The Protection And Mechanism Of Erythropoietin On The Inner Blood-Retinal Barrier Via Inhibiting Microglia Activation In Diabetic Retinopathy

Purpose:Diabetic retinopathy（DR）is one of the leading causes of blindness in the working-age population,but without effective treatments.The understanding of pathogenesis of DR in ophthalmology includes microangiopathy,neuropathy and low-grade inflammation.Erythropoietin（EPO）,as one of glycoprotein hormones,was mainly applied to the treatment of renal anemia due to its promotion for hematopoiesis.It has been found that it has effects of neurotrophy,anti-inflammation and anti-apoptosis recently.Our previous work revealed that EPO had protective effect on diabetic retina.The study of clinical case implied that EPO was effective to refractory diffuse diabetic macular edema.Microglia,as a special macrophage,is resident cell with phagocytosis in retina.It has not been clearly illustrated whether microglia were regulated by EPO to protect inner BRB（i BRB）as well as the potential mechanism.In this study,we will investigate whether the activation of DR microglia is involved in i BRB destruction and whether EPO can protect i BRB by regulating microglia as well as related mechanisms.This will provide new intervention strategies for the treatment of DR.Methods:Sprague-Dawley rats were rendered diabetic with intraperitoneal injection of streptozotocin（STZ）.Diabetic rats are utilized to evaluate the protective effect of EPO with intravitreally injection of EPO in right eye,while the left eye was injected with the equivalent volume of normal saline.The changes of microglia in different groups are evaluated by immunostaining of retinal flat-mount and retinal protein extraction.Meanwhile,intravenous injection of FITC-Dextran and fluorescein fundus angiography（FFA）were exploited to detect vascular leakage.The interaction of microglia and retinal vessels was evaluated by confocal imaging assay.Hypoxia-induced BV₂ cell is exploited to mimic the activated microglia in vivo.The regulatory effects of EPO on microglia are assessed by activation,phagocytosis and migration examination after exposure to hypoxia.Results:Microglia were activated with pronounced morphological changes in diabetic rat retinas,i.e.,the resting microglia with ramified morphology turned to the activated microglia with amoeboid morphology.It becomes more pronounced with disease progression.Activated microglia induce increased acellular capillary（AC）and retinal leakage,gathering around the blood vessels.High magnification revealed that activated microglia penetrated the basement membrane of retinal blood vessels and phagocytosed the endothelial cells,resulting in increased AC and vascular leakage.The results in vitro revealed that hypoxia can activate BV₂ cell.The cell model was utilized to reprint the phagocytosis of activated microglia on retinal endothelial cell by co-culture system.The activation of microglia was inhibited remarkably by EPO with disease alleviation.Under hypoxic condition,the phagocytosis of microglia was enhanced,where Src/Akt/cofilin pathway was inhibited with downregulation of phosphor-Src,phosphor-Akt and phosphor-cofilin.EPO could suppress the microglial phagocytosis via activating the pathway.Conclusions:In experimental DR,activated microglia penetrate the basement membrane of the i BRB and engulf endothelial cells,leading to i BRB breakdown and AC upregulation.EPO exerts its protective effect on maintaining i BRB integrity via inhibition of microglial phagocytosis through activating Src/Akt/cofilin signaling.This study provides a novel therapeutic strategy for regulating microglia in the treatment of DR.