Figure 4. GPR91 modulated VEGF secretion via ERK1/2/cyclooxygenase-2 (COX-2)/ Prostaglandin E₂ (PGE₂) signaling pathway in the retinas of STZ rats. A: Western blot analysis of the COX-2 protein in the retinas of diabetic rats in different time periods (from 1 week to 10 weeks). B: The levels of COX-2 protein were upregulated in the retinas of diabetic rats during the period of 2 weeks to 6 weeks after the induction of diabetes. Each column denotes the mean ± SD (n = 6). C: Immunofluorescence showed that COX-2 expression located in the retinal ganglion cell layer increased in the 4 week diabetic rats compared with the control. Scale bar, 100 μm. D: Changes in COX-2 in the retinas of the 4 week diabetic rats treated with scrambled shRNA lentiviral particles, GPR91 shRNA lentiviral particles, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor). E: The increases in COX-2 expression were significantly blocked by GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor). Each column denotes the mean ± SD (n = 6). F: qRT-PCR showed that the levels of COX-2 mRNA were decreased obviously in the retinas of the 4 week diabetic rats treated with scrambled shRNA lentiviral particles, GPR91 shRNA lentiviral particles, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) compared with the 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). G: Changes in vitreal PGE₂ release in each group. The increase of PGE₂ secretion was significantly blocked by GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) in the retinas of 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). H: The levels of VEGF mRNA (using qRT-PCR) were downregulated in the 4 week diabetic rats treated with GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5mM NS-398 (COX-2 inhibitor) compared with the STZ rats. Each column denotes the mean ± SD (n = 6). I: Enzyme-linked immunosorbent assay analysis of vitreal VEGF release in vitreous. The increase of VEGF secretion was significantly blocked in the 4 week diabetic rats treated with GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) compared with the 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). **p<0.01 versus control. #p<0.05 versus LV.shScrambled group rats. ##p<0.01 versus LV.shScrambled group rats. ψ<0.05 versus STZ group rats. ψψ<0.01 versus STZ group rats.