Harpagoside (HAR) is a natural compound isolated from Harpagophytum procumbens (devil's claw) that is reported to have anti-inflammatory effects; however, these effects have not been investigated in the context of bone development. The current study describes for the first time that HAR inhibits receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in vitro and suppresses inflammation-induced bone loss in a mouse model. HAR also inhibited the formation of osteoclasts from mouse bone marrow macrophages (BMMs) in a dose-dependent manner as well as the activity of mature osteoclasts, including filamentous actin (F-actin) ring formation and bone matrix breakdown. This involved a HAR-induced decrease in extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) phosphorylation, leading to the inhibition of Syk-Btk-PLCγ2-Ca(2+) in RANKL-dependent early signaling, as well as the activation of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), which resulted in the down-regulation of various target genes. Consistent with these in vitro results, HAR blocked lipopolysaccharide (LPS)-induced bone loss in an inflammatory osteoporosis model. However, HAR did not prevent ovariectomy-mediated bone erosion in a postmenopausal osteoporosis model. These results suggest that HAR is a valuable agent against inflammation-related bone disorders but not osteoporosis induced by hormonal abnormalities.

It is thought that calcium and magnesium may be related to metabolic disorders such as obesity and metabolic syndrome; however, to date, there have been few studies investigating the association between serum calcium and magnesium levels and metabolic syndrome in middle-aged male adults. We aimed to investigate the association between serum calcium and magnesium levels and metabolic syndrome in Korean middle-aged male adults. Study subjects included 213 men aged 30∼60 years. MetS risk score is determined by adding the number of risk factors, waist circumference, triacylglyceride (TG), HDL cholesterol, glucose, and blood pressure (BP). The study population was divided into three groups according to the MetS risk score: group I (MetS risk score ≤1; n = 106), group II (MetS risk score = 2; n = 51), and group III (MetS risk score ≥3; n = 56). The serum Ca, according to increase of MetS risk score, was significantly higher (p < 0.001), and there was no significant difference in serum Mg concentration among the three groups. Subjects with high TG and high BP had higher serum calcium levels than those without such abnormalities. Subjects with higher glucose had lower serum magnesium levels than those without such abnormality. The correlation analysis indicated that the serum Ca had positive correlations with the MetS risk score (r = 0.1769, p < 0.01), serum TG (r = 0.2516, p < 0.001), and DBP (r = 0.2246, p < 0.01). The correlation analysis indicated that the serum Mg had an inverse relationship with serum glucose (r = -0.2404, p < 0.001). In conclusion, serum Ca had positive association with TG and BP, while serum Mg had negative association with serum glucose after adjusting age and BMI among the middle-aged Korean male adults.

Lysyl oxidase (LOX) is an extracellular amine oxidase that mediates the formation of collagen fibers. Thus far, five LOX family genes [LOX, lysyl oxidase-like (LOXL)1, LOXL2, LOXL3 and LOXL4] have been identified in humans, each encoding the characteristic C-terminal domains that are required for amine oxidase activity. During osteoblastogenesis, collagen fibers function as a three-dimensional scaffold for organizing mineral deposition. In this study, to assess the functional roles of the LOX family members in osteoblastogenesis, we investigated the temporal expression of these genes as a function of phenotypic development during the osteoblast differentiation of primary cultured mouse calvaria cells. Of the LOX family members, only LOX was prominently expressed during osteoblast differentiation. LOX expression was highest on day 9 of differentiation, as shown by RT-PCR and western blot analysis. The expression pattern of collagen, type I, alpha 2 (COL1A2), which encodes the α2-chain of mouse collagen type I, was similar to that of LOX. The total amine oxidase activity of the differentiating calvaria cells exhibited a temporal pattern that paralleled LOX expression, reaching the highest level on day 9 of differentiation. We also noted that the inhibition of the amine oxidase activity of LOX significantly suppressed both mineral nodule formation and the expression of osteoblast marker genes during the differentiation of primary calvaria cells. Taken together, these findings suggest that the LOX-mediated organization of collagen fibers in the extracellular matrix is an important regulator of osteoblastogenesis.

Protocatechuic acid (PCA) plays a critical role in nutritional metabolism; it is a major metabolite of anthocyanins, which are flavonoids with a range of health benefits. PCA has a variety of biological activities including anti-oxidant, antiinflammatory, anti-apoptosis, and anti-microbial activities. However, the pharmacological effect of PCA, especially on osteoclastogenesis, remains unknown. We examined the effect of PCA on receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation and bone resorption. PCA dose-dependently inhibited RANKL-induced osteoclast differentiation in mouse bone marrow macrophages (BMMs) and suppressed the bone-resorbing activity of mature osteoclasts. At the molecular level, PCA suppressed RANKL-induced phosphorylation of JNK among MAPKs only, without significantly affecting the early signaling pathway. PCA also suppressed RANKL-stimulated expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1) at the mRNA and protein levels, without altering c-Fos mRNA expression. Additionally, PCA down-regulated the expression of downstream osteoclastogenesis-related genes including β3-integrin, DC-STAMP, OC-STAMP, Atp6v0d2, CTR, and CtsK. Mice treated with PCA efficiently recovered from lipopolysaccharide-induced bone loss in vivo. Thus, PCA inhibits RANKL-induced osteoclast differentiation and function by suppressing JNK signaling, c-Fos stability, and expression of osteoclastic marker genes. These results suggest that PCA could be useful in treatment of inflammatory bone disorders.

Auranofin, as a thioredoxin reductase (TrxR) inhibitor, has promising anti-cancer activity in several cancer types. However, little is known about the inhibitory effect of auranofin on lung cancer cell growth. We, therefore, investigated the antigrowth effects of auranofin in various lung cancer cells with respect to cell death, reactive oxygen species (ROS), and glutathione (GSH) levels. Treatment with 0~5 µM auranofin decreased cell proliferation and induced cell death in Calu-6, A549, SK-LU-1, NCI-H460, and NCI-H1299 lung cancer cells at 24 h. In addition, 0~5 µM auranofin increased ROS levels, including O2•−, and depleted GSH levels in these cells. N-acetyl cysteine (NAC) prevented growth inhibition and mitochondrial membrane potential (MMP, ∆Ψm) loss in 3 and 5 µM auranofin-treated Calu-6 and A549 cells at 24 h, respectively, and decreased ROS levels and GSH depletion in these cells. In contrast, L-buthionine sulfoximine (BSO) enhanced cell death, MMP (∆Ψm) loss, ROS levels, and GSH depletion in auranofin-treated Calu-6 and A549 cells. Treatment with 3 and 5 µM auranofin induced caspase-3 activation and poly (ADP ribose) polymerase (PARP) cleavage in Calu-6 and A549 cells, respectively. Both were prevented by NAC, but enhanced by BSO. Moreover, TrxR activity was reduced in auranofin-treated Calu-6 and A549 cells. That activity was decreased by BSO, but increased by NAC. In conclusion, these findings demonstrate that auranofin-induced cell death is closely related to oxidative stress resulted from increased ROS levels and GSH depletion in lung cancer cells.