Authors: Yang Ning, Lu Qiang, Yuan Mengke, Zhang Han, Cui Wei
Proline hydroxylase 2 (PHD2) is a degrading enzyme of hypoxia-inducible factor-1α (HIF-1α) and can regulate the expression of vascular endothelial growth factor (VEGF) and erythropoietin that promote hypoxia-induced blood vessel generation.Baicalein is an inhibitor of PHD2.Understanding the inhibitory effects of baicalein on overexpression of PHD2 in hypoxia-induced retinal glial cells is helpful for elucidating the pathogenesis of the ocular nevascular diseases.
This study aimed to observe the expression change of PHD2 in hypoxia-induced retinal glial cells and explore the inhibition of baicalein on PHD2.
The study protocol was approved by Experimental Animal Ethic Committee.Retinal glial cells were isolated from SPF neonatal SD rats (within 48 hours) and primarily cultured in DEME by explant cell culture.Cultured cells were identified by immunofluorescence staining of glial fibrillary acidic protein.The cells were divided into normal control group, CoCl2 group and CoCl2+ baicalein group.The cells in the normal control group were cultured in the conventional medium, and 200 μmol/L CoCl2 was added in the medium to establish the hypoxia cell models in the CoCl2 group, and 200 μmol/L CoCl2 and 50 μmol/L baicalein were added in the medium in the CoCl2+ baicalein group.The proliferation (absorbancy) of the cells was detected by cell counting kit-8 (CCK-8). The expression of PHD2 and VEGF in the cells was detected and located using immunofluorescence staining.The expressions of PHD2 mRNA and VEGF mRNA and their proteins in the cells were assayed by real-time quantitative PCR and Western blot, respectively.
The proliferation (absorbancy) of the cells was significantly different among the groups (F=132.05, P=0.00), and the absorbancy was considerably increased in the CoCl2 group compared with normal control group and CoCl2+ baicalein group, with significant differences between the groups (both at P<0.01). Immunofluorescence staining showed that the PHD2 and VEGF were weakly expressed in the cells of the normal control group, and the expression intensity was high in the CoCl2 group.The expression intensities of PHD2 and VEGF in the cells were weakened in the CoCl2+ baicalein group compared with those of the CoCl2 group.The relative expression levels of PHD2 mRNA were 0.366±0.034, 0.894±0.015 and 0.445±0.017 and those of PHD2 protein were 131.27±2.61, 140.12±4.29, 133.14±2.11; those of VEGF mRNA were 1.346±0.008, 3.465±0.048 and 2.264±0.073 and those of VEGF protein were 532.25±19.92, 601.13±10.21, 537.34±5.96 in the normal control group, CoCl2 group and CoCl2+ baicalein group, showing significant differences among the three groups (PHD2: F=905.89, 43.18, both at P<0.01.VEGF: F=27.13, 185.79, both at P<0.01), and the relative expression levels of PHD2 and VEGF mRNA and protein in the CoCl2group were higher than those in the normal control group and CoCl2+ baicalein group (all at P<0.01).
Hypoxia stimulates the proliferation of retinal glial cells and increase the expression of PHD2 and VEGF.Baicalein can effectively inhibit the overexpression of PHD2 and VEGF in retinal glial cells and proliferation of retinal glial cells induced by hypoxia.PHD2 is a potential provention and treatment target of hypoxia-induced retinal neovascularization.