The Effects Of Hydrogen Peroxide On The Proliferation And VEGF Expression In Human Lens Epithelial Cells

PurposeCataract is the leading cause of blindness in the world. It has shown that ROS plays important roles in formation of cataract. Hydrogen peroxide, an inducer of oxidative stress is found in the aqueous humor with high concentration in cataract patients. This implies that H₂O₂ may involve in cataractgenisis. It has also shown that H₂O₂ can induce the secretion of CTGF and LEDGF, and influence the biological behaviors of the cells. However, to our knowledge, we have not found the reports about upregulation of VEGF in lens epithelial cells induced by H₂O₂. The goals of this study are firstly to investigate the effects of hydrogen peroxide at various concentrations on the proliferation and expression of VEGF in human lens epithelial cells in vitro. AbstractMethodsHuman lens epithelial cell line SRA01/04 was used in this study. The cells were treated with H₂O₂ at various concentrations of 1nM, 10nM, 100nM, 1μM, 10μM,100μM and 1mM, respectively. The controls were cultured in the medium without H₂O₂. After treated for 40 min, the cells were then cultured in normalcondition for additional up to 24 h. The survival and proliferation of the cells was quantified by MTT assay before and 6h, 12h and 24h after the treatment of H₂O₂. By Weston blot assay, PCNA protein was observed in the cells at 6h after treatment with H₂O₂ for 40min. The concentration of VEGF in the supernatants was measured by ELISA at 24h after the 40min-treatment with H₂O₂.ResultThe proliferation rates (OD values) of the human lens epithelial cells treated with 1nM to 10μM of hydrogen peroxide, increased significantly compared to that of the controls, in a dose dependent manner. Of them, the highest OD value was in the cells treated with 10nM H₂O₂. However, high dose (100μM and 1mM) of hydrogen peroxide suppressed the proliferation of the cells. Consistent with this finding, the other proliferation marker, PCNA protein had higher expressions in the cells treated with 1nM to 10μM of hydrogen peroxide, and the expression peak was at 10nM H₂O₂ group. Moreover, high dose of hydrogen peroxide inhibited the expression of PCNA.By ELISA, we found that the VEGF levels were elevated in a dose dependent way at low dose (1nM– 10μM) of H₂O₂. The peak level appeared in the cells treated with 10nM and 10μM H₂O₂. There was no statistically significant between the 100μM and 1mM H₂O₂ groups and the control group.ConclusionHydrogen peroxide at low dose may stimulate the proliferation of lens epithelial cells and increase production of VEGF. It suggests that VEGF can be a product of oxidative stress and serve protection of lens epithelial cells from damage. To the best of our knowledge, this is the first evidence to show that low dose of H₂O₂ induce VEGF expression in human lens epithelial cells.