Protective Effect Of DHK On Diabetic Retinopathy Via Antioxidation And Antiinflammation

ObjectiveDiabetic retinopathy.(DR),one kind' of diabetic micro vascular complications,is the leading cause of vision loss.Previous studies showed that Danhong Huayu Koufuye(DHK)prevented DR in streptozotocin(STZ)-induced Sprague-Dawley(SD)diabetic rats and spontaneous Zucker diabetic fatty(ZDF)rats.The aim of the study was to explore whether antioxidation and antiinflammation were main mechanisms of DHK on DR in STZ-induced diabetic rats in vivo and high glucose-treated EA.hy926 cells in vitro.Methods(1)Mechanism study of DHK on DR in vivoOne week after STZ injection,SD rats with average fasting blood glucose higher than 14.0 mmol/L were randomly divided into two groups:(1)STZ+Vehicle group(distilled water,3.2 mL/kg,25 rats);(2)STZ+DHK group(DHK,3.2 mL/kg,26 rats).In addition,20 normal rats were regarded as Normal+Vehicle group(distilled water,3.2 mL/kg).All rats were orally administered once daily and consecutively for 16 weeks.Activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),and levels of malondialdehyde(MDA)and nitric oxide(NO)in serum were detected by commercial kits.Enzyme linked immunosorbent assay was used to evaluate levels of vascular endothelial growth factor(VEGF)and intercellular cell adhesion molecule-1(ICAM-1)in serum.Protein expressions of VEGF,VEGF receptor 1(VEGFR1),VEGFR2 and inducible nitric oxide synthase(iNOS)in retinas of rats were assessed by immunohistochemical method.Furthermore,permeability of blood-retinal barrier(BRB)was tested by Evans blue.(2)Mechanism study of DHK on DR in vitroCell viability was detected by MTT assay,and apoptotic rate was obtained by flow cytometry.Abilities of cell migration and angiogenesis were evaluated by transwell assay and tube formation assay,respectively.Intracellular reactive oxygen species(ROS)in EA.hy926 cells was evaluated using H2DCFDA probe.Activities of SOD and GSH-Px,and level of MDA in culture medium were measured by commercial kits.Protein expressions of nuclear transcription factor-?KB(NF-?B),ICAM-1,hypoxia-inducible factor-la(HIF-la),VEGF,VEGFR1 and VEGFR2 were determined by Western blot.Results(1)Mechanism study of DHK on DR in vivoSerum activities of SOD(P<0.01)and GSH-Px(P<0.01)were significantly decreased,and levels of NO(P<0.01),VEGF(P<0.01)and ICAM-1(P<0.05)were markedly increased in STZ-induced diabetic rats.Protein expressions of VEGF(P<0.01),VEGFR1(P<0.01),VEGFR2(P<0.01)and iNOS(P<0.01)were remarkably up-regulated in retinas of diabetic rats.High permeability of BRB was observed in DR rats.DHK improved activities of SOD and GSH-Px by 19.11%(P<0.01)and 545.28%(P<0.01),respectively.Levels of NO,VEGF and ICAM-1 in DHK treated rats were decreased by 55.11%(P<0.01),57.57%(P<0.01)and 25.45%(P<0.05),respectively.DHK down-regulated the protein expressions of VEGF and iNOS in retinas of diabetic rats.High permeability of BRB in diabetic rats was reversed by treatment of DHK.(2)Mechanism study of DHK on DR in vitroAs compared with normal group,cells cultured in 40 mmol/L glucose for 96 h showed the decreased cell viability and increased apoptotic rate.Cell migrating ability and activities of SOD and GSH-Px were decreased,while cell angiogenesis ability,levels of MDA,intracellular ROS and protein expressions of NF-?B,ICAM-1,HIF-la,VEGF,VEGFR1 and VEGFR2 were increased in high glucose-treated cells.As compared with model group,DHK medicated serum concentration-dependently increased cell proliferation and migration ability,inhibited apoptosis and neovascularization,reduced the levels of MDA and ROS,down-regulated protein expressions of NF-?B,ICAM-1,HIF-1?,VEGF,VEGFR1 and VEGFR2 in cells.Ten percent of DHK medicated serum increased cell viability and migrating ability by 40.99%(P<0.01)and 291.52%(P<0.01),respectively.Ten percent of DHK medicated serum reduced levels of ROS and MDA,apoptotic rate and angiogenesis ability by 164.68%(P<0.01),26.42%(P<0.01),14.07%(P<0.01)and 319.89%(P<0.01),respectively.Ten percent of DHK medicated serum increased the activities of SOD and GSH-Px by 57.91%(P<0.01)and 95.59%(P<0.05),respectively.As compared with 40 mmol/L glucose group,10%DHK medicated serum down-regulated protein expressions of NF-?B,ICAM-1,HIF-la,VEGF,VEGFR1 and VEGFR2 by 140.34%(P<0.01),186.67%(P<0.01),71.00%(P<0.01),97.33%(P<0.01),92.67%(P<0.01)and 150.34%(P<0.01),respectively.ConclusionAntioxidant and antiinflammatory activities of DHK contribute to the protective effect of DHK on DR.