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Visfatin, an adipokine secreted by various cell types, plays multifaceted pathophysiological roles in inflammatory conditions, including obesity, which is closely associated with osteoclastogenesis, a key process underlying bone loss and increased osteoporosis (OP) risk. However, the role of visfatin in osteoclastogenesis remains controversial. This study was conducted to investigate the effects of visfatin on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation from precursor cells in vitro. Our results demonstrated that although visfatin exhibited a modest osteoclast-inductive effect relative to that of RANKL, co-stimulation of bone marrow-derived macrophages (BMDMs) with visfatin and RANKL led to significantly enhanced osteoclast differentiation and activation compared to individual stimulation. Neutralization of visfatin activity using blocking antibodies before differentiation markedly suppressed RANKL-induced osteoclastogenesis, as evidenced by a near-complete absence of tartrate-resistant acid phosphatase-positive multinucleated osteoclasts, decreased levels of nuclear factor of activated T cells cytoplasmic 1 and osteoclast-specific proteins, inhibition of nuclear factor-κB and mitogen-activated protein kinase signaling pathways, and a decrease in resorption pit formation. Our findings underscore the critical role of visfatin in RANKL-induced osteoclastogenesis in vitro and highlight the RANKL/visfatin signaling axis as a potential therapeutic target for destructive bone loss-related diseases. [ABSTRACT FROM AUTHOR]

Bone regeneration is a highly dynamic and complex process that involves hematopoietic stem cells and mesenchymal cells, collagen fibers, non-collagenous proteins and biomolecules from extracellular matrices, and different cytokines and immune cells, as well as growth factors and hormones. Some phytochemicals due to antioxidant and anti-inflammatory effects can modulate the bone signaling pathways and improve bone healing and thus can be a good candidate for osteoregeneration. The aim of this study was to analyze the impact of Juglans regia L. extract compared to ellagic acid on bone neoformation in rats. The animals with a 5 mm calvaria defect were divided into four groups (n = 10): group 1 was treated with ellagic acid 1% (EA), group 2 was treated with Juglans regia L. extract 10% (JR), group 3 was treated with a biphasic mix of hydroxyapatite and tricalcium phosphate (Ceraform), and group 4 was treated with vehicle inert gel with carboxymethylcellulose (CMC). After 3 weeks of treatment, blood samples were collected for oxidative stress and inflammation assessment. Additionally, the receptor activator of nuclear factor kappa-Β ligand (RANKL) and hydroxyproline levels were quantified in blood. The skull samples were analyzed by scanning electron microscopy in order to detect the modifications in the four groups. The results suggested that JR extract had relevant anti-oxidant effect and bone protective activity and generated the accumulation of Ca and P, demonstrating the potential therapeutic abilities in bone regeneration. [ABSTRACT FROM AUTHOR]

Platelet-rich plasma (PRP) is utilized as an autologous blood product to encourage bone regeneration. The receptor activator of nuclear factor-NB ligand (RANKL) is a key and central regulator of osteoclast homeostasis. A rat model of experimentally generated periodontitis was used to assess the impact of PRP preparation on the expression of the osteoclastogenic and pro-inflammatory markers respectively; RANKL and IL-1β. To induce periodontitis by silk ligature, thirty-six adult male Sprague Dawleys rats were used and they were allocated into three equal groups (n = 12): group I consisted of intact periodontal tissue, group II; rat-induced periodontitis without treatment by PRP, and group III of periodontitis + 10 µL PRP injection. The rats were sacrificed after both experiment durations (7 and 30 days), and the incisor teeth were fixed and decalcified in HCl for a day and in 10 % EDTA solution for eight weeks at room temperature then samples were processed for H&E stain for bone healing scores and bone cells counting, and the samples were utilized by IHC for detection of both RANKL and IL-1β expression. The PRP enhanced the process of healing on days 7 and 30 showed (Score 10) vs. the control positive group that had a delay in alveolar bone regarded as (Score 4) significantly (P ≤ 0.05). The PRP group attenuated significantly (P ≤ 0.05) the alveolar bone loss by increasing the number of osteoblasts and declining the proliferation of osteoclast vs. the control positive group that revealed bone destruction due to rising osteoclast proliferation and decreasing the osteoblast proliferation significantly (P ≤ 0.05). PRP inhibited the IL-1β expression (score = 0) vs. the control positive group that showed moderate staining of positive cells detected in both inflammatory cells and endothelium (score = 4). Regarding the RANKL expression, the PRP reduced its expression in vs. the control positive group (score = 4 vs. 12 respectively). PRP is an anabolic agent that enhances proliferation of osteoblast and inhibit the osteoclast differentiation by downregulation of IL-1β and RANKL. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests a common physiological process for bone and coronary artery calcification (CAC), implying the role of bone metabolism markers in subclinical atherosclerosis development. However, the association between bone turnover markers and the development of CAC has remained controversial, as seen in various studies. Because CAC measurement has both financial burden and radiation exposure risk in individuals with suspected cardiovascular disease (CVD), applying the diagnostic role of osteogenic markers in predicting abnormal CAC would improve treatment adherence and reduce the rate of CVD mortality. In this review, we begin by describing the current understanding of the molecular mechanisms of bone markers in the etiology of CAC. Furthermore, we summarize bone-associated regulatory factors at the molecular level as novel therapeutic targets for CAC. In addition, we focused on the current results on the prognostic role of novel mediators of osteogenic activity in determining the risk of CAC as a preclinical factor of atherosclerotic CVD. Accumulating evidence suggests the role of bone marker-mediated pathways in the progression of CAC, which may lead to early diagnosis of CVD complications and the establishment of innovative targets for pharmacological therapy. Indeed, miRNAs and lncRNAs, as novel therapeutic interventions, can be a research priority in regulating bone metabolism at the gene expression level to attenuate high CAC and improve CVD outcomes. [ABSTRACT FROM AUTHOR]

Background: There is an increasing occurrence of periodontitis and metabolic syndrome (MetS), which is resulting in a decline in the overall quality of life. Both disorders can occur together since they are both linked to insulin resistance and systemic inflammation. However, evidence for a role of interleukin (IL)-20 in this comorbidity is very limited. This cross-sectional study aimed to comprehensively investigate, for the first time, the levels of RANKL/OPG, MMP-8 and OSI as well as the role of IL-20 in patients with MetS and periodontitis. Methods: The study included a total of 80 individuals, divided into four groups: 20 individuals who were healthy both systemically and periodontally, 20 individuals who were systemically healthy but had periodontitis, 20 individuals who had MetS but were periodontally healthy, and 20 individuals who had both MetS and periodontitis. Periodontal clinical parameters (plaque index, gingival index, bleeding on probing, clinical attachment loss, probing pocket depth) were evaluated. Gingival crevicular fluid (GGF) and serum samples were collected and used for biochemical assays. Enzyme-linked immunosorbent assay was used to determine the levels of IL-20, receptor activator of nuclear factor kappa B ligand (RANKL)/osteoprotegerin (OPG), matrix metalloproteinase-8 (MMP-8) and oxidative stress index (OSI). Results: IL-20 levels measured in serum and GCF were statistically significantly highest in patients with MetS and periodontitis (p = 0.001). Significant positive correlation was observed between serum and GCF IL-20 values and periodontal parameters (p < 0.05). There was a positive correlation between RANKL and RANKL/OPG levels and IL-20 and clinical parameters (p < 0.05). OSI values were found to be increased in the presence of both periodontitis and MetS (p = 0.001) and were positively correlated with serum and GCF IL-20 (p < 0.05). Conclusions: These data from the study suggest a correlation between IL-20 and both MetS and periodontitis. IL-20 may potentially worsen the condition of periodontal tissue by increasing both the oxidative stress levels, periodontal collagen degredation and the ratio of RANKL to OPG. Trial registration: This study was registered on ClinicTrials.gov (NCT06092853), 2023-10-10, retrospectively registered. [ABSTRACT FROM AUTHOR]

