Pleurotus sajor-caju (Fr.) Singer ß-1,3-Glucanoligosaccharide (Ps-GOS) Suppresses RANKL-Induced Osteoclast Differentiation and Function in Pre-Osteoclastic RAW 264.7 Cells by Inhibiting the RANK/NFκB/cFOS/NFATc1 Signalling Pathway.

Edible grey oyster mushroom, Pleurotus sajor-caju, ß (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 ß-glucan using Hevea ß-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.

Pathological progression of osteoarthritis: a perspective on subchondral bone.

Osteoarthritis (OA) is a degenerative bone disease associated with aging. The rising global aging population has led to a surge in OA cases, thereby imposing a significant socioeconomic burden. Researchers have been keenly investigating the mechanisms underlying OA. Previous studies have suggested that the disease starts with synovial inflammation and hyperplasia, advancing toward cartilage degradation. Ultimately, subchondral-bone collapse, sclerosis, and osteophyte formation occur. This progression is deemed as "top to bottom." However, recent research is challenging this perspective by indicating that initial changes occur in subchondral bone, precipitating cartilage breakdown. In this review, we elucidate the epidemiology of OA and present an in-depth overview of the subchondral bone's physiological state, functions, and the varied pathological shifts during OA progression. We also introduce the role of multifunctional signal pathways (including osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL)/receptor activator of nuclear factor-kappa B (RANK), and chemokine (CXC motif) ligand 12 (CXCL12)/CXC motif chemokine receptor 4 (CXCR4)) in the pathology of subchondral bone and their role in the "bottom-up" progression of OA. Using vivid pattern maps and clinical images, this review highlights the crucial role of subchondral bone in driving OA progression, illuminating its interplay with the condition.

Investigating the Effects and Mechanisms of Cyclomorusin on Osteoclasts in a High Glucose Environment.

Diabetes mellitus is an endocrine disease characterized by prolonged hyperglycemia. Prolonged high blood sugar levels interfere with the differentiation and maturation process of OBs and OCs, leading to the onset of osteoporosis. However, OCs differentiation and maturation is a complex regulatory process. In this study, we used a co-culture system of RAW264.7 and MC3T3-E1 cells under HG concentration to explore the effect of CYM on OCs in a HG environment. The effects of CYM on the formation and function of OCs were observed using TRAP-positive cell counts and bone resorption pits. Then, mRNA and protein expression levels of OCs-related genes were detected by real-time qPCR and western blotting. The results showed that CYM had an inhibitory effect on OCs differentiation and bone resorption, reduced mRNAs expression of OCs-associated genes, and downregulated RANKL/RANK/TRAF6 pathway that mediates OCs differentiation. CYM could be a promising natural compound against diabetic osteoporosis.

Interplay between hormonal and mechanical signals in mammary morphodynamics.

Mammographic density is a well-established risk factor for breast cancer. In a recent study, Northey et al. reveal that the associated increase in tissue stiffness elevates extracellular signal-regulated kinase (ERK) activity, promoting progesterone receptor-dependent receptor activator of nuclear factor κß (RANK) signaling. Thus, stiffness alters the context of hormonal signaling and increases mammary stem cells. This mechanism suggests potential treatments for breast cancer.

A20: a jack of all trades.

Mutations and polymorphisms in A20/TNFAIP3 have been linked to various inflammatory disorders. However, in addition to its well-known role in inflammation, A20 also controls EDAR- and receptor activator of NF-κB (RANK)-induced NF-κB signaling, regulating the development of epidermal skin appendages and bone, respectively. Furthermore, A20 regulates synapse remodeling through a mechanism dependent on NF-κB.

Mechanical Stress Induces Sodium Entry and Osmoprotective Responses in Murine Synovial Fibroblasts.

