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The aims of the present study were to investigate the effect of cadmium (Cd)-induced apoptosis and determine the protective effect of N-acetylcysteine (NAC) in BRL 3A cells. The BRL 3A cells were treated with 0, 10, 20 or 40 μmol/l cadmium acetate (CdAc2) for 12 h. Another two groups of cells were preincubated with 2 mmol/l NAC for 30 min, and then either incubated with 20 μmol/l CdAc2 for 12 h, or treated with NAC alone. The levels of apoptosis and mitochondrial membrane potential (ΔΨm) were measured using flow cytometry. Mitochondrial ultrastructural changes were detected using transmission electron microscopy. The protein levels of caspase-3, caspase-9, poly (ADP-ribose) polymerase (PARP), caspase-8, and Fas ligand (FasL) protein were measured using immunoblotting. As the dose of Cd increased, there was a significant increase in the apoptotic ratio, a significant decrease in ΔΨm, mitochondrial swelling and degeneration, and blurring, deformation and eventual collapse of the mitochondrial cristae. The protein levels of caspase-3, caspase-9 and PARP decreased, whereas the levels of cleaved caspase-3, cleaved caspase-9, cleaved caspase-8 and FasL increased dose-dependently in relation to Cd. NAC effectively inhibited these changes. Cd induced apoptosis through the mitochondrial and FasL pathways in the BRL 3A cells, and NAC exerted a protective effect against Cd-induced damage. [ABSTRACT FROM AUTHOR]

The present study aimed to determine whether the mitogen-activated protein kinase (MAPK) signaling pathway is involved in the osteoprotegerin (OPG)-mediated inhibition of osteoclast differentiation and maturation. RAW264.7 cells were incubated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) to stimulate osteoclastogenesis and treated with various concentrations of OPG, an inhibitor of osteoclast differentiation. The differentiation and activation of osteoclasts were monitored by tartrate-resistant acid phosphatase staining and bone resorption assays. The phosphorylation levels of p38-MAPK, c-Jun N-terminal kinase (JNK)-MAPK and extracellular signal-regulated kinase (ERK)-MAPK in the different treatment groups were determined by western blot analysis. The results confirmed that M-CSF + RANKL stimulated the differentiation and activation of osteoclasts as well as the phosphorylation of p38-MAPK, JNK-MAPK and ERK-MAPK in osteoclasts, which was attenuated by OPG treatment. These findings indicated that the MAPK signaling pathway is involved in the regulation of osteoclastogenesis and in the OPG-mediated inhibition of osteoclast differentiation and activation. [ABSTRACT FROM AUTHOR]

Macrophages, a type of immune cell, are the precursors of osteoclasts, and have important roles in bone remodeling and the immune system. In the present study, the RAW264.7 cell line was used as a macrophage model in order to study the macrophage changes during osteoclastogenesis. Receptor activator of nuclear factor ?B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) induce the formation of osteoclasts from several precursor cells. Observation of RAW264.7 macrophage osteoclastogenesis under the induction of RANKL and M-CSF revealed that except the few RAW264.7 macrophages that were differentiated into osteoclasts, almost all undifferentiated RAW264.7 macrophages underwent apoptosis. BRL-3A cells have no differentiation ability, and RANKL and M-CSF treatments did not induce BRL-3A cell apoptosis. When osteoprotegerin (OPG) was used to completely inhibit the differentiation of RAW264.7 macrophages to osteoclasts, apoptosis did not occur amongst the RAW264.7 macrophages despite the action of RANKL and M-CSF. Rac1, RhoA and RhoV are apoptosis-associated genes in the Rho guanosine triphosphate (GTP)ase family. Their expression levels were detected using quantitative polymerase chain reaction (qPCR). During the process of osteoclast differentiation, the mRNA expression of RhoV was significantly upregulated, while apoptosis occurred in a large proportion of macrophages. However, when macrophage apoptosis was inhibited by OPG, RhoV expression was significantly downregulated. Conversely, Rac1 and RhoA expression did not vary in correspondence with the apoptotic rate of the RAW264.7 macrophages. In conclusion, differentiation of RAW264.7 macrophages into osteoclasts resulted in their apoptosis. OPG inhibited RAW264.7 macrophage differentiation into osteoclasts, and thereby inhibited the apoptosis of RAW264.7 macrophages. RhoV mediated the apoptosis of RAW264.7 macrophages during osteoclast differentiation. [ABSTRACT FROM AUTHOR]