The Role Of GRP91 In Diabetic Retinopathy By Modulation Of MAPK Signaling Pathway

Objective: Diabetic retinopathy(DR) is one of the most serious microvascular complications of diabetes. The pathogenesis is still unclear. To investigate the effects of G protein-coupled receptor 91(GPR91) on regulating the microvascular pathological changes in retina of diabetic rats. To explore the molecular mechanism of GPR91 regulating VEGF expression by modulation of MAPK signaling pathway.Methods: The lentiviral vectors of rat GPR91 small hairpin RNA(sh RNA) and scrambled sh RNA lentiviral particles were constructed.(1). In vivo part: Healthy male Sprague Dawley(SD) rats were selected in this study. The diabetic models were induced by the peritoneal injection of streptozocin(STZ) at one time. Diabetic rats received an intravitreal injection of the scrambled sh RNA lentiviral particles or GPR91 sh RNA at 2 weeks after the induction of diabetes. Accumulation of succinate in retina of diabetic rats was assessed by gas chromatography-mass spectrometry(GC-MS). At 12 weeks after intravitreal injection, the ultrastructure and function of the retinal vessel were assessed using haematoxylin and eosin(HE) staining, transmission electron microscopy(TEM) and Evans blue dye permeability.(2). In vitro part: The RGC-5 cells and primary retinal ganglion cells were transduced with GPR91 sh RNA or GPR91 si RNA under the condition of high-glucose, succinate or hypoxia. Western blot was conducted to detect the expression of GPR91, p-ERK1/2, p-JNK, p-p38 MAPK, COX-2, C/EBPβ, C/EBPδ, c-Fos and VEGF. Immunofluorescence was used to detect the localization of GPR91, C/EBPβ, C/EBPδ and c-Fos. RT-PCR was used to detect the m RNA levels of the GPR91, COX-2, C/EBPβ, C/EBPδ, c-Fos and VEGF. ELISA was used to detect the secretion of PGE2 and VEGF. RGC-5 cells were co-cultured with RF/6A cells to examine the proliferation and migration of RF/6A cells. Luciferase reporter assay and chromatin immunoprecipitation(Ch IP) were used to assess the VEGF promoter and its transcriptional regulation by C/EBP and AP1.Results: The lentiviral particles of GPR91 sh RNA were successfully constructed.(1). In vivo part: Succinate exhibited abundant accumulation in diabetic rat retinas compared to the control group(P<0.01), however, the expression of GPR91 had no significant difference. The GPR91 sh RNA transduction attenuated the damage of the ultrastructure and function of the retinal vessels.(2). In vitro part: The expression of p-ERK1/2, p-JNK, p-p38 MAPK, COX-2, PGE2, C/EBPβ, C/EBPδ, c-Fos and VEGF were increased in RGC-5 cells and primary retinal ganglion cells under the condition of high-glucose, succinate or hypoxia(P<0.01). The expression of p-ERK1/2, p-JNK, COX-2, PGE2, C/EBPβ, C/EBPδ, c-Fos and VEGF were significantly blocked by GPR91 sh RNA or si RNA transduction. The expression of COX-2, PGE2, C/EBPβ, C/EBPδ, c-Fos and VEGF were decreased after cells were pre-treated with ERK1/2 inhibitor. The expression of PGE2 and VEGF were decreased after cells were pre-treated with COX-2 inhibitor. The proliferation and migration of RF/6A cells were also decreased by GPR91 sh RNA transduction(P<0.01). The expression of VEGF was decreased by C/EBPβ si RNA transfection. The results of luciferase reporter and Ch IP indicated that C/EBPβ modulated the transcriptional regulation of VEGF.Conclusions: Succinate exhibits accumulation in the retina of early stage of diabetic rats. The intravitreal injection of GPR91 sh RNA attenuates the damage of the retinal vessels. GPR91 regulates the VEGF release and the proliferation and migration of RF/6A cells under the condition of high-glucose/succinate probably through ERK1/2/COX-2/PGE2 signaling pathway. GPR91 regulated the transcriptional regulation of VEGF in primary retinal ganglion cells under the condition of hypoxia probably through ERK1/2/CEB/Pβ signaling pathway.