Background: Platelet-rich plasma (PRP) is utilized as an autologous blood product to encourage bone regeneration. The receptor activator of nuclear factor-NB ligand (RANKL) is a key and central regulator of osteoclast homeostasis. A rat model of experimentally generated periodontitis was used to assess the impact of PRP preparation on the expression of the osteoclastogenic and pro-inflammatory markers respectively; RANKL and IL-1β. Material and Methods: To induce periodontitis by silk ligature, thirty-six adult male Sprague Dawleys rats were used and they were allocated into three equal groups (n = 12): group I consisted of intact periodontal tissue, group II; rat-induced periodontitis without treatment by PRP, and group III of periodontitis + 10 µL PRP injection. The rats were sacrificed after both experiment durations (7 and 30 days), and the incisor teeth were fixed and decalcified in HCl for a day and in 10 % EDTA solution for eight weeks at room temperature then samples were processed for H&E stain for bone healing scores and bone cells counting, and the samples were utilized by IHC for detection of both RANKL and IL-1β expression. Results: The PRP enhanced the process of healing on days 7 and 30 showed (Score 10) vs. the control positive group that had a delay in alveolar bone regarded as (Score 4) significantly (P ≤ 0.05). The PRP group attenuated significantly (P ≤ 0.05) the alveolar bone loss by increasing the number of osteoblasts and declining the proliferation of osteoclast vs. the control positive group that revealed bone destruction due to rising osteoclast proliferation and decreasing the osteoblast proliferation significantly (P ≤ 0.05). PRP inhibited the IL-1β expression (score = 0) vs. the control positive group that showed moderate staining of positive cells detected in both inflammatory cells and endothelium (score = 4). Regarding the RANKL expression, the PRP reduced its expression in vs. the control positive group (score = 4 vs. 12 respectively). Conclusion: PRP is an anabolic agent that enhances proliferation of osteoblast and inhibit the osteoclast differentiation by downregulation of IL-1β and RANKL.

Increasing evidence suggests a common physiological process for bone and coronary artery calcification (CAC), implying the role of bone metabolism markers in subclinical atherosclerosis development. However, the association between bone turnover markers and the development of CAC has remained controversial, as seen in various studies. Because CAC measurement has both financial burden and radiation exposure risk in individuals with suspected cardiovascular disease (CVD), applying the diagnostic role of osteogenic markers in predicting abnormal CAC would improve treatment adherence and reduce the rate of CVD mortality. In this review, we begin by describing the current understanding of the molecular mechanisms of bone markers in the etiology of CAC. Furthermore, we summarize bone-associated regulatory factors at the molecular level as novel therapeutic targets for CAC. In addition, we focused on the current results on the prognostic role of novel mediators of osteogenic activity in determining the risk of CAC as a preclinical factor of atherosclerotic CVD. Accumulating evidence suggests the role of bone marker-mediated pathways in the progression of CAC, which may lead to early diagnosis of CVD complications and the establishment of innovative targets for pharmacological therapy. Indeed, miRNAs and lncRNAs, as novel therapeutic interventions, can be a research priority in regulating bone metabolism at the gene expression level to attenuate high CAC and improve CVD outcomes.

Abstract Background There is an increasing occurrence of periodontitis and metabolic syndrome (MetS), which is resulting in a decline in the overall quality of life. Both disorders can occur together since they are both linked to insulin resistance and systemic inflammation. However, evidence for a role of interleukin (IL)-20 in this comorbidity is very limited. This cross-sectional study aimed to comprehensively investigate, for the first time, the levels of RANKL/OPG, MMP-8 and OSI as well as the role of IL-20 in patients with MetS and periodontitis. Methods The study included a total of 80 individuals, divided into four groups: 20 individuals who were healthy both systemically and periodontally, 20 individuals who were systemically healthy but had periodontitis, 20 individuals who had MetS but were periodontally healthy, and 20 individuals who had both MetS and periodontitis. Periodontal clinical parameters (plaque index, gingival index, bleeding on probing, clinical attachment loss, probing pocket depth) were evaluated. Gingival crevicular fluid (GGF) and serum samples were collected and used for biochemical assays. Enzyme-linked immunosorbent assay was used to determine the levels of IL-20, receptor activator of nuclear factor kappa B ligand (RANKL)/osteoprotegerin (OPG), matrix metalloproteinase-8 (MMP-8) and oxidative stress index (OSI). Results IL-20 levels measured in serum and GCF were statistically significantly highest in patients with MetS and periodontitis (p = 0.001). Significant positive correlation was observed between serum and GCF IL-20 values and periodontal parameters (p

Background/purpose: Cementum shares many properties with bone; however, in contrast to bone, it is not innervated or vascularized and has a limited capacity for remodeling. Osteocytes located in the lacunae-canalicular system of bone tissue play a central role in bone remodeling by communicating with osteoblasts and osteoclasts. Although cementocytes are present in cellular cementum and are morphologically similar to osteocytes, it remains unclear whether they are involved in the dynamic functional regulation of metabolism in cementum. The present study focused on the extracellular vesicles (EVs) secreted by cementocytes and examined their effects on osteoclasts and osteoblasts. Materials and methods: EVs were extracted from the mouse cementocyte cell line, IDG-CM6. The effects of EVs on recombinant RANKL-induced osteoclastogenesis and recombinant Bone morphogenetic protein (BMP)-2-mediated osteoblastogenesis were investigated using the mouse osteoclast progenitor cell line, RAW264.7 and mouse pre-osteoblast cell line, MC3T3-E1, respectively. Results: EVs enhanced the formation of tartrate-resistant acid phosphatase activity-positive cells. Real-time PCR revealed that EVs up-regulated the expression of osteoclast-related genes. On the other hand, the cell culture supernatant of cementocytes significantly inhibited the differentiation of osteoclasts. Regarding osteoblastogenesis, EVs suppressed the expression of alkaline phosphatase, bone sialoprotein, and osteocalcin induced by recombinant BMP-2 at the gene and protein levels. Conclusion: A network of cementocytes, osteoblasts, and osteoclasts may exist in cellular cementum, which suggests the involvement of cementocytes in dynamic metabolism of cementum through EVs.

Abstract Background Nitrogen-containing bisphosphonate(N-BP)had been found to inhibit the osteogenic differentiation and calcification in vascular smooth muscle cells (VSMCs), but the mechanism is not clear. We intend to verify that N-BP induces enhancement of OPG expression and inhibition of RANKL expression via inhibition of farnesyl pyrophosphate synthase(FPPS) to inhibit the osteogenic differentiation and calcification in VSMCs. Methods β-glycerophosphate (β-GP) was used to induce the osteogenic differentiation and calcification in VSMCs. VSMCs were treated with N-BP or pretreated with downstream products of farnesyl pyrophosphate synthase(FPPS) in mevalonate pathway, such as farnesol (FOH) or geranylgeraniol (GGOH). Alizarin red S staining and determination of calcium content were used to detect calcium deposition.Western Blotting were used to detect expressions of proteins(OPG and RANKL ) and osteogenic marker proteins (Runx2 and OPN). Results β-GP induced the osteogenic differentiation and calcification in VSMCs, increased RANKL protein expression and had no significant effect on OPG protein expression. With the treatment of N-BP, the expression of OPG protein was increased and expression of RANKL protein was decreased in VSMCs undergoing osteogenic differentiation and calcification. In addition, N-BP reduced the osteogenic marker proteins (Runx2 and OPN) expression and calcium deposition in VSMCs undergoing osteogenic differentiation and calcification. These effects of N-BP on the osteogenic differentiation and calcification in VSMCs were concentration-dependent, which could be reversed by the downstream products of FPPS, such as FOH or GGOH. Conclusion N-BP increases OPG expression and decreases RANKL expression via inhibition of FPPS to inhibit the osteogenic differentiation and calcification in VSMCs.

