Resveratrol inhibits neural apoptosis and regulates RAX/P-PKR expression in retina of diabetic rats.

PURPOSE: This study was to investigate the effect of resveratrol (RSV) administration on diabetes-induced neural apoptosis and on RNA-dependent-protein-kinase (PKR)-associated protein X (RAX), PKR and phosphorylated PKR (P-PKR) expression and distribution in retina of diabetic rats. METHODS: Retina was obtained from normal and diabetic Sprague-Dawley rats with or without RSV (5 and 10 mg/kg/d) treatment at 30-, 32-, 34- and 36-weeks. Apoptosis of retinal neural cells and distribution of RAX/P-PKR was assessed by TUNEL and immunofluorescence methods. Expression of RAX, PKR and P-PKR was evaluated by qRT-PCR and western-blotting methods. RESULTS: Our study showed that the TUNEL-positive cells were mainly localized in ganglion cells layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL) of the diabetic rat's retina at 30-, 32-, 34- and 36-weeks. RSV administration effectively suppressed the neural apoptosis in GCL, INL and ONL. Almost no TUNEL-positive cells were observed in retina of normal control and RSV-treated normal control rats. Our study also showed that the expression level of RAX, P-PKR in diabetic rats retina at 30-, 32-, 34-, and 36-weeks was elevated. With supplementation of 5 and 10 mg/kg/d RSV, the expression level of RAX and P-PKR was decreased (P < 0.05). The expression level of RAX and P-PKR in the retina of normal control rats was not altered by RSV. The expression level of PKR was not altered by streptozotocin injection and RSV treatment. CONCLUSIONS: Our results suggested that RSV attenuates retinal neural apoptosis in diabetic rats retina may be via regulation RAX/P-PKR expression.

Resveratrol Inhibits Diabetic-Induced Müller Cells Apoptosis through MicroRNA-29b/Specificity Protein 1 Pathway.

The aim of this study was to evaluate the anti-apoptosis effects of resveratrol (RSV) on diabetic rats retinal Müller cells in vivo and in vitro and to further investigate the roles of microRNA-29b (miR-29b)/specificity protein 1 (SP1) in the anti-apoptosis mechanism of RSV. Retina was obtained from normal and diabetic rats with or without RSV (5 and 10 mg/kg/day) treatments at 1-7 months. TdT-mediated dUTP-biotin nick end labeling (TUNEL) and Annexin V/PI staining were used to detect apoptosis. Immunofluorescence was used to assess distribution of SP1 in retina. MiR-29b and SP1 messenger RNA (mRNA) expression was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). SP1, Bax, and bcl-2 protein expression was evaluated by western blotting. Caspase-3 activity was detected by assay kit. Our study showed that the TUNEL-positive cells were mainly localized in the inner nuclear layer (INL) of retina and RSV administration effectively suppressed streptozotocin (STZ)-induced apoptosis of retinal cells in INL in vivo (P < 0.001). Our study also showed that RSV administration effectively suppressed high glucose (HG)-induced retinal Müller cells' apoptosis in vitro (P < 0.001). Furthermore, our study revealed that the diabetes-induced downregulated expression of miR-29b and upregulated expression of SP1 could be rescued by RSV in vivo and in vitro (P < 0.05). The anti-apoptosis effect and downregulated SP1 expression effect of RSV was prevented by miR-29b inhibitor (P < 0.05). MiR-29b mimic increased the above-mentioned effects of RSV (P < 0.001). These findings indicate that RSV is a potential therapeutic option for diabetic retinopathy (DR) and that miR-29b/SP1 pathway play roles in the anti-apoptosis mechanism of RSV.

Resveratrol Prevents Retinal Dysfunction by Regulating Glutamate Transporters, Glutamine Synthetase Expression and Activity in Diabetic Retina.

