Nuclear factor-κB Ligand Research Articles

The influence of ESR1 polymorphisms on selected hormonal, metabolic and mineral balance markers in women with hyperandrogenism

Hyperandrogenism is the most common endocrine disorder in women, characterized by an imbalance in normal estrogen and androgen levels in the blood. Androgens influence bone mineral density, body mass composition, muscle mass, mental state, and the regulation of sexual function.. The aim of the study was to assess the effect of estrogen receptor α gene (ESR1) polymorphisms on selected markers of bone metabolism and hormonal parameters in women with hyperandrogenism. The study group included 80 young women with hyperandrogenism who underwent measurements of bone mineral density (BMD), and determination of hormonal and metabolic parameters. Enzyme immunoassays were used to measure leptin, sRANKL (soluble receptor activator of nuclear factor-kB ligand), osteoprotegerin and 25-OH vitamin D total levels. An analysis of ESR1 gene polymorphisms was performed using the real-time PCR method. A relationship was demonstrated between the concentration of free estradiol (FEI) and the concentration of 17-OH-progesterone, and the ESR1 gene polymorphisms: rs3020314 (p = 0.031, p = 0.026 respectively) and rs1884051 (p = 0.033, p = 0.026 respectively). In conclusion, the ESR gene polymorphisms may be associated with hormonal disturbances in the concentration of estrogens and androgens, in hyperandrogenism in young women which may indirectly affect bone mineral density. However, no statistically significant relationships between the studied polymorphisms and the selected parameters of mineral metabolism have been demonstrated..

Isoorientin ameliorates osteoporosis and oxidative stress in postmenopausal rats

Context Isoorientin has many biological activities, including antioxidant, anti-inflammatory, antitumor. However, the effect of isoorientin on postmenopausal osteoporosis remains unclear. Objective To evaluate the effect of isoorientin on postmenopausal osteoporosis. Materials and methods Sprague-Dawley rats were divided into five groups (n = 5): sham, model, 17-β-oestradiol (E2, 10 μg/kg/day), low-dose isoorientin (L-Iso, 50 mg/kg), and high-dose isoorientin (H-Iso, 100 mg/kg). The rats were ovariectomized, treated by gavage daily for 12 weeks, and serum and femur samples were collected. Bone mineral density, bone metabolism, and oxidative stress were assessed. H&E staining, immunohistochemistry, and western blotting were employed. Results Isoorientin improved the bone mineral density of the lumbar vertebrae (2.01 ± 0.05 g/cm3 in H-Iso group vs. 1.74 ± 0.07 g/cm3 in model group) and femur (1.46 ± 0.06 g/cm3 vs. 1.19 ± 0.03 g/cm3), increased the trabecular bone number (1.97 ± 0.03 vs. 1.18 ± 0.13) and thickness (0.27 ± 0.02 vs. 0.16 ± 0.03 mm). Isoorientin decreased the separation degree of trabecular bone, ameliorated bone histomorphology changes, and significantly improved the mechanical properties. Isoorientin diminished MDA (by 60%) and increased SOD (by 49.2%), and GSH-Px (by 159%) activity. Furthermore, osteoprotegerin (OPG), nuclear factor erythroid 2-like 2 (Nrf2), haem oxygenase (HO-1), NAD(P)H quinone dehydrogenase 1(NQO1), and oestrogen receptor 1(ESR1) protein expression increased, while receptor activator of nuclear factor-κB ligand (RANKL) protein expression decreased after treatment. Conclusions Isoorientin ameliorates osteoporosis via upregulating OPG and Nrf2/ARE signalling, suggesting isoorientin maybe a potential therapeutic drug for PMOP.

Osteoporosis in the USA: prevention and unmet needs

The past 30 years have witnessed tremendous progress in our ability to diagnose and treat osteoporosis. Once considered an inevitable consequence of ageing, osteoporosis can now be accurately diagnosed using dual-energy x-ray absorptiometry (DXA). Fundamental advances in the understanding of the pathogenesis of osteoporosis have driven the development of highly effective treatments, including four bisphosphonates (alendronate, risedronate, ibandronate, and zole-dronate), a human monoclonal anti-body to the receptor activator of nuclear factor κB ligand (denosumab), parathyroid hormone/parathyroid hormone-related peptide analogues (teriparatide and abaloparatide), and a humanised monoclonal antibody to sclerostin (romosozumab).

