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X. Cao, D. Shen, J. Tuo, Y. Cho, C.-C. Chan; The Protective Effect of Quercetin Against Oxidative Stress in Cultured Human RPE and Retinal Lesions of Ccl2/cx3cr1 Double Deficient Mice. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1839.
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Oxidative stress in retinal pigment epithelium (RPE) plays a role in age-related macular degeneration (AMD) development. The flavonoid quercetin has been extensively studied for its antioxidant effect. This study investigates the mechanisms of the protective effect of quercetin on cultured human RPE and in Ccl2-/-/Cx3cr1-/- (DKO) mice, which spontaneously develop retinal lesions mimicking AMD.
Cultured ARPE-19 cells were exposed to 1mM hydrogen peroxide (H2O2) with or without 50µM quercetin for 2 hours. Cellular viability was measured by MTT assay. Apoptosis was detected by TUNEL and Comet assay. Expression of Bcl-2, Bax, Caspase-3, Caspase-9 and p53 mRNA were measured by RQ-PCR. DKO and wild type (WT) mice were randomly divided into quercetin-treated and vehicle control groups. Quercetin in 40% v/v DMSO/PBS was injected intraperitoneally (25mg/kg/day) for two months. Equivalent volume of 40% v/v DMSO/PBS was given intraperitoneally as the control vehicle. Funduscopy was performed monthly. After treatment for two months, the mice were sacrificed. Serum from each mouse was collected for COX (fluorescent activity assay), PGE2 (EIA kit) and nitrite (Griess colorimetric reaction) measurement. The eyes were harvested for histology.
H2O2-treated RPE cells showed a significant decrease in cell viability, whereas introduction of quercetin into the system diminished this decrease hence increasing cell survival. H2O2-induced apoptosis was significantly attenuated by quercetin as shown by TUNEL and Comet assay. The ratio of Bcl-2/Bax mRNA expression decreased after H2O2 treatment (0.77-fold), but this was reversed by quercetin treatment (3.08-fold). H2O2 increased caspase-3 and -9 levels. This effect was abolished by quercetin, from 2.09- and 1.83- to 1.26- and 1.32-fold, respectively. Furthermore, quercetin stabilized p53 mRNA expression in H2O2-treated cells. Funduscopic evaluation of the DKO mice showed that the retinal lesions were less progressed than that of control group after quercetin treatment. Quercetin lowered serum COX from 0.53±0.23 to 0.27±0.21nmol/min/ml (p<0.05), PGE2 from 95.41±5.34 to 85.98±8.44pg/ml (p<0.05), and nitrite from 24.49±13.94 to 11.32±3.04µM (p<0.05).
The data demonstrate that quercetin is able to protect RPE cells against oxidative damage in vitro. It can also slow the progression of retinal lesions in the DKO mice, which may act via suppression of the COX/PGE2 pathway and the intrinsic apoptotic signal pathway. Therefore, we recommend taking antioxidative dietary flavonoids such as quercetin to prevent the development of AMD.
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