Tea Polyphenols Attenuate Ultraviolet B Induced Damage To Human Retinal Pigment Epithelial Cells In Vitro

Age-related macular degeneration (ARMD) is a major eye disease in the world. Ultraviolent B (UVB) light is considered to be a source of oxidative stress, which induces morphological changes and alteration in size and movement of retinal pigment epithelial (RPE) cells. Many deleterious effects on the skin have been associated with the UVB of the solar spectrum, which is believed to be an important factor inducing the retinal diseases such as ARMD. This study investigated the damage induced by UVB and protective effect of tea polyphenol (TP) on RPE cells. Although the detailed molecular mechanisms of cell apoptosis induced by UVB are not fully clear, it is believed that the intracellular oxidative stress is one of the important factors. In this study, in vitro RPE cells were used to investigae the protective and healing effect of TP on UVB-irradiated RPE cells.MTT test and photomicroscopy revealed that RPE cells were damaged by UVB irradiation at 100μW/cm2 for 2h. The morphology of the cells deformed, changing from spindle shape to clustered round shape. TP could alleviate the damage caused by UVB-irradiation. The protective effect of low level TP on cell viability showed a dose-dependent manner. However, high level TP had cytotoxic effect.Transmittance electronic microscopy confirmed that significant abnormalities in RPE cell microstructures, such as microvilli shedding, nucleolus degeneration, mitochondria deformity and formation of vesicular structures in cytoplasm, were observed after UVB irradiation. Before UVB irradiation, the intact cytoplasm of RPE cell was evenly distributed and nucleolus (Nu) located the center of the cell. The outer cell membrane microvilli (MV) were observed. The mitochondria were full of regular disposition cristae. The RPE cells pretreated with 70 and 140 mg/L GTP before UVB irradiation had less changes in the cell microstructures. The microvilli were partially seen. There were less vesicular structures. Most of the mitochondria looked normal at GTP level 140 mg/L though a few mitochondria became dumbbell-shaped at GTP level 70 mg/L. The GTP post-treatments showed the same tendency as the GTP pretreatments, but its protective effects were less than GTP pretreatments. The dumbbell-shaped and long mitochondria were seen in the GTP post-treatments of both GTP levels (Fig.2, UVB+GTP). These suggest that GTP attenuated UVB-induced RPE cell damages by both pretreatment and post-treatment.Single band was observed in the RPE cell DNA samples before UVB irradiation. There were small DNA fragments on the electrophoresis gels after the RPE cells were irradiated by UVB. Pretreatment with both 140 mg/L and 70 mg/L GTP levels partially inhibited the DNA fragmentation. However, the post-treatment with GTP had less protective effect against the DNA fragmentation, especially at level 70 mg/L. It is considered that GTP suppressed the DNA damage induced by UVB and promoted the repair of the damaged DNA.GTP regulated the expression of genes Survivin and c-fos. Both GTP pretreatment and post-treatment attenuated the UVB suppression of Survivn gene expression. Survivin is a member of the inhibitor of apoptosis (IAP) family. The Survivin protein functions to inhibit caspase activation therefore leading to negative regulation of apoptosis or programmed cell death. Disruption of Survivin induction pathways leads to increase in apoptosis. This suggests that the protective effect of GTP against UVB damage to RPE cells might be related to its regulation of Survivin gene expression. The expression of c-fos gene was up-regulated by UVB irradiation. Both TP pretreament and TP post-treatment inhibited the increase in c-fos gene expression induced by UVB.This study showed that TP affords protection against the UVB-induced stress via interacting with UVB-induced reactive oxygen species (ROS), attenuating mitochondrion-mediated apoptosis, protecting DNA from damage and regulating expressions of gene Survivin and c-fos. It is considered that TP is a potential candidate to be further developed as a chemoprotective factor for the primary prevention of age-related eye diseases such as ARMD.