Lophatherum gracile Bronghiart Suppresses Receptor Activator of Nuclear Factor Kappa-B Ligand-Stimulated Osteoclastogenesis and Prevents Ovariectomy-Induced Osteoporosis.

Lophatherum gracile Bronghiart, used in traditional herbal medicine, has many biological properties including antiviral, antipyretic, antitumor, vasorelaxation, and neutrophilic inflammatory effects. However, its modulatory effects on bone metabolism have not been investigated previously. In this study, we examined the effects of a water extract of the leaves of L. gracile (WELG) on osteoclast differentiation and bone loss, and explored its underlying mechanisms. We found that WELG inhibits osteoclastogenesis by suppressing both receptor activator of nuclear factor-κB ligand (RANKL)-induced early activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB)- and RANKL-induced modulation of the positive and negative regulators of osteoclastogenesis in osteoclast precursors. In vivo study demonstrated that WELG protects against bone loss, weight gain, and fat accumulation without affecting uterine atrophy in an ovariectomy-induced postmenopausal osteoporosis mice model. In addition, photochemical analysis of WELG identified active constituents known to have bone-protective effects. Overall, the results of this study suggest that WELG can be a potential candidate for therapy and prevention of postmenopausal osteoporosis.

Interleukin-13 reduces bone erosion in rheumatoid arthritis by up-regulating osteoprotegerin expression in fibroblast-like synoviocytes : an in vitro and in vivo study.

Bone erosion in rheumatoid arthritis (RA) is partly caused by excessive activation of osteoclasts. Osteoclasts can be derived from RA synovium and their differentiation can be inhibited by osteoprotegerin (OPG), a decoy receptor of the osteoclastogenesis-promoting cytokine receptor activator of nuclear factor κB ligand (RANKL). Fibroblast-like synoviocytes (FLSs) are the main stromal cells in the synovium that can secret OPG. The OPG secretion of FLSs can be modulated by various cytokines. Interleukin (IL)-13 can alleviate bone erosion in RA mouse models, but the mechanisms remain unclear. Therefore, we aimed to investigate whether IL-13 can induce OPG secretion by RA-FLSs, thus ameliorating bone destruction in RA by inhibiting osteoclast differentiation. OPG, RANKL, and IL-13 receptors expression by RA-FLSs were evaluated by RT-qPCR. OPG secretion was determined by ELISA. Western blot was performed to analyse OPG expression and the activation of the STAT6 pathway. IL-13 and (or) OPG siRNA pre-treated RA-FLSs conditioned medium were used in osteoclast induction to test if IL-13 can inhibit osteoclastogenesis by up-regulating OPG in RA-FLSs. Micro-CT and immunofluorescence were performed to determine if IL-13 can induce OPG expression and alleviate bone erosion in vivo. IL-13 can promote OPG expression of RA-FLSs, and the promotion can be overcome by IL-13Rα1 or IL-13Rα2 siRNA transfection, or STAT6 inhibitor. Osteoclast differentiation can be inhibited by IL-13 pre-treated RA-FLSs conditioned medium. The inhibition can be reversed by OPG siRNA transfection. IL-13 injection can increase OPG expression in the joints while reducing bone destruction in collagen-induced arthritis mice. IL-13 can inhibit osteoclastogenesis by up-regulating OPG in RA-FLSs through IL-13 receptors via the STAT6 pathway, thus may ameliorate bone erosion in RA.

Bone-sparing effects of tocilizumab in rheumatoid arthritis: a monocentric observational study.

Bone loss is a common feature in several autoimmune and chronic inflammatory diseases, such as rheumatoid arthritis (RA). Indeed, the high levels of pro-inflammatory cytokines seem to enhance bone resorption and to diminish bone formation, thus producing an uncoupling between osteoclast and osteoblast function and favoring the onset of juxtarticular as well as systemic osteoporosis. Many papers underline the high prevalence of osteoporosis in RA, as well as the negative correlation between interleukin 6 (IL-6) serum levels and bone mineral density (BMD). The aim of this study was to assess the effectiveness of one-year treatment with tocilizumab (TCZ), the first approved IL-6 receptor inhibitor, in reducing bone loss in RA. We enrolled 18 patients fulfilling 2010 ACR and EULAR criteria for RA from our arthritis outpatient clinic, assessing clinical and biochemical parameters during a 12-month period. The patients received TCZ 8 mg/kg i.v. every 4 weeks and underwent dual energy X-ray absorptiometry (DXA) for the measurement of bone mineral density (BMD) at baseline and at the end of study. Serum levels of C-reactive protein (CRP), erythrocytes sedimentation rate (ESR), IL-6, serum CrossLaps, osteoprotegerin (OPG), receptor activator of nuclear factor κβ ligand (RANK-L) and dickkopf-1 (DKK-1) were measured at baseline, at 6 months and 1 year. No significant difference in IL-6, RANK-L, DKK-1, OPG and serum CrossLaps levels between baseline, 6 months and 1 year were found. A significant increase of lumbar spine BMD was evidenced after 1 year of TCZ treatment. No difference in total body and femoral neck BMD was documented the end of the study. This study suggest the bone-sparing effect of TCZ in RA affected individuals.

