Sirtuin (Sirt) 3 Overexpression Prevents Retinopathy in Streptozotocin-Induced Diabetic Rats.

BACKGROUND Sirtuin (Sirt) 3 could promote autophagy by downregulating the expression of genes related to neovascularization in retinal endothelial cells. In this study, we aimed to investigate the effect of Sirt3 overexpression on retinopathy in streptozotocin (STZ)-induced diabetic rats, and to assess its mechanisms. MATERIAL AND METHODS Ntraperitoneal injection of STZ in rats was used to produce a diabetic model. The study rats were divided into 4 groups (n=6 for each group): a control group; a model group; a model+scrambled adenovirus group; and a model+Sirt3 overexpression group. Hematoxylin and eosin (H&E) staining determined the pathological changes of retina tissues. Immunohistochemistry, fluorescence quantitative polymerase chain reaction, and western blotting were used to detect the expression of Sirt3, vascular endothelial growth factor (VEGF), and microtubule-associated protein 1A/1B-light chain 3 (LC3). RESULTS In the model group, the inner limiting membrane was swollen, uneven and thickened, and the capillary endothelial cells occasionally protruded into the inner limiting membrane. These abnormalities were prevented by Sirt3 overexpression. Compared with the control group, the expression of Sirt3 at both mRNA and protein levels in the model group was significantly reduced, while the expression of VEGF was increased versus the control group (P.

miR-204-5p promotes diabetic retinopathy development via downregulation of microtubule-associated protein 1 light chain 3.

Diabetic retinopathy (DR) is a chronic and progressive complication of diabetes mellitus. DR impairs sight due to neuronal and vascular dysfunction in the retina. It is critical to investigate the pathogenesis of DR to develop effective treatment. In the present study, a streptozotocin (STZ)-induced diabetic rat model was constructed and the expression of microRNA (miR)-204-5p and vascular endothelial growth factor (VEGF) were determined. Immunohistochemistry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were employed to detect the effects of miR-204-5p on the expression of microtubule-associated protein 1 light chain 3 (LC3B). RT-qPCR analysis demonstrated that miR-204-5p and VEGF were significantly upregulated in the retina tissue of diabetic rats compared with the control group (P<0.01). Immunohistochemistry and western blotting revealed that the protein expression levels of LC3B-II and the ratio of LC3B-II/LC3B-I were significantly suppressed in the diabetes group compared with the control (P<0.01). In retinal tissues, anti-miR-204-5p treatment significantly enhanced the protein expression levels of LC3B-II and the ratio of LC3B-II/LC3B-I and these levels were significantly reduced in response to miR-204-5p mimic treatment compared with the negative miR control (P<0.01). In rat retinal endothelial cells isolated from diabetic rats, anti-miR-204-5p treatment increased the number of autophagic vacuoles, and significantly promoted LC3B-II expression and the LC3B-II/LC3B-I ratio compared with the negative control (P<0.01). The results of the present study revealed that miR-204-5p downregulated the expression of LC3B-II to inhibit autophagy in DR. Therefore, miR-204-5p may be considered as a novel effective therapeutic target during the development of DR.

Targeting human 8-oxoguanine DNA glycosylase to mitochondria protects cells from high glucose-induced apoptosis.

PURPOSE: Diabetic retinopathy (DR) is a major vision threatening disease mainly induced by high glucose. Despite great efforts were made to explore the etiology of DR, the exact mechanism responsible for its pathogenesis remains elusive. METHODS: In our study, we constructed diabetic rats via Streptozotocin (STZ) injection. TUNEL assay was employed to examine retinal cell apoptosis. The levels of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were analyzed via flow cytometry. The mRNA and protein levels of mitochondrial respiratory chain were investigated by RT-qPCR and western blot. RESULTS: Compared with normal rats, the retinal cell apoptosis rate in diabetic rats was significantly upregulated. What's more, the signals of 8-OHdG and the levels of Cytochrome C in diabetic rats were enhanced; however, the MnSOD signals and NADPH-1 levels were reduced. We investigated the effect of mitochondrialy targeted hOGG1 (MTS-hOGG1) on the primary rRECs under high glucose. Compared with vector-transfected cells, MTS-hOGG1-expressing cells blocked high glucose-induced cell apoptosis, the loss of MMP and the overproduction of ROS. In addition, under high glucose, MTS-hOGG1 transfection blocked the expression of Cytochrome C, but enhanced the expression of cytochrome c oxidase subunit 1 and NADPH-1. CONCLUSIONS: These findings indicated that high glucose induced cell apoptosis by causing the loss of MMP, the overproduction of ROS and mtDNA damage. Targeting DNA repair enzymes hOGG1 in mitochondria partly mitigated the high glucose-induced consequences, which shed new light for DR therapy.

Potential suppression of the high glucose and insulin-induced retinal neovascularization by Sirtuin 3 in the human retinal endothelial cells.

Retinal neovascularization generally play roles in the formation of various severe eye diseases, such as age-related macular degeneration and diabetic retinopathy. The regulation of neovascularization-related pathways by Sirtuin 3 (Sirt3), a major mitochondrial NAD+-dependent deacetylase, give us a cue that Sirt3 may participate in the retinal neovascularization. However, the mechanism remains unclear. Here, we established a retinal neovascularization model by using human retinal endothelial cells (HRECs) under the induction of high glucose and insulin (HGI). With this model, Sirt3-expressing lentivirus was constructed and then used to investigate the effect of Sirt3 overexpression on the expression of migration-, neovascularization- and autophagy-related genes. After the treatment of HGI on HRECs, the mRNA and protein levels of migration-related genes, including matrix metalloproteinase-2 (MMP-2) and MMP-9, were significantly upregulated. Meanwhile, angiogenesis-related genes, including vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1α (HIF-1α), and insulin-like growth factor-1 (IGF-1) were promoted at both mRNA and protein levels. However, HGI had no clear effect on the mRNA and protein levels of microtubule associated protein 1 light chain 3 (LC3), an autophagy-related gene. When Sirt3 was overexpressed by lentivirus infection after HGI, the upregulation of MMP-2, MMP-9, VEGF, HIF-1α, and IGF-1 were suppressed at both transcription and translation levels. At the same time, LC3 mRNA and LC3-II protein increased. These results suggest that Sirt3 may inhibit retinal neovascularization by regulating the migration-, neovascularization- and autophagy-related factors expression. Thus we argue that Sirt3 may be a potential candidate drug for curing various eye diseases induced by retinal neovascularization.