Osteoporosis is widely recognized as a major health problem caused by an inappropriate rate of bone resorption compared to bone formation. Previously we showed that d-pinitol inhibits osteoclastogenesis but has no effect on osteoblastogenesis. However, the effect on osteoblast differentiation of its isomer, l-quebrachitol, has not yet been reported. The purpose of this study was, therefore, to investigate whether l-quebrachitol promotes the osteoblastogenesis of pre-osteoblastic MC3T3-E1 cells. Moreover, the molecular mechanism of action of l-quebrachitol was further explored. Here, it is shown for the first time that l-quebrachitol significantly promotes proliferation and cell DNA synthesis. It also enhances mineralization accompanied by increases in mRNA expression of bone matrix proteins including alkaline phosphatase (ALP), collagen type I (ColI), osteocalcin (OCN), and osteopontin (OPN). In addition, l-quebrachitol upregulates the mRNA and protein expression of bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor-2 (Runx2), while down-regulating the receptor activator of the nuclear factor-κB ligand (RANKL) mRNA level. Moreover, the expression of regulatory genes associated with the mitogen-activated protein kinase (MAPK) and wingless-type MMTV integration site (Wnt)/β-catenin signaling pathways are also upregulated. These findings indicate that l-quebrachitol may promote osteoblastogenesis by triggering the BMP-2-response as well as the Runx2, MAPK, and Wnt/β-catenin signaling pathway.

Osteoporosis is a leading world health problem that results from an imbalance between bone formation and bone resorption. β-glucans has been extensively reported to exhibit a wide range of biological activities, including antiosteoporosis both in vitro and in vivo. However, the molecular mechanisms responsible for β-glucan-mediated bone formation in osteoblasts have not yet been investigated. The oyster mushroom Pleurotus sajor-caju produces abundant amounts of an insoluble β-glucan, which is rendered soluble by enzymatic degradation using Hevea glucanase to generate low-molecular-weight glucanoligosaccharide (Ps-GOS). This study aimed to investigate the osteogenic enhancing activity and underlining molecular mechanism of Ps-GOS on osteoblastogenesis of pre-osteoblastic MC3T3-E1 cells. In this study, it was demonstrated for the first time that low concentrations of Ps-GOS could promote cell proliferation and division after 48 h of treatment. In addition, Ps-GOS upregulated the mRNA and protein expression level of bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor-2 (Runx2), which are both involved in BMP signaling pathway, accompanied by increased alkaline phosphatase (ALP) activity and mineralization. Ps-GOS also upregulated the expression of osteogenesis related genes including ALP, collagen type 1 (COL1), and osteocalcin (OCN). Moreover, our novel findings suggest that Ps-GOS may exert its effects through the mitogen-activated protein kinase (MAPK) and wingless-type MMTV integration site (Wnt)/β-catenin signaling pathways.

Excessive osteoclast-mediated bone resorption is a critical cause of osteoporosis affecting many aging people worldwide. 5'-Methylthioadenosine (MTA) is a natural sulfur-containing nucleoside normally produced in prokaryotes, plants, yeast, and higher eukaryotes via polyamine metabolism. MTA affects various physiological responses particularly the inflammatory pathway in both normal and cancerous cells and modulates the activation of nuclear factor-κB involved in the osteoclastogenesis signalling process. While several studies have reported that natural products possess anti-osteoclastogenesis phenolics and flavonoids, the effect of nucleoside derivatives on osteoclastogenesis remains limited. Therefore, this study aimed to explore the molecular mechanisms by which MTA affects pre-osteoclastic RAW 264.7 cells as a potential alleviation compound for inflammation-mediated bone loss. Osteoclasts were established by incubating RAW264.7 macrophage cells with receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor, the vital cytokines for activation of osteoclast differentiation. Cell viability was measured using MTT assays at 24, 48, and 72 h. The suppressive effect of MTA on RANKL-induced osteoclast differentiation and function was assessed using tartrate-resistant acid phosphatase (TRAP) analysis, qRT-PCR, and pit formation, Western blot, and immunofluorescence assays. MTA showed dose-dependent anti-osteoclastogenic activity by inhibiting TRAP-positive cell and pit formation and reducing essential digestive enzymes, including TRAP, cathepsin K, and matrix metallopeptidase 9. MTA was observed to suppress the osteoclast transduction pathway through (RANKL)-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NFƘB); it attenuated NFƘB-P65 expression and down-regulated cFos proto-oncogene and nuclear factor of activated T cell c1 (NFATc1), the main regulators of osteoclasts. Moreover, the suppression of RANK (the initial receptor triggering several osteoclastogenic transduction pathways) was observed. Thus, this study highlights the potential of MTA as an effective therapeutic compound for restoring bone metabolic disease by inhibiting the RANK-NFATc1 signal pathway.