This study investigated the effects of resveratrol (RSV) on retinal functions, glutamate transporters (GLAST) and glutamine synthetase (GS) expression in diabetic rats retina, and on glutamate uptake, GS activity, GLAST and GS expression in high glucose-cultured Müller cells. The electroretinogram was used to evaluate retinal functions. Müller cells cultures were prepared from 5- to 7-day-old Sprague-Dawley rats. The expression of GLAST and GS was examined by qRT-PCR, ELISA and western-blotting. Glutamate uptake was measured as (3)H-glutamate contents of the lysates. GS activity was assessed by a spectrophotometric assay. 1- to 7-month RSV administrations (5 and 10 mg/kg/day) significantly alleviated hyperglycemia and weight loss in diabetic rats. RSV administrations also significantly attenuated diabetes-induced decreases in amplitude of a-wave in rod response, decreases in amplitude of a-, and b-wave in cone and rod response and decreases in amplitude of OP2 in oscillatory potentials. 1- to 7-month RSV treatments also significantly inhibited diabetes-induced delay in OP2 implicit times in scotopic 3.0 OPS test. The down-regulated mRNA and protein expression of GLAST and GS in diabetic rats retina was prevented by RSV administrations. In high glucose-treated cultures, Müller cells' glutamate uptake, GS activity, GLAST and GS expression were decreased significantly compared with normal control cultures. RSV (10, 20, and 30 mmol/l) significantly inhibited the HG-induced decreases in glutamate uptake, GS activity, GLAST and GS expression (at least P < 0.05). These beneficial results suggest that RSV may be considered as a therapeutic option to prevent from diabetic retinopathy.

Taurine prevents high glucose-induced angiopoietin-2/tie-2 system alterations and apoptosis in retinal microvascular pericytes.

Previously, we confirmed that taurine prevented diabetes-induced apoptosis in retinal glial cells via its anti-oxidation and anti-glutamate excitotoxicity mechanisms. The aim of this study is to investigate the effects of taurine on angiopoietin-2 (Ang-2)/Tie-2 system expressions and apoptosis in high glucose-treated retinal microvascular pericytes (RMPs). Also, the possible mechanism involved in the inhibition of taurine on RMPs apoptosis is investigated. The expressions of Ang-2, Tie-2 were detected by qRT-PCR and ELISA. The level of phosphorylated Tie-2 (P-Tie-2) was examined by ELISA. Hoechst 33342 and Annexin V/PI staining were used to detect RMPs apoptosis. The activity of caspase-3 was detected by assay kit. In 25 mM high glucose group, the expression of Ang-2 was increased significantly, taurine down-regulated Ang-2 in a dose (0.1, 1, and 10 mM)-dependent manner (P < 0.05). The Tie-2 expression and P-Tie-2 level were decreased in high glucose group (P < 0.05). Interestingly, taurine at 1 and 10 mM showed significant increase in Tie-2 expression and P-Tie-2 level (P < 0.05). The number of apoptotic RMPs and the activity of caspase-3 increased in the presence of high glucose (P < 0.05). Treatment with taurine at 1 mM decreased the number of apoptotic RMPs and the activity of caspase-3 (P < 0.05). Blocking antibody and small interfering RNA (siRNA) treatment showed that taurine required Tie-2 to perform its anti-apoptotic effect. Taken together, our data suggest that high glucose-induced Ang-2/Tie-2 system expressions alteration can be reversed by taurine, and that taurine can inhibit high glucose-induced RMPs apoptosis via Tie-2.

Development and validation of a nutritional and nursing risk assessment method for diabetic patients.

