The Oxidative Stress On RPE Cells And Mechanism Of Genistein In Hypoxia

Neovascular glaucoma is a serious complication caused by ocular ischemia or hypoxia, but no effective means have been developed for its treatment so far.Some studies have indicated that when ischemia or hypoxia occurs, the retina may suffer damage mediated by free radicals which may be at the same time involved in the signal transduction.In different cells, oxidative stress can regulate many physiological functions and biochemical reactions through the mediation of free radicals, and cell proliferation, differentiation or apoptosis may occur, producing new blood vessels. Many researches show that Genistein (Gen) extracted from soybeans is a neogenetic blood vessel inhibitor. It has been confirmed that Gen can restrain protein tyrosine kinase ( PTK ) activity and type â…¡ topoisomerase activity, and produce the effect of weak estrogens/antiestrogens and of antioxidation. Now, we investigate its impact on oxidative stress of RPE in hypoxia: When hypoxia, a certain concentration of Gen how to influence the proliferation of RPE and what is the mechanism of action in thecell growth when treated with Gen.In the present study, a RPE in hypoxia model was established to investigate the impact of Gen of a certain concentration on the cell growth and oxidative stress of RPE in hypoxia. We hope to reveal some pharmacologic action of Gen in hypoxia. Two parts experiments were performed. First, we observed how a certain concentration of Gen effect the growth of RPE; the second, we observed how a certain concentration of Gen effect the oxidative stress in RPE under oxygen deficiency.As well known, in the couse of cell proliferation, tyrosine protein kinase-RAS singnal path way is one of most important singnal channel. When cell proliferation just as tumor cell amplification, increased RAS mRNA can be detected by RT-PCR.When cells under oxidative stress, the free radicals and antioxidase activity can be used to indicate the degree of the stress. However, the NO, can diffuse quickly, so in tissues it is difficult to measure it directly as well as the O₂ ·. The NO gas in tissues can be oxidized by peroxide anion to toxic ONOO which modifies the tyrosine residue in protein into nitrotyrosine (3-NT). Using Enzyme-linked immunoadsordent assay (ELISA) for 3-NT, the amount of the NO and O₂· can be detected indirectly to indicate the situation of oxidative stress.On the basis of above, Several Experiments were carried about: On the first part, the RPE cell were cultured. Two models of RPE cells were set up, one under anoxic conditions which the cells were cultured by an airtight container, and the other under normal conditions as a control. Fourgroups of RPE cells in the first model were treated with Gen 0,50,100,200μmol-L-1 respectively. After 24 hours, thiazolyl (MTT) metheod was used to evaluate the cell proliferation under anoxic circumstance, the OD value of MTT of RPE cells was measured, and the expression of Ras mRNA was detected with RT-PCR.In the basis of part one, on the second part, the specific 3-NT antibody was used for ELISA to detect 3-NT; The activity of superoxide dismutase (SOD), one of antioxidase , was determined by Xanthine Oxidase Enzymic method.The findings of the experiments are listed below:In anoxic environment, with stronger genistein, there was increased expression of Ras mRNA in the cells. Compared with the control group there was a significantly bigger increase of 3-NT(P<0.05), and a better expression of SOD(P<0.05). Compared with the RPE cells not treated with Gen in the anoxic environment, the OD value of MTT showed a significant increase in the RPE cells treated with Gen100, 200μmol·L-1. (P<0.05), indicating a proliferation of RPE cells in 24 hours.Based on the experimental results, the following conclusions are drawn:1. Under anoxia, the RPE cells can proliferate when treated with Genof a certain concentration ( 100, 200μmol·L-l).2. Under Anoxia, the RAS mRNA expression goes up in the RPE cellstreated with Gen of a certain concentration.3. Under anoxia, the oxidative stress is increased in RPE c...