Exogenous EPO Protects Inner Blood-retinal Barrier Through Regulation Of Mircroglia In Dibetic Retinopathy

PURPOSE:Investigate the protection on inner blood-retinal barrier(iBRB),regulation on microglial cells by intravitreal injection of exogenous erythropoietin(EPO)in early diabetic retinopathy;and explore the relationship between iBRB broken and activation of microglial cells in diabetic retinopathy in vitro.METHODS:Diabetic rat model was established by intraperitoneal injection of streptozotocin(STZ).At the same time,the diabetic rats were divided into two groups for intravitreal injection of EPO and Phosphate buffered saline(PBS).Two weeks after onset of diabetes,FITC-Dextran perfusion was used to detect the retina vessel leakage,the distribution,number and morphology of microglia in the retina were observed by immunofluorescence and Intercellular cell adhesion molecule-1(ICAM-1)expression was detected by immunofluorescence and Western blot.BV2 microglial cells were treated by oxidized low density lipoprotein(oxLDL)as cell model,select an appropriate oxLDL concentration and treatment time by CCK-8 method to detect cell viability.Human umbilical vein endothelial cells(HUVEC)were cultured with different microglial culture medium,and the integrity of HUVEC barrier was measured by FITC leakage and trans-epithelial electric resistance(TEER)test.The change of microglial morphology and inflammatory factors were investigated by immunofluorescence,real-time PCR and Western blot.RESULTS:Compared to the normal control group,2-week diabetic rat retina show an increased FITC-Dextran leakage,increased expression and wider distribution of ICAM-1;microglia was activated with increased number,morphology changed from ramified to ameboid and migration to outer retina.After the intravitreal injection of EPO,the FITC leakage of the retina was alleviated,and the expression of the adhesion molecule ICAM-1 was decreased;the cell number decreased,the change of microglial morphology decreased and migration reduced.The cell viability of BV2 microglia decreased in a dose-and time-dependent manner under oxLDL cultured condition,cells became larger,the inflammatory cytokines transcriptional level increased,and the level of activation-associated proteins increased.The microglial cell culture medium treated with oxLDL enhanced the FITC leakage and the TEER value of the HUVEC barrier in vitro.And the EPO treated group showed a decrease result in the above detections.Real-time PCR and Western blot results showed that the expression level of inflammatory cytokines treated with oxLDL was increased while the expression of these factors in EPO treated group decreased.CONCLUSIONS:1.Intravitreal injection of EPO alleviates the broken of iBRB in early diabetic retinopathy and inhibits the activation of microglia.2.EPO inhibits oxLDL-induced microglial activation and alleviates the broken of the vascular endothelial barrier by activated microglial cells.