UCLA Center for Excellence in Pancreatic Disease, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA. These authors contributed equally to this work. Objective: Apoptosis and autophagy of retinal cells, which may be induced by oxidative stress, are tightly associated with the pathogenesis of diabetic retinopathy (DR). The autophagy induced by oxidative stress is considered as excessively stimulated autophagy, which accelerates the progression of DR. This study aims to investigate the protective effect of GLP-1 treatment on alleviating apoptosis and autophagy of retinal cells in type 2 diabetic rats and reveals its possible mechanism. Methods: Type 2 diabetic rats were induced by fed with high sugar, high fat diet and followed with streptozotocin injection. GLP-1 was applied to treat the diabetic rats for one week after the onset of diabetes. The expressions of oxidative stress-related enzymes, retinal GLP-1R, mitochondria-dependent apoptosis- related genes, autophagy markers, and autophagy-associated pathway genes were studied by Western blotting or immunohistochemistry analysis. Conclusions: GLP-1 treatment can alleviate autophagy which may be induced by oxidative stress; this protective effect is likely through GLP-1R-ERK1/2-HDAC6 signaling pathway.
Keywords: retinopathy, GLP-1, oxidative stress, autophagy, HDAC6. However, all these strategies have undesirable side effects. Therefore, options for treating DR remain limited and more effective methods are urgently needed. However, the mechanism of DR is still not entirely clear. However, the molecular mechanism of GLP-1 treatment in diabetic retinopathy has not been clarified clearly. Thus, the molecular mechanism of GLP-1 treatment in diabetic retinopathy may be tightly associated with its antioxidant effect. However, it may cause autophagic death in retinal cells due to excessively stimulated autophagy. Herein, we hypothesize that GLP-1 may exert its antioxidant effect to protect the retinal cells through preventing the ROS-induced autophagy. The mechanism of Medic GLP Supplement-1treatment on preventing the ROS-induced autophagy remains unclear. In present study, we first investigated the effects of GLP-1on improving diabetic retinopathy in type 2 diabetic rats. We also revealed the possible mechanism of GLP-1 on alleviating apoptosis and autophagy in diabetic retinal cells.
Streptozotocin (STZ) was purchased from Sigma (St. Louis, MO, USA). Formaldehyde and xylene were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China). Caspase3 antibody, LC3B antibody, p-Akt antibody, p-ERK1/2 and HDAC6 antibody were purchased from Cell Signaling Technology. Inc. (Danvers, Massachusetts, USA). 24 male SD rats (aged 6-8weeks, weighing 160-180g) were purchased from Experiment Animal Center of Southern Medical University (Guangzhou, China). All rats were housed in 12 hour light/dark cycle with providing water and food ad libitum. All animal experiments were carried out in accordance with the guidelines of the Animal Ethic Committee at Southern Medical University. 8). The diabetic rats were established by fed with high fat and sugar (20% sucrose, 10% lard, 2.5% cholesterol, 1% cholic acid and 66.5% conventional feed) for a month and intraperitoneally injected with STZ once with a dose of 40 mg/kg body weight. Animals were considered diabetic when glucose levels were higher than 16.7 mM for 3 consecutive days (reference range, 5-8 mM).
The normal rats were fed with the standard rodent chow. GLP-1). In GLP-1 treatment group, GLP-1 was given via subcutaneous osmotic pump (30pmol/kg/min) for 7 days in diabetic rats. Meanwhile, in DR and normal control groups, equal amount of normal saline were given to the rats for the same period. After treatment, all rats were anesthetized by pentobarbital (0.1mg/g intraperitoneal injection) and sacrificed. Then, the eyeballs were removed and retina tissues were separated for HE staining and immunohistochemistry analysis. Retinal tissues were selected randomly from each group. The samples were fixed with 10% paraformaldehyde, decalcified with 20% EDTA solution, dehydrated with ethanol by gradient, and embedded in paraffin. 5.0μm thickness), Medic GLP Supplement stained with Hematoxylin-Eosin (HE) by a series of standard techniques. Histological changes were observed under light microscope. Retinal tissues were collected and homogenized in RIPA buffer with PMSF. Thirty micrograms of total cell lysate was applied to an SDS-PAGE (10%) for separation and then transferred to a PVDF membrane.
The membranes were then probed with primary antibodies (1:1000 dilution) at 4℃ overnight, followed with a secondary antibody (1:5000 dilution) conjugated with the horseradish peroxidase (HRP) for 1 hour at room temperature, and then visualized by enhanced chemiluminescence reagents (ECL; Pierce, Rockford, USA). Bands of interest were quantified by a densitometry, using Gel-Pro analysis software. The protein expression levels of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the house keeping control. Retinal tissues were selected randomly from the remaining tissues of each group. After formalin-fixed and paraffin-embedded, specimens were cut into sections with 4 micron, dewaxed and hydrated, incubated with 3% hydrogen peroxide solution in dark at room temperature (RT) for 25 minutes, washed with PBS. Subsequently, the sections were incubated in 3% BSA for 30 minutes at RT, and incubated with cleaved caspase 3 antibody (1:100), LC3B antibody (1:100), p-Akt antibody (1:100), p-ERK (1:100) and HDAC6 antibody (1:100) overnight, respectively. After that, the sections were washed 3 times with PBS, each time for 5 minutes, and stained by DAB using a secondary antibody kits. The staining time was controlled under the microscope and stopped by water washing. Finally, sections were re-stained by Harris hematoxylin for 3 minutes, and then washed, dehydrated, treated with xylene and coverslipped. The results were evaluated by light microscopy. As shown in table 1, blood glucose levels of all rats were normal before the diabetic model established. After that, the blood glucose levels of diabetic rats were significantly higher than that of the normal group (23.8±2.2 mmol/L vs.