Purpose: To investigate the role of elastase in corneal epithelial barrier dysfunction caused by the exoproteins secreted by Pseudomonas aeruginosa.
Methods: Exoproteins obtained from Pseudomonas aeruginosa culture supernatant were analyzed by shotgun proteomics approach. In vitro multilayered rabbit corneal epithelial barrier model prepared by air-liquid interface technique (CECs-ALI) were treated with 2 µg/ml exoproteins and/or 8 mM elastase inhibitor. Then the epithelial barrier function was evaluated by transepithelial electrical resistance (TEER) assay and tight junction proteins immunofluorescence. Cell viability and the apoptosis rate were examined by CCK8 assay and flow cytometry. TNF-α, IL-6, IL-8, and IL-1β levels were measured by ELISA. Mice cornea treated with exoproteins and/or elastase inhibitor were evaluated in vivo and in vitro.
Results: Elastase (24.2%) is one of the major components of exoproteins. After 2 µg/ml exoproteins were applied to CECs-ALI for two hours, TEER decreased from 323.2 ± 2.7 to 104 ± 6.8 Ω/cm2 (P < 0.001). The immunofluorescence results showed a distinct separation in tight junction and significant degradation of ZO-1 and occludin (P < 0.05). Elastase inhibitor (8 mM) alleviated the decrease in TEER value (234 ± 6.8 Ω cm2) induced by exoproteins. Inhibition of elastase decreased the apoptosis rate of CECs treated with exoproteins from 30.2 ± 3.8% to 7.26 ± 1.3% and the levels of inflammatory factors (P < 0.05). Mice corneal epithelium defect could be induced by exoproteins and protected by elastase inhibitor.
Conclusions: Elastase plays a critical role in corneal epithelial barrier dysfunction caused by Pseudomonas aeruginosa exoproteins via damaging tight junctions. The inhibition of elastase could protect the corneal epithelial barrier via reducing virulence and inflammation.