Chitosan promoted the corneal epithelial wound healing via activation of ERK MAPK Pathway

Abstract

 

Purpose

 

Chitosan has ability to promoting the viability of rabbits corneal epithelial cells(RCECs) are estimated by MTT assay. However, the molecular mechanism by which chitosan functions is unknown. We investigated the possible effects of chitosan on extracellular regulated kinase (ERK) and p38 pathways in rabbit corneal wound healing.

 

Methods

 

Establishing the corneal epithelium mechanical damaged model, followed by the respectively treatments with normal saline(group A), 0.5%chitosan(group B), 20μg/ml recombinant human epidermal growth factor (group C), Observing the corneal wound area by corneal fluorescein staining at timing series. RCECs were cultured in vitro, epithelial cell proliferation was evaluated with Ki67 antibody. To measure in vitro wound closure, confluent RCECs were wounded by a linear scraping with a sterile plastic tip in the center of the well. Cells were incubated for 24 hours with or without chitosan.Phosporylation of ERK and p38 were analyzed by western blot in the presence or absence of inhibitors. 8 mm epithelial debridement wounds were made in rabbits corneas in organ culture in the presence of chitosan with or without ERK pathway inhibitor.

 

Results

 

The corneal unhealing area of Group B was statistically significant smaller than group A (p<0.05) and no significant difference with group C (p>0.05) in 24hour, 48hour and 72hour. Immunostaining with Ki67 and migrating assay showed that chitosan could up-regulate the cell proliferation and promote the cell migration. Chitosan activated ERK in 5min to 30min but not p38 and PD98059 inhibited the chitosan-stimulated ERK phosphorylation. Chitosan increased corneal epithelial wound closure in organ culture and ERK inhibition with PD98059 blocked the effect of chitosan on wound healing.

 

Conclusions

 

Chitosan promoted corneal epithelial proliferation and migration during the wound healing in rabbits eyes. Chitosan-stimulated epithelial wound healing is partially mediated through the activation of ERK pathway but not p38 pathway.

 

 

Phosporylation of ERK and p38 were analyzed by western blot in the presence of chitosan. Chitosan activated ERK in 5min to 30min but not p38.

 

 

Epithelial debridement wounds were made in rabbits corneas in organ culture in the presence of chitosan with or without ERK pathway inhibitor.Chitosan increased corneal epithelial wound closure and ERK inhibition blocked the effect of chitosan on wound healing.

 

Keywords: 480 cornea: basic science • 482 cornea: epithelium