Abstract
Genistein, a phytoestrogen, has been demonstrated to have a bone-sparing and antiresorptive effect. Genistein can inhibit the osteoclast formation of receptor activator of nuclear factor-κB ligand (RANKL)-induced RAW 264.7 cells by preventing the translocation of nuclear factor-κB (NF-κB), a redox-sensitive factor, to the nucleus. Therefore, the suppressive effect of genistein on the reactive oxygen species (ROS) level during osteoclast differentiation and the mechanism associated with the control of ROS levels by genistein were investigated. The cellular antioxidant capacity and inhibitory effect of genistein were confirmed. The translation and activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox1), as well as the disruption of the mitochondrial electron transport chain system were obviously suppressed by genistein in a dose-dependent manner. The induction of phase II antioxidant enzymes, such as superoxide dismutase 1 (SOD1) and heme oxygenase-1 (HO-1), was enhanced by genistein. In addition, the translational induction of nuclear factor erythroid 2-related factor 2 (Nrf2) was notably increased by genistein. These results provide that the inhibitory effects of genistein on RANKL-stimulated osteoclast differentiation is likely to be attributed to the control of ROS generation through suppressing the translation and activation of Nox1 and the disruption of the mitochondrial electron transport chain system, as well as ROS scavenging through the Nrf2-mediated induction of phase II antioxidant enzymes, such as SOD1 and HO-1.
Highlights
The bone remodeling process consists of the resorption of mineralized bone by osteoclasts and bone formation by osteoblasts [1]
The cells were treated with 2',7'-dichlorodihydrofluorescein-diacetate (DCFH-DA), which is a fluorescent probe that detects reactive oxygen species (ROS), for 30 min to measure the intracellular oxidative stress induced by azobis(2-amidinopropane) dihydrochloride (AAPH) or H2O2
Catalase expression significantly (p < 0.05) reduced by receptor activator of nuclear factor kappa-B ligand (RANKL) treatment was not significantly (p < 0.05) changed by genistein at 1–20 μm when compared to the treated control. These results indicate that genistein enhances superoxide dismutase 1 (SOD1) and heme oxygenase-1 (HO-1) expression to result in the suppression of ROS and superoxide anion production in RANKL-treated RAW 264.7 cells
Summary
The bone remodeling process consists of the resorption of mineralized bone by osteoclasts and bone formation by osteoblasts [1]. According to previous in vitro studies, genistein stimulates osteoblastic differentiation and mineralization and inhibits osteoclast formation from pre-osteoclast cells and the bone resorption activity of osteoclasts [18,19]. During osteoclast differentiation from pre-osteoclast RAW 264.7 cells activated by RANKL, genistein can inhibit RANKL-induced inhibitor-κB (I-κB) degradation and nuclear factor-κB (NF-κB) translocation to the nucleus, resulting in the prevention of osteoclast formation [20]. This inhibitory effect of genistein on osteoclast differentiation may be associated with its potent antioxidant activity, because the NF-κB/I-κB signaling pathway is known to be redox sensitive [21]. In this study, the suppressive effect of genistein on the ROS level during osteoclast differentiation of RANKL-induced RAW 264.7 cells was investigated after confirming its cellular antioxidant capacity in HepG2 cells, and it was determined how the ROS level can be controlled in view of its production and scavenging by genistein
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