Abstract:

Objective To investigate the oxidative mechanism of homocysteine (Hcy)-induced apoptosis in endothelial progenitor cells (EPCs). Methods Total mononuclear cells were isolated from mouse bone marrow by Ficoll density gradient centrifugation and were cultured in vitro for 7 d. Adherent cells were harvested and identified by fluorescence microscopy. EPCs were cultured with Hcy (0, 50, 100 and 500 μmol/L) for 12, 24 and 48 h, or pretreated with NAC(1 mmol/L), DPI(10 μmol/L) or SB203580 (10 μmol/L) for 30 min, then cultured with 500 μmol/L Hcy for 24 h. Apoptosis was detected by Annexin-V/PI flow cytometry, levels of reactive oxygen species (ROS) in cells were measured using H₂DCF-DA as a fluorescence probe, NADPH oxidases were evaluated with lucigenin-enhanced chemiluminescence, and NO in the supernatant was determined by nitrate reductase assay. Results Hcy induced EPCs apoptosis, ROS accumulation, NADPH oxidase activation and decrease of NO in a time-dose dependent manner（P<0.05 or P<0.01）. Pretreatment with NAC, DPI and SB203580 could inhibit these effects （P<0.05 or P<0.01）. Conclusion Hcy could activate NADPH oxidase, induce ROS increase and NO decrease, and activate p38MAPK to enhance EPCs apoptosis.

Key words: homocysteine, endothelial progenitor cells, apoptosis, reactive oxygen species, NADPH oxidase, p38MAPK