晚期糖基化终末产物对人视网膜色素上皮细胞铁死亡的诱导作用

童俊; 解正高; 雷黄依; 包延波; 黄振平

doi:10.3760/cma.j.cn115989-20240318-00074

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中华实验眼科杂志

• 实验研究 •

ENGLISH ABSTRACT

晚期糖基化终末产物对人视网膜色素上皮细胞铁死亡的诱导作用

作者及单位信息

出版日期： 2025 -01 -10 ·

DOI： 10.3760/cma.j.cn115989-20240318-00074

Inducing effect of advanced glycation end products on ferroptosis in human retinal pigment epithelial cells

Authors Info & Affiliations

Tong Jun

Department of Ophthalmology, Nanjing Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210002, China

Xie Zhenggao

Department of Ophthalmology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China

Lei Huangyi

Department of Ophthalmology, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing 210029, China

Bao Yanbo

Department of Ophthalmology, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing 210029, China

Huang Zhenping

Department of Ophthalmology, Nanjing Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210002, China

Published： 2025 -01 -10 ·

DOI： 10.3760/cma.j.cn115989-20240318-00074

APP内阅读

摘要

目的探讨晚期糖基化终末产物(AGEs)对体外培养的人视网膜色素上皮(ARPE-19)细胞铁死亡的影响。

方法ARPE-19细胞系在含10%胎牛血清的DMEM培养基中培养并传代，选取第3～6代的细胞进行研究。分别在培养板中加入0、50、100、200、400 μg/ml AGEs，培养48 h，采用细胞计数试剂盒8检测各组细胞活性。选取200 μg/ml AGEs培养细胞48 h，采用脂质过氧化试剂盒(Bodipy 581/591 C11)联合流式细胞术测定细胞脂质过氧化水平；采用实时荧光定量PCR(qRT-PCR)和Western blot检测铁死亡标志蛋白溶质载体家族7成员11(SLC7A11)及谷胱甘肽过氧化物酶4(GPX4)的相对表达水平；采用透射电子显微镜观察各组细胞线粒体形态。

结果ARPE-19细胞活性随着AGEs浓度的增加逐渐下降，0、50、100、200、400 μg/ml AGEs组细胞活性总体比较差异有统计学意义( F＝6.21， P<0.01)，200、400 μg/ml AGEs组ARPE-19细胞活性均低于对照组，差异均有统计学意义(均 P<0.05)。流式细胞仪检测结果显示，AGEs组荧光强度为622.0±11.3，明显高于对照组的487.7±12.8，差异有统计学意义( t＝6.809， P＝0.002)。qRT-PCR检测结果显示，AGEs组细胞中SLC7A11、GPX4 mRNA和蛋白相对表达量明显低于对照组，差异均有统计学意义(mRNA： t＝3.72、7.14，均 P<0.05；蛋白： t＝6.20、5.15，均 P<0.05)。透射电子显微镜观察结果显示，AGEs组中线粒体皱缩，体积较对照组明显缩小，线粒体脊较对照组减少，线粒体膜密度较对照组增加。

结论AGEs能诱导体外培养的ARPE-19细胞发生铁死亡。

关键词：铁死亡;晚期糖基化终末产物;人视网膜色素上皮细胞;糖尿病视网膜病变

ABSTRACT

ObjectiveTo investigate the effect of advanced glycation end products (AGEs) on ferroptosis in human retinal pigment epithelium (RPE) cell cultured in vitro.

MethodsARPE-19 cell lines were cultured in DMEM medium containing 10% fetal bovine serum (FBS), and the 3rd to 6th generations of cells were used for further study.Cell activity was detected by using the cell counting kit 8 after ARPE-19 were cultured with AGEs at 0, 50, 100, 200, 400 μg/ml for 48 hours.The cells were cultured with 200 μg/ml AGEs for 48 hours.Cell lipid peroxidation level was measured by Lipid Peroxidation Assay Kit (Bodipy 581/591 C11) combined with flow cytometry.The relative mRNA and protein expression levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were determined by quantitative real-time PCR (qRT-PCR) and Western blot.The morphology of mitochondria was observed by transmission electron microscope.

