Ceramide-Induced Apoptosis in Cultured Keratocyte

Purpose: We investigated the effect of ceramide on keratocyte apoptosis and pathway of ceramide-induced keratocyte apoptosis. Methods: Cultured Newzealand White Rabbit keratocytes were exposed to various concentrations of ceramide type II, VI and phytoceramide type II, VI. LDH level was measured for the evaluation of time and concentration related apoptosis. Keratocytes were preincubated in various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK, diffuse caspase inhibitor), specific caspase-8 inhibitor (IETD-CHO) and specific caspase-9 inhibitor (Z-LEHD-FMK), then were exposed to 20 M of 4 types of ceramide. Cytochome C immune stainining was done after exposure of keratocyte to 4 types of ceramide. Results: The lower effective dose of 4 types of ceramide was 20 M. Apoptosis of keratocytes was dependent on ceramide exposure time. Ceramide induced keratocyte apoptosis was inhibited by CPP32-like protease inhibitor, specific caspase-8 inhibitor and specific caspase-9 inhibitor. Apoptotic keratocytes induced by ceramide were immune-stained with cytochrome C antibody. Conclusions: Ceramide induced apoptosis in cultured corneal keratocytes. This apoptosis developed according caspase cascade, especially via mitochondria.