The Role And Mechanism Of Nogo-B On Blood-Retinal Barrier In Diabetic Retinopathy

Objective: According to the related changes of clinical diabetic retinopathy(DR),using experimental DR rat models,to explore the effects and mechanisms of Nogo-B(Reticulons-4B,RTN4B)on blood-retinal barrier(BRB)throughout the whole process of DR,especially in the course of diabetic macular edema(DME),which leads to severe visual loss.The aim is to elucidate the occurrence of DME more comprehensively and find new intervention targets according to its pathogenic mechanisms,which will open up new avenues for the development of therapeutic drugs for DME and DR.Methods: Sprague-Dawley(SD)rats were injected with streptozotocin(STZ)intraperitoneally to induce type I diabetes as a disease model of DR.Adeno-associated virus(AAV)which induced si Nogo-B was subsequently injected intravitreally in rats to reduce Nogo-B expression in the retina,and AAV induced green fluorescent protein(GFP)as negative control.The Rhodamine-B-dextran leakage assay was used to measure the permeability of the blood-retinal barrier.In vitro experiments,lentivirus(LV)containing Nogo-B interference fragment was further constructed and infected into human retinal microvascular endothelial cells(HRMECs).The cell viability,cell number,migration ability and the effect of intercellular barrier function were detected after glyoxal treatment.The expression of Nogo-B,tight junctional proteins,inflammatory factors and related signaling pathways were detected by immunofluorescence(IF),Western blot(WB)and quantitative real-time polymerase chain reaction(q RT-PCR)in vitro and in vivo.Enzyme linked immunosorbent assay(ELISA)was used to measure the concentration of Nogo-B in the aqueous humor,vitreous and plasma samples of the patients included in this study,and patients with rhegmatogenous retinal detachment or epimacular membrane without diabetes regard as the negative control group.Results: Nogo-B expression was significantly upregulated in experimental DR models and ocular tissue samples from clinical diabetes patients.Under normal conditions,knockdown of Nogo-B led to increased retinal vascular permeability in rats and decreased intercellular barrier function of HRMECs.While in DR,up-regulation of inflammatory factors and down-regulation of tight junctional proteins leading to the BRB leakage was significantly increased,and knockdown of Nogo-B could partially prevent the disruption of BRB function by increasing the expression of p/t-Src(Try529)and p/t-Akt(Ser473)and reducing the expression of p/t-ERK1/2.The results in vitro have shown that the down-regulation of Nogo-B leads to changes in the statement of HRMECs,and the intercellular barrier function was destroyed.After glyoxal treatment and then knockdown of Nogo-B could also partially upregulated the expression of Occludin and ZO-1 by regulating the ERK pathway,preventing the destruction of intercellular barrier.Conclusion: Nogo-B may be one of the essential proteins for HRMECs to balance the barrier functions under normal physiological conditions.However,its expression was significantly upregulated in both experimental DR models and clinical disease samples,and blocking Nogo-B upregulation improved the integrity of BRB in experimental DR.Nogo-B is expected to be a potential target for the treatment of DR.