Osteoarthritis (OA) is a multifactorial disease depending on molecular, genetic, and environmental factors like mechanical strain. Next to the cartilage and the subchondral bone, OA also affects the synovium, which is critically involved in the maintenance of joint homeostasis. As there is a correlation between the extracellular sodium content in the knee joint and OA, this study investigates the impact of sodium on OA-associated processes like inflammation and bone remodeling without and with mechanical loading in synovial fibroblasts. For that purpose, murine synovial fibroblasts from the knee joint were exposed to three different extracellular sodium chloride concentrations (-20 mM, ±0 mM and +50 mM NaCl) in the absence or presence of compressive or intermittent tensile strain. In addition to the intracellular Na+ content and gene expression of the osmoprotective transcription factor nuclear factor of activated T cells 5 (Nfat5), the gene and protein expression of inflammatory mediators (interleukin-6 (IL6), prostaglandin endoperoxide synthase-2 (Ptgs2)/prostaglandin E2 (PGE2)), and factors involved in bone metabolism (receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG)) were analyzed by qPCR and ELISA. Mechanical strain already increased intracellular Na+ and Nfat5 gene expression at standard salt conditions to levels obtained by exposure to increased extracellular Na+ content. Both high salt and compressive strain resulted in elevated IL6 and PGE2 release. Intermittent tensile strain did not increase Il6 mRNA expression or IL6 protein secretion but triggered Ptgs2 expression and PGE2 production. Increased extracellular Na+ levels and compressive strain increased RANKL expression. In contrast, intermittent tension suppressed RANKL expression without this response being subject to modification by extracellular sodium availability. OPG expression was only induced by compressive strain. Changes in extracellular Na+ levels modified the inflammatory response and altered the expression of mediators involved in bone metabolism in cells exposed to mechanical strain. These findings indicate that Na+ balance and Nfat5 are important players in synovial fibroblast responses to mechanical stress. The integration of Na+ and Na+-dependent signaling will help to improve the understanding of the pathogenesis of osteoarthritis and could lead to the establishment of new therapeutic targets.

Immunomodulatory Effects of RANK/RANKL Blockade in Patients with Cancer.

In cancer, multiple factors converge upon receptor activator of nuclear factor κB (RANK) and its ligand (RANKL) signaling to promote the development of bone metastases; agents that inhibit RANKL signaling reduce skeletal-related events (SRE) in patients with cancer. In addition, RANKL signaling is important in augmenting the ability of dendritic cells (DC) to stimulate both naïve T-cell proliferation and the survival of RANK+ T cells. In this issue, Chang and colleagues using high-dimensional cytometry to evaluate immunomodulatory effects of denosumab in patients with advanced solid, observe early on treatment changes in multiple compartments, and greater effects in patients receiving concurrent chemotherapy or steroids. See related article by Chang et al., p. 453 (4).

Icariin regulates RANKL-induced osteoclast differentiation via the ERα/c-Src/RANK signaling.

Osteoporosis (OP) is a common metabolic bone disease. Excessive osteoclastic activity significantly contributes to the development of OP. Icariin (ICA) is a flavonol glycoside derived from herbal plants and possesses curative effects on postmenopausal OP and bone fracture. This study aimed to investigate the effects of ICA on osteoclast differentiation induced by receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) and the involvement of estrogen receptorα(ERα) and RANK signaling cascade in this process. RANKL was used to induce the differentiation of RAW264.7 cells to into osteoclasts. Small interfering RNA technique was used to knockdown ERαin cells. Cell counting kit-8 assay was performed to determine the cytotoxicity of ICA. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells was quantified by TRAP staining. RANKL induced the differentiation of RAW264.7 cells into osteoclasts, while ICA abolished the pro-osteoporotic effect of RANKL. Moreover, ERαknockdown abolished the effects of ICA on RANKL-induced osteoclastogenesis. Further exploration revealed that ICA inhibited the phosphorylation ofc-Src in osteoclasts via regulating ERα, while inactivation ofc-Src reversed ERαknockdown-promoted osteoclastogenesis. Lastly, ICA inhibited the activation of the mitogen-activated protein kinase signaling pathway and downregulated the expressions of target osteoclastogenic proteins in RANKL-treated RAW 264.7 cells, while ERαknockdown almost completely diminished the effects of ICA. ICA inhibited RANKL-induced osteoclast differentiation via regulating the ERα/c-Src/RANK signaling. These findings elucidated a novel mechanism by which ICA exerts an anti-osteoporotic effect.

RANK/RANKL axis promotes migration, invasion, and metastasis of osteosarcoma via activating NF-κB pathway.

