Edible grey oyster mushroom, Pleurotus sajor-caju, β (1,3), (1,6) glucan possesses a wide range of biological activities, including anti-inflammation, anti- microorganisms, and antioxidant. However, its biological activity is limited by their low water solubility resulting from the 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 RANKL-induced osteoclastogenesis in pre-osteoclastic RAW 264.7 cells. Cell cytotoxicity of Ps-GOS on RAW 264.7 cells was determined by the MTT assay and its effect on osteoclast differentiation was determined by TRAP staining. Additionally, its effect on osteoclast bone-resorptive ability was detected by the pit formation assay. The osteoclastogenic-related factors were assessed by 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 NFB-P65 expression and its subsequent master osteoclast modulators including NFATc1 and cFOS via the NF-B 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, MMP-9, and 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. Current 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 the receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis in pre-osteoclastic RAW 264.7 cells. The results revealed that L-quebrachitol suppressed RANK-mediated signalling, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Fos proto-oncogene (cFOS) pathways, at both 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. These findings demonstrate the dose-dependent inhibitory effect of L-quebrachitol on osteoclastogenesis through the modulation of receptor activator of nuclear factor κB (RANK)-mediated signalling pathways. However, further exploration of L-quebrachitol's potential as a therapeutic approach for osteoporosis is warranted.