AQP5 Promotes/Facilitates Corneal Epithelial Wound Healing And Nerve Regeneration By Reactivating Akt Signaling Pathway

Purpose:Aquaporins（AQPs）,ordinarily regarded as a transmembrane protein,plays an important role in maintaining cell water homeostasis.In recent years,it has been confirmed that AQPs participate in other cellular functions such as cell-to-cell adhesion,cell migration,cell proliferation etc.The current investigation was undertaken to find out whether AQP5water channel plays a role in corneal epithelial wound healing.Methods:AQP5 knockout(AQP5^-/-)mice were constructed by using CRISPR/Cas9technology.An in vivo corneal wound healing model was performed using epithelial debridement on corneas of wild-type(AQP5^+/+)and AQP5^-/-mice.All the experimental mice were 8-10 weeks old.The corneas were stained using fluorescein sodium,β-tubulin III,and Ki-67,as markers of corneal epithelial defect,corneal nerve,and corneal epithelial cell proliferation,respectively.The reactivation of epithelial nerve regeneration-related signaling,including phosphorylated Phospho-Akt（p-Akt）and nerve growth factor（NGF）were assessed with western blot and immunofluorescence staining.The corneal epithelial and nerve regeneration rate was evaluated in AQP5^-/-mice after the treatment with NGF,or Akt inhibitor（Akti）.Results:（1）AQP5 was widely expressed in the cell membrane of AQP5^+/+mouse corneal epithelial cells,but hardly expressed in AQP5^-/-mouse corneal epithelial cells.Time to corneal epithelial and nerve regeneration in vivo was significantly delayed in AQP5^-/-mice compared to AQP5^+/+mice.（2）The nerve staining and sensitivity measurement of the cornea showed that the nerve fiber density and corneal sensitivity of AQP5^-/-mice decreased significantly compared with AQP5^+/+mice.（3）Compared with AQP5^+/+mice,the number of Ki-67 positive cells and the expression levels of NGF and p-Akt in the corneal epithelium of AQP5^-/-mice were significantly decreased,no matter whether the mice were treated with corneal epithelial scraping or not.These results suggest that the proliferation ability and nerve regeneration ability of corneal epithelial cells in AQP5^-/-mice may be defective after AQP5 gene knockout.It may affect the activation of Akt to inhibit the repair speed of corneal epithelial injury and nerve regeneration.（4）Upon central epithelial debridement,the epithelial and nerve regeneration rate was significantly promoted in AQP5^-/-mice with the treatment of NGF than that of PBS control,which accompanied with the recovered levels of p-Akt.（5）NGF treatment also improved the recovery of corneal nerve fiber density and sensitivity that was impaired in AQP5^-/-mice.（6）The promotion of NGF induced corneal epithelial and nerve regeneration rate and Akt reactivation was completed reversed by Akt inhibitor.Conclusion:The significant impairment in corneal wound healing in AQP5^-/-mice results from distinct defects in corneal epithelial cell proliferation and nerve regeneration.It may affect the activation of Akt signaling pathway and NGF expression,and then act on the process of corneal injury repair and nerve regeneration.The results provide evidence for involvement of an aquaporin in cell proliferation and suggest AQP5 induction as a possible therapy to accelerate the resurfacing of corneal defects.