Investigation of the effects of resveratrol, lutein, and crocetin on ARPE-19 cells induced with oxidative damage by H2O2

IF 2.5 4区生物学 Q1 ANATOMY & MORPHOLOGY

Tissue & cell Pub Date : 2025-09-09 DOI:10.1016/j.tice.2025.103133

Ayşegül Yabaş , Veysel Levent Karabaş , Selenay Furat , Ecem Önder Tokuç , Ahmet Öztürk , Candan Altuntaş , Özgür Doğa Özsoy , Gökhan Duruksu , Yusufhan Yazır

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引用次数: 0

Abstract

Objective

This study aimed to evaluate and compare the antioxidant effects, influence on autophagy and mitophagy, and impact on cell viability of resveratrol, lutein, and crocetin in hydrogen peroxide (H₂O₂)-induced oxidative damage in ARPE-19 cells as an in vitro model of age-related macular degeneration (AMD).

Methods

Oxidative damage was induced in ARPE-19 cells by exposure to 800 μM H₂O₂ for 1 h. Cell viability was assessed using the WST-1 assay. Subsequently, ARPE-19 cells were treated with lutein (5 and 10 μM), crocetin (10 and 20 μM), or resveratrol (100 μM), and the levels of oxidative damage biomarkers including malondialdehyde (MDA), glutathione (GSH), and nitric oxide (NO) were quantified via spectrophotometry. The autophagy- and mitophagy-related markers, LC3B, PINK1, and PARKIN, were visualized using confocal microscopy, and LC3B and PARKIN were further evaluated by western blotting (WB).

Results

Cell viability results were 100 % in the control group, decreased to 73.5 % and 69.1 % with 10 and 20 μM crocetin, 62.7 % and 59.3 % with 5 and 10 μM lutein, and 52.7 % with 100 μM resveratrol, respectively, while H₂O₂ exposure reduced viability to 0.04 %. Exposure to H₂O₂ (800 µM, 1 h) induced significant oxidative damage in ARPE-19 cells, as indicated by a reduction in GSH levels (p < 0.01) and an increase in MDA (p < 0.001) and NO (p < 0.001) compared to the control group, along with a notable decrease in WST-1 viability. Among the interventions, 10 µM crocetin significantly decreased MDA (p = 0.019) and NO (p = 0.05) levels compared to those in the damage group, although the 20 µM concentration also reduced these markers without achieving statistical significance. 5 µM Lutein significantly reduced NO levels compared to the damage group, whereas reductions in MDA at concentrations of 5–10 µM were not statistically significant. GSH levels exhibited a numerical, albeit non-significant, increase with 10 µM lutein (p = 0.09), and showed modest, non-significant increases with crocetin and resveratrol. The highest LC3B expression was observed in the 5 μM lutein group compared to control and other treatment groups, while PARKIN expression was significantly elevated in the 10 μM lutein, 20 μM crocetin, and 100 μM resveratrol groups, with 20 μM crocetin and resveratrol levels also exceeding lutein 5 μM.

Conclusions

10 μM Crocetin demonstrated the strongest antioxidant protection, while 5 μM lutein primarily improved cell survival, likely through autophagy activation and 100 μM resveratrol also activated both autophagy and mitophagy. These results highlighted the complementary concentration-dependent mechanisms of natural antioxidants in protecting RPE cells from oxidative stress related to AMD.

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白藜芦醇、叶黄素和西红花素对H2O2诱导的ARPE-19细胞氧化损伤的影响

目的评价和比较白藜芦醇、叶黄素和crocetin在过氧化氢（H₂O₂）诱导的ARPE-19细胞氧化损伤中的抗氧化作用、对细胞自噬和自噬的影响以及对细胞活力的影响，以此作为年龄相关性黄斑变性（AMD）的体外模型。方法800 μM H₂O₂照射1 H，诱导ARPE-19细胞氧化损伤。采用WST-1法测定细胞活力。随后，分别用叶黄素（5和10 μM）、西红花素（10和20 μM）或白藜芦醇（100 μM）处理ARPE-19细胞，并通过分光光度法定量氧化损伤生物标志物丙二醛（MDA）、谷胱甘肽（GSH）和一氧化氮（NO）的水平。共聚焦显微镜观察自噬和有丝自噬相关标志物LC3B、PINK1和PARKIN， western blotting （WB）进一步检测LC3B和PARKIN。ResultsCell可行性结果100 %在对照组,下降到73.5 % 69.1 % 10和20 μM藏花酸, % 62.7和59.3 % 5和10 μM叶黄素,和52.7 % 100 μM白藜芦醇,分别,而H₂O₂暴露生存能力降低到0.04 %。与对照组相比，暴露于H₂O₂（800 µM, 1 H）诱导ARPE-19细胞显著氧化损伤，GSH水平降低（p <； 0.01），MDA （p <； 0.001）和NO （p <； 0.001）增加，WST-1活力显著降低。在干预措施中，与损伤组相比，10 µM crocetin显著降低MDA （p = 0.019）和NO （p = 0.05）水平，尽管20 µM浓度也降低了这些标志物，但没有达到统计学意义。与损伤组相比，5 µM叶黄素显著降低NO水平，而5 - 10 µM浓度的MDA降低无统计学意义。10 µM叶黄素（p = 0.09）使谷胱甘肽水平有了数值上的，但不显著的增加（p p = 0.09），而番石榴素和白藜芦醇则有了适度的，不显著的增加。与对照组和其他处理组相比，5 μM叶黄素组LC3B表达量最高，10 μM叶黄素、20 μM豆黄素和100 μM白藜芦醇组PARKIN表达量显著升高，20 μM豆黄素和白藜芦醇的表达量也超过5 μM叶黄素。结论10 μM Crocetin表现出最强的抗氧化保护作用，5 μM叶黄素主要通过激活自噬来提高细胞存活率，100 μM白藜芦醇同时激活自噬和有丝自噬。这些结果强调了天然抗氧化剂在保护RPE细胞免受AMD相关氧化应激中的互补浓度依赖机制。

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来源期刊

Tissue & cell 医学-解剖学与形态学

CiteScore

3.90

自引率

0.00%

发文量

234

期刊介绍： Tissue and Cell is devoted to original research on the organization of cells, subcellular and extracellular components at all levels, including the grouping and interrelations of cells in tissues and organs. The journal encourages submission of ultrastructural studies that provide novel insights into structure, function and physiology of cells and tissues, in health and disease. Bioengineering and stem cells studies focused on the description of morphological and/or histological data are also welcomed. Studies investigating the effect of compounds and/or substances on structure of cells and tissues are generally outside the scope of this journal. For consideration, studies should contain a clear rationale on the use of (a) given substance(s), have a compelling morphological and structural focus and present novel incremental findings from previous literature.