Abstract Introduction Osteoporosis, a systemic skeletal disease, is characterized by a quantitative and qualitative, and progressive decrease in bone mass, which is related to inflammation. Since a cytokine storm is triggered in Coronavirus disease 2019 (COVID-19), this study aims to evaluate pro-inflammatory cytokines (TNF-α, IL-1β), Receptor activator of nuclear factor-κB ligand (RANKL)/serum osteoprotegerin (OPG) ratio, and their relationship in mild and severe COVID-19. Methods This study was performed on 48 adult patients (18 mild, 18 severe COVID-19, and 12 healthy subjects as a control group). Serum OPG, RANKL, TNF-α, IL-1β, 25-OH vitamin D, and ALKp were measured by ELISA and colorimetric assay. Results COVID-19 patients had a significant increase in RANKL, and RANKL/OPG in mild and severe form (p

Purpose: Chordomas are malignant tumors that destroy bones, compress surrounding nerve tissues and exhibit phenotypes that recapitulate notochordal differentiation in the axial skeleton. Chordomas recur frequently, as they resist radio-chemotherapy and are difficult to completely resect, leading to repeated bone destruction and local expansion via unknown mechanisms. Here, using chordoma specimens and JHC7 chordoma cells, we asked whether chordoma cells possess bone-dissolving activity. Methods: CT imaging and histological analysis were performed to evaluate the structure and mineral density of chordoma-invaded bone and osteolytic marker expression. JHC7 cells were subjected to immunocytochemistry, imaging of cell fusion, calcium dynamics and acidic vacuoles, and bone lysis assays. Results: In patients, we found that the skull base invaded by chordoma was highly porous, showed low mineral density and contained brachyury-positive chordoma cells and conventional osteoclasts both expressing the osteolytic markers tartrate-resistant acid phosphatase (TRAP) and collagenases. JHC7 cells expressed TRAP and cathepsin K, became multinucleated via cell-cell fusion, showed spontaneous calcium oscillation, and were partly responsive to the osteoclastogenic cytokine RANKL. JHC7 cells exhibited large acidic vacuoles, and nonregulatory bone degradation without forming actin rings. Finally, bone-derived factors, calcium ions, TGF-β1, and IGF-1 enhanced JHC7 cell proliferation. Conclusion: In chordoma, we propose that in addition to conventional bone resorption by osteoclasts, chordoma cells possess bone-dissolving activity at the tumor-bone boundary. Furthermore, bone destruction and tumor expansion may occur in a positive feedback loop. [ABSTRACT FROM AUTHOR]

Cementum shares many properties with bone; however, in contrast to bone, it is not innervated or vascularized and has a limited capacity for remodeling. Osteocytes located in the lacunae-canalicular system of bone tissue play a central role in bone remodeling by communicating with osteoblasts and osteoclasts. Although cementocytes are present in cellular cementum and are morphologically similar to osteocytes, it remains unclear whether they are involved in the dynamic functional regulation of metabolism in cementum. The present study focused on the extracellular vesicles (EVs) secreted by cementocytes and examined their effects on osteoclasts and osteoblasts. EVs were extracted from the mouse cementocyte cell line, IDG-CM6. The effects of EVs on recombinant RANKL-induced osteoclastogenesis and recombinant Bone morphogenetic protein (BMP)-2-mediated osteoblastogenesis were investigated using the mouse osteoclast progenitor cell line, RAW264.7 and mouse pre-osteoblast cell line, MC3T3-E1, respectively. EVs enhanced the formation of tartrate-resistant acid phosphatase activity-positive cells. Real-time PCR revealed that EVs up-regulated the expression of osteoclast-related genes. On the other hand, the cell culture supernatant of cementocytes significantly inhibited the differentiation of osteoclasts. Regarding osteoblastogenesis, EVs suppressed the expression of alkaline phosphatase, bone sialoprotein, and osteocalcin induced by recombinant BMP-2 at the gene and protein levels. A network of cementocytes, osteoblasts, and osteoclasts may exist in cellular cementum, which suggests the involvement of cementocytes in dynamic metabolism of cementum through EVs. [ABSTRACT FROM AUTHOR]

Background: Osteoarthritis (OA) is the most common degenerative joint disease and affects millions of people worldwide, particularly the elderly population. The pathophysiology of OA is complex and involves multiple factors. Methods: Several studies have emphasized the crucial role of inflammation in this process. The receptor activator of NF-κB ligand (RANKL), the receptor activator of NF-κB (RANK), and osteoprotegerin (OPG) trigger a signaling cascade that leads to the excessive production of RANKL in the serum. Conclusions: The aim of this narrative review is (i) to assess the role of the RANK/RANKL/OPG signaling pathway in the context of OA progression, focusing especially on the physiopathology and on all the mechanisms leading to the activation of the inflammatory cascade, and (ii) to evaluate all the potential therapeutic strategies currently available that restore balance to bone formation and resorption, reducing structural abnormalities and relieving pain in patients with OA. [ABSTRACT FROM AUTHOR]

Background: Osteoporosis (OP) is a chronic disease characterized by decreased bone mass, loss of skeletal structural integrity and increased susceptibility to fracture. Available studies have shown that the pyruvate dehydrogenase kinase (PDK) family is associated with osteoclastogenesis and bone loss, but the specific role of Pdk3 in bone pathology has not been systematically investigated. Methods: A cell OP model was established in receptor activator for nuclear factor-κB Ligand (RANKL)-induced bone marrow macrophages (BMMs). Hereafter, the expression levels of Pdk3 and osteoclastogenesis feature genes including nuclear factor of activated T cells 1 (Nfatc1), Cathepsin K (Ctsk), osteoclast associated Ig-like receptor (Oscar) in BMMs-derived osteoclasts were examined based on real-time quantitative PCR and western blotting methods. Further, the phosphorylation of ERK, P65 and JAK/STAT and their correlation was Pdk3 was gauged. In particular, changes in the activity of these signaling pathways were observed by silencing experiments of the Pdk3 gene (using small interfering RNA). Finally, the effects of Pdk3 gene silencing on signaling pathway activity, osteoclastogenesis, and related inflammatory and apoptotic indicators were observed by transfection with PDK3-specific siRNA. Results: Following RANKL exposure, the levels of Pdk3 and osteoclastogenesis feature genes were all elevated, and a positive correlation between Pdk3 and osteoclastogenesis feature genes was seen. Meanwhile, ERK, P65 and JAK/STAT phosphorylation was increased by RANKL, and Pdk3 was confirmed to be positively correlated with the phosphorylation of ERK, P65 and JAK/STAT. Additionally, in RANKL-exposed osteoclasts, Pdk3 knockdown diminished the phosphorylation of ERK, P65 and JAK/STAT, reduced the expressions of osteoclastogenesis feature genes. Importantly, knockdown of Pdk3 also reduced the expression of inflammatory cytokines and resulted in elevated levels of Bax and Casp3 expression, as well as downregulation of Bcl2 expression. Conclusion: This study reveals for the first time the role of Pdk3 in RANKL-induced osteoclastogenesis and OP. These findings provide a foundation for future studies on the role of Pdk3 in other bone diseases and provide new ideas for the development of OP therapeutics targeting Pdk3. [ABSTRACT FROM AUTHOR]

