Dynamic determination of Ox-LDL-induced oxidative/nitrosative stress in single macrophage by using fluorescent probes

Increased oxidative/nitrosative stress, resulting from generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) appears to play an important role in the inflammatory responses to atherosclerosis. By using MitoTracker Orange CM-H2TMRos, CM-H2DCFDA (DCF-DA), Dihydrorhodamine 123 (DHR123), DAF-FM, Dihydroethidium (DHE) and JC-1 alone or in all combinations of red and green probes, the present study was designed to monitor the ROS and RNS generation in acute exposure of single monocyte U937-derived macrophage to oxidized low density lipoprotein (Ox-LDL). Acute Ox-LDL (100 μg/ml) treatment increased time-dependently production of intracellular nitric oxide (NO), superoxide (O2−), hydrogen peroxide (H2O2) and peroxynitrite (ONOO−), and decreased mitochondrial membrane potential (Δψ) in single cell. Pretreatment of aminoguanidine (an inhibitor of inducible nitric oxide synthase (iNOS), 10 μM) and vitamin C (an antioxidant agent, 100 μM) for 2 h, reduced significantly the Ox-LDL-induced increase of NO and O2−, and vitamin C completely inhibited increase of intracellular NO and O2−. In contrast to aminoguanidine, Vitamin C pretreatment significantly prevented Ox-LDL-induced overproduction of NO and O2− (P < 0.01), indicating that antioxidant may be more effective in therapeutic application than iNOS inhibitor in dysfunction of ROS/RNS. By demonstrating a complex imbalance of ROS/RNS via fluorescent probes in acute exposure of single cell to Ox-LDL, oxidative/nitrosative stress might be more detected in the early atherosclerotic lesions.