The Effects Of High-concentration Glucose And High-concentration High With Hypoxia On The Expression Of HIF-1α And The Investigation Of ShRNA Inhibitting The Expression Of HIF-1α In Human RPE Cells

Objective To determine whether the retina of diabetic rats at early stage express hypoxia-inducible factor lα (HIF-1α) and vascular endothelial growth factor (VEGF), to investigate the effects of high-concentration glucose and high-concentration high with hypoxia on the expression of HIF-lα and VEGF in human retinal pigment epithelial (RPE) cell, thus to illustrate the roles of high-concentration glucose and hypoxia played in retinal neovascularization. To explore the effect of small hairpin loop RNA (shRNA) keeping HIF-1α silencing on the expression of VEGF and pigment epithelium derived factor (PEDF), thus to illustrate whether shRNA inhibitting the expression of HIF-1α could reduce retinal neovascularization by downregulation of VEGF and upregulation of PEDF in human RPE cells. Methods 1) Thirty male Wistar rates were randomly divided into the diabetic group (n=20) and normal control group (n=10). Diabetic rats were induced by injection of streptozotocin (STZ) intraperitondally. One month after the model being builded, the eyeballs were removed for making the retinal vascular network, PAS stained, HE stained and immunohistochemically stained. 2) 150μmol CoCl₂ is used as hypoxic environment of Human RPE cells, RPE cells are cultured under 5.56 mM glucose with 0 μMCoCl₂ (control group), 5.56mM glucose with 150 μM CoCl₂ (hypoxic group) , 25 mM glucose with 0 μM CoCl₂ (high glucose group) and 25 mM glucose with 150 μM CoCl₂(combination group). RT-PCR was used to examine the expression of HIF-1α and VEGF mRNAs, Western blot analysis was used to measure the levels of HIF-1α and VEGF proteins. 3) Utilizing 150 μM CoCl₂ to model the hypoxia environment of RPE cells. Two target sites of HIF-1α mRNA were chosen by certain design principle. In vitro, two kind of shRNA were designed and synthesised by the two target sites, and transfected into human RPE cells in vitro. Then these cells were cultivated under hypoxia condition (150 μM CoCl₂). The mRNA expressions of HIF-1α, VEGF and PEDF were tested by RT-PCR. And the protein levels of HIF-1α, VEGF and PEDF were tested by western blot.Results 1) The reaction for HIF-1α and VEGF was negative in normal retina. However, HIF-1α and VEGF express in the retina of diabetic rats at early stage. 2) As compared with under 5.56 mM glucose condition, the expression of the HIF-1α mRNA of RPE cells under 25 mM glucose condition is not different, the HIF-1α protein is able to be detected out, but the quantity of the HIF-1α protein is small, however the HIF-1α protein under 5.56 mM glucose condition is not able to be detected out; the mRNA expression and the protein synthesis of VEGF are up-regulated. As compared with hypoxic group, the expression of the HIF-1α mRNA of RPE cells under 25 mM glucose with hypoxic condition is not different, but the protein synthesis of HIF-1α is more obviously up-regulated; meanwhile, the mRNA expression and the protein synthesis of VEGF are more obviously up-regulated too. 3) After the two kind of HIF-1α-specific shRNA were respectively transfected into RPE cells, the expression of HIF-1α mRNA and the levels of HIF-1α protein both significantly decreased in RPE cells under hypoxia condition. Moreover, the expression of VEGF mRNA and the levels of protein significantly decreased too. However, the levels of PEDF protein was significantly increased, but the expression of PEDF mRNA is no significant change.Conclusion 1) The results indicate that diabetes (or high-concentration boold glucose) increases the expression of HIF-1α in retina may play a part in retinal neovascularization. 2) high-concentration glucose mainly influence the protein synthesis of HIF-1α of RPE cell, and under high concentration glucose, HIF-1α protein is able to be accumulated; however, with under hypoxia condition, the HIF-1α protein induced by high concentration glucose is more stable, and meanwhile, the expression of VEGF is obvious increases. These suggest high concentration glucose may play a large part in retinal neovascularization, especially at ischemia stage of diabetic retinopathy. 3) Under hypoxia condition, HIF-1α-specific shRNA can effectively keep the HIF-1α gene silencing , downregulate VEGF and upregulate PEDF. These results reveal HIF-1 is concerned with posttranslational mechanism for downregulating PEDF under hypoxia condition and provide an explanation for hypoxia-provoked increases in VEGF/PEDF ratios. These results suggest HIF-1 is the most key cytokine to retinal neovascularization too.