Oxysterols induced inflammation and oxidation in primary porcine retinal pigment epithelial cells
Oxysterols induced inflammation and oxidation in primary porcine retinal pigment epithelial cells
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DOI：
10.1080/02713680601187951
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发表时间：
2007-01-01
影响因子：
2
通讯作者：
Creuzot-Garcher, Catherine
中科院分区：
文献类型：
--
作者：
Joffre, Corinne;Leclere, Laurent;Creuzot-Garcher, Catherine
目的：衰老与胆固醇酯在布鲁赫膜中的积累有关。胆固醇酯易于发生氧化并产生具有细胞毒性和促炎性质的氧固醇。我们研究了三种氧固醇对猪视网膜色素上皮（RPE）细胞原代培养物中线粒体功能障碍、炎症和氧化应激的影响。研究方法：将RPE细胞与氧固醇（50 μ M的24-羟基胆固醇、25-羟基胆固醇或7-酮基胆固醇）孵育24小时和48小时。线粒体脱氢酶活性测定线粒体活性。用荧光探针DCFH-DA检测RPE细胞内活性氧的生成，ELISA检测培养上清中IL-8的含量，RT-PCR半定量检测细胞内相应转录本的含量。结果如下：在RPE细胞和相应的媒体中的氧固醇含量的分析表明，细胞摄取率高，虽然观察到一些差异之间的7-酮胆固醇一方面和24-羟基胆固醇和25-羟基胆固醇另一方面。所有氧化固醇诱导轻微的线粒体功能障碍，但与对照相比，活性氧（ROS）的产生显着增加2至4倍。它们还以以下降序增强IL-8基因表达和IL-8蛋白分泌：25-羟基胆固醇> 24-羟基胆固醇> 7-酮胆固醇。结论：我们的结论是，在融合的原代猪RPE细胞，24-羟基胆固醇，25-羟基胆固醇，和7-酮胆固醇是氧化和炎症的有效诱导剂。
Purpose: Aging is associated with an accumulation of cholesterol esters in the Bruch membrane. Cholesterol esters are prone to undergo oxidation and generate oxysterols that have cytotoxic and proinflammatory properties. We investigated the effects of three oxysterols on mitochondrial dysfunctions, inflammation, and oxidative stress in primary cultures of porcine retinal pigment epithelial (RPE) cells. Methods: RPE cells were incubated with oxysterols (50 mu M of 24-hydroxycholesterol, 25-hydroxycholesterol, or 7-ketocholesterol) for 24 hr and 48 hr. Oxysterol content was determined in cells and in corresponding media by gas chromatography. Mitochondrial activity was measured by mitochondrial dehydrogenase activity. The intracellular formation of reactive oxygen species in RPE cells was detected by using the fluorescent probe DCFH-DA. IL-8 was assayed in the supernatants by ELISA, and the corresponding cellular transcripts were semiquantified by RT-PCR. Results: Analyses of the oxysterols content in the RPE cells and corresponding media suggested a high rate of cellular uptake, although some differences were observed between 7-ketocholesterol on the one hand and 24-hydroxycholesterol and 25-hydroxycholesterol on the other hand. All oxysterols induced slight mitochondrial dysfunctions but a significant 2- to 4-fold increase in reactive oxygen species (ROS) production compared with the control. They also enhanced IL-8 gene expression and IL-8 protein secretion in the following decreasing order: 25-hydroxycholesterol > 24-hydroxycholesterol > 7-ketocholesterol. Conclusions: We conclude that in confluent primary porcine RPE cells, 24-hydroxycholesterol, 25-hydroxycholesterol, and 7-ketocholesterol are potent inducers of oxidation and inflammation.