The present study aimed to develop and evaluate a nutritional and nursing risk assessment method for diabetic inpatients to improve healthcare and risk management. Diabetic inpatients diagnosed according to the World Health Organization guidelines, together with their nursing staff, were divided into two groups for nutritional and nursing risk assessment. Data from one group were used to establish the assessment method, and data from the other group were used to evaluate the reliability and effectiveness of the method. To establish the method, various risk variables in the nutritional and nursing processes were evaluated by logistic regression analysis; the score and probability of the risk variables were determined based on odds ratios. The overall nutritional and nursing risk for individual inpatients was then judged by the accumulated scores. The analysis showed that there were a number of risk factors, including age and body mass index. The risk was shown to increase with increasing score for the inpatients, and the χ2 test (P<0.01) was used to indicate a significant association. When the score was 50, the sensitivity and specificity of the method used to detect the nutritional and nursing risk were 88.3 and 66.5%, respectively, with predictive positive and negative rates of 12.83 and 98.53%, respectively. Therefore, the method is simple, cost-effective and fast; it can be used to screen a large number of patients by nursing staff and can also be used by patients themselves. Overall, the method is an effective and practicable nutritional and nursing risk assessment and educational tool.

Effects of taurine on glutamate uptake and degradation in Müller cells under diabetic conditions via antioxidant mechanism.

Glutamate is the excitatory neurotransmitter in the retina, but it is neurotoxic in excessive amounts. A decrease in the ability of Müller cells to remove glutamate from the extracellular space may play a crucial role in the disruption of glutamate homeostasis that occurs in the diabetic retina. Previously we have shown that taurine has protective effects against diabetes-induced glutamate dysmetabolism in retinal Müller cells. The aim of this study is to examine the effects and underlying mechanism of taurine on high glucose-induced alterations of Müller cells glutamate uptake and degradation. Müller cells cultures were prepared from 5- to 7-day-old Sprague-Dawley rats. Glutamate uptake was measured as (3)H-glutamate content of the lysates. Glutamine synthetase (GS) activity was assessed by a spectrophotometric assay. The expressions of glutamate transporters (GLAST) and GS were examined by RT-PCR and western-blot. In 25 mmol/l high glucose-treated cultures, Müller cells glutamate uptake, GS activity and GLAST, GS expressions were decreased significantly compared with 5 mmol/l normal glucose cultures (p<0.05). Taurine (1 and 10 mmol/l) significantly inhibited the high glucose-induced decreases in glutamate uptake, GS activity and GLAST, GS expressions (p<0.05). The generation of TBARS, ROS and NO in Müller cells increased significantly after treatment with high glucose compared with normal glucose. However, treatment of 1 and 10 mmol/l taurine resulted in a significant decrease in TBARS, ROS and NO levels (p<0.05). The high glucose treatment decreased antioxidant enzyme (catalase, SOD and GSH-px) activities compared with normal glucose. Taurine treatment increased the catalase, SOD and GSH-px activity in a dose-dependent manner. These findings suggest that taurine may regulate Müller cells' glutamate uptake and degradation under diabetic conditions via its antioxidant mechanism.

Effects of taurine on glial cells apoptosis and taurine transporter expression in retina under diabetic conditions.

Taurine, a ß-aminosulfonic acid, has been reported to reduce the risk of a number of diseases, including cardiovascular disease, diabetes, and also perhaps to reduce neurodegeneration in the elderly. The transport of taurine is known to be mediated by taurine transporter (TauT). The purpose of this study is to examine the effects of taurine on glial cells apoptosis and on TauT expression in retina of diabetic rats and retinal glial cells cultured with high glucose. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining analysis showed that the number of TUNEL-positive cells in taurine treated diabetic rats was significantly lower than those of untreated diabetic rats over the 8-, and 12-week time courses, respectively (all P < 0.001). No TUNEL-positive cells were observed in retina of control groups and taurine treated control groups. In cultured retinal glial cells, the apoptosis in high glucose-treated cells was significantly increased vs the control. When the cells were incubated with high glucose and taurine at 0.1, 1.0 and 10 mmol/l, the percentage of apoptosis was significantly decreased to 16.4, 5.7 and 7.6% respectively (all P < 0.05). With supplementation of taurine in diet and culture medium, higher expression of TauT in retina of diabetic rats and cultured retinal glial cells under diabetic conditions were detected by western-blotting (P < 0.05). Taken together, our data suggest that diabetes or high glucose induced retinal glial cells apoptosis can be inhibited by taurine, and that taurine reverses the diabetes-induced or high glucose-induced decrease in TauT expression.

