The Mechanism Of DJ-1 Regulating Oxidative Stress In RGCs Injury Of Diabetic Mice

Objective:This study is aim to reveal the role and mechanism of DJ-1 in regulating oxidative stress in retinal ganglion cells（RGCs）injury of diabetic mice.Methods:1.Bioinformatics screened the differentially expressed genes（DEGs）in the vitreous fibrovascular membrane of patients with proliferative sugar reticulum and the retina of healthy people and explored their cellular location and function.The DEGs related to oxidative stress and mitochondrial function were screened,RT-q PCR detected the gene expression level of R28 cells,and cell apoptosis was detected by flow cytometry.The miRNA targeting DJ-1 was screened,and RT-q PCR detected its expression.2.In vivo experiment:We constructed an STZ-induced type 1 diabetes（T1DM）mouse model,and selected 4 weeks,12 weeks and 24 weeks of wild type（WT）,DJ-1^-/-,WT T1DM and DJ-1^-/-T1DM mice for the following experiment.OCT and paraffin section staining was used to observe retinal structure;an electroretinogram was used to detect retinal function;whole retinal smears were used to count RGCs;Western blot（WB）was used to detect protein expression levels.AAV was used to overexpress DJ-1 in the mouse retina,and the changes in retinal structure and function were detected after 12 weeks.3.In vitro experiments:Normal glucose（5.6 m M）was used as a control,high glucose（30 m M）was used to intervene with R28 cells;CCK-8 was used to detect cell viability;TUNEL staining was used to detect apoptosis;CellROXDeepRed was used to detect reactive oxygen species（ROS）content;JC-1 was used to detect mitochondrial membrane potential;protein expression was detected by WB;mitochondrial ultrastructure was observed by transmission electron microscope.DJ-1 was overexpressed,and miR-122 inhibitor was transfected in R28,and the phenotypic changes of the cells were detected.4.Statistical analysis:Graph Pad Prism software was used for statistical analysis.Continuous variables were described by mean±standard deviation.The t-test was used to compare the means between two groups,and the analysis of variance and Tukey’s correction for multiple comparisons were used to compare the means among multiple groups.A P value less than 0.05 was considered statistically significant.Results:1.Bioinformatics analysis screened out five differentially expressed genes related to oxidative stress and five related to mitochondrial function.In the high glucose group,TP53,CASP3,CDKN2A,and CASP1expressions increased,while the expressions of PARK7,Nrf2,and SOD2decreased.The expression of miR-122 in R28 cells in the high glucose group increased.2.DJ-1^-/-further exacerbated RGC loss and GCL thinning in T1DM mice,and the a-wave and b-wave amplitudes of DJ-1^-/-T1DM mice were lower than those of other groups of mice of the same age.DJ-1^-/-inhibits the expression of anti-oxidative and anti-apoptotic proteins such as Nrf2and Bcl2 in mice.Overexpression of DJ-1 can improve the damage of retinal structure and function in T1DM mice,effectively increase the expression of retinal antioxidant enzymes,protect mitochondrial function,and resist apoptosis.The expression of miR-122 in mouse retina decreased after overexpression of DJ-1.3.Compared with the control group,high glucose inhibited R28 cell viability,increased ROS production,caused mitochondrial dysfunction and apoptosis,inhibited the expression of DJ-1 and antioxidant enzymes,promoted the expression of PTEN and apoptotic protein,and increased the ratio of LC3II/LC3I,damaging the mitochondrial structure.Overexpression of DJ-1 can effectively increase the expression of antioxidant enzymes,protect mitochondrial function,and resist apoptosis.Transfection of miR-122 inhibitor can increase the expression of DJ-1 and play the same role as overexpression of DJ-1.Conclusions:1.Processes such as oxidative stress,mitochondrial dysfunction,neuronal injury,apoptosis,and ageing are all involved in the pathogenesis of DR.2.In retinopathy of T1DM mice,hyperglycemia increased the expression of miR-122,inhibited DJ-1,reduced the content of RGC antioxidant enzymes,impaired mitochondrial membrane potential and membrane homeostasis,and aggravated RGC oxidative stress.3.High glucose-induced oxidative stress,mitochondrial damage and apoptosis in R28 cells by inhibiting the expression of DJ-1.miR-122impairs mitochondrial function by inhibiting DJ-1 expression,causing oxidative stress and apoptosis in R28 under high glucose.