Objective: To investigate the relationship between circulating receptor activator of nuclear factor-kappa B ligand (RANKL) levels and marrow adipose tissue in postmenopausal females. Methods: A total of 164 postmenopausal females were included in the study. Serum levels of osteoprotegerin (OPG) and RANKL were measured using ELISA kits. Body composition and bone mineral density (BMD) were assessed using dual-energy X-ray absorptiometry. Complex-based chemical shift imagingbased MRI was employed to evaluate the vertebral marrow proton density fat fraction (PDFF). A multivariate linear regression model was utilized to analyze the predictive effects of PDFF and BMD on circulating levels of OPG and RANKL. Results: Simple regression analysis showed significant associations among the marrow PDFF, BMD at either site, serum RANKL, and the RANKL/OPG ratio. In multivariate linear regression models, marrow PDFF was found to have a positive correlation (b = 3.15, 95% CI 2.60 to 3.70) and BMD had negative correlations (b = -0.200, 95% CI -0.348 to -0.051 for vertebral BMD; b = -0.383, 95% CI -0.589 to -0.177 for total hip BMD; and b =-0.393, 95% CI -0.598 to -0.188 for femoral neck BMD, all p < 0.01) with circulating soluble RANKL levels after adjusting for age, body mass index, physical activity, total fat mass, android/gynoid ratio, and lean mass. Similar results were observed for the RANKL/OPG ratio. Additionally, multivariate linear regression analyses revealed that marrow PDFF was a significant independent contributor of circulating soluble RANKL (b = 1.34, 95% CI 1.10 to 1.58, p < 0.001) after further controlling for BMD. However, marrow PDFF or BMD had no associations with circulating levels of OPG after adjusting for all potential confounders mentioned above. Conclusions: Vertebral marrow fat fraction is independently associated with circulating soluble RANKL levels in postmenopausal females. [ABSTRACT FROM AUTHOR]

Introduction: Osteoporosis, a systemic skeletal disease, is characterized by a quantitative and qualitative, and progressive decrease in bone mass, which is related to inflammation. Since a cytokine storm is triggered in Coronavirus disease 2019 (COVID-19), this study aims to evaluate pro-inflammatory cytokines (TNF-α, IL-1β), Receptor activator of nuclear factor-κB ligand (RANKL)/serum osteoprotegerin (OPG) ratio, and their relationship in mild and severe COVID-19. Methods: This study was performed on 48 adult patients (18 mild, 18 severe COVID-19, and 12 healthy subjects as a control group). Serum OPG, RANKL, TNF-α, IL-1β, 25-OH vitamin D, and ALKp were measured by ELISA and colorimetric assay. Results: COVID-19 patients had a significant increase in RANKL, and RANKL/OPG in mild and severe form (p < 0.001) while OPG decreased significantly in severe form compared to healthy controls (p < 0.05). Inflammatory cytokines (TNF-α and IL-1β) increased in both groups of patients whereas Alkaline phosphatase (ALKp) increased only in severe patients (p < 0.001). Both groups had 25-OH vitamin D deficiency in comparison to healthy ones (p < 0.001). Pearson's correlation coefficient was performed to determine the relationship between RANKL, OPG, ALKp, and 25-OH vitamin D with TNF-α and IL-1β in mild and severe COVID-19, which was statistically significant. Conclusion: Serum RANKL/OPG ratio was elevated in COVID-19 individuals and is assumed to be a risk factor for BMD reduction and osteoporosis in these patients. Correlations between IL-1β, TNF-α, ALKp, 25-OH vitamin D, OPG, RANKL, and RANKL/OPG ratio offered the potential role of these proinflammatory markers in the mechanism of osteoporosis in COVID-19 patients. Summary: Since cytokine storm, can stimulate osteoclastic activity, favoring bone resorption, in this study, we decided to evaluate its molecular mechanisms in COVID-19. We found an elevation of RANKL/OPG ratio with a positive correlation with inflammatory cytokines in COVID-19 which can be assumed as a risk factor for osteoporosis. [ABSTRACT FROM AUTHOR]

Background: Nitrogen-containing bisphosphonate(N-BP)had been found to inhibit the osteogenic differentiation and calcification in vascular smooth muscle cells (VSMCs), but the mechanism is not clear. We intend to verify that N-BP induces enhancement of OPG expression and inhibition of RANKL expression via inhibition of farnesyl pyrophosphate synthase(FPPS) to inhibit the osteogenic differentiation and calcification in VSMCs. Methods: β-glycerophosphate (β-GP) was used to induce the osteogenic differentiation and calcification in VSMCs. VSMCs were treated with N-BP or pretreated with downstream products of farnesyl pyrophosphate synthase(FPPS) in mevalonate pathway, such as farnesol (FOH) or geranylgeraniol (GGOH). Alizarin red S staining and determination of calcium content were used to detect calcium deposition.Western Blotting were used to detect expressions of proteins(OPG and RANKL) and osteogenic marker proteins (Runx2 and OPN). Results: β-GP induced the osteogenic differentiation and calcification in VSMCs, increased RANKL protein expression and had no significant effect on OPG protein expression. With the treatment of N-BP, the expression of OPG protein was increased and expression of RANKL protein was decreased in VSMCs undergoing osteogenic differentiation and calcification. In addition, N-BP reduced the osteogenic marker proteins (Runx2 and OPN) expression and calcium deposition in VSMCs undergoing osteogenic differentiation and calcification. These effects of N-BP on the osteogenic differentiation and calcification in VSMCs were concentration-dependent, which could be reversed by the downstream products of FPPS, such as FOH or GGOH. Conclusion: N-BP increases OPG expression and decreases RANKL expression via inhibition of FPPS to inhibit the osteogenic differentiation and calcification in VSMCs. [ABSTRACT FROM AUTHOR]

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Periapical lesions are common pathologies affecting the alveolar bone, often initiated by intraradicular lesions resulting from microbial exposure to dental pulp. These microorganisms trigger inflammatory and immune responses. When endodontic treatment fails to eliminate the infection, periapical lesions persist, leading to bone loss. The RANK/RANKL/OPG pathway plays a crucial role in both the formation and the destruction of the bone. In this study, the objective was to inhibit the RANK/RANKL pathway in vitro within exposed Thp-1 macrophages to endodontic microorganisms, specifically Enterococcus faecalis, which was isolated from root canals of 20 patients with endodontic secondary/persistent infection, symptomatic and asymptomatic, and utilizing an α-IRAK-4 inhibitor, we introduced endodontic microorganisms and/or lipoteichoic acid from Streptococcus spp. to cellular cultures in a culture plate, containing thp-1 cells and/or PBMC from patients with apical periodontitis. Subsequently, we assessed the percentages of RANK+, RANKL+, and OPG+ cells through flow cytometry and measured the levels of several inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8, IL-10, and IL-12p70) in the cellular culture supernatant through a CBA kit and performed analysis by flow cytometry. A significant difference was observed in the percentages of RANK+RANKL+, OPG+ RANKL+ cells in thp-1 cells and PBMCs from patients with apical periodontitis. The findings revealed significant differences in the percentages of the evaluated cells, highlighting the novel role of the IRAK-4 inhibitor in addressing this oral pathology, apical periodontitis, where bone destruction is observed. [ABSTRACT FROM AUTHOR]