Hypoxia-inducible factor 1α enhances RANKL-induced osteoclast differentiation by upregulating the MAPK pathway

Hypoxia (low-oxygen tension) and excessive osteoclast activation are common conditions in many bone loss diseases, such as osteoporosis, rheumatoid arthritis (RA), and pathologic fractures. Hypoxia-inducible factor 1 alpha (HIF1α) regulates cellular responses to hypoxic conditions. However, it is not yet known how HIF1α directly affects osteoclast differentiation and activation. This study sought to. explore the effects of HIF1α on osteoclast differentiation and it's molecular mechanisms. L-mimosine, a prolyl hydroxylase (PHDs) domain inhibitor, was used to stabilize HIF1α in normoxia. In the presence of receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL), RAW264.7 cells were cultured and stimulated by treatment with L-mimosine at several doses to maintain various levels of intracellular HIF1α. The multi-nucleated cells were assessed by a tartrate-resistant acid phosphatase (TRAP) and F-actin ring staining assays. The osteoclast-specific genes, such as Cathepsin K, β3-Integrin, TRAP, c-Fos, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and matrix metallo-proteinase 9 (MMP9), were analyzed by real time-polymerase chain reaction (RT-PCR). The expression of relevant proteins was analyzed by Western blot. L-mimosine increased the content of intracellular HIF1α in a dose-dependent manner, which in turn promoted RANKL-induced osteoclast formation and relevant protein expression by upregulating the mitogen-activated protein kinase (MAPK) pathways. Our findings suggest that HIF1α directly increases the osteoclast differentiation of RANKL-mediated RAW264.7 cells in vitro by upregulating the MAPK pathways.

Parkin Inhibits RANKL-Induced Osteoclastogenesis and Ovariectomy-Induced Bone Loss

Osteoporosis and osteoporotic fractures comprise a substantial health and socioeconomic burden. The leading cause of osteoporosis is an imbalance in bone formation and bone resorption caused by hyperactive osteoclasts. Therefore, a new strategy to suppress osteoclastogenesis is needed. Parkin is likely closely associated with bone metabolism, although its role in osteoclastogenesis is unclear. In this study, the Parkin protein inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation, osteoclast-specific gene expression, F-actin ring formation, and bone resorption pit formation in vitro. Moreover, depletion of Parkin enhanced RANKL-induced osteoclast formation, osteoclast-specific gene expression, F-actin ring formation, and bone resorption pit formation. Reactive oxygen species (ROS) activity was suppressed, while autophagy was upregulated with the presence of the Parkin protein. ROS activity was upregulated and autophagy was decreased due to Parkin knockdown. In addition, intravenous administration of Parkin rescued ovariectomy-induced bone loss and reduced osteoclastogenesis in vivo. Collectively, Parkin has therapeutic potential for diseases associated with overactive osteoclasts.

Circulating Extracellular Vesicles Express Receptor Activator of Nuclear Factor κB Ligand and Other Molecules Informative of the Bone Metabolic Status of Mouse Models of Experimentally Induced Osteoporosis