Osteosarcoma (OS) is one of the most prevalent primary bone tumors with a high degree of metastasis and poor prognosis. Epithelial-to-mesenchymal transition (EMT) is a cellular mechanism that contributes to the invasion and metastasis of cancer cells, and OS cells have been reported to exhibit EMT-like characteristics. Our previous studies have shown that the interaction between tumor necrosis factor superfamily member 11 (TNFRSF11A; also known as RANK) and its ligand TNFSF11 (also known as RANKL) promotes the EMT process in breast cancer cells. However, whether the interaction between RANK and RANKL enhances aggressive behavior by inducing EMT in OS cells has not yet been elucidated. In this study, we showed that the interaction between RANK and RANKL increased the migration, invasion, and metastasis of OS cells by promoting EMT. Importantly, we clarified that the RANK/RANKL axis induces EMT by activating the nuclear factor-kappa B (NF-κB) pathway. Furthermore, the NF-κB inhibitor dimethyl fumarate (DMF) suppressed migration, invasion, and EMT in OS cells. Our results suggest that the RANK/RANKL axis may serve as a potential tumor marker and promising therapeutic target for OS metastasis. Furthermore, DMF may have clinical applications in the treatment of lung metastasis in patients with OS.

Iguratimod suppresses sclerostin and receptor activator of NF-κB ligand production via the extracellular signal-regulated kinase/early growth response protein 1/tumor necrosis factor alpha pathway in osteocytes and ameliorates disuse osteoporosis in mice.

Disuse osteoporosis is a prevalent complication among patients afflicted with rheumatoid arthritis (RA). Although reports have shown that the antirheumatic drug iguratimod (IGU) ameliorates osteoporosis in RA patients, details regarding its effects on osteocytes remain unclear. The current study examined the effects of IGU on osteocytes using a mouse model of disuse-induced osteoporosis, the pathology of which crucially involves osteocytes. A reduction in distal femur bone mass was achieved after 3 weeks of hindlimb unloading in mice, which was subsequently reversed by intraperitoneal IGU treatment (30 mg/kg; five times per week). Histology revealed that hindlimb-unloaded (HLU) mice had significantly increased osteoclast number and sclerostin-positive osteocyte rates, which were suppressed by IGU treatment. Moreover, HLU mice exhibited a significant decrease in osteocalcin-positive cells, which was attenuated by IGU treatment. In vitro, IGU suppressed the gene expression of receptor activator of NF-κB ligand (RANKL) and sclerostin in MLO-Y4 and Saos-2 cells, which inhibited osteoclast differentiation of mouse bone marrow cells in cocultures. Although IGU did not affect the nuclear translocation or transcriptional activity of NF-κB, RNA sequencing revealed that IGU downregulated the expression of early growth response protein 1 (EGR1) in osteocytes. HLU mice showed significantly increased EGR1- and tumor necrosis factor alpha (TNFα)-positive osteocyte rates, which were decreased by IGU treatment. EGR1 overexpression enhanced the gene expression of TNFα, RANKL, and sclerostin in osteocytes, which was suppressed by IGU. Contrarily, small interfering RNA-mediated suppression of EGR1 downregulated RANKL and sclerostin gene expression. These findings indicate that IGU inhibits the expression of EGR1, which may downregulate TNFα and consequently RANKL and sclerostin in osteocytes. These mechanisms suggest that IGU could potentially be used as a treatment option for disuse osteoporosis by targeting osteocytes.

MCP-5 suppresses osteoclast differentiation through Ccr5 upregulation.

Human monocyte chemoattractant protein-1 (MCP-1) in mice has two orthologs, MCP-1 and MCP-5. MCP-1, which is highly expressed in osteoclasts rather than in osteoclast precursor cells, is an important factor in osteoclast differentiation. However, the roles of MCP-5 in osteoclasts are completely unknown. In this study, contrary to MCP-1, MCP-5 was downregulated during receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation and was considered an inhibitory factor in osteoclast differentiation. The inhibitory role of MCP-5 in osteoclast differentiation was closely related to the increase in Ccr5 expression and the inhibition of IκB degradation by RANKL. Transgenic mice expressing MCP-5 controlled by Mx-1 promoter exhibited an increased bone mass because of a decrease in osteoclasts. This result strongly supported that MCP-5 negatively regulated osteoclast differentiation. MCP-5 also prevented severe bone loss caused by RANKL.

Assessment of osteoprotegerin and RANKL levels and several cardiovascular risk scoring systems in acromegaly.

