Hyperglycemia or high-glucose (HG)-induced apoptosis in human retinal pigment epithelial (RPE) cells is a characteristic process in diabetic retinopathy. In our study, we examined whether microRNA-29 (miR-29) may regulate HG-induced RPE cell apoptosis. Human RPE cell line, ARPE-19 cells, was treated with various high concentration of glucose in vitro. HG-induced RPE cell apoptosis was examined by terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay and miR-29 gene expression by quantitative RT-PCR (qRT-PCR). miR-29 was then downregulated in RPE cells, and its effect on HG-induced apoptosis was examined by TUNEL assay and western blot assay on caspase-7 protein. Association of miR-29 on its downstream target, PTEN, in HG-induced RPE cell apoptosis was evaluated by dual-luciferase assay and qRT-PCR. PTEN was silenced in RPE cells. The effects of PTEN downregulation on miR-29-mediated HG-induced RPE cell apoptosis were also examined by TUNEL and western blot assays. HG induced significant apoptosis in RPE cells in a dose-dependent manner. miR-29 was upregulated by HG in RPE cells. miR-29 downregulation protected HG-induced apoptosis and reduced the production of caspase-7 protein in RPE cells. PTEN was shown to be directly downregulated by HG and then upregulated by miR-29 downregulation in RPE cells. Small interfering RNA (siRNA)-mediated PTEN downregulation reversed the protective effect of miR-29 downregulation on HG-induced RPE cell apoptosis. This study demonstrates that miR-29, through inverse association of PTEN, plays an important role in the process of HG-induced apoptosis in RPE cells.

Abstract.Retinal pigmented epithelial cell (RPE) oxidative stress is known to have a vital role in the etiology of age-related macular degeneration (AMD). The present study aimed to investigate whether allicin, a natural product with antioxidant activity, was able to protect RPEs (ARPE-19) from hydrogen peroxide (H 2 O 2 )-induced damage, and to determine the underlying mechanisms. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to determine cellular viability, and reactive oxygen species (ROS) were detected using a ROS Assay kit. The results demonstrated that allicin was able to protect ARPE-19 cells from H 2 O 2 -induced damage in a dose-dependent manner. In addition, allicin attenuated oxidative stress by reducing the levels of intracellular ROS and malondialdehyde (MDA), and enhancing the glutathione/glutathione disulfide (GSSG) ratio. With regards to the underlying mechanism, allicin was able to markedly modulate the expression levels of ROS-associated enzymes, including superoxide dismutase, NADPH oxidase 4 and NAD(P)H dehydrogenase quinone 1, and elevate the activity of nuclear factor erythroid 2-related factor 2 in the H 2 O 2 -stimulated ARPE-19 cells. These results suggested that allicin may exert protective effects against H 2 O 2 -induced cytotoxicity in RPEs via ROS regulation.

The purpose was to investigate the protective effects of Vitamin C (Vit C) and the regulatory mechanism between Vit C and sirtuin 1 (SIRT1) in PREs during oxidative stress as Vit C and SIRT1 exerted famous effects as antioxidants. We found that moderate Vit C (100 µM) prevented ARPE-19 cells from damages induced by H2O2, including increasing viability, reducing apoptosis, and attenuating intracellular ROS levels. But lower and higher concentration of Vit C had no effects. Further results indicated that Vit C caused the dysregulation of some stress responses factors (SIRT1, p53 and FOXO3) in ARPE-19 cells response to H2O2. Moreover we found that SIRT1 activator resveratrol (SRV) stimulated significantly the protective effects of moderate Vit C, provided the property of antioxidative stress for the lower and higher concentration of Vit C in ARPE-19 cells as well. Consistently, nicotinamide (NA) relieved the protective functions of moderate Vit C. Interestingly, data also revealed the dysregulation of p53 and FOXO3 was dependent on the regulation of SIRT1 rather than Vit C. Summarily, the protective effect of Vit C against oxidative stress was involved in regulation of SIRT1. It suggested that combined application of Vit C and RSV might be a promising therapeutic method for AMD.

Abstract.It has been previously demonstrated that there are interactions between sirtuin 1 (SirT1) and signal transducer and activator of transcription 3 (STAT3), which have versatile roles in various microenvironments. However, whether or not there is crosstalk between these two molecules during oxidative stress, and what mechanism of crosstalk occurs in retinal pigmented epithelium cells (RPEs), the protection of which may delay the process of age-related macular degeneration (AMD), has required further elucidation. The present study aimed to investigate the interactions between SirT1 and STAT3 in RPEs, following exposure to oxidative stress. The rates of proliferation and apoptosis, levels of intracellular reactive oxygen species and cell senescence of RPEs, induced by oxidants [H 2 O 2 and oxidized low density lipoprotein (oxLDL)], were evaluated. The results revealed a downregulation of SirT1 expression, and an upregulation of STAT3 expression during oxidative stress. Further investigation indicated that SirT1 protected RPEs from oxidative stress-induced damage. Furthermore, gain-and loss-of-function experiments indicated that SirT1 had negative effects on the regulation of STAT3 expression in RPEs during oxidative stress. Notably, STAT3 directly protected the cells from oxidative stress, rather than depending on SirT1. Additionally, the protective effects of STAT3 had no association with the modulation of cell senescence during oxidative stress. In conclusion, SirT1 had negative effects on the regulation of STAT3 expression during oxidative stress. However, SirT1 and STAT3 demonstrated protective roles against oxidative stress in RPEs. These results therefore suggested that there was an equilibrium mechanism between SirT1 and STAT3 against oxidative stress, meaning that an equilibrium mechanism is required to be considered when combined application of STAT3 and SirT1 were performed to treat AMD.

Our results suggest that LEKR1-CCNL1 and IGSF21-KLHDC7A influence the development of DR.