Extracellular vesicles (EVs) are potent means of cell-to-cell communication. They are released in biological fluids, including blood, urine, and saliva, and can be exploited to identify new biomarkers of diseases. We hypothesized that EVs contain molecular cargos involved in bone metabolism, possibly mirroring biological differences between postmenopausal and disuse osteoporosis. We tested this hypothesis in primary murine osteoblasts subjected to steroid depletion or to unloading, and in the serum of animal models of osteoporosis induced by ovariectomy or hindlimb tail suspension. EVs were isolated by ultracentrifugation and analysed by transmission electron microscopy, cytofluorimetry, immunoblotting and RT-PCR. Large-scale analyses were performed by Real-Time arrays and Proteome Profiler™ Antibody arrays. Finally, precise titration of analytes was carried out by ELISA assay. In vitro, we confirmed an increased release of EVs enriched in surface RANKL by primary mouse osteoblasts subjected to steroid depletion or simulated microgravity compared to controls. In vivo, circulating EVs isolated from the sera of control female mice expressed RANKL along with other genes associated with bone metabolism. Serum EVs from ovariectomized or hindlimb tail-suspended mice showed distinct molecular profiles. They expressed RANKL with different kinetics, while transcriptomic and proteomic profiles uncovered unique molecular signatures that discriminated the two conditions, unveiling exclusive molecules expressed in time- and osteoporosis type-dependent manner. These results suggest that circulating EVs could represent a new tool for monitoring the onset and the progression of diverse types of the disease in mice, paving the way for their exploitation to diagnose human osteoporosis in liquid biopsies.

Inhibitory effect of infliximab on orthodontic tooth movement in male rats

ObjectiveThis study aimed to investigate the effect of infliximab on orthodontic tooth movement in rats. Materials and methodsSixty male Sprague-Dawley rats were randomly divided into two groups: saline group and infliximab group. The two groups of rats received weekly intraperitoneally injection of saline or infliximab (5 mg/kg), respectively. After four weeks of injection, five rats in each group were euthanized and orthodontic appliances were placed in the other twenty-five rats each. On days 1, 3, 7, 14 and 21, five rats from each group were euthanised. Maxillae of all the rats were collected and examined by micro-computed tomography, haematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, and immunohistochemical staining of tumour necrosis factor (TNF)-α, receptor activator of nuclear factor κB ligand (RANKL), receptor activator of nuclear factor κB (RANK), and osteoprotegerin (OPG). All data were analysed with Mann-Whitney test. ResultsInfliximab inhibited orthodontic tooth movement and decreased osteoclastogenesis on the compression side during orthodontic tooth movement. The elevated TNF­α level, induced by orthodontic force, was decreased by infliximab. Furthermore, infliximab reduced the expression of RANKL and RANK, while increased the expression of OPG on the compression side. ConclusionInfliximab inhibits orthodontic tooth movement by reducing levels of TNF-α, RANKL, and RANK, while increasing level of OPG, and decreasing osteoclastogenesis on the compression side of periodontium.

Serum fetuin-A and RANKL levels in patients with early stage breast cancer.

Breast cancer (BC) is the primary cause of mortality due to cancer in females around the world. Fetuin-A is known to increase metastases over signals and peroxisomes related with growing. Receptor activator of nuclear factor-kB ligand (RANKL) takes part in cell adhesion, and RANKL inhibition is used in the management of cancer. We aimed to examine the relationship between serum fetuin-A, RANKL levels, other laboratory parameters and clinical findings in women diagnosed with early stage BC, in our population. Women having early stage BC (n=117) met our study inclusion criteria as they had no any anti-cancer therapy before. Thirty-seven healthy women controls were also confirmed with breast examination and ultrasonography and/or mammography according to their ages. Serum samples were stored at -80°C and analysed via ELISA. Median age of the patients was 53 (range: 57-86) while it was 47 (range: 23-74) in the healthy group. Patients had lower high-density lipoprotein levels (p=0.002) and higher neutrophil counts (p=0.014). Fetuin-A and RANKL levels did not differ between the groups (p=0.116 and p=0.439, respectively) but RANKL leves were found to be lower in the favorable histological subtypes (p=0.04). In this study, we found no correlation between serum fetuin-A levels and clinical findings in patients diagnosed with early stage BC. However, RANKL levels are found to be lower in subgroups with favorable histopathologic subtypes such as tubular, papillary and mucinous BC and there was statistically significant difference.

Bone vitality and vascularization of mandibular and maxillary bone grafts in maxillary sinus floor elevation: A retrospective cohort study.

