The Role And Molecular Mechanism Of Ciliary Neurotrophic Factor CNTF And Axon Guiding Factor Slit2 In Diabetic Corneal Lesions

Objective China has become the country with the largest number of patients with diabetes mellitus. Approximately half of diabetic patients develop primary diabetic keratopathy, mainly characterized by corneal epithelial regeneration delay and neurotrophic disorders.We detected by gene chip, the expression of ciliary neurotrophic factor CNTF and axon guidance factors Slit2 in diabetic mice cornea significantly down-regulated.In this study, we explore the effect and the mechanism of CNTF and Slit2 in diabetic mice corneal epithelium and nerve repair therapeutic through in vivo and in vitro.Methods C57 BL / 6 mice more than 8 weeks old were selected, diabetes mellitus were induced by continuous intraperitoneal injection of streptozotocin(STZ). Animal models of 3mm corneal epithelial scrape were made,CNTF and Slit2 were injected under the conjunctiva, and observe the effects of CNTF and Slit2 in corneal epithelium and nerve damage repair. To take the mouse corneal epithelial stem / progenitor cell line(TKE2) as an in vitro model, observe the efferts of CNTF in corneal epithelial stem cell activity and activation of signaling pathways of epithelial regeneration. High glucose induced cell model was established by using mouse stem / progenitor cells(TKE2). Signaling pathways activated by Slit2 on corneal epithelial cells were detected by western blot. The effect of Slit2 on primary cultured trigeminal ganglion cell axons growth was measured by calculating neurite length.Results Through the establishment of murine corneal epithelial wound healing model, both Slit2 and CNTF can significantly promote normal / diabetic corneal epithelium and corneal nerve injury repairing.TKE2 cells under high glucose environment, exogenously added CNTF can significantly promote corneal epithelial stem cell activation.By immunofluorescence and western blot detected CNTF activates the regeneration of corneal epithelial cells and TKE2 Akt and STAT3 signaling pathway, blocking CNTF-STAT3 signaling pathway can lead to a significant delay in the repair of corneal epithelium, corneal epithelial stem cell activation decreased ability. Slit2 could activate EGFR, ERK, and β-catenin in regenerated diabetic corneal epithelium, and significantly enhanced Ki67 expression. Slit2 could significantly promote the trigeminal ganglion cell axon growth.Conclusion 1. Subconjunctival injection of CNTF could significantly promote corneal epithelial wound healing and accelerate corneal nerve regeneration. Under high glucose environment, exogenous CNTF can significantly promote corneal epithelial stem cell activation.CNTF activates Akt and STAT3 signaling pathway in corneal epithelium and corneal epithelial stem cells, blocking the CNTF-STAT3 signaling pathway, which can lead to significantly delayed epithelial repair and corneal epithelial stem cell activation. 2. Subconjunctival injection of Slit2 could significantly promote corneal epithelial wound healing and accelerate corneal nerve regeneration. Slit2 could activate EGFR, ERK, and β-catenin in regenerated diabetic corneal epithelium, and significantly enhanced Ki67 expression. Slit2 could significantly promote the trigeminal ganglion cell axon growth.