Osteoporosis, a metabolic bone disorder characterized by decreased bone mass per unit volume, poses a significant global health burden due to its association with heightened fracture risk and adverse impacts on patients' quality of life. This review synthesizes the current understanding of the pathophysiological mechanisms underlying osteoporosis, with a focus on key regulatory pathways governing osteoblast and osteoclast activities. These pathways include RANK/RANKL/OPG, Wingless-int (Wnt)/β-catenin, and Jagged1/Notch1 signaling, alongside the involvement of parathyroid hormone (PTH) signaling, cytokine networks, and kynurenine in bone remodeling. Pharmacotherapeutic interventions targeting these pathways play a pivotal role in osteoporosis management. Anti-resorptive agents, such as bisphosphonates, estrogen replacement therapy/hormone replacement therapy (ERT/HRT), selective estrogen receptor modulators (SERMs), calcitonin, anti-RANKL antibodies, and cathepsin K inhibitors, aim to mitigate bone resorption. Conversely, anabolic agents, including PTH and anti-sclerostin drugs, stimulate bone formation. In addition to pharmacotherapy, nutritional supplementation with calcium, vitamin D, and vitamin K2 holds promise for osteoporosis prevention. However, despite the availability of therapeutic options, a substantial proportion of osteoporotic patients remain untreated, highlighting the need for improved clinical management strategies. This comprehensive review aims to provide clinicians and researchers with a mechanistic understanding of osteoporosis pathogenesis and the therapeutic mechanisms of existing medications. By elucidating these insights, this review seeks to inform evidence-based decision-making and optimize therapeutic outcomes for patients with osteoporosis. [ABSTRACT FROM AUTHOR]

Orthodontic relapse, the return to a pre-treatment position after orthodontic correction, is driven by the RANKL/TNF-α-mediated ROS/Keap1/Nrf2 signaling axis. This mechanism triggers aseptic inflammation and oxidative stress, influencing bone resorption and formation. Antioxidants can mitigate oxidative stress, potentially improving post-orthodontic outcomes. This study explores the efficacy of antioxidant compounds derived from green seaweed/algae in managing orthodontic relapse. Green seaweed/algae extracts were prepared via sonication, and bioactive compounds were identified using ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) analysis. Compounds underwent bioactivity prediction, toxicity assessment, and drug-likeness evaluation, revealing significant therapeutic potential. Network pharmacology and molecular docking identified key proteins associated with orthodontic relapse, including IL-1β, STAT3, ESR1, MAPK1, JAK2, and HMOX1. Molecular docking simulations indicated favorable binding energies for green seaweed compounds, particularly the alkaloids adenosine (ΔG −6.9 to −7.3 kcal/mol) and lycopodine (ΔG −6.3 to −8.5 kcal/mol), against targeted proteins, matching or outperforming standard drugs such as s-ibuprofen (ΔG −6.7 kcal/mol). In vitro assays confirmed the antioxidant activity of these compounds, with EC50 dose of 52.2–54.2 μg/mL for ABTS radical scavenging capacities. Protein expression analysis in tibial-femoral bone marrow cells further demonstrated the potential of green seaweed/algae compounds to suppress osteoclastogenesis by modulating the RANKL/TNF-α-mediated ROS/Keap1/Nrf2 pathway. This research highlights the promise of green seaweed-derived antioxidants in reducing oxidative stress and managing orthodontic relapse, providing a foundation for future therapeutic developments.

Abstract Background Skeletal Stem Cells (SSCs) are required for skeletal development, homeostasis, and repair. The perspective of their wide application in regenerative medicine approaches has supported research in this field, even though so far results in the clinic have not reached expectations, possibly due also to partial knowledge of intrinsic, potentially actionable SSC regulatory factors. Among them, the pleiotropic cytokine RANKL, with essential roles also in bone biology, is a candidate deserving deep investigation. Methods To dissect the role of the RANKL cytokine in SSC biology, we performed ex vivo characterization of SSCs and downstream progenitors (SSPCs) in mice lacking Rankl (Rankl −/− ) by means of cytofluorimetric sorting and analysis of SSC populations from different skeletal compartments, gene expression analysis, and in vitro osteogenic differentiation. In addition, we assessed the effect of the pharmacological treatment with the anti-RANKL blocking antibody Denosumab (approved for therapy in patients with pathological bone loss) on the osteogenic potential of bone marrow-derived stromal cells from human healthy subjects (hBMSCs). Results We found that, regardless of the ossification type of bone, osteochondral SSCs had a higher frequency and impaired differentiation along the osteochondrogenic lineage in Rankl −/− mice as compared to wild-type. Rankl −/− mice also had increased frequency of committed osteochondrogenic and adipogenic progenitor cells deriving from perivascular SSCs. These changes were not due to the peculiar bone phenotype of increased density caused by lack of osteoclast resorption (defined osteopetrosis); indeed, they were not found in another osteopetrotic mouse model, i.e., the oc/oc mouse, and were therefore not due to osteopetrosis per se. In addition, Rankl −/− SSCs and primary osteoblasts showed reduced mineralization capacity. Of note, hBMSCs treated in vitro with Denosumab had reduced osteogenic capacity compared to control cultures. Conclusions We provide for the first time the characterization of SSPCs from mouse models of severe recessive osteopetrosis. We demonstrate that Rankl genetic deficiency in murine SSCs and functional blockade in hBMSCs reduce their osteogenic potential. Therefore, we propose that RANKL is an important regulatory factor of SSC features with translational relevance.

Abstract Background Postmenopausal osteoporosis is a chronic metabolic bone disease caused by excessive osteoclast formation and function. Targeting osteoclast differentiation and activity can modulate bone resorption and alleviate osteoporosis. Cirsilineol, an active constituent of Vestita Wall, has shown numerous biological activities and has been used to treat many metabolic diseases. However, whether cirsilineol inhibits osteoclast activity and prevents postmenopausal osteoporosis still remain unknown. Materials and methods Primary bone marrow macrophages (BMMs) and RAW264.7 cells were used. Osteoclast activity was measured by TRAP staining, F-actin staining, and bone resorption assay after BMMs were treated with cirsilineol at concentrations of 0, 1, 2.5 and 5 µM. RT-PCR and western blotting were performed to evaluate the expression of osteoclast-related genes. In addition, female C57BL/6 mice underwent OVX surgery and were treated with cirsilineol (20 mg/kg) to demonstrate the effect of cirsilineol on osteoporosis. Results Cirsilineol significantly inhibited receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast differentiation in a concentration- and time-dependent manner, respectively. Additionally, cirsilineol inhibited F-actin ring formation, thus reducing the activation of bone resorption ability. Cirsilineol suppressed the expression of osteoclast-related genes and proteins via blocking nuclear factor (NF)-κb, ERK, and p38 signaling cascades. More importantly, cirsilineol treatment in mice with osteoporosis alleviated osteoclasts hyperactivation and bone mass loss caused by estrogen depletion. Conclusion In this study, the protective effect of cirsilineol on osteoporosis has been investigated for the first time. In conclusion, our findings prove the inhibitory effect of cirsilineol on osteoclast activity via NF-κb/ERK/p38 signaling pathways and strongapplication of cirsilineol can be proposed as a potential therapeutic strategy.