Edible grey oyster mushroom, Pleurotus sajor-caju, β (1,3), (1,6) glucan possesses a wide range of biological activities, including anti-inflammation, anti-microorganism and antioxidant. However, its biological activity is limited by low water solubility resulting from its high molecular weight. Our previous study demonstrated that enzymatic hydrolysis of grey oyster mushroom β-glucan using Hevea β-1,3-glucanase isozymes obtains a lower molecular weight and higher water solubility, Pleurotus sajor-caju glucanoligosaccharide (Ps-GOS). Additionally, Ps-GOS potentially reduces osteoporosis by enhancing osteoblast–bone formation, whereas its effect on osteoclast–bone resorption remains unknown. Therefore, our study investigated the modulatory activities and underlying mechanism of Ps-GOS on Receptor activator of nuclear factor kappa-Β ligand (RANKL) -induced osteoclastogenesis in pre-osteoclastic RAW 264.7 cells. Cell cytotoxicity of Ps-GOS on RAW 264.7 cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and its effect on osteoclast differentiation was determined by tartrate-resistant acid phosphatase (TRAP) staining. Additionally, its effect on osteoclast bone-resorptive ability was detected by pit formation assay. The osteoclastogenic-related factors were assessed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), Western blot and immunofluorescence. The results revealed that Ps-GOS was non-toxic and significantly suppressed the formation of mature osteoclast multinucleated cells and their resorption activity by reducing the number of TRAP-positive cells and pit formation areas in a dose-dependent manner. Additionally, Ps-GOS attenuated the nuclear factor kappa light chain-enhancer of activated B cells’ P65 (NFκB-P65) expression and their subsequent master osteoclast modulators, including nuclear factor of activated T cell c1 (NFATc1) and Fos proto-oncogene (cFOS) via the NF-κB pathway. Furthermore, Ps-GOS markedly inhibited RANK expression, which serves as an initial transmitter of many osteoclastogenesis-related cascades and inhibited proteolytic enzymes, including TRAP, matrix metallopeptidase 9 (MMP-9) and cathepsin K (CTK). These findings indicate that Ps-GOS could potentially be beneficial as an effective natural agent for bone metabolic disease.

Inositol is a natural carbocyclic sugar that plays an essential role in regulating the vital cellular functions of plants and animals. Existing research has explored methyl derivatives of inositol, reporting on their various biological activities, including antitumor, anti-inflammatory, and anti-osteoporosis activities. Our previous study demonstrated that L-quebrachitol, a methyl derivative of inositol, enhances osteoblastogenesis and bone formation; however, its effect on osteoclastogenesis remains unclear. Consequently, we aimed to investigate the effect of L-quebrachitol on receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis in pre-osteoclastic RAW 264.7 cells, and bone resorption in an ovariectomized rat model. The results revealed that L-quebrachitol suppressed RANK-mediated signaling, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Fos proto-oncogene (cFOS) pathways, at both the gene and protein levels. Moreover, the critical transcription factor for osteoclastogenesis, nuclear factor of activated T cells c1 (NFATc1), was downregulated. Inhibition of osteoclast-associated marker genes encoding proteolytic enzymes, such as tartrate-resistant acid phosphatase (TRAP), matrix metallopeptidase 9 (MMP-9), and cathepsin K, led to reduced formation of TRAP-positive multinucleated cells and resorption pits. In addition, proteasome subunit alpha type-5 (PSMA5), which is involved in the degradation of the NF-κB inhibitor, was also suppressed. In particular, the animal study clearly supported the bone homeostasis property of the agent by increasing the BV/TV (bone volume/total volume) and Tb.Th (trabecular thickness) in ovariectomized rats. These findings demonstrate the dose-dependent inhibitory effect of L-quebrachitol on osteoclastogenesis through the modulation of RANK-mediated signaling pathways and prevention of bone loss in an animal model. However, further exploration of the potential of L-quebrachitol as an effective approach for osteoporosis is required.