Role Of DJ-1/PTEN/Nrf2 Signaling Pathway In Corneal Epithelial Injury In Diabetic Mice

Background:Diabetes mellitus(DM)is a chronic metabolic disorder characterized by abnormally increased blood glucose level or decreased insulin sensitivity.Diabetes keratopathy(DK)is one of the major ocular complications of diabetes patients,which seriously affects the visual quality of diabetes patients.With the global prevalence of diabetes mellitus over recent decades,DK has attracted more and more attention.Oxidative stress is an important factor in the progression of DK.As a multifunctional protein,DJ-1 plays a very important role in the resistance to oxidative stress,regulation of signaling pathways and mitochondrial homeostasis.However,the molecular mechanism of DJ-1 in the diabetic corneal epithelial injury remains unclear.Purpose:This study aimed to explore the regulatory molecular mechanism of DJ-1 in diabetic corneal epithelial injury,providing a new direction for the treatment of DK.Method:1.Establishment and corresponding changes of diabetic keratopathy model in DJ-1 knockout mice:In this part,we evaluated the impact of diabetic keratopathy on corneal epithelial cell morphology,corneal sensitivity,corneal epithelial integrity,tear secretion,and corneal nerve fiber density in WT mice、WT DM mice、DJ-1 knockout mice and DJ-1knockout DM mice,and detected the content of corneal ROS,investigated the characteristics and mechanism of diabetic keratopathy in DJ-1 knockout mice.2.Effect of DJ-1 on cornea epithelial injury and regeneration in diabetic mice:The corneal epithelial scraping model was created on WT、WT DM、DJ-1 knockout and DJ-1 knockout DM mice when the diabetic model was established for 16 weeks.By fluorescein staining,the healing rate of corneal epithelium was observed at postoperative 12,24 and 48 hours under the microscope.The recovery of corneal sensitivity was detected by Cochet-Bonnet corneal esthesiometer.At postoperative 14 days,the corneal nerve immunofluorescence staining was used to evaluate the corneal nerve regeneration.3.Role of DJ-1/PTEN/Nrf2 signaling pathway in cornea epithelial injury in diabetic mice: To explore the role of DJ-1 in oxidative stress induced by high glucose in HCECs.Western blot was used to detect the Mn SOD,CAT expression of HCECs in different groups,including control group(17.5 m M glucose medium),high glucose group(50 m M glucose),empty vector control group(50 m M glucose),DJ-1overexpression group(50 m M glucose).And detected the changes of reactive oxygen species(ROS)in each group.We also detected the protein expression of PTEN,Nrf2 and its downstream signaling pathways HO-1 and NQO1 in HCECs and DJ-1 overexpressed HCECs both treated with high glucose.4.The datas were presented as mean ± standard error of mean(SEM)and All experiments were performed independently at least three times.Data analysis was performed using Graph Pad Prism Version 8.0were analyzed using independent samples t-test.The differences among multiple groups were analyzed using one-way ANOVA.P<0.05 was considered statistically significant.Results:1.Establishment and corresponding changes of diabetic keratopathy model in DJ-1 knockout mice: In this part,we evaluated the corneal epithelial cell morphology,corneal sensitivity,corneal epithelial integrity,tear secretion and corneal nerve fiber density in DJ-1 knockout DM mice,DJ-1 knockout mice,WT DM mice and WT mice.We found that DJ-1knockout DM mice had significantly lower body weight and higher blood glucose elevation than WT DM mice at 16 weeks.The corneal epithelial thickness was decreased,epithelial cell arrangement was disordered,the corneal sensitivity was significantly reduced,the epithelial integrity was decreased,the tear secretion was decreased,and the corneal nerve fiber density was decreased.The signs of diabetic keratopathy in DJ-1knockout DM mice is more serious than WT DM mice,and the content of ROS in DJ-1 knockout DM mice is higher than WT DM mice.The expression of DJ-1 in corneal epithelium of WT mice and WT DM mice was also observed.The results showed that the expression of DJ-1 in corneal epithelium and trigeminal ganglion of WT DM mice was significantly reduced.2.Effect of DJ-1 on corneal epithelial wound healing and regeneration in diabetic mice: In the corneal epithelial injury model,the corneal epithelial healing rate and corneal nerve regeneration rate of DJ-1knockout DM mice were slower than WT DM mice.The recovery of corneal sensitivity lagged behind that of WT DM mice at the same age.3.Role of DJ-1/PTEN/Nrf2 signaling pathway in corneal epithelial injury in diabetic mice: Compared with the control group,ROS level increased and Mn SOD,CAT protein expression decreased in the high glucose group.Compared with the high glucose group,ROS decreased and Mn SOD,CAT protein expression increased in the DJ-1overexpression group.Compared with the control group,the expression of PTEN protein in the high glucose group increased,while the expression of Nrf2 decreased.Compared with the high glucose group,the expression of PTEN protein in DJ-1 overexpression group was decreased,and the expression of Nrf2 and its downstream proteins HO-1 and NQO1 was increasedConclusions:1.DJ-1 knockout aggravates diabetic keratopathy by aggravating oxidative stress.2.DJ-1 knockout inhibits corneal epithelial regeneration and nerve recovery in diabetic mice.3.DJ-1 protects corneal epithelial cells from oxidative stress induced by high glucose by regulating the activity and protein expression of Mn SOD,CAT and improving mitochondrial function.4.DJ-1 can regulate diabetic keratopathy through PTEN/ Nrf2 signaling pathway.