Abstract
Purpose:
To investigate the effects of Advanced Glycation End Products (AGE) on apoptosis in human corneal epithelial cells and the underlying mechanisms in vitro.
Methods:
Telomerase-immortalized human corneal epithelial cells (THCEs) were treated with 0-400 μg/ml AGE-modified bovine serum albumin (BSA) for various times. THCEs apoptosis was analyzed by annexin-V-FITC and propidium iodide (PI) stain. The production of reactive oxygen species (ROS) was measured with 2', 7'- dichlorofluorescein diacetate (DCFH-DA) dye assay and imaged on laser scanning confocal fluorescence microscope. NADPH oxidase activity was examined by lucigenin-enhanced chemiluminescence assay. The protein levels of cytochrome c, caspase-3, Bax, Bcl-2, p47phox, p67phox, phosphorylated/total proteins of ERK, JNK, and p38 MAPK were determined by Western blot.
Results:
Treatment of THCEs with AGE-BSA resulted in activation of the caspase 3, increase of Bax synthesis and the release of cytochrome c, whereas, the expression of anti-apoptosis factor Bcl-2 were decreased markedly. AGE-BSA induced THCEs apoptosis was accompanied by production of ROS. AGE-BSA-induced apoptosis was inhibited by pretreatment with ROS quencher N-acetylcysteine (NAC). AGE-BSA activates NADPH oxidase through translocation of p47phox and p67phox, cytosolic subunits of NADPH oxidase, to the cell membrane. Furthermore, inhibition of NADPH oxidase by its specific inhibitors (DPI or apocynin) significantly prevented AGE-BSA induced ROS production and subsequent apoptosis. We also found that AGE-BSA stimulation activated JNK and p38 MAPK. JNK inhibitor SP600125 and p38 inhibitor SB203580 effectively blocked AGE-BSA-induced apoptosis. In addition, inhibition of ROS generation with NAC completely blocked phosphorylation of JNK and p38 MAPK induced by AGE-BSA.
Conclusions:
AGE-BSA induced THCEs apoptosis via ROS generation and JNK/p38 MAPK pathway activation, providing a new mechanism for AGE-induced cell apoptosis in human corneal epithelial cells. The results suggest that regulation of ROS production and JNK/p38 MAPK pathway may be a new strategy for prevention of AGE-induced corneal epithelial diseases.
Keywords: 426 apoptosis/cell death •
482 cornea: epithelium •
498 diabetes