SOX9 depletion attenuates retinal ganglion cell ferroptosis through blocking ERK/p38 signaling.

BACKGROUND: Retinal ischemia-refusion (I/R) is a leading cause of irreversible blindness worldwide. This study aims to explore the regulatory role of SOX9 in retinal I/R injury, and attempts to elucidate its potential regulatory mechanism. METHODS: Retinal I/R injury model was established in vivo, and the histological changes was examined by hematoxylin and eosin (H&E) staining and immunofluorescent assay was performed to examine SOX9 expression. Oxygenation-glucose deprivation/reoxygenation (OGD/R)-induced retinal ischemia/reperfusion (I/R) injury in 661 W cells was constructed as an in vitro cellular model of glaucoma. The production of cytokines, lactate dehydrogenase (LDH) and the antioxidant enzymes were assessed by their commercial kits. Cellular reactive oxygen species (ROS) and lipid ROS was detected using DCFH-DA and C11-BODIPY 581/591 staining, respectively. Lipid peroxidation and Fe2+ level were detected to assess the ferroptosis level. Protein expression was examined by western blot. LM22B-10, the agonist of ERK signaling, was used to pretreat 661 W cells for mechanism investigation. RESULTS: SOX9 was aberrantly upregulated following retinal I/R injury both in vivo and in vitro. SOX9 knockdown exerted a protective role against OGD/R-triggered oxidative stress, inflammatory response and ferroptosis in 661 W cells. Further, ERK/p38 signaling was activated in 661 W cells following OGD/R induction, which was repressed by SOX9 knockdown, and the ERK signaling agonist partially counteracted the protective role of SOX9 knockdown against oxidative stress, inflammatory response and ferroptosis in OGD/R-induced 661 W cells. CONCLUSION: Collectively, inhibiting SOX9 to block oxidative stress, inflammation and ferroptosis by inactivating ERK/p38 signaling might be effective to prevent retinal I/R injury, thereby alleviating glaucoma.

Association between LEKR1-CCNL1 and IGSF21-KLHDC7A gene polymorphisms and diabetic retinopathy of type 2 diabetes mellitus in the Chinese Han population.

BACKGROUND: Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes. The present study aimed to identify a possible connection between gene polymorphisms and the risk of developing DR. MATERIALS AND METHODS: A total of 319 patients with type 2 diabetes mellitus (T2DM) were selected. All patients underwent a complete eye examination. Based on this, the patients with T2DM were divided into two subgroups: 175 patients with retinopathy (DR) and 144 patients without retinopathy (NDR). We calculated the genotype frequencies of case and control subjects using the chi-squares test. The odds ratio (OR) and 95% confidence intervals (CIs) were calculated by unconditional logistic regression adjusted for age and sex. RESULTS: The finding by analysis is that the mean of duration of diabetes, total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), glomerular filtration rate and C-peptide were significantly different between DR and NDR. We found significant differences in cystatin-C concentrations with LEKR1-CCNL1 rs13064954 and NOS3 rs3918227 of different genotypes. Significant differences in serum TG levels were seen among the three genotypes of MTHFR rs1537516. Subjects carried the T allele of IGSF21-KLHDC7A rs3007729 had higher serum LDL concentrations (p = 0.015). In the allele model, LEKR1-CCNL1 rs13064954 decreased the risk of DR (OR =0.57, 95% CI = 0.34-0.96, p = 0.032). Under the dominant model, the IGSF21-KLHDC7A rs3007729 CT-TT genotype increased the risk of DR (OR =1.84, 95% CI = 1.14-2.99, p = 0.013). CONCLUSIONS: Our results suggest that LEKR1-CCNL1 and IGSF21-KLHDC7A influence the development of DR.

MicroRNA-29 regulates high-glucose-induced apoptosis in human retinal pigment epithelial cells through PTEN.

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.

Allicin attenuates H2O2-induced cytotoxicity in retinal pigmented epithelial cells by regulating the levels of reactive oxygen species.

Retinal pigmented epithelial cell (RPE) oxidative stress is known to have a vital role in the etiology of age­related macular degeneration (AMD). The present study aimed to investigate whether allicin, a natural product with antioxidant activity, was able to protect RPEs (ARPE­19) from hydrogen peroxide (H2O2)­induced damage, and to determine the underlying mechanisms. The 3-(4,5-dimethylthiazol-2-yl)-2,5­diphenyl 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 ARPE­19 cells from H2O2­induced damage in a dose­dependent 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 ROS­associated enzymes, including superoxide dismutase, NADPH oxidase 4 and NAD(P)H dehydrogenase quinone 1, and elevate the activity of nuclear factor erythroid 2­related factor 2 in the H2O2­stimulated ARPE­19 cells. These results suggested that allicin may exert protective effects against H2O2­induced cytotoxicity in RPEs via ROS regulation.

SirT1 and STAT3 protect retinal pigmented epithelium cells against oxidative stress.

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 [H2O2 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.

Vitamin C protected human retinal pigmented epithelium from oxidant injury depending on regulating SIRT1.

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.