Heme Oxygenase-1Mediated Protection Of Neurons And Vascular Endothelial Cells In Diabetic Retinopathy

Heme Oxygenase-1Mediated Protection of Neurons and Vascular Endothelial Cells in Diabetic RetinopathyObjective Heme oxygenase-1(HO-1) is a novel enzyme with potent anti-inflammatory, anti-oxidant and anti-proliferative effects. The expression of HO-1via Nrf2/ERK pathway has been shown to play a key role in some oncoma and hematologic diseases. This research aims to investigate the protective effects of HO-1in streptozotocin(STZ)-induced diabetic rats’retina and vascular endothelial cells. Explore the potential mechanism in both gene level and microRNA level. Methods Sprague-Dawley (SD) rats were induced to diabetes by intraperitoneal injection of STZ (60mg/kg). Later, some of the rats were given intraperitoneal injections of hemin (20mg/kg) to induce expression of HO-1. The protective effects of hemin were evaluated by examining the hemoglobin concentration (Hb) and the glycosylated hemoglobin (HbAlc) level of blood from the rats, including the TUNEL positive retinal ganglion cells (RGCs). We also documented the expressions of HO-1, HIF-1α, SOD-1, VEGF, p53and bcl-2by Western blot analysis and real-time quantitative PCR. Expressions of Nrf2, tERK1/2and pERK1/2were detected only by Western blot analysis. HO-1, Nrf2, pERK and GFAP proteins were detected by immunofluorescence. We also identified hemin/ZnPP-IX as inducer/inhibitor of heme oxygenase-1(HO-1) in muller cells which were isolated from Sprague-Dawley (SD) rats’eyes and cultured in vitro. The expression of HO-1, Nrf-2, HIF-1α, SOD-1and bcl-2were confirmed by real-time PCR, Western immunoblot analysis and immunohistochemistry in Muller cells cultured by DMEM contained high and normal concentrations of glucose. VEGF were detected only by ELISA. HUVECs were co-cultured with muller cells pretreated by hemin and ZnPP-IX and the expression of ZO-1and VEGF were detected. A normal-disease-treatment model for detection of differential gene expression in small microarray experiments was performed in STZ-induced and hemin-treated rat retina. And the multiclass differences were analyzed. Results The Hb level increased in hemin-treated rat blood, while the HbAlc level decreased. Hemin significantly activated HO-1expression in the full retinal layer of diabetic rats, combined with accordant changes of Nrf2/pERK protein expression, and further up-regulated the expression of GFAP in Miiller cells. Retinal ganglion cells displayed greater sensitivity to apoptosis when the HO-1level was lower. Overexpression of HO-1was associated with an increase in the activation of SOD-1and bcl-2, and suppression of the expression of HIF-la, VEGF and p53. hemin/ZnPP-IX significantly increased/blocked HO-1expression combined with accordant changes of Nrf2, HIF-la, VEGF, SOD-1and bcl-2gene expression in Miiller cells and further down-regulated/up-regulated the expression of VEGF, up-regulated/down-regulated the expression of ZO-1in vascular endothelial cells. miR-214and miR-181b were the two key microRNA in the differences of function and pathway. Conclusions HO-1is an important positive modulator of the Nrf2/ERK-dependent signaling that counteracts diabetic retinopathy-mediated injuries in microvascular and retinal neurons, acting through anti-inflammatory, anti-apoptotic and anti-proliferative effects which related to the induction of SOD-1and bcl-2and to the suppression of HIF-la, p53and VEGF. Miiller cell is the cell which response to the regulation of HO-1level and its protection of neurons and vascular endothelial cells in diabetic retinopathy. miR-214and miR-181b may be the new research target for HO-1-mediated protection.