Mandibular retromolar (predominantly cortical) and maxillary tuberosity (predominantly cancellous) bone grafts are used in patients undergoing maxillary sinus floor elevation (MSFE) for dental implant placement. The aim of this retrospective cohort study was to investigate whether differences exist in bone formation and vascularization after grafting with either bone source in patients undergoing MSFE. Fifteen patients undergoing MSFE were treated with retromolar (n= 9) or tuberosity (n= 6) bone grafts. Biopsies were taken 4months postoperatively prior to dental implant placement, and histomorphometrically analyzed to quantify bone and osteoid area, number of total, apoptotic, and receptor activator of nuclear factor-κB ligand (RANKL)-positive osteocytes, small and large-sized blood vessels, and osteoclasts. The grafted area was divided in three regions (caudal-cranial): RI, RII, and RIII. Bone volume was 40% (RII, RIII) higher and osteoid volume 10% (RII) lower in retromolar compared to tuberosity-grafted areas. Total osteocyte number and number of RANKL-positive osteocytes were 23% (RII) and 90% (RI, RII) lower, but osteoclast number was higher (retromolar: 12, tuberosity: 0) in retromolar-grafted areas. The total number of blood vessels was 80% (RI) to 60% (RIII) lower, while the percentage of large-sized blood vessels was 86% (RI) to 25% (RIII) higher in retromolar-grafted areas. Number of osteocyte lacunae and apoptotic osteocytes were similar in both bone grafts used. Compared to the retromolar bone, tuberosity bone showed increased bone vitality and vascularization in patients undergoing MSFE, likely due to faster bone remodeling or earlier start of new bone formation. Therefore, tuberosity bone grafts might perform better in enhancing bone regeneration.

Study on mechanism of Rehmanniae Radix Praeparata for treatment of osteoarthritis based on network pharmacology and molecular docking

The mechanism of Rehmanniae Radix Praeparata against osteoarthritis was investigated based on network pharmacology, molecular docking, and in vitro experiments in the present study. Osteoclast models were established via receptor activator of nuclear factor-κB ligand(RANKL) and macrophage colony-stimulating factor(M-CSF) inducing RAW264.7 cells. Further, the influence of Rehmanniae Radix Praeparata on the activity of tartrate-resistant acid phosphatase(TRAP) was evaluated and the efficacy of Rehmanniae Radix Praeparata in the treatment of osteoarthritis was verified. The active components of Rehmanniae Radix Praeparata were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and literature, and the potential targets of the components were collected from SwissTargetPrediction. Osteoarthritis disease targets were searched in Online Mendelian Inheritance in Man(OMIM), Therapeutic Target Database(TTD), GeneCards, and DisGeNET. The intersection targets of Rehmanniae Radix Praeparata and osteoarthritis were obtained by Venny platform. The protein-protein interaction(PPI) network was constructed by Cytoscape 3.8.2, and key targets were obtained based on topology algorithm. The Database for Annotation, Visualization and Integrated Discovery(DAVID) was used to perform Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. Finally, the mRNA expression of the key targets was determined by RT-qPCR and the binding activity between the components and key targets was validated by molecular docking. The results showed that Rehmanniae Radix Prae-parata inhibited the TRAP activity, thus inhibiting bone resorption by osteoclasts and treating osteoarthritis. By network pharmacology, 14 active components of Rehmanniae Radix Praeparata and 126 intersection targets were obtained. The network pharmacology enrichment results revealed 432 biological processes and 139 signaling pathways. Key targets such as proto-oncogene tyrosine-protein kinase Src(SRC), signal transducer and activator of transcription 3(STAT3) and transcription factor p65(RELA) were obtained according to the degree in topological analysis. SRC was highly expressed in osteoclasts, which accelerated the development of osteoarthritis. Therefore, SRC was selected for subsequent verification, and Rehmanniae Radix Praeparata decreased the gene expression level of SRC. The molecular docking showed that acteoside, isoacteoside, raffinose had good bonding activity with SRC, suggesting that they might be the critical components in treating osteoarthritis. In conclusion, Rehmanniae Radix Praeparata can inhibit bone resorption by osteoclasts and balance the metabolism of articular cartilage and subchondral bone via acting on SRC, thus playing a therapeutic role in osteoarthritis. In addition, Rehmanniae Radix Praeparata may exert overall efficacy on osteoarthritis through other targets such as STAT3 and RELA, and other related pathways such as PI3 K-AKT and IL-17 signaling pathways.