Introduction: Exercise training is likely to be a non-pharmacological intervention to improve bone health in children and the elderly. The aim of this study was to investigate the effect of resistance-aerobic training on RUX2, CTX, RANKL and ALP biomarkers in rats of different ages. Methods: In the present experimental study, 30 male Wistar rats were obtained from Instituto Pasteur of Iran and were divided into three groups according to their age: immature (2 weeks old), mature (6 weeks old) and elderly (96 weeks old). In each age group, the mice were randomly divided into two training (n=5) and control (n=5) groups. Six weeks of combined training included three sessions of aerobic training and three sessions of resistance training per week. Extracting genes by PCR method was used. One-way analysis of variance and Tukey's post hoc test and SPSS16 software were used. The significance level P≥0.05 was considered. Results: One-way analysis of variance showed that resistance-aerobic training has had a significant effect on RUNX2, RANKL and ALP variables of rats in different age groups (P < 0.05). This significance was not observed in CTX variable (P > 0.05). On the other hand, the results of Tukey's RUNX2 test were significantly different between the adult training and elderly training groups (p = 0.019) as well as between the elderly control groups (p = 0.01) and adult training (p = 0.002). Regarding the RANKL variable, it showed that there was a significant difference only between the adult exercise group and the elderly control group (p = 0.01). Conclusion: Resistance-endurance training probably increases absorption and reabsorption biomarkers such as RUX2, RANKL and ALP in immature, mature and elderly male rats.

Infection of ruminants such as cattle with Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, a disease characterized by chronic inflammation of the small intestine and diarrhoea. Infection with MAP is acquired via the faecal-to-oral route and the pathogen initially invades the epithelial lining of the small intestine. In this study we used an in vitro 3D mouse enteroid model to determine the influence of M cells in infection of the gut epithelia by MAP, in comparison with another bacterial intestinal pathogen of veterinary importance, Salmonella enterica serovar Typhimurium. The differentiation of M cells in the enteroid cultures was induced by stimulation with the cytokine receptor activator of nuclear factor-βB ligand (RANKL), and the effects on MAP and Salmonella uptake and intracellular survival were determined. The presence of M cells in the cultures correlated with increased uptake and intracellular survival of Salmonella, but had no effect on MAP. Interestingly neither pathogen was observed to preferentially accumulate within GP2- positive M cells. [ABSTRACT FROM AUTHOR]

Background: Skeletal Stem Cells (SSCs) are required for skeletal development, homeostasis, and repair. The perspective of their wide application in regenerative medicine approaches has supported research in this field, even though so far results in the clinic have not reached expectations, possibly due also to partial knowledge of intrinsic, potentially actionable SSC regulatory factors. Among them, the pleiotropic cytokine RANKL, with essential roles also in bone biology, is a candidate deserving deep investigation. Methods: To dissect the role of the RANKL cytokine in SSC biology, we performed ex vivo characterization of SSCs and downstream progenitors (SSPCs) in mice lacking Rankl (Rankl−/−) by means of cytofluorimetric sorting and analysis of SSC populations from different skeletal compartments, gene expression analysis, and in vitro osteogenic differentiation. In addition, we assessed the effect of the pharmacological treatment with the anti-RANKL blocking antibody Denosumab (approved for therapy in patients with pathological bone loss) on the osteogenic potential of bone marrow-derived stromal cells from human healthy subjects (hBMSCs). Results: We found that, regardless of the ossification type of bone, osteochondral SSCs had a higher frequency and impaired differentiation along the osteochondrogenic lineage in Rankl−/− mice as compared to wild-type. Rankl−/− mice also had increased frequency of committed osteochondrogenic and adipogenic progenitor cells deriving from perivascular SSCs. These changes were not due to the peculiar bone phenotype of increased density caused by lack of osteoclast resorption (defined osteopetrosis); indeed, they were not found in another osteopetrotic mouse model, i.e., the oc/oc mouse, and were therefore not due to osteopetrosis per se. In addition, Rankl−/− SSCs and primary osteoblasts showed reduced mineralization capacity. Of note, hBMSCs treated in vitro with Denosumab had reduced osteogenic capacity compared to control cultures. Conclusions: We provide for the first time the characterization of SSPCs from mouse models of severe recessive osteopetrosis. We demonstrate that Rankl genetic deficiency in murine SSCs and functional blockade in hBMSCs reduce their osteogenic potential. Therefore, we propose that RANKL is an important regulatory factor of SSC features with translational relevance. [ABSTRACT FROM AUTHOR]

Bone disease associated with multiple myeloma (MM) is characterized by osteolytic lesions and pathological fractures, which remain a therapeutic priority despite new drugs improving MM patient survival. Antiresorptive molecules represent the main option for the treatment of MM-associated bone disease (MMBD), whereas osteoanabolic molecules are under investigation. Among these latter, we here focused on the myokine irisin, which is able to enhance bone mass in healthy mice, prevent bone loss in osteoporotic mouse models, and accelerate fracture healing in mice. Therefore, we investigated irisin effect on MMBD in a mouse model of MM induced by intratibial injection of myeloma cells followed by weekly administration of 100 μg/kg of recombinant irisin for 5 wk. By micro-Ct analysis, we demonstrated that irisin improves MM-induced trabecular bone damage by partially preventing the reduction of femur Trabecular Bone Volume/Total Volume (P  = .0028), Trabecular Number (P  = .0076), Trabecular Fractal Dimension (P  = .0044), and increasing Trabecular Separation (P  = .0003) in MM mice. In cortical bone, irisin downregulates the expression of Sclerostin, a bone formation inhibitor, and RankL, a pro-osteoclastogenic molecule, while in BM it upregulates Opg, an anti-osteoclastogenic cytokine. We found that in the BM tibia of irisin-treated MM mice, the percentage of MM cells displays a reduction trend, while in the femur it decreases significantly. This is in line with the in vitro reduction of myeloma cell viability after 48 h of irisin stimulation at both 200 and 500 ng/mL and, after 72 h already at 100 ng/mL rec-irisin. These results could be due to irisin ability to downregulate the expression of Notch 3, which is important for cell-to-cell communication in the tumor niche, and Cyclin D1, supporting an inhibitory effect of irisin on MM cell proliferation. Overall, our findings suggest that irisin could be a new promising strategy to counteract MMBD and tumor burden in one shot. [ABSTRACT FROM AUTHOR]

Functional M cells are differentiated by receptor activator of NF-κB ligand (RANKL) and capture of luminal antigens to initiate immune responses. We aimed to use postbiotic-based recombinant chicken RANKL (cRANKL) to promote M cell differentiation and test the efficacy of oral vaccines. Chicks were divided into three groups that were administered phosphate-buffered saline (PBS), cell extracts of wild-type Lactococcus lactis subsp. lactis IL1403 (WT_CE), or cell extracts of recombinant L. lactis expressing cRANKL (cRANKL_CE). The expression of the M cell marker was measured, and the gut microbiome was profiled. The efficiency of the infectious bursal disease (IBD) vaccine was tested after 12 consecutive days of administering cRANKL_CE. The chickens that were administered cRANKL_CE (p = 0.038) had significantly higher Annexin A5 (ANXA5) mRNA expression levels than those in the PBS group (PBS vs. WT_CE, p = 0.657). In the gut microbiome analysis, no significant changes were observed. However, the relative abundance of Escherichia-Shigella was negatively correlated (r = − 0.43, p = 0.019) with ANXA5 mRNA expression in Peyer's patches. cRANKL_CE/IBD (p = 0.018) had significantly higher IBD-specific faecal IgA levels than PBS/IBD (PBS/IBD vs. WT_CE/IBD, p = 0.217). Postbiotic-based recombinant cRANKL effectively improved the expression of M cell markers and the efficiency of oral vaccines. No significant changes were observed in the gut microbiome after administration of postbiotic-based recombinant cRANKL. This strategy can be used for the development of feed additives and adjuvants. Key points: • Postbiotic-based recombinant cRANKL enhanced the expression of ANXA5 in chicken. • The relative abundance of Escherichia-Shigella was negatively correlated with ANXA5 expression. • Postbiotic-based recombinant cRANKL effectively improved the efficiency of oral vaccine. [ABSTRACT FROM AUTHOR]

