Abstract
Purpose :
Hyperosmolar-induced ocular surface cell death is a mitochondria-mediated event in inflammatory eye diseases such as dry eye. Hyperosmolarity is a key element of dry eye disease pathophysiology. It has been reported that hyperosmolarity induces apoptosis of human corneal epithelial cells through a cytochrome c-mediated death pathway, which may be dependent upon, or modulated by, JNK and ERK MAPK signaling pathways. Histatin peptides are well established antimicrobial and wound healing agents and have minimal to no immunogenicity or side effects. The mechanisms of action of histatin peptides are poorly understood. We studied the role of Histain 5 (H5) and its involvement in hyperosmolarity-stimulated apoptotic cell death in human corneal epithelial (HCE) cells.
Methods :
HCE cells were stimulated in hyperosmolar media (450 mOsM) by adding 100 mM NaCl, with or without H5 (20 µM). For measuring apoptotic activity levels, we checked cleavage of caspase-3 and PARP-1 with a Western blot assay. For quantitative measurements, caspase-3 activity was assayed with the Caspase-Glo reagent (Promega, Madison, WI) in HCE cells stimulated with Hosm, and treated with H5. Additionally, the activity of p38, JNK and ERK were analyzed using phospho-specific antibodies.
Results :
H5 inhibited the apoptosis stimulated by hyperosmolar solution (450 mOsM) in HCE cells. Cleavage of active caspase, caspase-3 and PARP-1 was observed in hyperosmolar solution in HCE cells, the treatment of H5 inhibited their activation at 8 and 24 hrs. Quantification of caspase-3 activity revealed significant inhibition of caspase-3 after co- treatment with H5 compared to treatment with hyperosmolar solution alone at 16 hr. in HCE cells. Phosphorylation of p38 and JNK1/2 MAPKs was inhibited at 30 min after treatment with H5; however, Pro-survival AKT and MAPK-ERK1/2 proteins were activated in HCE cells.
Conclusions :
H5 can reduce apoptosis induced by hyperosmolar solution in HCE cells. Activation of survival pathways and reduction of activity of apoptotic pathways was noted with H5 exposure. Taken together these results suggest that H5 may have utility in reducing corneal epithelial cell apoptosis and enhancing survival in hyperosmolar solutions.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.