Involvement Of G6PD In Diabetic Retinopathy And Hypoxia Induced Endothelial Lesion And Its Underlying Mechanism

OBJECTIVE:Pentose Phosphate Pathway(PPP),also known as hexose monophosphate shunt,is an important shunt of glucose metabolism.Many researches have demonstrated its important role in tumorigenesis,cell proliferation and antioxidation.Our preliminary experiments show that in the non-proliferative diabetic retinopathy(NPDR),G6PD,the key enzyme of pentose phosphate pathway,decreased significantly at mRNA level.N-acetyl cysteine(NAC),a well-known medicine with excellent antioxidant and anti-inflammatory effect has been widely applied in treatment of acute cough respiratory diseases to dissolve phlegm for many years.Previous studies have shown that NAC can enhance the activity of G6PD.This present study aims at exploring the change of G6PD in diabetic retinopathy and hypoxia induced endothlial cell injury and the underlying mechanism is discussed.METHODS:In this study,STZ-induced type 1 diabetic animal model was established.We observed retinal leakage using fluorescence microscope by injecting FITC-dextran from tail vein and pericyte loss using retinal whole mount staining.In vitro EA.Hy926 were simulated with CoCl2.We detected G6PD protein expression by Western Blot,G6PD activity and NADPH/NADP+ by relative assay kit.Cell viability,proliferation ability and tube formation ability were measured by CCK-8 assay kits and EdU assay kit and matrigel tube formation assay.Expression of inflammatory/angiogenic cytokines were determined by Real-Time PCR.RESULTS:1)G6PD mRNA was decreased significantly in the non-proliferative diabetic retinopathy,indicating that G6PD could be involved in the pathogenesis of diabetic retinopathy.Of note,in vitro study hypoxia stimulation could also decrease G6PD protein level and activity,cell proliferation,viability and tube formation,and increase the level of VEGF,IL-1?,IL-6.Treatment with 6-AN,an inhibitor of G6PD activity,could also inhibit cell proliferation,viability and tube formation ability,and could increase the level of VEGF,IL-1?,IL-6.2)NAC treatment increase G6PD activity without affecting G6PD expression;3)NAC could significantly abrogate decrease of cell proliferation,viability,tube formation ability and increase of VEGF,IL-1?,IL-6 induced by hypoxia;4)Furthermore,addition of 6-AN could efficiently abrogate the effect of NAC to rescue hypoxia induced cell lesions including decrease of cell viability,proliferation,tube formation and increase of VEGF,IL-1?,IL-6.CONCLUSIONS:This research demonstrate that hypoxia could induced decrease of G6PD expression and activity in endothelial cells,which further leaded to reduction of cell proliferation,viability,tube formation and increase of VEGF/IL-1?/IL-6.NAC can rescue the decrease of G6PD activity under hypoxia stimulation,so as to improve cell damage.These results suggested that G6PD may play an important role in the pathogenesis of diabetic retinopathy,NAC could be a potential approach for therapy of diabetic retinopathy targeting G6PD.