For humans to explore and colonize the universe, both engineering and physiological obstacles must be successfully addressed. A major physiological problem is that humans lose bone rapidly in microgravity. Understanding the underlying mechanisms for this bone loss is crucial for designing strategies to ameliorate these effects. Because bone physiology is entangled with other organ systems, and bone loss is a component of human adaptation to microgravity, strategies to reduce bone loss must also account for potential effects on other systems. Here, we consider the receptors involved in normal bone remodeling and how this regulation is altered in low-gravity environments. We examine how single cells, tissues and organs, and humans as a whole are affected by low gravity, and the role of receptors that have been implicated in responses leading to bone loss. These include receptors linking cells to the extracellular matrix and to each other, alterations in the extracellular matrix associated with changes in gravity, and changes in fluid distribution and fluid behavior due to lack of gravity that may have effects on receptor-based signaling shared by bone and other regulatory systems. Inflammatory responses associated with the environment in space, which include microgravity and radiation, can also potentially trigger bone loss. [ABSTRACT FROM AUTHOR]

Vascular calcification has a global health impact that is closely linked to bone loss. The Receptor Activator of Nuclear Factor Kappa B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system, fundamental for bone metabolism, also plays an important role in vascular calcification. The Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4), a novel receptor for RANKL, regulates bone remodeling, and it appears to be involved in vascular calcification. Besides RANKL, LGR4 interacts with R-spondins (RSPOs), which are known for their roles in bone but are less understood in vascular calcification. Studies were conducted in rats with chronic renal failure fed normal or high phosphorus diets for 18 weeks, with and without control of circulating parathormone (PTH) levels, resulting in different degrees of aortic calcification. Additionally, vascular smooth muscle cells (VSMCs) were cultured under non-calcifying (1 mM phosphate) and calcifying (3 mM phosphate) media with different concentrations of PTH. To explore the role of RANKL in VSMC calcification, increasing concentrations of soluble RANKL were added to non-calcifying and calcifying media. The effects mediated by RANKL binding to its receptor LGR4 were investigated by silencing the LGR4 receptor in VSMCs. Furthermore, the gene expression of the RANK/RANKL/OPG system and the ligands of LGR4 was assessed in human epigastric arteries obtained from kidney transplant recipients with calcification scores (Kauppila Index). Increased aortic calcium in rats coincided with elevated systolic blood pressure, upregulated Lgr4 and Rankl gene expression, downregulated Opg gene expression, and higher serum RANKL/OPG ratio without changes in Rspos gene expression. Elevated phosphate in vitro increased calcium content and expression of Rankl and Lgr4 while reducing Opg. Elevated PTH in the presence of high phosphate exacerbated the increase in calcium content. No changes in Rspos were observed under the conditions employed. The addition of soluble RANKL to VSMCs induced genotypic differentiation and calcification, partly prevented by LGR4 silencing. In the epigastric arteries of individuals presenting vascular calcification, the gene expression of RANKL was higher. While RSPOs show minimal impact on VSMC calcification, RANKL, interacting with LGR4, drives osteogenic differentiation in VSMCs, unveiling a novel mechanism beyond RANKL-RANK binding. [ABSTRACT FROM AUTHOR]

Background: Postmenopausal osteoporosis is a chronic metabolic bone disease caused by excessive osteoclast formation and function. Targeting osteoclast differentiation and activity can modulate bone resorption and alleviate osteoporosis. Cirsilineol, an active constituent of Vestita Wall, has shown numerous biological activities and has been used to treat many metabolic diseases. However, whether cirsilineol inhibits osteoclast activity and prevents postmenopausal osteoporosis still remain unknown. Materials and methods: Primary bone marrow macrophages (BMMs) and RAW264.7 cells were used. Osteoclast activity was measured by TRAP staining, F-actin staining, and bone resorption assay after BMMs were treated with cirsilineol at concentrations of 0, 1, 2.5 and 5 µM. RT-PCR and western blotting were performed to evaluate the expression of osteoclast-related genes. In addition, female C57BL/6 mice underwent OVX surgery and were treated with cirsilineol (20 mg/kg) to demonstrate the effect of cirsilineol on osteoporosis. Results: Cirsilineol significantly inhibited receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast differentiation in a concentration- and time-dependent manner, respectively. Additionally, cirsilineol inhibited F-actin ring formation, thus reducing the activation of bone resorption ability. Cirsilineol suppressed the expression of osteoclast-related genes and proteins via blocking nuclear factor (NF)-κb, ERK, and p38 signaling cascades. More importantly, cirsilineol treatment in mice with osteoporosis alleviated osteoclasts hyperactivation and bone mass loss caused by estrogen depletion. Conclusion: In this study, the protective effect of cirsilineol on osteoporosis has been investigated for the first time. In conclusion, our findings prove the inhibitory effect of cirsilineol on osteoclast activity via NF-κb/ERK/p38 signaling pathways and strongapplication of cirsilineol can be proposed as a potential therapeutic strategy. [ABSTRACT FROM AUTHOR]

Background: Bone tissue homeostasis relies on the coordinated activity of the bone‐forming osteoblasts and bone‐resorbing osteoclasts. Osteomesopyknosis is considered a distinctive rare sclerosing skeletal disorder of unelucidated pathophysiology and presumably autosomal dominant transmission. However, the causal genes are unknown. Methods: We present a case report encompassing clinical assessments, imaging studies, and whole‐exome sequencing analysis, complemented by functional in vitro experiments. Results: This new case of osteomesopyknosis was associated with a missense ALOX5 variant predicted to induce protein misfolding and proteasomal degradation. Transfection experiments demonstrated that the variant was associated with reduced protein levels restored by proteasomal inhibition with bortezomib. Likewise, gene expression analysis showed that the mutated gene was associated with a decreased RANKL/OPG ratio, which is a critical driver of osteoclast precursor differentiation. Conclusion: Our data indicate impaired bone resorption as the underlying mechanism of this rare osteosclerosis, implicating ALOX5 pathogenic variants as potential etiological factors. [ABSTRACT FROM AUTHOR]

Adult bones are continuously remodeled by the balance between bone resorption by osteoclasts and subsequent bone formation by osteoblasts. Many studies have provided molecular evidence that bone remodeling is under the control of circadian rhythms. Circadian fluctuations have been reported in the serum and urine levels of bone turnover markers, such as digested collagen fragments and bone alkaline phosphatase. Additionally, the expressions of over a quarter of all transcripts in bones show circadian rhythmicity, including the genes encoding master transcription factors for osteoblastogenesis and osteoclastogenesis, osteogenic cytokines, and signaling pathway proteins. Serum levels of calcium, phosphate, parathyroid hormone, and calcitonin also display circadian rhythmicity. Finally, osteoblast- and osteoclast-specific knockout mice targeting the core circadian regulator gene Bmal1 show disrupted bone remodeling, although the results have not always been consistent. Despite these studies, however, establishing a direct link between circadian rhythms and bone remodeling in vivo remains a major challenge. It is nearly impossible to repeatedly collect bone materials from human subjects while following circadian changes. In addition, the differences in circadian gene regulation between diurnal humans and nocturnal mice, the main model organism, remain unclear. Filling the knowledge gap in the circadian regulation of bone remodeling could reveal novel regulatory mechanisms underlying many bone disorders including osteoporosis, genetic diseases, and fracture healing. This is also an important question for the basic understanding of how cell differentiation progresses under the influence of cyclically fluctuating environments. [ABSTRACT FROM AUTHOR]

