The Effects Of Geinstein On Interleukin-1β-induced Chemokine Expression In Retina And Blood-retinal Barrier Breakdown

Objective. To study the effects of genistein, a protein tyrosine kinase (PTK) inhibitor on interleukin(IL)-induced interleukin(IL)-8 and monocyte chemotactic protein(MCP)-1 gene expression in human retinal pigment epithelial(hRPE) cells and rat neuroretina. And to study the effects of genistein on interleukin(IL) -induced blood-retinal barrier breakdown in rat.Methods. (1) Reverse transcriptase polymerase chain reaction (RT-PCR) was employed to determined the induced IL-8 and MCP-1 gene expression of untreated hRPE cultures or cultures exposed to 0.1-10ng/m1IL-1 and effects of genistein on it. MTT method was used to analyze the influence of IL-1 to hRPE proliferation. (2) RT-PCR was employed to determined the induced IL-8 and MCP-1 gene expression in neuroretina after intravitreous injection of IL-1 3 . MCP-1 protein expression was further localized by immunohistochemical method. The effects of genistein on those changes above were determined with same methods. (3) The changes of vessels and infiltration of leukocyte were observed with HE stain. Evans Blue method was used to analyze the permeability changes of BRB with the stimulation of IL-1 3 . The effects of genistein on those changes above were determined with same methods.Results. (1) RT-PCR results indicated that hRPE could express IL-8mRNA obviously with the stimulation of Ing/ml , 10ng/mlIL-1 and express MCP-1mRNA obviously with the stimulation of 10ng/m1IL-1 . 10ug/ml 50ug/ml genistein eliminated most of the stimulated production of IL-8 and MCP-1. 0.1-10ng/ml IL-1 3 stimulated the growth of hRPE cells obviously. (2) RT-PCR results indicated that neuroretina expressed IL-8 and MCP-lmRNA obviously 2h after IL-1 3 injection and persisted to 8h. The expression declined to control level within 24h. Immunohistochemical results showed that MCP-1 protein expression was found in neuroretina 2h post IL-1 3 injection and achieved maximum after 8h. After 72h, the protein expression declined to control level. Immunoreactive MCP-1 was detectedmainly in the ganglion cell layer, inner nuclear layer and outer plexiform layer. Genistein could eliminate most of the stimulated production of IL-8 and MCP-1. (3) The vessel of retina was dilated 2h after injection of IL-1 (3 and most obviously after 4 to 8h and then lightened gradually. Leukocyte infiltration was appeared at 4h, and persisted to 48h. With Evans blue method, the difference of BRB permeability was significant at 2,4 and 48h after IL-1 injection compared to control group. Compared to IL-1 group, the differences of BRB permeability of lug and 5ug group were significant with the intervention of genistein at 4h, the differences of 0.2ug, lug and 5ug group were all significant at 48h. Conclusion. (1) IL-1 3 induced IL-8 and MCP-1 expression in hRPE cells in a concentration-dependent manner. Genistein could inhibit IL-1 3 -induced IL-8 and MCP-1 expression in hRPE cells. This study indicated that PTK pathway plays an important role in IL-1 3 -induced IL-8 and MCP-1 expression. (2) IL-8 and MCP-1 could expressed in neuroretina with the stimulation of IL-1 3 . MCP-1 protein expressed mainly in the ganglion cell layer, inner nuclear layer and outer plexiform layer. Genistein could inhibit IL-1 induced IL-8 and MCP-1 expression in neuroretina. (3) The IL-1 -induces BRB permeability increase had two peaks at 4h and 48h. BRB permeability increase could inhibit by genistein in a dose-dependent manner.