The effects and mechanisms of G protein-coupled receptor 91 on blood-retinal barrier in diabetic rats
10.3760/cma.j.issn.1005-1015.2015.02.012
- VernacularTitle:G蛋白偶联受体91对早期糖尿病大鼠血视网膜屏障的影响及机制
- Author:
Jianyan HU
;
Tingting LI
;
Qiang WU
- Publication Type:Journal Article
- Keywords:
Diabetic retinopathy/physiopathology;
RNA,small interfering;
Receptors,G-protein-coupled;
Vascular endothelial growth factors;
p38 Mitogen-activated protein kinases;
Animal experimentation
- From:
Chinese Journal of Ocular Fundus Diseases
2015;31(2):157-161
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effects and mechanisms of G protein-coupled receptor 91 (GPR91) on blood-retinal barrier (BRB) in diabetic rats.Methods A lentiviral vector of shRNA targeting rat GPR91 and scrambled shRNA were constructed.Healthy male Sprague-Dawley (SD) rats were selected in this study.The 60 rats were randomized into 4 groups and treated as follows:(1) control group (Group A,n=15),the rats received injections of an equal volume of 0.1％ citrate buffer;(2) streptozocin (STZ) group (Group B,n=15),the rats received injections of STZ;(3) LV.shScrambled group (Group C,n=15),diabetic rats received an intravitreal injection of 1 μl 1 × 10s TU/ml scrambled shRNA lentiviral particles at 2 weeks after the induction of diabetes;(4) LV.shGPRg1 group (Group D,n=15),diabetic rats received an intravitreal injection of 1 μl 1 × 108 TU/ml pGCSIL-GFP-shGPR91 lentiviral particles.At 12 weeks after intravitreal injection,immunohistochemistry and Western blot were used to assess the expression of GPRg1,p-extracellular signal-regulated kinase(ERK)1/2,t-ERK1/2,p-Jun N-terminal kinase (JNK),t-JNK,p-p38 mitogen-activated protein kinase (MAPK) and t-p38 MAPK.Haematoxylin and eosin (HE) staining and Evans blue dye were used to assess the structure and function of the retinal vessel.Immunohistochemistry enzyme-linked immunosorbent assay (ELISA) was used to test the protein level of VEGF.Results Immunohistochemistry staining showed that GPR91 was predominantly localized to the cell bodies of the ganglion cell layer.Western blot showed that GPR91 expression in Group D decreased significantly compared with Group C (F=39.31,P＜0.01).HE staining showed that the retina tissue in Group B and C developed telangiectatic vessels in the inner layer of retina,while the telangiectatic vessels attenuated in Group D.It was also demonstrated in Evans blue dye that the microvascular leakage in Group D decreased by (33.8±4.11)％ compared with Group C and there was significant difference (F =30.35,P＜0.05).The results of ELISA showed the VEGF secretion of Group B and C increased compared with Group A and the VEGF expression in Group D was significantly down regulated after silencing GPR91 gene (F=253.15,P＜0.05).The results of Western blot indicated that compared with Group A,the expressions of p-ERK1/2,p-JNK and p-p38 MAPK were significantly upregulated (q=6.38,2.94,3.45;P＜0.05).Meanwhile,the activation of ERK1/2 was inhibited by GPR91 shRNA and the difference was statistically significant (F=22.50,P＜0.05).Conclusions The intravitreal injection of GPR91 shRNA attenuated the leakage of BRB in diabetic rats.GPR91 regulated the VEGF release and the leakage of BRB possibly through the ERK1/2 signaling pathway.
