Characterize And Signaling Pathway Of Diabetic Keratopathy In The BKS Db/Db Mouse Model Of Type Ⅱ Diabetes Mellitus

PURPOSE. This study seeks to characterize diabetic keratopathy in the BKS db/db mouse model of type Ⅱ diabetes mellitus (DM) and to examined changes in global gene expression to define pathways regulated by diabetes in cornea.METHODS. Corneal morphology and histology of24-week-old BKS db/db and db/+mouse cornea were evaluated with slit lamp, corneal confocal microscopy and hematoxylin and eosin staining. corneal sensitivity was determined with an esthesiometer. Protein expression and distribution were assessed with Western blotting and immunohistochemistry. Wound healing was determined using an in vivo corneal epithelial debridement model. Total RNA were extracted for microarray assay and(or) qRT-PCR. Microarray data for24-week-old BKS db/db and db/+mouse cornea were analyzed to define significantly differentially expressed genes (DEGs) by GENESPRING12.0, DEGs were further analyzed to identify regulated biological processes and pathways with DAVID(the Database for Annotation, Visualization, and Integrated Discovery) and Pathway database.RESULTS. Compared with the BKS db/+mouse, the BKS db/db mouse are significantly higher levels of fasting blood glucose and GHb than the BKS db/+mouse at24weeks of age. BKS db/db mouse showed stronger Rose Bengal staining, and fuorescein staining, slightly attenuated sensitivity, less innervation, delayed epithelial wound healing, increased peroxynitrite and MIP2, and decreased SP.2,423differentially expressed genes(DEG) were identified by GENESPRING12.0, out of which1125genes were upregulated and1298genes were downregulated. Annotation using DAVID of the DEGs revealed that the significant enrichment GO_BP,(GO biological process) were "Wnt receptor signaling pathway" and "phosphate metabolic process" for the upregulated genes, and "response to DNA damage stimulus","DNA repair","DNA recombination","DNA metabolic process","cell cycle","regulation of cell proliferation","cell division" and "stem cell division" for the downregulated genes, that the significant enrichment GO_CC(GO cellular component) were "neuron projection" for the upregulated genes, and "chromosome" and "Mitochondrion" for the downregulated genes, and that the significant enrichment pathway were "Wnt signaling pathway" for the upregulated genes, and "DNA replication" and "Homologous recombination" for the downregulated genes.CONCLUSIONS.24-week-old type Ⅱ diabetes BKS-db/db mouse maintains to metabolic syndrome of diabetes. Pathogenesis of keratopathy involves multiple tissues and several events including corneal epithelial structure and function abnormalities, impaired innervation, and altered wound responses in type II diabetes BKS-db/db mouse.24-week-old type Ⅱ diabetes BKS-db/db mouse can be used as an animal model to to study diabetic keratopathy, because corneal complications of type Ⅱ diabetes BKS-db/db mouse is consistent with diabetic keratopathy of type Ⅱ diabetes mellitus. Dates of microarray shows mitochondrial dysfunction, abnormal metabolic of peroxynitrite, activation of wnt signaling pathway, decreased ability to repair DNA damage, and cell proliferation. This results which are form dates of microarray can give a reasonable explanation based on molecular level. This study could predict Wnt signaling pathway and peroxynitrite involved in diabetic keratopathy, and further study of Wnt signaling pathway and peroxynitrite will supplement current understanding about the pathogenesis of diabetic keratopathy.