PURPOSE: The OPG/RANKL (osteoprotegerin/receptor activator of nuclear factor kappa-B) system, which plays a crucial role in bone metabolism, is also associated with vascular calcification. Acromegaly is characterized by excessive secretion of growth hormone and insulin-like growth factor, and studies have demonstrated an elevated risk of cardiovascular disease in individuals with acromegaly. In this study, our objective was to investigate the relationship between OPG/RANKL and various cardiovascular risk scoring systems. METHODS: We recruited 44 consecutive acromegaly patients and 41 healthy controls with a similar age and gender distribution for this study. RESULTS: While RANKL levels were significantly higher in the acromegaly group compared to the controls, OPG levels were not found to be significantly different between the two groups. Furthermore, within the acromegaly group, RANKL levels were significantly higher in patients with active acromegaly compared to those with controlled acromegaly. Osteoprotegerin levels showed a positive correlation with the Framingham risk score (FRS) in the acromegaly group. Linear regression analysis revealed an association of OPG with FRS (adjusted R2 value of 21.7%). CONCLUSION: OPG and RANKL may serve as potential markers for assessment of cardiovascular calcification and prediction of the cardiovascular risk status in acromegalic patients.

NOTCH2 promotes osteoclast maturation and metabolism and modulates the transcriptome profile during osteoclastogenesis.

Notch signaling plays a key regulatory role in bone remodeling and NOTCH2 enhances osteoclastogenesis, an effect that is mostly mediated by its target gene Hes1. In the present study, we explored mechanisms responsible for the enhanced osteoclastogenesis in bone marrow-derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice. Notch2tm1.1Ecan mice are osteopenic and have enhanced osteoclastogenesis. Bulk RNA-Seq and gene set enrichment analysis of Notch2tm1.1Ecan BMMs cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand revealed enrichment of genes associated with enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis. These pathways were not enhanced in the context of a Hes1 inactivation. Analysis of single cell RNA-Seq data of pooled control and Notch2tm1.1Ecan BMMs treated with M-CSF or M-CSF and receptor activator of NF-κB ligand for 3 days identified 11 well-defined cellular clusters. Pseudotime trajectory analysis indicated a trajectory of clusters expressing genes associated with osteoclast progenitors, osteoclast precursors, and mature cells. There were an increased number of cells expressing gene markers associated with the osteoclast and with an unknown, albeit related, cluster in Notch2tm1.1Ecan than in control BMMs as well as enhanced expression of genes associated with osteoclast progenitors and precursors in Notch2tm1.1Ecan cells. In conclusion, BMM cultures display cellular heterogeneity, and NOTCH2 enhances osteoclastogenesis, increases mitochondrial and metabolic activity of osteoclasts, and affects cell cluster allocation in BMMs.

TIFA contributes to periodontitis in diabetic mice via activating the NF­κB signaling pathway.

Diabetic periodontitis (DP) refers to destruction of periodontal tissue and absorption of bone tissue in diabetic patients. Tumor necrosis factor receptor­associated factor (TRAF)­interacting protein with forkhead­associated domain (TIFA) as a crucial regulator of inflammation activates the NF­κB signaling pathway to regulate cell biological behavior. However, the function and mechanism of TIFA on DP suffer from a lack of research. In the present study, TIFA was upregulated in the periodontal tissue of a DP mouse model. In addition, the expression of TIFA in RAW264.7 cells was induced by high glucose (HG) culture and increased by lipopolysaccharide (LPS) from Porphyromonas gingivalis treatment in a time­dependent manner. Knockdown of TIFA significantly reduced the levels of inflammatory cytokines, including TNF­α, IL­6, IL­1ß and monocyte chemoattractant protein­1, in HG and LPS­induced RAW264.7 cells. The nuclear translocation of NF­κB p65 was induced by HG and LPS and was clearly suppressed by absence of TIFA. The expression of downstream factors Nod­like receptor family pyrin domain­containing 3 and apoptosis­associated speck­like protein was inhibited by silencing TIFA. Moreover, TIFA was increased by receptor activator of NF­κB (RANK) ligand (RANKL) in a concentration dependent manner. The expression of cathepsin K, MMP9 and nuclear factor of activated T cells cytoplasmic 1 was downregulated by depletion of TIFA. RANKL­induced osteoclast differentiation was inhibited by silencing of TIFA. Meanwhile, the decrease of TIFA blocked activation of the NF­κB pathway in RANKL­treated RAW264.7 cells. In conclusion, TIFA as a promoter regulates the inflammation and osteoclast differentiation via activating the NF­κB signaling pathway.