Oat Seedlings Extract Inhibits RANKL-Induced c-Fos/NFATc1 Transcription Factors in the Early Stage of Osteoclast Differentiation

Osteoporosis is a common disease that increases the risk of fractures due to decreased bone density and weakens the bone microstructure. Preventing and diagnosing osteoporosis using the available drugs can be a costly affair with possible side effects. Therefore, natural product-derived therapeutics are promising alternatives. Our study demonstrated that the oat seedlings' extract (OSE) inhibited the receptor activator of the nuclear factor κB ligand (RANKL)-induced osteoclastogenesis from the bone marrow-derived macrophages (BMMs). The OSE treatment significantly attenuated the RANKL-mediated induction of the tartrate-resistant acid phosphatase (TRAP) activity as well as the number of TRAP-positive (TRAP+) multinucleated cells (MNCs) counted through the TRAP staining in a dose-dependent manner. It was also confirmed that the OSE suppressed the formation of the TRAP + MNCs in the early stage of differentiation and not in the middle and late stages. The results of the real-time quantitative polymerase chain reaction (qPCR) and the western blotting showed that the OSE dramatically inhibited the mRNA and protein expressions of the osteoclastogenesis-mediated transcription factors such as the c-Fos and the nuclear factor-activated T cells c1 (NFATc1). In addition, the OSE strongly attenuated the mRNA induction of the c-Fos/NFATc1-dependent molecules such as the TRAP, the osteoclast-associatedimmunoglobulin-like receptor (OSCAR), the dendritic cell-specific transmembrane protein (DC-STAMP), and the cathepsin K. These results suggest that the naturally derived OSE may be useful for preventing bone diseases.

Inhibition of Neddylation Suppresses Osteoclast Differentiation and Function In Vitro and Alleviates Osteoporosis In Vivo.

Neddylation, or the covalent addition of NEDD8 to specific lysine residue of proteins, is a reversible posttranslational modification, which regulates numerous biological functions; however, its involvement and therapeutic significance in osteoporosis remains unknown. Our results revealed that during the soluble receptor activator of nuclear factor-κB ligand (sRANKL)-stimulated osteoclast differentiation, the neddylation and expression of UBA3, the NEDD8-activating enzyme (NAE) catalytic subunit, were dose- and time-dependently upregulated in RAW 264.7 macrophages. UBA3 knockdown for diminishing NAE activity or administering low doses of the NAE inhibitor MLN4924 significantly suppressed sRANKL-stimulated osteoclast differentiation and bone-resorbing activity in the macrophages by inhibiting sRANKL-stimulated neddylation and tumor necrosis factor receptor-associated factor 6 (TRAF6)-activated transforming growth factor-β-activated kinase 1 (TAK1) downstream signaling for diminishing nuclear factor-activated T cells c1 (NFATc1) expression. sRANKL enhanced the interaction of TRAF6 with the neddylated proteins and the polyubiquitination of TRAF6’s lysine 63, which activated TAK1 downstream signaling; however, this process was inhibited by MLN4924. MLN4924 significantly reduced osteoporosis in an ovariectomy- and sRANKL-induced osteoporosis mouse model in vivo. Our novel finding was that NAE-mediated neddylation participates in RANKL-activated TRAF6–TAK1–NFATc1 signaling during osteoclast differentiation and osteoporosis, suggesting that neddylation may be a new target for treating osteoporosis.

A bibliometric research based on hotspots and frontier trends of denosumab.

Denosumab is a monoclonal antibody that targets and inhibits the osteoclast activating factor receptor activator for nuclear factor-κB ligand (RANKL). It has been widely used in the treatment of osteoporosis, giant cell tumors of bone, and in the prevention of malignant skeletal-related events (SREs). We collected the research results and related MeSH terms of denosumab from 2011 to 2021 through the Web of Science and PubMed, respectively. The literature was visualized and analyzed by CiteSpace and bibliometric online analysis platforms. The MeSH terms were biclustered using the Bibliographic Co-Occurrence Analysis System (BICOMB) and graph clustering toolkit (gCLUTO). The results show that the number of denosumab-related annual publications had increased from 51 to 215, with the United States leading and Amgen Inc. being the most influential in the past 10 years. Articles published in the Journal of Bone and Mineral Research had the highest total citations. Three scholars from Shinshu University in Matsumoto, Yukio Nakamura, Takako Suzuki, and Hiroyuki Kato, joined the field relatively late but produced the most. The clinical comparison and combination of denosumab with other drugs in the treatment of osteoporosis was the most significant focus of research. Drug withdrawal rebound and management strategies have gained more attention and controversy recently. MeSH analysis revealed eight major categories of research hotspots. Among them, exploring the multiple roles of the RANK-RANKL-OPG system in tumor progression, metastasis, and other diseases is the potential direction of future mechanism research. It is a valuable surgical topic to optimize the perioperative drug administration strategy for internal spinal fixation and orthopedic prosthesis implantation. Taken together, the advantages of denosumab were broad and cost-effective. However, there were still problems such as osteonecrosis of the jaw, severe hypocalcemia, a high recurrence rate of giant cells in the treatment of bone and individual sarcoidosis, and atypical femoral fractures, which need to be adequately solved.

