Inhibitory Effects Of Genistein On Experimental Retinal Neovascularization

Objective: To establish a quantifying mice model of oxygen-induced retinopathy, learn the method of judging vascular proliferating, investigate CD105 expression in retina.Then to explore the inhibitory effects of genistein on the retinal neovascularization and the expression of VEGF,VEGFR-2(flk-1),CD105, investigate the potential mechanism of its effect and its value in clinic.Methods: 1 Twenty 7-day-old C57BL/6 mice were devided into two groups randomly that include air group and high concentration oxygen group.Ten mice were exposed to 75% oxygen for 5 days,followed by 5 days room air recovery, the other ten mice of the same age were kept in room air as colltrol.All animals were killed on day 17 of life and both eyes were enucleated,and embedded in paraffin.The proliferative neovascular response was quanified by counting the endothelial cell nuclei of new vessels above the internal limiting membrane in sections stained with HE and to observe the changes of retinal vessels of mice by making retinal flat mounted for ADPase staining.The expression of retinal CD105 were detected with immunohistochemistry.2 Thirty 7-day-old C57BL/6 mice were devided into three groups randomly that include genistein treatment group, DMSO group and high concentration oxygen group. All mice were put into the environment with 75% oxygen for 5 days and then to room air to establish a model of vascular proliferation retinopathy. Each mouse in the treatment group was received hypodermic injection of genistein(25 mg/kg/d)for 5 days. The other animals received the same dosage of DMSO and normal sodium injection. All animals were killed on day 17 of life and both eyes were enucleated, making retinal flat mounted for ADPase staining or embedded in paraffin, serial axial sections of the retina were obtained, staining with HE or doing immunohistochemistry of VEGF,VEGFR-2(flk-1),CD105, investigated the effects of genistein on expression of them and the retinal neovascularization.Results:1 There were large retinal neovascularization in the high concentration oxygen group compared with those in air condition, and the density and shape of retinal neovascularization were disordered. There was a mean of 36.33±1.66 neovascular nuclei per cross-section in hyperoxia compared to less than 1 nuclei per cross-secton in the air control group (P<0.01). These results indicated that the animal model of Oxygen-Induced Retinopathy was successful. Immunohistochemistry of the retinal sections revealed CD105 overexpression in the retina of the oxygen treated mice. 2 Comparing with high concentration oxygen group, regular distributions and reduced density of retinal neovascularization were observed in the treatment group.There was a mean of 17.56±2.65 neovascular nuclei per cross-section in the genistein treatment group, and compared to high concentration oxygen group and DMSO group, the difference were significant (P<0.01). The difference of the number of new blood vessels endotheliocyte nuclei in high concentration oxygen and DMSO groups were not significant(P>0.05). Immunohistochemistry of the retinal sections shown VEGF,VEGFR-2(flk-1),CD105 expression in the retina were partially inhibited by genistein injection.Conclusion:1 Hypoxia induced the expression of CD105 in retina, derivned mice emerging process of OIR,and CD105 became a specificity marker of new vessels. 2 Retinal neovascularization can be successfully inhibited by hypodermic injection of genistein, the VEGF,VEGFR-2( flk-1 ),CD105 expression in the hyperoxia retina were partial inhibited.These results suggest that genistein may have potential therapeutic benifits in retinal vascular disease.