Exploring The Effect Of FUNDC1 On Diabetic Retinopathy In Mice Based On Retinal Mitochondrial Dysfunction And Oxidative Stress

Objective:Diabetic retinopathy（DR）is a ophthalmic complications of diabetes,Mitochondrial dysfunction is an important factor in the development of DR.FUN14 domain containing 1（FUNDC1）is an membrane protein located on the outer membrane of mitochondria,which can participate in various mitochondrial-related processes such as mitochondrial autophagy and oxidative stress by regulating the interaction between mitochondria and endoplasmic reticulum.This study was designed to investigate the intervention effect of FUNDC1 gene deletion on mouse diabetes retinopathy model by observing mouse retinal nerve function,pathological structure and vascular disease,and to analyze the differential genes related to FUNDC1 and DR with transcriptome sequencing technology,to clarify the specific mechanism of FUNDC1 participating in DR.Methods:C57BL/6J male mice and FUNDC1 knockout(FUNDC1^-/-)male mice were selected.（1）Genotype identification:the genomic DNA of mice was extracted,PCR amplification and agarose electrophoresis were performed to verify the genotypes of mice.（2）Animal model construction:type I diabetes model was established by intraperitoneal injection of STZ solution,and the blood glucose level and body weight of mice were recorded every two weeks.（3）Detection of flash-electroretinogram（f-ERG）:the changes of amplitudes of a wave,b wave and oscillatory potentials（OPs）were compared by f-ERG test,and the retinal function of each group was evaluated.（4）HE staining of paraffin sections:by taking out mouse eyeballs,paraffin-embedded sections and HE staining,the cells of retinal ganglion cell layer were counted and quantified,and the changes of retinal thickness were counted.（5）Frozen section immunofluorescence:platelet endothelial cell adhesion molecule-1（PECAM-1/CD31）,a specific marker of vascular endothelial cells,was used for immunofluorescence experiment.The fluorescence intensity of CD31 was counted and the expression of CD31 protein was evaluated.（6）RNA-Seq:isolation of mouse retinal tissue,screening of differential genes by high-throughput sequencing,combined with GO analysis,KEGG enrichment analysis and other analysis methods to explore the mechanism of FUNDC1 involved in DR.Results:（1）Identification of genotypes of FUNDC1 knockout mice:agarose gel electrophoresis experiments show that all are purely congenic.（2）Body weight and blood glucose levels in diabetic mice:The DM group showed the typical manifestation of diabetes with no weight gain and elevated blood glucose compared with the normal group,and the differences in body weight and blood glucose levels between the FUNDC1^-/-DM group and the C57BL/6J DM group at 0w,2w,4w,6w,8w,10w,and 12w after modeling were not statistically significant（P>0.05）.（3）f-ERG detection:In C57BL/6J mice,compared with the normal group,the amplitudes of a wave and b wave in the model group did not change significantly in DM8w,but decreased at 24w,and there was significant difference between the two groups（P<0.05）,and the amplitudes of OPs-OS2 in DM8w and 24w were statistically significant（P<0.05）.In FUNDC1^-/-mice,compared with the normal group,the amplitudes of a wave,b wave and OPs-OS2 in the model group were statistically significant at DM8w and 24w（P>0.05）.Compared with C57BL/6J mice,the amplitudes of a-wave and b-wave in FUNDC1^-/-mice were significantly different from those in C57BL/6J mice at DM8w and 24w,while OPs only had statistical significance in DM 8w.（4）HE sections showed that the thickness of retina became thinner,the number of RGCs decreased,nuclear shrinkage and chromatin aggregation in DM group than that in normal group.Compared with C57BL/6JDM group,the above pathological changes were more obvious in DM8w,12w and24w in FUNDC1^-/-DM group.（5）Expression of CD31 molecule:CD31was mainly expressed in nerve fiber layer,ganglion cell layer,inner and outer subordinate layer of retina.Compared with normal group,the fluorescence intensity of CD31 in DM group increased significantly,and with the extension of modeling time,the fluorescence intensity increased gradually,and there was significant difference in DM8w and 12w CD31fluorescence intensity between FUNDC1^-/-DM group and C57BL/6JDM group.（6）RNA-seq:through the enrichment analysis of differential genes,it was found that the differential genes in FUNDC1^-/-DM16w group were concentrated in mitochondrial plasmid pump transport and oxidative stress-related pathways compared with C57BL/6JDM16w group.Conclusion:FUNDC1 gene deletion affects the pathophysiological process of DR mice and exacerbates diabetic retinal neuropathy and vasculopathy,probably by affecting mitochondrial proton pump transport and oxidative stress.