The Protective Function And Mechanism Of Glutamine On Diabetic Corneal Endothelial Cells

Objective:To investigate the function and mechanism of glutamine(Gln)on diabetic corneal endothelial cells using human corneal endothelial cells(HCECs)treated with high glucose and streptozotocin(STZ)-induced type I diabetic mice model.Methods:1.High performance liquid chromatography-mass spectrometry was used to determine the content of neurotransmitters in the aqueous humor(AH)of patients with age-related cataract and diabetic cataract;Glutamine kit was used to detect the level of glutamine in the AH of normal mice and diabetic mice;Corneal endothelial injury model was established in mice with anterior chamber perfusion,and the experimental group was perfused with a glutamine-containing irrigation fluid.Slit lamp,optical coherent tomography(OCT),corneal whole-mount staining,and endothelial cell counting were performed to observe corneal edema,corneal thickness,and expression of endothelial barrier and pump function in mice,to assess the protective effects of glutamine on corneal endothelium in diabetic mice.2.Transmission electron microscopy was used to observe the mitochondrial ultrastructure of the corneal endothelium in normal and diabetic mice;RT-q PCR was done to detect the changes of mitochondrial DNA(mt DNA)copy number in the endothelium of diabetic mice;8-OHd G,a marker of oxidative damage,was detected by immunohistochemistry in mice corneal endothelium,and GSH in human AH was detected by Enzyme-linked immunosorbent assay(ELISA).3.To detect reactive oxygen species(ROS),glutathione(GSH)fluorescence intensity and total GSH content in normal and high glucose treated HCECs;Western Blot analysis of changes in expression of endothelial functional proteins ZO-1 and ATP1a1,apoptosisrelated signaling proteins Caspase-3,Bcl-2,Cytochrome C in HCECs,for the observation of Alanyl-glutamine(Ala-Gln)on oxidative stress-related parameters in HCECs under high glucose environment.4.Mito-tracker probe staining was performed to detect changes in mitochondrial membrane potential in HCECs;Western Blot was conducted to measure the expression of the proteins DRP1 and MFN1 associated with mitochondrial dynamics in HCECs;Seahorse analyzer detects changes in the mitochondrial energy metabolic spectrum of HCECs for evaluating the influence of Ala-Gln on mitochondrial function in HCECs under high glucose environment.Results:1 Reduction in glutamine levels in the AH of diabetic patients and miceCompared to non-diabetic and normal mice,glutamine levels in atrial water were significantly declined in both diabetic and diabetic mice.2 Gln promotes functional recovery after corneal endothelial injury in diabetic mice(1)Compared with normal control mice,diabetic mice showed corneal clouding,increased central corneal thickness,and disrupted structure and arrangement of endothelial barrier and pump function-related proteins ZO-1 and ATP1a1 after 10 d of corneal endothelial injury.The results showed that hyperglycemia could significantly affect corneal endothelial function in mice under the injury conditions.(2)After 10 d of corneal endothelial injury,corneal transparency was restored in experimental mice,with no significant loss of expression of endothelial barrier and pump function proteins.The results indicated that Ala-Gln could promote functional recovery after corneal endothelial injury in normal and diabetic mice.3 Redox imbalance and mitochondrial damage in diabetic corneal endothelial cellsTransmission electron microscopy showed mitochondrial vacuolization and cristae deletion in the corneal endothelium of diabetic mice;RT-q PCR revealed a significant decrease in mt DNA copy number expression in the corneal endothelium of diabetic mice compared with normal mice;immunohistochemistry showed enhanced positive expression of 8-OHd G in the corneal endothelium of diabetic mice compared with normal mice;ELISA revealed that the GSH content of AH was lower in diabetic patients than in patients with age-related cataract.4 Gln protects the corneal endothelium from oxidative damage caused by high glucose via upregulation of GSH synthesisAfter treatment with Ala-Gln,the cellular ROS fluorescence intensity was reduced and GSH was enhanced in the high glucose group of HCECs.The total glutathione content assay revealed that the total GSH content of HCECs in the high glucose group decreased,and the total GSH synthesis of HCECs increased after Ala-Gln treatment.Western Blot results indicated that Ala-Gln increased the expression of barrier function and pump function proteins ZO-1 and ATP1a1,decreased the expression of pro-apoptotic signaling-related proteins Caspase-3 and Cytochrome C,and increased the expression of apoptosis inhibitory signaling-related protein Bcl-2 in HCECs of high glucose group.5 Gln protects corneal endothelial cells through the mitochondrial pathway(1)The Mito-tracker fluorescence probe demonstrated that the fluorescent intensity of HCECs in the high glucose group was reduced and the mitochondrial membrane potential decreased compared with that of the normal group.After Ala-Gln treatment,the Mito-tracker fluorescence intensity was enhanced and the mitochondrial membrane potential depolarization was reduced in HCECs of the high glucose group.(2)Cellular mitochondrial function was detected using Seahorse analyzer,and it was found that mitochondrial bioenergy was reduced in HCECs in the high glucose group compared with the normal group,while Ala-Gln treatment significantly improved mitochondrial function.Conclusion:Hyperglycemia leads to decreased glutamine levels in aqueous humor.Exogenous supplementation of glutamine promotes functional recovery after diabetic corneal endothelial injury by regulating redox homeostasis and mitochondrial function in diabetic corneal endothelial cells.