Protective Effects And Its Mechanisms Of Exendin-4 In Early Diabetic Retinopathy Of GK Rats

Purpose To explore the protective effects and elucidate its mechanisms of exendin-4 on early DR of type II diabetic GK rats.Methods (1) Exendin-4 was injected intravitreally with different doses to find the optimal one of protective effects for early DR of type II diabetic GK rats. Expression of GLP-1R was detected at protein levels and verified by immunohistochemistry (IHC). (2) 12 Wistar rats and 24 GK rats were included in the experiment, the age of starting animals is 8 weeks.The rats are divided into 3 groups:normal wistar control group(Control), diabetic GK control with normal saline treatment group (GK+ NS),diabetic GK control with Exendin-4 treatment group (GK+E4), each group contained 12 animals. For GK+E4 group, the rats were treated with E4 (ii, 0.1μg/eye);for GK+NS and Control groups, the rats were treated with normal saline (NS,ii), accordingly. Fasting blood glucose levels, non-fasting blood glucose levels and body weight were measured weekly. The animals were treated with E4 or normal saline once at 12 weeks of age. Electroretinogram (ERG) was performed at 8 weeks, 12 weeks and 16 weeks of age. The retinal cell counts in each layer were evaluated under light microscopy after ERG examination.(3) Expression of gliosis marker, glial fibrillary acidic protein(GFAP) was detected in protein levels by western blot and verified by IHC, as well as another gliosis protein Vimentin.(4) Mitochondrial related pro-apoptosis and anti-apoptosis proteins Bax,Bcl-2 and Bcl-xL were detected by western blot.(5) Expression of inflammatory factors IL-1β,MCP-1,ICAM-1,vascular endothelium growth factors (VEGF), placental growth factor (PIGF) were detected by western blot.(6) The blood retinal barrier (BRB) related proteins, tight junction protein occludin and claudin-5 were detected by western blot and immunostaining in retina. Additionally, retinal vascular permeability and leakage was detected by Evans blue perfusion through iliac vein. (7) Related possible inner cellular signal pathway activated by GLP-1R though E4 stimulation was also explored.(8) Primary Muller cells was cultured in high glucose with or without E4. GLP-1R and Bcl-2 expression in primary Muller cells was detected by western blot after culture.Results (1) From 8 weeks to 16 weeks of age, fasting blood glucose level, non-fasting glucose levels of GK rats were much higher than those of wistar controls (P<0.05). Body weight of GK rats was little less than that of witar controls (P>0.05). And there was no significant difference of blood glucose and body weight between GK+NS group and GK+E4 group. (2) At 8 weeks of age, GK rats ERG ops wave amplitudes are much lower compared with wistar control, while ERG b wave amplitudes are similar with the control group. When grew up to 12 weeks of age, GK rats ERG ops wave amplitudes and b wave amplitudes decreased so much with the progress of diabetes that they had significant difference compared with wistar controls (Ops P<0.01, b wave P<0.05). From 8 weeks to 16 weeks of age, b-wave amplitudes and OPs decreased with the progress of diabetes. E4 injected intravitreally prevented the loss of b-wave amplitude and OPs caused by diabetic GK rats (P<0.05). (3) Immunostaining of the rat retina revealed that GLP-1R was extensively expressed in the whole neural retina. The cell counts of ONL, INL and GCL were reduced accordingly in diabetic GK rats at 16 weeks of age.E4 prevented the cell loss of each layer, especially the ONL and GCL. The anti-apoptosis/pro-apoptosis balance, Bcl-2/Bax and Bcl-xL/Bax ration was maintained by E4 while decrease in diabetic GK rats.The protein level of cleaved caspase-3 was decresed by E4. The expression of GFAP and Vimentin in GK rats were also reduced by E4.(4) The tight junction proteins occludin and claudin-5 were decreased in GK rats and verified by immunostaining in retina, while was maintained by E4. Inflammatory factors ICAM-1 and IL-1β, cytokines VEGF and PIGF were increased in GK rats while were reduced by E4.Retinal vascular permeability and leakage was increased in GK rats and E4 reversed the trends. (5) The phosphorylation of signal modulators ERK1/2, AKT(Ser473) and STAT3 which were up-regulated in GK rats were down-regulated to normal levels by E4. (6) GLP-1R was expressed a lot in the membrane of normal primary Muller cells and its expression was decreased by high glucose culture which was reversed by E4. So was the expression of Bcl-2.Conclusion GK rat is a good model for early diabetic retinopathy.GLP-1R was expressed in the whole neural retina. E4 protects retinal cell through reducing retinal reactive gliosis, increasing the Bcl-2/Bax and Bcl-xL/Bax ratio, decreasing cleaved caspase-3 protein level and maintains BRB function. And the protective effects of E4 are through modulating ERK1/2, AKT1/2 and STAT3 phosphorylation. Therefore, E4 is a promising approach for treatment of early DR.