Expression and functional characterization of bovine receptor activator of NF-κB ligand (RANKL).

Receptor activator of nuclear factor Kappa-B Ligand (RANKL) is a member of the tumor necrosis factor ligand (TNF) family involved in immune responses and immunomodulation. Expressed in various cells types around the body, RANKL plays a crucial role in bone remodeling and development of the thymus, lymph nodes and mammary glands. Research in other species demonstrates that RANKL is required for the development of microfold cells (M cells) in the gut, however limited information specific to cattle is available. Cloning and expression of bovine RANKL (BoRANKL) was carried out and bioactivity of the protein was demonstrated in the induction of osteoclast differentiation from both bovine and ovine bone marrow cells. The effects of BoRANKL on particle uptake in bovine enteroids was also assessed. The production of cross-reactive bovine RANKL protein will enable further investigations into cell differentiation using the available ruminant organoid systems, and their role in investigating host-pathogen interactions in cattle and sheep.

Vascular Pericyte-Derived Exosomes Inhibit Bone Resorption via Traf3.

Purpose: Blood vessels distribute cells, oxygen, and nutrients throughout the body to support tissue growth and balance. Pericytes and endothelial cells form the inner wall of blood vessels, crucial for organ development and tissue homeostasis by producing paracrine signaling molecules. In the skeletal system, pericyte-derived vascular factors along with angiogenic factors released by bone cells regulate angiogenesis and bone formation. Although the involvement of angiogenic factors and skeletal blood vessels in bone homeostasis is relatively clear, the role of pericytes and the underlying mechanisms remain unknown. Here, our objective was to elucidate the significance of pericytes in regulating osteoclast differentiation. Methods: We used tissue staining to detect the coverage of pericytes and osteoclasts in femoral tissues of osteoporotic mice and mice of different ages, analyzing their correlation. We developed mice with conditionally deleted pericytes, observing changes in bone mass and osteoclast activity using micro-computer tomography and tissue staining to detect the regulatory effect of pericytes on osteoclasts. Pericytes-derived exosomes (PC-EVs) were collected and co-cultured with monocytes that induce osteoclast differentiation to detect the effect of the former on the exosomes. Finally, the specific mechanism of PC-EVs regulating osteoclast differentiation was verified using RNA sequencing and Western blotting. Results: Our study indicates a significant correlation between pericytes and age-related bone resorption. Conditional deletion of pericytes activated bone resorption and led to osteopenia in vivo. We discovered that PC-EVs inhibited the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, which is mediated by tumor necrosis factor receptor-associated factor 3 (Traf3), negatively regulating osteoclast development and bone resorption. Silencing Traf3 in PC-EVs canceled their inhibitory effect on osteoclast differentiation. Conclusion: Our study provides a novel perspective into the regulatory role of pericytes on bone resorption and may provide potential strategies for developing novel anti-bone resorption therapies.

Downregulation of osteoprotegerin in colorectal cancer cells promotes liver metastasis via activating tumor-associated macrophage.

Osteoprotegerin (OPG) is a secreted cytokine that functions as a decoy receptor for receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL). Anti-RANKL treatment for bone metastasis has been widely accepted for solid tumors. However, the mechanism of OPG-RANKL-RANK signaling in systemic colorectal cancer (CRC) metastasis remains unclear. In this study, we investigated the relevance and function of OPG expression in CRC liver metastasis. First, we performed in silico analysis using The Cancer Genome Atlas public database and found that lower OPG expression in CRC was associated with poor overall survival. Immunohistochemistry analyses using resected specimen from patients with CRC in our institute confirmed the result. Patient-matched primary CRC and liver metastases showed a significant downregulation of OPG expression in metastatic lesions. In CRC cell lines, OPG expression did not suppress cell proliferation and migration. However, OPG expression inhibited macrophage migration by suppressing the RANKL-RANK pathway. Moreover, in vivo mouse liver metastasis models showed that OPG expression in CRC cells suppressed liver metastases. In addition, treatment with an anti-RANKL neutralizing antibody also suppressed liver metastases. These results showed that downregulation of OPG expression in CRC cells promotes liver metastasis by activating tumor-associated macrophage, which can become a candidate for targeted therapy with anti-RANKL neutralizing antibody for CRC liver metastasis.

