Study On IGF-1-mediated β-catenin Signaling Involving In High Glucose-impaired Corneal Epithelial Cells Wound Healing

Part I:Role of IGF-1in high-glucose impaired corneal epithelial cells wound healingPurposeTo investigate the expression and role of insulin-like growth factor-1(IGF-1) in high glucose-impaired corneal epithelial cells wound healing.Methods1. THCE cells were treated with glucose solutions (5mM-45mM) for24h or48h and cell viability was measured by MTT to determine the best concentration of glucose and time. THCE cells were treated with IGF-1(10-200ng/ml) for48h and cell viability was measured by MTT to determine the best concentration of IGF-1.2. Respectively, with a concentration of5mM or25mM D-glucose treatment THCE cells for48h, then cells were growth factors starved for12h. After wounding, cells were harvested and real-time PCR, ELISA, western blot and immunofluorescence staining were performed to detect IGF-1and IGF-1R mRNA and protein expression.3. In wounded corneal epithelium of streptozotocin induced diabetic rats, IGF-1and IGF-1R mRNA and protein expression were measured by real-time PCR and immunofluorescence.4. THCE cells were pretreated with25mM D-glucose. For experiments,50ng/ml IGF-1was added into high glucose medium. Then, three groups of cells were formed: cells grown in normal glucose medium, high glucose medium, or high glucose medium plus50ng/ml IGF-1. The effects of IGF-1in the presence of high glucose on cell proliferation, migration and apoptosis were evaluated by MTT, cell restitution assay and flow cytometry after PI-Annexin-V staining. 5. The possible molecular mechanisms were detected by the levels of ki-67, MMP-2, bax, and bcl-2expression using real-time PCR, immunohistochemistry or western blot.Results1. For experiments, the proper concentration was25mM glucose or50ng/ml IGF-1.2. After wounding, both IGF-1and IGF-1R mRNA and protein expression were increased. At12and24h, the expressions were significantly lower in high glucose group than that in normal group. As for IGF-1and IGF-1R protein, the expressions were significantly lower in high glucose group than that in normal group at24and48h.3. Delayed wound healing was observed in wounded corneal epithelium of diabetic rats which were accompanied with reduced IGF-1and IGF-1R mRNA and protein expression.4. High glucose reduced THCE cells proliferation and migration, induced cell apoptosis via downregulating ki-67, MMP-2, bcl-2mRNA and protein expression, upregulating bax expression. Treatment with IGF-1could reverse these high glucose effects.ConclusionsHigh glucose-inhibited IGF-1/IGF-1R expression caused THCE cell dysregulation including decreased cell proliferation, migration and increased cell apoptosis, which might result, at least in part, in delayed corneal epithelial cell wound healing. Part Ⅱ:Role of β-catenin Signaling in high-glucose impaired corneal epithelial cells wound healingPurposeTo investigate the expression and role of P-catenin signaling in high glucose-impaired corneal epithelial cells wound healing.Methods 1. THCE cells were pretreated with25mM D-glucose and growth factors starved. After wounding, real-time PCR and western blot were performed to detect J3-catenin> cyclin D1、c-Myc mRNA and protein expression.2. THCE cells were pretreated with25mM D-glucose and growth factors starved. For experiments,0.5mM Licl was added into high glucose medium. Then, three groups of cells were formed:cells grown in normal glucose medium, high glucose medium, or high glucose medium plus0.5mM Licl. The effects of Licl in the presence of high glucose on cell proliferation, migration and apoptosis were evaluated by MTT, cell restitution assay and flow cytometry after PI-Annexin-V staining.3. The possible molecular mechanisms were detected by the levels of ki-67, MMP-2, bax, and bcl-2expression using real-time PCR, immunohistochemistry or western blot.ResultsHigh glucose downregulated β-catenin、cyclin D1、c-Myc mRNA and protein expression while Licl upregulated these factors expression in wounded THCE cells cultured in high glucose. Licl improved high glucose-impaired THCE cells proliferation, migration and apoptosis via upregulating ki-67, MMP-2,’bcl-2and downregulating bax mRNA and protein expression.ConclusionsHigh glucose decreased THCE cell proliferation, migration and increased cell apoptosis through inhibited β-catenin signaling. Part Ⅲ:IGF-1mediated β-catenin signaling in high glucose-impaired corneal epithelial cells wound healingPurposeTo investigate whether IGF-1mediated β-catenin signaling in high glucose-impaired corneal epithelial cell wound healing.Methods THCE cells were pretreated with25mM D-glucose and growth factors starved. After wounding, IGF-1was added into high glucose medium. Real-time PCR and western blot were performed to detect β-catenin, cyclin D1mRNA and protein expression.ResultsThe addition of IGF-1significantly increased β-catenin、cyclin D1mRNA and protein expression compaired with high glucose-treated control.ConclusionsIGF-1mediated β-catenin signaling to modulate high glucose-impaired corneal epithelial cell wound healing.