Abstract
Purpose :
To explore novel role and molecular mechanism of a natural osmoprotectant ectoine in protecting corneal barrier function through promoting anti-inflammatory cytokine IL-37 in human corneal epithelial cells (HCECs) under hyperosmolarity stress, an in vitro dry eye model.
Methods :
Primary HCECs were established from donor limbal tissue. The confluent cultures in iso-osmolar medium were switched to hyperosmotic media (400-500 mOsM), without or with prior incubation of ectoine at different concentrations (1-40 mM) for 2-48 hours. Cell viability and proliferation were evaluated by WST assay. The integrity of apical barrier junction proteins and the expression of cytokines and cathepsin S were evaluated by RT-qPCR, ELISA, immunostaining and confocal microscopy.
Results :
HCECs were survived well in 450mOsM but partially damaged in 500mOsM media. Ectoine well protected HCEC survival and proliferation in 450 and 500mOsM media. The integrity of corneal epithelial barrier was largely disrupted in HCECs exposed to 450 mOsM, as shown by 3D confocal images of immunofluorescent staining of tight junction proteins ZO-1 and occludin. Ectoine at 10-20mM well protected the barrier proteins under hyperosmotic stress. The expression of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were dramatically stimulated by hyperosmolarity but significantly suppressed by Ectoine at 5-40 mM as quantified by RT-qPCR and ELISA. The protease cathepsin S, which was stimulated by hyperosmolarity and TNF-α, directly disrupted epithelial barrier while its inhibitor LY3000328 restored the barrier integrity. Interestingly, IL-37 decreased significantly in HCECs by hyperosmolarity, but prior-incubation of ectoine increased IL-37 at mRNA and protein levels, suppressed TNF-α and cathepsin S activity, and protected cells from barrier disruption under hyperosmolarity. Furthermore, rhIL-37 was observed to suppress pro-inflammatory cytokines and TNF-α-induced cathepsin S in HCECs exposed to hyperosmolarity.
Conclusions :
Our findings demonstrate that the hyperosmotic stress disrupts corneal epithelial barrier through stimulating pro-inflammatory cytokines and cathepsin S while suppressing IL-37. Ectoine inhibited the pathological course to protect the corneal epithelium. It provides new insight into pathogenesis and therapeutic potential for dry eye disease.
This is a 2021 ARVO Annual Meeting abstract.