还显着增加 RPE 细胞中的活性氧水平并降低抗氧化蛋白（超氧化物歧化酶 2）水平，可能导致氧化应激和 DNA 损伤并损害增殖。Western blotting显示，RPE细胞长期高糖暴露会诱导自噬应激、内质网应激和基因毒性应激。尽管使用膜联蛋白 V 染色法检测到中等程度的凋亡细胞群，但衰老相关蛋白 p53 和 p21 以及 SA-β-gal 阳性细胞的增加表明，长期高葡萄糖暴露主要使 RPE 细胞对过早衰老敏感。全面的下一代测序表明，氧化应激和 DNA 损伤相关途径的上调导致应激诱导的 ARPE-19 细胞过早衰老。我们的研究结果阐明了高血糖相关视网膜疾病的病理生理学，应该有利于预防药物的未来开发。

图形概要

长时间的高血糖暴露可通过调节超氧化物歧化酶 2 (SOD2) 表达来增加活性氧 (ROS) 的产生，从而下调葡萄糖摄取和氧化应激。自噬应激、内质网应激和 DNA 损伤应激（基因毒性应激）也会因 RPE 细胞长期暴露于高葡萄糖而诱发。因此，多重应激会诱导衰老相关蛋白 p53 和 p21 的上调。虽然细胞凋亡和过早衰老都有助于高糖暴露诱导的 RPE 细胞的抗增殖，但目前的研究表明，过早衰老而不是细胞凋亡是 RPE 退化的主要原因，最终导致 DR 的发病机制。