Taurine protects transformed rat retinal ganglion cells from hypoxia-induced apoptosis by preventing mitochondrial dysfunction.

Hypoxia-induced apoptosis of retinal ganglion cells (RGCs) is the major cause of progressive vision loss in numerous retinal diseases, including glaucoma and diabetic retinopathy. Taurine is a naturally occurring free amino acid that has been shown to have neurotrophic and neuroprotective properties in the retina. We investigated the specific potential for taurine to be protective for immortalized rat retinal ganglion cells (RGC-5) exposed to hypoxia (5% O(2)). Pretreatment of RGC-5 cells with 0.1 mM taurine significantly reduced the extent of apoptosis detected by DAPI staining, MTT, and Annexin V-FITC/PI assays. To further study the mechanism underlying the beneficial effect of taurine, interactions between taurine and the process of mitochondria-mediated apoptosis were examined. Taurine treatment of RGC-5 cells suppressed the induction of the mitochondrial permeability transition (mPT) by reducing intracellular calcium levels and inhibiting the opening of mitochondrial permeability transition pores (mPTPs). Moreover, the loss of mitochondrial membrane potential, a decline in cellular ATP levels, a reduction in the amount of cytochrome c translocated to the cytoplasm and caspase-3 activation were observed in taurine-treated cultures. These results demonstrate the potential for taurine to protect RGCs against hypoxic damage in vivo by preventing mitochondrial dysfunction.

Dietary taurine supplementation prevents glial alterations in retina of diabetic rats.

The preventive effect of dietary taurine supplementation on glial alterations in retina of streptozotocin-induced diabetic rats was examined in this study. Blood glucose content, content of taurine, glutamate and -amino butyric acid (GABA) and expression of glial fibrillary acid protein (GFAP), vascular endothelial growth factor (VEGF), glutamate transporter (GLAST), glutamine synthetase (GS) and glutamate decarboxylase (GAD) in retina were determined in diabetic rats fed without or with 5% taurine in a controlled trial lasting 12 weeks, with normal rats fed without or with 5% taurine served as controls. Dietary taurine supplementation could not lower glucose concentration in blood (P > 0.05), but caused an elevation of taurine content and a decline in levels of glutamate and GABA in retina of diabetic rats (P < 0.05). The content of GABA in normal control group was not altered by taurine supplementation. With supplementation of taurine in diet, lower expression of GFAP and VEGF while higher expression of GLAST, GS and GAD in retina of diabetic rats were determinated by RT-PCR, Western-blotting and immunofluorescence (P < 0.05). GFAP, VEGF, GLAST, GS and GAD expressions in normal controls were not altered by taurine treatment. This may have prospective implications of using taurine to treat complications in diabetic retinopathy.

Dietary taurine supplementation ameliorates diabetic retinopathy via anti-excitotoxicity of glutamate in streptozotocin-induced Sprague-Dawley rats.

The purpose of this study was to investigate whether taurine ameliorate the diabetic retinopathy, and to further explore the underlying mechanisms. The Sprague-Dawley rats were injected with streptozotocin to establish experimental diabetic model, then fed without or with 1.2% taurine for additional 4-12 weeks. After that, the protective effects of dietary taurine supplementation on diabetic retinopathy were estimated. Our results showed that chronic taurine supplement effectively improved diabetic retinopathy as changes of histopathology and ultrastructure. The supplementation could not lower plasma glucose concentration (P > 0.05), but caused an elevation in taurine content and a decline in levels of glutamate and gamma-aminobutyric acid (GABA) in diabetic retina (P < 0.05). Moreover, chronic taurine supplementation increased glutamate transporter (GLAST) expression (P < 0.05), decreased intermediate filament glial fibrillary acidic protein (GFAP) and N-methyl-D: -aspartate receptor subunit 1 (NR1) expression in diabetic retina (P < 0.05). These results demonstrated that chronic taurine supplementation ameliorates diabetic retinopathy via anti-excitotoxicity of glutamate in rats.