Protective Effect And Mechanism Of Melatonin On Inner Blood-retinal Barrier In Experimental Diabetic Retinopathy

Aims/hypothesis:The pathophysiological process of diabetic retinopathy（DR）includes the breakdown of blood-retinal barrier（BRB）and diabetic macular edema（DME）,the mechanisms involve the activation of glia cells and their increased production of the inflammatory cytokines,growth factors,and etc.,that aggravate vascular injury.Studies showed that melatonin could protect neurons,retinal pigment epithelial cells,Müller cells and BRB in various retinal diseases with its protective mechanisms not been fully elucidated.In this study,different models were used to explore the protective effect and mechanism of melatonin on retinal pericytes,endothelial cells as well as BRB,so as to find a therapy for early intervention of DR with optimal efficacy.Methods:Male Sprague-Dawley rats were injected intraperitoneally with streptozotocin（STZ）to establish the diabetic rat model.At 2 hours after STZ injection,the right eye was injected intravitreally with melatonin（30μg/eye,2μL）and the left eye was injected with an equivalent volume of normal saline.At 8 weeks after onset of diabetes,electroretinogram（ERG）was used to detect retinal function,and FITC-dextran leakage assay and albumin immunostaining were used to detect the i BRB leakage.Immunofluorescence and Western blot were used to detect retinal microglia,pericyte and vascular endothelial cells.Western blot was used to detect the expressions of inflammatory factors and tight junctions of endothelial cells.The in vitro experiment was divided into two parts,as in first part,a co-culture system of human brain vascular pericytes（HBVPs）and human retinal microvascular endothelial cells（HRMECs）was established,and BV2 cells（mouse microglia cell line）were incubated with Co Cl₂or melatonin.The supernatant of activated microglia was added into HBVP/HRMEC co-culture system to study the regulation of melatonin on microglia and indirect protection of i BRB.The barrier function of i BRB was detected by transepithelial electrical resistance（TEER）and FITC-Dextran.Pericyte survival was observed by cell counting kit-8（CCK-8）and flow cytometry,and the changes of tight junction protein between endothelial cells were detected by immunofluorescence.The pericyte and microglial-related molecular markers,as well as PI3K/Akt,Stat3 and NF-κB signaling pathways were assessed by Western blot and RT-q PCR,and validated with related inhibitors such as melatonin receptor antagonist（luzindole）as well as Akt inhibitor or NF-κB inhibitor.In addition,we adopted glyoxal or melatonin treated-HRMECs to simulate endothelial cell injury in DR,and studied the direct protective effect of melatonin on the endothelial cells.The permeability in HRMECs was quantified with FITC-dextran permeability assay and TEER.Cell viability was assayed with CCK-8.The changes of tight junction proteins were detected by immunofluorescence and Western blot.The protein changes of inflammatory factors and related signaling pathways were measured with Western blot and enzyme linked immunosorbent assay（ELISA）,and further validated with related inhibitors such as melatonin receptor antagonist（luzindole）as well as p38 inhibitor or NF-κB inhibitor.Results:Compared with normal control group,the destruction of i BRB was obvious in diabetic rats,manifested by the increased FITC-dextran leakage in retinal capillaries,significant loss of pericytes and endothelial cells,and disruption of tight junctions（ZO-1,Occludin,JAM-A and Claudin-5）.Meanwhile,activated microglia proliferated and migrated from inner retina to the outer retina,accompanied with the obvious morphological changes.Interestingly,significant leakage of albumin was evidenced at the site of close interaction between activated microglia and the damaged pericytes and endothelial cells.In addition,the inflammatory factors（VEGF,TNF-α,IL-1β,ICAM-1,MMP-9）increased significantly in diabetic rat retinas,while melatonin therapy can effectively prevent these changes.The in vivo data were recapitulated with the in vitro experiments.Activated microglia overexpressed inflammatory factors,i.e.,TNF-α,i NOS,and IL-1β,inducing pericytes and endothelial cell apoptosis as well as disrupted the junctional proteins between endothelial cells,which impaired barrier function.Melatonin suppressed the microglial over-activation via decreasing the inflammatory factors and increasing the anti-inflammatory factors release,alleviated the pericyte loss and increased the tight junctions,further remaining the i BRB integrity.Besides,elevated inflammatory factors,lost tight junction protein,and impaired barrier function was evident in glyoxal-treated HRMECs.Melatonin inhibited the release of inflammatory factors and restored tight junction protein,thus protected barrier function.Conclusions/interpretation:In experimental DR,inflammatory factors induced endothelial cell loss,tight junction proteins disruption,as well as vascular leakage.Meanwhile,activated microglia migrated to the deep capillary network and secreted pro-inflammatory cytokines,causing pericyte and endothelial cell loss and aggravating i BRB breakdown.Melatonin directly maintains the i BRB integrity by restoring tight junctions and decreasing the production of inflammatory factors via inhibition of p38/TXNIP/NF-κB pathways in endothelial cell.In addition,melatonin exerts its protective effect to regulate microglial overactivation and promote the production of anti-inflammatory factors via inhibition of PI3K/Akt/Stat3/NF-κB signaling pathways in microglia,and thus maintains the integrity of i BRB.