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A rat model of diabetes mellitus was established by intraperitoneal injection of streptozotocin. Three days later, the rats were intraperitoneally administered 140 mg puerarin/kg daily, for a total of 60 successive days. DNA ladder results showed increased apoptosis over time in retinal pigment epithelial cells from rats with streptozotocin-induced diabetes mellitus. West blot analysis, Reverse transcription-PCR, immunohistochemistry, and flow cytometry results showed increased expression of 3-nitrotyrosine, a peroxyntrite marker, as well as inducible nitric synthase and Fas/FasL, in retinal pigment epithelial cells. Puerarin reversed these changes, and results demonstrated that puerarin inhibited Fas/FasL expression and alleviated peroxyntrite injury to retinal pigment epithelial cells. These results suggested that puerarin inhibited production of inducible nitric oxide synthase and directly antagonized peroxyntrite injury in retinal pigment epithelial cells.