The time-dependent changes of hypoxia-inducible factor-1alpha (HIF-1alpha) expression induced by hypoxia and CoCl2 treatment and the effects of genistein on the level of HIF-1alpha expression in human retinal pigment epithelium cells were examined. Judging by relative fluorescence using a confocal scanning laser microscope coupled to a computer, HIF-1alpha expression was determined. It was found that hypoxia could markedly increase the expression of HIF-1alpha. The highest expression of HIF-1alpha was detected at 1 h, which was 313.9% +/- 38.2% of the control level. After pretreatment with genistein (50, 100, and 200 micromol/l), the hypoxia-evoked HIF-1alpha expression was concentration-dependently inhibited. CoCl2 treatment could significantly elevate the level of HIF-1alpha expression. At 0.5 h after CoCl2 treatment, the highest level was observed, which was 141.4% +/- 14.1% of the control level. Genistein 50, 100, 200 micromol/L could also suppress HIF-1alpha expression in a concentration-dependent manner. These results suggested that the inhibition of HIF-1alpha protein expression by genistein may partly account for its effect on retinal neovascularization in vivo.
Recent studies showed that interleukin-8 (IL-8) played an important role in retinal neovascularization. In this study, the effects of genistein on the expression of IL-8 in the arising retinal pigment epithelia-19 cells were studied. The levels of IL-8 protein expression in supernatants were punctually detected by ELISA. When the cells were treated with hypoxia (5% CO2, 95% N2), IL-8 secretion increased from 0.29 +/- 0.04 to 2.59 +/- 0.42 ng/ml. To study calcium-dependent IL-8 expression, cells were treated with KCl at 25 mM, norepinephrine (NE) at 10 nM, and glutamate (Glu) at 1 microM for 8 h. As a result, the levels of IL-8 protein in supernatants were significantly increased compared with that in the controls. When the cells are treated with genistein (50, 100, 200 microM) for 30 min before hypoxia or stimulations by KCl, NE, and Glu, the elevated expression of IL-8 protein was all suppressed in a concentration-dependent manner. These results suggested that suppression of IL-8 expression in retinal pigment epithelial cells might partly account for the inhibitive effect of genistein on retinal neovascularization.
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