Protective effects of Slit2 on corneal epithelium and nerves in diabetic mice and its mechanism

Objective

To explore the promoting effects of slit guidance ligand 2 (Slit2) on the repair of corneal epithelium and nerve damage in diabetic mice and possible molecular mechanism.

Methods

Sixty SPF C57BL/6 mice aged 5-6 weeks were divided into normal control group, diabetes model group and Slit2 injection group according to the random number table method, 20 for each group.Diabectic model was prepared by intraperitoneal injection of streptozotocin in the diabetes model group and Slit2 injection group.A mouse corneal epithelial injury repair model was established using electric epithelial scraper, and Slit2 recombinant protein was subconjunctivally injected immediately following modeling in the Slit2 injection group.The equal volume of phosphate buffer saline (PBS) was used in a same way in the diabetes model group.No intervention was performed in the normal control group.Corneal epithelial healing were examined at 24, 48 and 72 hours after corneal epithelial defect by corneal fluorescin staining.Real-time fluorescent quantitative PCR was used to detect the expression of Slit2 and its related receptors in the corneal epithelium of normal and diabetic model mice.Fluorescence staining of corneal wholemount with β-tubulin Ⅲ was used to observe the changes in corneal nerve morphology.Immunofluorescence staining was performed to detect the expression and distribution of Slit2 in mouse corneal epithelium in normal control group and diabetes model group, as well as the expression and distribution of Slit2, epidermal growth factor receptor (EGFR), extracellular-signal-regulated kinase (ERK), threonine protein kinase (AKT), β-catenin and Ki67 in the healing corneal epithelium of mice after corneal epithelium damage in different groups.The mouse corneal epithelial stem/progenitor cell line (TKE2) was divided into normal control group, high-glucose group and Slit2 treatment group.Western blot was performed to detect the expression of p-EGFR/EGFR and p-AKT/AKT in the TKE2 of the three groups.The expression of p-EGFR/EGFR and p-AKT/AKT in high glucose-cultured TKE2 with 0.01, 0.1 and 0.5 μg/ml Slit2 treatment for 10 minutes, and before and 10, 20, 30, 60, 120 minutes after 0.5 μg/ml Slit2 treatment was detected by Western blot.The effects of Slit2 on the axon regeneration of mouse trigeminal ganglion cells (TGs) were observed by immunofluorescence staining.The use and care of animals complied with the ARVO statement.This study protocol was approved by an Ethics Committee of Qingdao Eye Hospital of Shandong First Medical University (No.[2020]57).

Results

At 48 and 72 hours after corneal epithelial scraping, the speed of corneal epithelial repair was significantly slowed down in diabetes model group in comparison with the normal control group and Slit2 injection group.The relative expression levels of Slit2 and its receptors Robo1, Robo2 and Robo4 mRNA in the normal corneal epithelium in the diabetes model group were significantly higher than those of the normal control group (all at P<0.05). The fluorescence intensity of Slit2 in normal corneal epithelium in diabetes model group was similar to the normal control group, and the fluorescence intensity of Slit2 in damaged corneal epithelium in diabetic mice was significantly weaker than that in normal control group.Corneal nerve plexus was denser at 7 days after corneal epithelial injury and the nerve fibers were increased with more branches in Slit2 injection group compared with diabetic group.The fluorescence intensity of p-EGFR, p-ERK, β-catenin and Ki67 in damaged corneal epithelium in normal control group and Slit2 injection group was stronger than that of the diabetes model group.The relative expression levels of p-EGFR/EGFR, p-AKT/AKT, and β-catenin in TKE2 in high-glucose group were significantly lower than those in normal control group and Slit2 treatment group (all at P<0.05). The relative expression levels of p-EGFR/EGFR and p-AKT/AKT in high glucose-cultured TKE2 after Slit2 treatment were significantly increased in comparison with before Slit2 treatment (both at P<0.05), and the relative expression levels of p-EGFR/EGFR and p-AKT/AKT in TKE2 were elevated as the increase of Slit2 concentration.The activation effect of 0.5 μg/ml Slit2 on EGFR and AKT pathways was most obvious.The synapse length of TGs cultured by high glucose was (40.52±5.44) μm, which was significantly shortened than (72.14±9.48) μm in normal control group and (73.04±4.66) μm in Slit2 injection group (both at P<0.05).

Conclusions

Slit2 can protect the corneal epithelium by activating EGFR signaling pathway and play a protective role to neurons by increasing the density of corneal subepithelial plexus and promoting the growth of TGs axons in diabetic mice.

Axon guidance factor; Corneal epithelial cells; Trigeminal ganglia; Diabetic corneal disease