ResultsThe activity of ARPE-19 cells decreased with increasing AGEs concentration, and the overall difference of ARPE-19 activity among 0, 50, 100, 200, 400 μg/ml AGEs groups was statistically significant ( F=6.21, P<0.01).The cell activity of ARPE-19 cells in 200 and 400 μg/ml AGEs groups was lower than that in control group (both P<0.05).Flow cytometry showed that the fluorescence intensity in AGEs group was 622.0±11.3, which was significantly higher than 487.7±12.8 in control group ( t=6.809, P=0.002).qRT-PCR showed that the mRNA and protein relative expression levels of SLC7A11 and GPX4 were lower in AGEs group than those in control group (mRNA: t=3.72, 7.14, both P<0.05; protein: t=6.20, 5.15, both P<0.05).Transmission electron microscopy showed that mitochondria in AGEs group shrank with significantly reduced volume, decreased mitochondrial cristae, and increased mitochondrial membrane density.

ConclusionsAGEs can induce ferroptosis in ARPE-19 cultured in vitro.

KEYWORDS： Ferroptosis;Advanced glycation end products;Human retinal pigment epithelial cell;Diabetic retinopathy

Corresponding author: Huang Zhenping, Email: mocdef.6ab2133691pzh

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引用本文

童俊,解正高,雷黄依,等. 晚期糖基化终末产物对人视网膜色素上皮细胞铁死亡的诱导作用[J]. 中华实验眼科杂志,2025,43(01)：32-37.

DOI:10.3760/cma.j.cn115989-20240318-00074

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糖尿病视网膜病变(diabetic retinopathy，DR)是糖尿病的主要眼部并发症，约占糖尿病患者的30%～40% ^{[
1
,

2
]}。全球DR患者超过1亿，是导致工作人群视力损伤和盲的主要原因 ^{[
1
,

2
,

3
]}。晚期糖基化终末产物(advanced glycation end products，AGEs)是DR中导致神经血管损伤的主要病理因素之一。有研究显示，糖尿病患者皮肤及玻璃体液中AGEs水平高于非糖尿病患者，且较高的AGEs水平与DR的进展独立相关 ^{[
4
,

5
]}。此外，较高的AGEs水平也是增生性DR的危险因素 ^{[
4
]}。AGEs在体内产生后优先积累在结构蛋白上，Bruch膜中的AGEs水平与视网膜色素上皮(retinal pigment epithelium，RPE)细胞功能密切相关 ^{[
6
]}。因此，研究AGEs对RPE细胞的影响及其发生机制具有重要意义。铁死亡作为一种新型的程序性细胞死亡方式，主要由铁依赖性的脂质过氧化物积累引起，与传统的凋亡或坏死机制不同，其特征在于细胞内铁离子的过量积聚和抗氧化能力下降 ^{[
7
]}。在DR中，高糖诱导下产生的大量活性氧可促进脂质过氧化物生成，从而触发铁死亡；另一方面，缺乏抗氧化酶，如谷胱甘肽过氧化物酶4(glutathione peroxidase 4，GPX4)，可使细胞更易受到氧化应激影响，加速DR的发展 ^{[
8
,

9
]}。一些研究还发现，抑制铁死亡可以显著减轻DR模型小鼠中的视网膜损伤，并改善其视觉功能 ^{[
10
,

11
]}。AGEs作为糖尿病患者体内常见的一类有害物质，影响细胞的抗氧化能力，从而加剧了铁死亡的发生 ^{[
12
]}。在DR中，AGEs能否诱导RPE细胞发生铁死亡尚不清楚。本研究探讨AGEs能否诱导RPE细胞发生铁死亡，从而为DR的发病机制研究及治疗方法探索提供新的思路。

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参考文献

参考文献

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Yau JW , <x>Ro</x> <x>gers</x> SL , Kawasaki R ,et al. Global prevalence and major risk factors of diabetic retinopathy[J]. Diabetes Care， 2012，35(3)∶556-564. DOI： 10.2337/dc11-1909 .