Structural insights into the binding of zoledronic acid with RANKL via computational simulations.

Zoledronic acid (ZOL) inhibits receptor activator of nuclear factor-κB ligand (RANKL) and reduces bone turnover. This plays an important role in the development of bisphosphonate-related osteonecrosis of the jaw (BRONJ). Previous reports have shown that ZOL binds to the enzyme farnesyl pyrophosphate synthase (FPPS) to block its activity. However, the mechanism of action of ZOL and its interaction with RANKL is still unclear. In this study, we confirmed that ZOL significantly suppressed the bone remodeling in ZOL-treated rats, investigated whether ZOL could bind to RANKL and examined the interactions between these molecules at the atomic level. Surface plasmon resonance (SPR) assay was performed to validate that ZOL could directly bind to RANKL in a dose dependent manner, and the equilibrium constant was calculated (KD = 2.28 × 10−4 M). Then, we used molecular docking simulation to predict the binding site and analyze the binding characteristics of ZOL and RANKL. Through molecular dynamics simulation, we confirmed the stable binding between ZOL and RANKL and observed their dynamic interactions over time. Binding free energy calculations and its decomposition were conducted to obtain the binding free energy −70.67 ± 2.62 kJ/mol for the RANKL–ZOL complex. We identified the key residues of RANKL in the binding region, and these included Tyr217(A), Val277(A), Gly278(A), Val277(B), Gly278(B), and Tyr215(C). Taken together, our results demonstrated the direct interaction between ZOL and RANKL, indicating that the pharmacological action of ZOL might be closely related to RANKL. The design of novel small molecules targeting RANKL might reduce the occurrence of BRONJ.

Evaluation of the oxytocin effect in a rat model with experimental periodontitis.

The aim of the present study was to evaluate the inhibitory effects of oxytocin on the development of periodontitis based on its properties against bone loss and resorption. Thirty-two Wistar albino rats were divided into four equal groups: control, periodontitis + saline, periodontitis + 0.5mg/kg/day oxytocin, and periodontitis + 1mg/kg/day oxytocin. Periodontitis groups received 4.0 silk ligatures around their cervixes of the right and left mandibular incisors in an "8" shape, kept for 14days. Animals in oxytocin groups were injected once every day during 14days with oxytocin. The mandibles were fixed and scanned using microcomputed tomography to quantify bone resorption and volumetric measurements. Blood samples were collected to analyze the concentrations of macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor-κΒ ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase-8 (MMP-8), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, glutathione peroxidase (GPx), superoxide dismutase (SOD), and malondialdehyde (MDA). Histopathological evaluations were conducted to examine the gingiva and alveolar bone. Oxytocin prevented the development of periodontitis by decreasing ligament deteriorations and leukocytes in the gingival connective tissue and promoting reintegration with the alveolar bone. Bone resorption in all regions was less in the periodontitis + 1mg/kg/day oxytocin group than in the periodontitis + saline group. Although TNF-α, IL-6, and RANKL values were lower in the periodontitis + 1mg/kg/day oxytocin group, OPG was higher than that in the periodontitis + saline group. M-CSF, MMP-8, and MDA were lower in the oxytocin groups than in the periodontitis + saline group. Oxytocin may be an effective agent for periodontal diseases because it decreased bone resorption, oxidative stress, and inflammation in an experimental periodontitis.

Ershiwuwei Lvxue Pill alleviates rheumatoid arthritis by different pathways and produces changes in the gut microbiota

