Oxidized LDL, homocysteine, homocysteine thiolactone and advanced glycation end products act as pro-oxidant metabolites inducing cytokine release, macrophage infiltration and pro-angiogenic effect in ARPE-19 cells
Oxidized LDL, homocysteine, homocysteine thiolactone and advanced glycation end products act as pro-oxidant metabolites inducing cytokine release, macrophage infiltration and pro-angiogenic effect in ARPE-19 cells
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DOI：
10.1371/journal.pone.0216899
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发表时间：
2019-05-14
期刊：
影响因子：
3.7
通讯作者：
Angayarkanni, Narayanasamy
中科院分区：
文献类型：
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作者：
AnandBabu, Kannadasan;Sen, Parveen;Angayarkanni, Narayanasamy
视网膜相关性黄斑变性（AMD）是威胁视力的主要眼病之一。氧化应激是AMD发病和发展的关键因素之一。在该研究中，基于在人视网膜色素上皮细胞（ARPE-19细胞）中的体外研究，在局部眼部环境中评价了与AMD病理学相关的代谢物（在全身水平上更是如此），即氧化LDL（oxLDL）、同型半胱氨酸（Hcy）、同型半胱氨酸硫内酯（HCTL）、晚期糖基化终产物（AGE））的促氧化性质。用促氧化剂50 pg/mL oxLDL、500 pM Hcy、500 nM HCTL、100 pg/mL AGE、200 pM H2 O2和200 pM H2 O2处理人ARPE-19细胞，用和不用5 mM N-乙酰半胱氨酸（NAC）预处理。ELISA法检测促氧化剂作用下ARPE-19细胞分泌的细胞因子IL-6、IL-8和血管内皮生长因子（VEGF）。在明胶-基质胶包被的96孔板中用促氧化剂处理的ARPE-19细胞的条件培养基进行体外血管生成测定。将人急性单核细胞白血病细胞系（THP-1）诱导分化为巨噬细胞，用transwell迁移实验研究其对促氧化剂损伤的ARPE-19细胞条件培养液的迁移反应。Western blot检测Bax、Bcl-2和NF-κ B蛋白表达，观察细胞凋亡情况。研究中涉及的化合物显示出ARPE-19细胞中活性氧（ROS）产生的显著增加（oxLDL; Hcy; AGE：p < 0.001和HCTL：p < 0.05）。NAC预处理显著降低了由促氧化剂引起的氧化应激，如通过降低细胞中的ROS和MDA水平所看到的。用促氧化剂处理显著增加IL-6（oxLDL：p < 0.05; Hcy、HCTL和AGE：p < 0.01）和IL-8细胞因子（oxLDL：p < 0.05; HCTL：p < 0.01）的分泌。
Age-related Macular Degeneration (AMD) is one of the major vision-threatening diseases of the eye. Oxidative stress is one of the key factors in the onset and progression of AMD. In this study, metabolites associated with AMD pathology more so at the systemic level namely, oxidized LDL (oxLDL), homocysteine (Hcy), homocysteine thiolactone (HCTL), advanced glycation end product (AGE) were evaluated for their pro-oxidant nature in a localized ocular environment based on in vitro studies in human retinal pigment epithelial cells (ARPE-19 cells). Human ARPE-19 cells were treated with pro-oxidants 50 pg/mL oxLDL, 500 pM Hcy, 500 nM HCTL, 100 pg/mL AGE, 200 pM H202 and 200 pM H2O2 with and without pre-treatment of 5 mM N-acetyl cysteine (NAC). The cytokines IL-6, IL-8 and vascular endothelial growth factor (VEGF) secreted from ARPE-19 cells exposed to pro-oxidants were estimated by ELISA. In vitro angiogenesis assay was performed with conditioned media of the pro-oxidant treated ARPE-19 cells in Geltrex-Matrigel coated 96-well plate. The human acute monocytic leukemia cell line (THP-1) was differentiated into macrophages and its migration in response to conditioned media of ARPE-19 cells insulted with the pro oxidants was studied by transwell migration assay. Western blot was performed to detect the protein expression of Bax, Bcl-2 and NF-KB to assess apoptotic changes. The compounds involved in the study showed a significant increase in reactive oxygen species (ROS) generation in ARPE-19 cells (oxLDL; Hcy; AGE: p < 0.001 and HCTL: p < 0.05). NAC pre-treatment significantly lowered the oxidative stress brought about by pro-oxidants as seen by lowered ROS and MDA levels in the cells. Treatment with pro-oxidants significantly increased the secretion of IL-6 (oxLDL: p < 0.05; Hcy, HCTL and AGE: p < 0.01) and IL-8 cytokines (oxLDL: p < 0.05; HCTL: p