The effects of quercetin in cultured human RPE cells under oxidative stress and in Ccl2/Cx3cr1 double deficient mice.
The effects of quercetin in cultured human RPE cells under oxidative stress and in Ccl2/Cx3cr1 double deficient mice.
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DOI：
10.1016/j.exer.2010.03.016
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发表时间：
2010-07
影响因子：
3.4
通讯作者：
Chan, Chi-Chao
中科院分区：
文献类型：
--
作者：
Cao, Xiaoguang;Liu, Melissa;Tuo, Jingsheng;Shen, Defen;Chan, Chi-Chao
关键词：
槲皮素是类黄酮家族的一员，是最重要的膳食抗氧化剂之一。本研究探讨了槲皮素对培养的人类 RPE 细胞和 Ccl2/Cx3cr1 双敲除 (DKO) 小鼠的影响机制，这些小鼠会自发地出现类似于年龄相关性黄斑变性 (AMD) 的进行性视网膜病变。在体外实验中，将培养的 ARPE-19 细胞暴露于含或不含 50μM 槲皮素的 1 mM H2O2 中 2 小时。分别使用结晶紫染色、MTT 测定和彗星测定评估细胞活力、线粒体功能和细胞凋亡。通过RQ-PCR测量BCL-2、BAX、FADD、CASPASE-3和CASPASE-9的凋亡分子转录物。测定培养基上清液中的 COX 活性和一氧化氮 (NO) 水平。槲皮素处理可保护 ARPE-19 细胞免受 H2O2 诱导的氧化损伤，增强 BCL-2 转录水平，增加 BCL-2/BAX 比值，抑制 BAX、FADD、CASPASE-3 和 CASPASE-9 等促凋亡因子的转录，抑制 TNF-α、COX-2 和 INOS 等炎症因子的转录，并降低培养基中 COX 和 NO 的水平。在体内实验中，DKO和C57/B6小鼠每天腹腔注射25mg/kg/天槲皮素，持续两个月。每月进行一次眼底检查。两个月后，收集血清以测量 NADP+/NADPH、COX、PGE-2 和 NO 水平。摘取眼睛用于组织学和 A2E 测量。通过RQ-PCR检测Bcl-2、Bax、Cox-2、Inos、Tnf-α、Fas、FasL和Caspase-3的眼部转录物。从眼底镜或组织学角度来看，槲皮素治疗并不能逆转 DKO 小鼠视网膜病变的进展。虽然槲皮素治疗可以恢复全身抗氧化能力，抑制NO、COX和PGE-2的全身表达，降低眼部A2E水平，但不能有效抑制DKO小鼠眼部炎症因子Tnf-α、Cox-2和Inos或促凋亡因子Fas、FasL和Caspase-3的转录。我们的数据表明，槲皮素可以通过抑制促炎分子和直接抑制内在凋亡途径，在体外保护人 RPE 细胞免受氧化应激。然而，槲皮素（25mg/kg/天）并不能改善 Ccl2−/−/Cx3cr1−/− 小鼠的视网膜 AMD 样病变，可能是由于其对眼部炎症和细胞凋亡途径的抑制不足。
Quercetin, a member of the flavonoid family, is one of the most prominent dietary antioxidants. This study investigates the mechanisms for the effects of quercetin on cultured human RPE cells and in Ccl2/Cx3cr1 double knock-out (DKO) mice, which spontaneously develop progressive retinal lesions mimicking age-related macular degeneration (AMD). In the in vitro experiment, cultured ARPE-19 cells were exposed to 1mM H2O2 with or without 50μM quercetin for 2 hours. Cellular viability, mitochondrial function, and apoptosis were assessed using crystal violet staining, MTT assay, and comet assay, respectively. Apoptotic molecular transcripts of BCL-2, BAX, FADD, CASPASE-3 and CASPASE-9 were measured by RQ-PCR. COX activity and nitric oxide (NO) level were determined in the supernatant of the culture medium. Quercetin treatment protected ARPE-19 cells from H2O2-induced oxidative injury, enhanced BCL-2 transcript levels, increased the BCL-2/BAX ratio, suppressed the transcription of pro-apoptotic factors such as BAX, FADD, CASPASE-3 and CASPASE-9, inhibited the transcription of inflammatory factors such as TNF-α, COX-2 and INOS, and decreased the levels of COX and NO in the culture medium. In the in vivo experiment, DKO and C57/B6 mice were treated with 25mg/kg/day quercetin by intraperitoneal injection daily for two months. Funduscopy was performed monthly. After two months, serum was collected to measure NADP+/NADPH, COX, PGE-2, and NO levels. The eyes were harvested for histology and A2E measurement. Ocular transcripts of Bcl-2, Bax, Cox-2, Inos, Tnf-α, Fas, FasL and Caspase-3 were detected by RQ-PCR. Quercetin treatment did not reverse the progression of retinal lesions in DKO mice funduscopically or histologically. Although quercetin treatment could recover systemic anti-oxidative capacity, suppress the systemic expression of NO, COX and PGE-2, and decrease ocular A2E levels, it could not effectively suppress the transcripts of the ocular inflammatory factors Tnf-α, Cox-2 and Inos, or the pro-apoptotic factors Fas, FasL and Caspase-3 in DKO mice. Our data demonstrate that quercetin can protect human RPE cells from oxidative stress in vitro via inhibition of pro-inflammatory molecules and direct inhibition of the intrinsic apoptosis pathway. However, quercetin (25mg/kg/day) does not improve the retinal AMD-like lesions in the Ccl2−/−/Cx3cr1−/− mice, likely due to its insufficient suppression of the inflammatory and apoptosis pathways in the eye.
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