BackgroundRheumatoid arthritis (RA) is a systemic autoimmune disease that often results in joint destruction. Ershiwuwei Lvxue Pill (ELP), a prescription of Tibetan medicine, has been used for centuries for the clinical treatment of RA in Tibet, China. In a previous study, we reported that ELP could ameliorate RA symptoms in CIA rats by inhibiting the inflammatory response and inducing apoptosis in synovial tissues. It is still needed further to clarify the mechanisms of action of ELP in mitigating RA. PurposeIn this study, we aim to elucidate the mechanism of action of ELP to improve RA joint damage and explore the changes in the intestinal flora and host metabolites. MethodsFirstly, we analyzed the main absorbed constituents of ELP in the serum of rats by ultra-performance liquid chromatography quadrupole-time-flight mass spectrometry (UPLC-Q-TOF/MS). Then, we verified the alleviating effects of ELP on cartilage injury and bone erosion as well as the inflammatory response in CIA rats by microCT, H&E staining, safranin-O staining, and ELISA. Moreover, we investigated the main factors that mediate joint damage, including the production of matrix metalloproteinases (MMPs) and osteoclast activity in the ankle of rats by immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) staining. Further, we explored the molecular mechanisms of the MMPs production and osteoclast activity in CIA rats treated with ELP through various experiments such as ELISA, qRT-PCR, western blotting, and immunofluorescence assay. Besides, we investigated gut microbiota composition by 16S rDNA sequencing and serum metabolites through untargeted metabolomics. In addition, we analyzed the correlation between gut microbiota and metabolites by Spearman correlation analysis. ResultsIn this study, we identified 20 compounds from rat serum samples, which could be the ELP components that improve RA. Moreover, we found that ELP could alleviate cartilage and bone injury by reducing MMP-1, MMP-3, and MMP-13 expression and osteoclast activity in CIA rats. Further studies demonstrated that ELP could reduce joint damage by inhibiting osteoprotegerin (OPG)/receptor activator for nuclear factor-κB ligand (RANKL) /nuclear factor-κB (NF-κB) and extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinases (JNK) signal pathways. The 16S rDNA sequencing analysis indicated that there was a significant difference in the gut microbiota composition between the normal and CIA rats, and these differences were changed after ELP administration. ELP could alter the gut microbiota by increasing the abundance of the genus Lactobacillus and decreasing the abundance of Dorea, [Eubacterium]_ventriosum_group, Anaerostipes, Collinsella, Coprococcus_1, Ruminiclostridium_5, Ruminococcus_1, Family_XIII_UCG-001, Butyricicoccus, Erysipelotrichaceae_UCG-003, Lachnoclostridium, Faecalibacterium, Lachnospiraceae_UCG-010, Roseburia, Rs-E47_termite_group_norank, Treponema_2 genera. Non-targeted metabolomics analysis showed that ELP reduced arachidonic acid levels. The serum arachidonic acid level was significantly correlated with the abundance of 41 genera, particularly Collinsella and Lactobacillus. ConclusionOur study shows that ELP can improve RA joint damage by inhibiting MMPs production and osteoclast activity, and regulating intestinal flora and host metabolites, which provides a novel insight into the ELP in alleviating RA.

MCU Inhibitor Ruthenium Red Alleviates the Osteoclastogenesis and Ovariectomized Osteoporosis via Suppressing RANKL-Induced ROS Production and NFATc1 Activation through P38 MAPK Signaling Pathway.

Osteoporosis is a disorder of bone metabolism that is extremely common in elderly patients as well as in postmenopausal women. The main manifestation is that the bone resorption capacity is greater than the bone formation capacity, which eventually leads to a decrease in bone mass, increasing the risk of fracture. There is growing evidence that inhibiting osteoclast formation and resorption ability can be effective in treating and preventing the occurrence of osteoporosis. Our study is the first time to explore the role of the mitochondrial calcium uniporter (MCU) and its inhibitor ruthenium red (RR) in bone metabolism, clarifying the specific mechanism by which it inhibits osteoclast formation in vitro and plays a therapeutic role in osteoporosis in vivo. We verified the suppressive effects of RR on the receptor activator of nuclear factor-κB ligand (RANKL-)-induced differentiation and bone resorption function of osteoclasts in vitro. The reactive oxygen species (ROS) production stimulated by RANKL and the expression level of P38 MAPK/NFATc1 were also found to be inhibited by RR. Moreover, the promotion of RR on osteogenesis differentiation was investigated by alkaline phosphatase (ALP) and alizarin red S (ARS) staining and the detection of osteogenesis-specific gene expression levels by quantitative polymerase chain reaction (qPCR) and western blotting. Moreover, in ovariectomy (OVX-)-induced osteoporosis models, RR can downregulate the expression and function of the MCU, relieving bone loss and promoting osteogenesis to present a therapeutic effect on osteoporosis. This new finding will provide an important direction for the study of RR and MCU in the study of bone metabolism therapy targets.