| Background and objectives:Dry eye(DE)is a multifactorial ocular surface disease whose core pathological mechanisms include tear film instability and hyperosmolarity,ocular surface inflammation and damage,and neurosensory abnormalities.Matrix metalloproteinase(MMP)-9 damages the corneal epithelial barrier,causes tear film instability and ocular surface damage,and plays an important role in the pathogenesis of DE.The prevalence of DE in diabetic patients is significantly higher than that in non-diabetic patients,and abnormal ocular surface metabolism is one of the important reasons.The Keap1/Nrf2 pathway can respond to various stress stimuli including metabolic changes and regulate the expression of more than 600 downstream genes,affecting physiological and pathological processes such as oxidative stress,proliferation,differentiation,and inflammation.However,there is a lack of research on the changes in corneal glycolysis in diabetic patients and how the downstream influences the pathogenesis of diabetic DE.This research aims to investigate the changes of corneal glycolysis in diabetic DE,the correlation between glycolytic activity and MMP-9 expression,and whether glycolysis regulates MMP-9 expression through the Keap1/Nrf2 pathway,and to further study the feasibility of reducing the expression of MMP-9 by interfering with key molecules of glycolysis to alleviate diabetic DE.Methods:Animal experiments:Mice were intraperitoneally injected with streptozocin(STZ)and fed on a high-sugar and high-fat diet to establish a diabetic mouse model.The left eye of each diabetic mouse received subconjunctival injection of 3PO,and the right one was injected with the same volume of DMSO.The weight and blood glucose of mice were measured before and 1,2,and 4 weeks after STZ treatment.The lactate was detected by microplate reader.Corneal fluorescein sodium staining and phenol red cotton thread test were performed to evaluate DE.Western blotting and immunostaining were used to detect glucose transporter 1(GLUT1),phosphofructokinase-1(PFK-1),6-phosphofructo-2-kinase/fructose-2,6bisphosphatase 3(PFKFB3)and MMP-9 expression changes.Cell experiment:The primary mouse corneal epithelial cells(pMCECs)were divided into control group,high glucose group(30 mM glucose),and 3PO group(30 mM glucose+3PO).The lactate was detected by microplate reader.Cell immunofluorescence staining,RT-PCR,and Western blotting were used to detect the expressions of GLUT1,PFK-1,PFKFB3,Keap1,Nrf2,and MMP-9.Cells with high glucose were used to be transfected with si-Nrf2,and the protein levels of Nrf2 and MMP-9 were detected by Western blotting.Results:1.Compared to the control group,the diabetic mice lost weight,the random blood glucose was>16.7 mM,and showed reduced tear production,increased fluorescein sodium staining score,increased corneal lactate,and increased expression of GLUT1,PFK-1,PFKFB3,and MMP-9.2.Compared to the control group,the lactate was increased and the expressions of GLUT1,PFK-1,PFKFB3,and MMP-9 were elevated in pMCECs with high glucose.3.Subconjunctival injection of 3PO resulted in decreased corneal lactate,decreased MMP-9 expression,increased tear production,and decreased sodium fluorescein staining score in diabetic mice.4.3PO treatment decreased lactate and the expression of MMP-9 in pMCECs with high glucose.5.Compared with the control group,the expression of Keapl was lower and the expression and nuclear translocation of Nrf2 were increased in pMCECs with high glucose.3PO treatment increased the expression of Keapl and decreased the expression and nuclear translocation of Nrf2.The expressions of MMP-9 decreased after si-Nrf2 transfection in cells with high glucose compared to the untransfected cells with high glucose.Conclusion:Corneal glycolysis is enhanced in mice with diabetic DE,up-regulating MMP-9 expression through the Keap1/Nrf2 pathway.3PO,an inhibitor of PFKFB3 which is one of the key molecules of glycolysis,inhibits glycolysis and reduces MMP-9 expression,thereby alleviating DE. |
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