Expression and ratio of receptor activator of nuclear factor kappa-B ligand and osteoprotegerin following application of Nigella sativa/bovine bone graft combination in post tooth extraction sockets.

Aims: The aim of this study was to analyze the induction effect of a combination of N. sativa and bovine bone graft on the expression and ratio of receptor activator of nuclear factor kappa-B ligand expression (RANKL) and osteoprotegerin (OPG) on alveolar bone socket preservation on days 7 and 14. Settings and Design: The research incorporated a posttest-only control group design. A total of 56 Cavia cobaya were divided into four groups: a control group, an N. sativa group, a bovine bone graft group, and a combined N. sativa and bovine bone graft group. Materials and Methods: The lower incisors of the C. cobaya were extracted with material subsequently being applied to the resulting socket. After the 7th and 14th days, the experimental animals were terminated to enable observation of the socket. Following processing, the tissue was subjected to immunohistochemistry staining consisting of RANKL and OPG antibodies before being observed under a light microscope at × 400. Statistical Analysis Used: Statistical analysis was carried out using the one-way ANOVA and Tukey's honestly significant difference tests. Results: A combination of N. sativa and bovine bone graft reduced both RANKL expression and the RANKL/OPG ratio while increasing OPG expression in comparison to the other groups. In all the results obtained, the N. sativa and bovine bone graft combination was significant (P < 0.05) when compared to the control group on both the 7th and 14th days. Conclusion: A combination of N. sativa and bovine bone graft reduced both RANKL expression and the RANKL/OPG ratio while increasing OPG expression.

In vivo and in vitro action mechanism of treatment of glucocorticoid-induced osteoporosis by regulation of osteoprotegerin/receptor activator of nuclear factor-κB pathways by denshensu.

The research aimed to discuss the action mechanism of the treatment of glucocorticoid-induced osteoporosis (GIOP) by denshensu. In the research, 60 rats were purchased and divided into a control group, model group, estradiol group, and denshensu treatment group. Except for the control group, GIOP models were established for all other groups, and then the structural changes of osseous tissues as well as osteoprotegerin (OPG), expression of receptor activator of nuclear factor-κB ligands (RANKL) were detected. Besides, the changes in osteoclasts were observed by bone marrow-derived mononuclear phagocytes in vitro. The results showed that the micro-structure of bone trabeculae, bone mineral density (BMD), and bone metabolic markers of rats in the denshensu treatment group were enhanced significantly, while trabecular separation and structural model index were reduced (P<0.05). OPG messenger ribonucleic acid (mRNA) and protein levels in the hypothalamus and femur tissues were increased, while RANKL content was remarkably decreased (P<0.05). In addition, in vitro experiments revealed that denshensu inhibited the differentiation of positive osteoclasts, and osteoclast-related genes were reduced (P<0.05). To conclude, denshensu might inhibit the expressions of OPG and RANKL and further play a role in treating GIOP.

Development of 3-acetylindole derivatives that selectively target BRPF1 as new inhibitors of receptor activator of NF-κB ligand (RANKL)-Induced osteoclastogenesis.

Bromodomain and PHD finger-containing (BRPF) proteins function as epigenetic readers that specifically recognize acetylated lysine residues on histone tails. The acetyl-lysine binding pocket of BRPF has emerged as an attractive target for the development of protein interaction inhibitors owing to its potential druggability. In this study, we identified 3-acetylindoles as bone antiresorptive agents with a novel scaffold by performing structure-based virtual screening and hit optimization. Among those derivatives, compound 18 exhibited potent and selective inhibitory activities against BRPF1B (IC50 = 102 nM) as well as outstanding inhibitory activity against osteoclastogenesis (73.8% @ 1 µM) and differentiation (IC50 = 0.19 µM) without cytotoxicity. Besides, cellular mechanism assays demonstrated that compound 18 exhibited a strong bone antiresorptive effect by modulating the RANKL/RANK/NFATc1 pathway. Structural and functional studies on BRPF1 inhibitors aid in making advances to understand the epigenetic mechanisms of bone cell development and create innovative therapeutics for treating bone metastases from solid tumors and other bone erosive diseases.