咖啡酸苯乙酯与贝伐单抗联合应用对过氧化氢诱导的人视网膜色素上皮细胞氧化应激的保护作用
Protective Effect of Combined Caffeic Acid Phenethyl Ester and Bevacizumab Against Hydrogen Peroxide-Induced Oxidative Stress in Human RPE Cells.
作者信息
Dinc Erdem, Ayaz Lokman, Kurt Akif Hakan
机构信息
a Department of Ophthalmology , Faculty of Medicine , Mersin University , Mersin , Turkey.
b Department of Biochemistry , Faculty of Pharmacy , Trakya University , Edirne , Turkey.
出版信息
Curr Eye Res. 2017 Dec;42(12):1659-1666. doi: 10.1080/02713683.2017.1368085. Epub 2017 Sep 22.
PURPOSE
This study aimed to evaluate the protective effects of caffeic acid phenethyl ester (CAPE) and combined CAPE-bevacizumab against oxidative stress induced by hydrogen peroxide (HO) in human retinal pigment epithelium.
METHODS
ARPE-19 cells were pretreated with 5, 10, and 30 μM CAPE alone and in combination with bevacizumab for 3 h, then exposed to HO for 16 h. Cell viability was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Vascular endothelial growth factor (VEGF) protein levels in the medium were measured using a human VEGF ELISA kit. Total antioxidant status (TAS) and total oxidant status (TOS) were measured in ARPE-19 cells using the test kit from Rel Assay. Expression levels of VEGF, Bax, Bcl-2, cytochrome c, apoptotic protease activating factor-1 (apaf-1), and caspase-3 were determined using reverse transcription polymerase chain reaction.
RESULTS
Pretreatment of ARPE-19 cells with 30 μM CAPE and combined CAPE-bevacizumab reduced HO mediated cell death. HO-induced oxidative stress increased TOS and VEGF production, which was significantly inhibited by CAPE and the CAPE-bevacizumab combination. VEGF, Bax, cytochrome c, apaf-1, and caspase-3 gene expressions were significantly decreased in cells pretreated with 5, 10, and 30 μM CAPE and combined CAPE-bevacizumab compared to the HO group. In addition, Bcl-2 expression was significantly increased in both the CAPE and CAPE-bevacizumab combination groups compared to the HO group.
CONCLUSIONS
CAPE has a protective effect on ARPE-19 cells against oxidative stress, and VEGF protein level and expression can be decreased by incubation with different concentrations of CAPE. These results demonstrate that CAPE suppresses the mitochondria-mediated apoptosis in ARPE-19 cells under oxidative stress. In addition, the use of CAPE in combination with bevacizumab has an additive effect.
目的
本研究旨在评估咖啡酸苯乙酯（CAPE）以及CAPE与贝伐单抗联合使用对过氧化氢（H₂O₂）诱导的人视网膜色素上皮细胞氧化应激的保护作用。
方法
将ARPE - 19细胞分别用5、10和30 μM的CAPE单独处理以及与贝伐单抗联合处理3小时，然后暴露于H₂O₂中16小时。采用3 -（4,5 - 二甲基噻唑 - 2 - 基）- 2,5 - 二苯基四氮唑溴盐（MTT）法评估细胞活力。使用人血管内皮生长因子（VEGF）ELISA试剂盒测定培养基中VEGF蛋白水平。使用Rel Assay检测试剂盒测定ARPE - 19细胞中的总抗氧化状态（TAS）和总氧化状态（TOS）。采用逆转录聚合酶链反应测定VEGF、Bax、Bcl - 2、细胞色素c、凋亡蛋白酶激活因子 - 1（apaf - 1）和半胱天冬酶 - 3的表达水平。
结果
用30 μM CAPE以及CAPE与贝伐单抗联合预处理ARPE - 19细胞可减少H₂O₂介导的细胞死亡。H₂O₂诱导的氧化应激增加了TOS和VEGF的产生，而CAPE以及CAPE与贝伐单抗联合使用可显著抑制这种增加。与H₂O₂组相比，用5、10和30 μM CAPE以及CAPE与贝伐单抗联合预处理的细胞中，VEGF、Bax、细胞色素c、apaf - 1和半胱天冬酶 - 3基因表达显著降低。此外，与H₂O₂组相比，CAPE组和CAPE与贝伐单抗联合组中Bcl - 2表达均显著增加。
结论
CAPE对ARPE - 19细胞的氧化应激具有保护作用，不同浓度的CAPE孵育可降低VEGF蛋白水平和表达。这些结果表明，CAPE在氧化应激下可抑制ARPE - 19细胞中线粒体介导的凋亡。此外，并使用CAPE与贝伐单抗具有相加作用。
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