The global incidence of Type 2 diabetes (T2D) is on the rise, fueled by factors such as obesity, sedentary lifestyles, socio-economic factors, and ethnic backgrounds. T2D is a multifaceted condition often associated with various health complications, including adverse effects on bone health. This study aims to assess key biomarkers linked to bone health and remodeling—Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL), and Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB)—among individuals with diabetes while exploring the impact of ethnicity on these biomarkers. A cross-sectional analysis was conducted on a cohort of 2083 individuals from diverse ethnic backgrounds residing in Kuwait. The results indicate significantly elevated levels of these markers in individuals with T2D compared to non-diabetic counterparts, with OPG at 826.47 (405.8) pg/mL, RANKL at 9.25 (17.3) pg/mL, and GPNMB at 21.44 (7) ng/mL versus 653.75 (231.7) pg/mL, 0.21 (9.94) pg/mL, and 18.65 (5) ng/mL in non-diabetic individuals, respectively. Notably, this elevation was consistent across Arab and Asian populations, except for lower levels of RANKL observed in Arabs with T2D. Furthermore, a positive and significant correlation between OPG and GPNMB was observed regardless of ethnicity or diabetes status, with the strongest correlation (r = 0.473, p < 0.001) found among Arab individuals with T2D. Similarly, a positive and significant correlation between GPNMB and RANKL was noted among Asian individuals with T2D (r = 0.401, p = 0.001). Interestingly, a significant inverse correlation was detected between OPG and RANKL in non-diabetic Arab individuals. These findings highlight dysregulation in bone remodeling markers among individuals with T2D and emphasize the importance of considering ethnic variations in T2D-related complications. The performance of further studies is warranted to understand the underlying mechanisms and develop interventions based on ethnicity for personalized treatment approaches. [ABSTRACT FROM AUTHOR]

Background Osteoporosis (OP) is a chronic disease characterized by decreased bone mass, loss of skeletal structural integrity and increased susceptibility to fracture. Available studies have shown that the pyruvate dehydrogenase kinase (PDK) family is associated with osteoclastogenesis and bone loss, but the specific role of Pdk3 in bone pathology has not been systematically investigated. Methods A cell OP model was established in receptor activator for nuclear factor-κB Ligand (RANKL)-induced bone marrow macrophages (BMMs). Hereafter, the expression levels of Pdk3 and osteoclastogenesis feature genes including nuclear factor of activated T cells 1 (Nfatc1), Cathepsin K (Ctsk), osteoclast associated Ig-like receptor (Oscar) in BMMs-derived osteoclasts were examined based on real-time quantitative PCR and western blotting methods. Further, the phosphorylation of ERK, P65 and JAK/STAT and their correlation was Pdk3 was gauged. In particular, changes in the activity of these signaling pathways were observed by silencing experiments of the Pdk3 gene (using small interfering RNA). Finally, the effects of Pdk3 gene silencing on signaling pathway activity, osteoclastogenesis, and related inflammatory and apoptotic indicators were observed by transfection with PDK3-specific siRNA. Results Following RANKL exposure, the levels of Pdk3 and osteoclastogenesis feature genes were all elevated, and a positive correlation between Pdk3 and osteoclastogenesis feature genes was seen. Meanwhile, ERK, P65 and JAK/STAT phosphorylation was increased by RANKL, and Pdk3 was confirmed to be positively correlated with the phosphorylation of ERK, P65 and JAK/STAT. Additionally, in RANKL-exposed osteoclasts, Pdk3 knockdown diminished the phosphorylation of ERK, P65 and JAK/STAT, reduced the expressions of osteoclastogenesis feature genes. Importantly, knockdown of Pdk3 also reduced the expression of inflammatory cytokines and resulted in elevated levels of Bax and Casp3 expression, as well as downregulation of Bcl2 expression. Conclusion This study reveals for the first time the role of Pdk3 in RANKL-induced osteoclastogenesis and OP. These findings provide a foundation for future studies on the role of Pdk3 in other bone diseases and provide new ideas for the development of OP therapeutics targeting Pdk3.

Background/purpose: The number of middle-aged and elderly orthodontic patients is increasing due to changes in age composition. It is important to investigate the detailed mechanisms of bone remodeling in orthodontic tooth movement (OTM) in the elderly. However, there are few reports on the mechanism of tooth movement in the elderly. The purpose of the present study was to analyze OTM and osteoclastogenesis in aged mice and to elucidate the mechanism. Materials and methods: It has been reported that tumor necrosis factor (TNF)-α plays an important role in osteoclast formation and OTM. First, 8-week-old and 78-week-old male C57BL/6J mice were subcutaneously injected with TNF-α into the calvaiae, and micro-CT, tartrate-resistant acid phosphatase (TRAP) staining, and real-time PCR were performed to evaluate osteoclast formation and bone resorption. Furthermore, osteoclastogenesis by TNF-α and receptor activator of nuclear factor-kappa B ligand (RANKL) using bone marrow cells was evaluated in vitro. Finally, a nickel-titanium closed-coil spring was attached, mesial movement of the maxillary left first molar was performed, and tooth movement distance and osteoclast formation were evaluated. Results: Compared to 8-week-old mice, 78-week-old mice had decreased TNF-α-induced bone resorption, osteoclastogenesis, and TRAP and cathepsin K expression in the calvariae. In vitro osteoclast formation also decreased in 78-week-old mice. Furthermore, tooth movement distance and osteoclastogenesis were reduced. Conclusion: OTM decreased in aged mice, which was shown to be caused by a decrease in osteoclastogenesis. Therefore, it was suggested that it is necessary to keep in mind that tooth movement may be suppressed when treating elderly patients.

Background/objective: As the pivotal cellular mediators of bone resorption and pathological bone remodeling, osteoclasts have emerged as a prominent target for anti-resorptive interventions. Pinocembrin (PIN), a predominant flavonoid found in damiana, honey, fingerroot, and propolis, has been recognized for its potential therapeutic effects in osteolysis. The purpose of our project is to investigate the potential of PIN to prevent bone resorption in ovariectomized (OVX) mice by suppressing osteoclast production through its underlying mechanisms. Methods: The study commenced by employing protein-ligand molecular docking to ascertain the specific interaction between PIN and nuclear factor-κB (NF-κB) ligand (RANKL). Subsequently, PIN was introduced to bone marrow macrophages (BMMs) under the stimulation of RANKL. The impact of PIN on osteoclastic activity was assessed through the utilization of a positive TRAcP staining kit and a hydroxyapatite resorption assay. Furthermore, the study investigated the generation of reactive oxygen species (ROS) in osteoclasts induced by RANKL using H2DCFDA. To delve deeper into the underlying mechanisms, molecular cascades triggered by RANKL, including NF-κB, ROS, calcium oscillations, and NFATc1-mediated signaling pathways, were explored using Luciferase gene report, western blot analysis, and quantitative real-time polymerase chain reaction. Moreover, an estrogen-deficient osteoporosis murine model was established to evaluate the therapeutic effects of PIN in vivo. Results: In this study, we elucidated the profound inhibitory effects of PIN on osteoclastogenesis and bone resorption, achieved through repression of NF-κB and NFATc1-mediated signaling pathways. Notably, PIN also exhibited potent anti-oxidative properties by mitigating RANKL-induced ROS generation and augmenting activities of ROS-scavenging enzymes, ultimately leading to a reduction in intracellular ROS levels. Moreover, PIN effectively abrogated the expression of osteoclast-specific marker genes (Acp5, Cathepsin K, Atp6v0d2, Nfatc1, c-fos, and Mmp9), further underscoring its inhibitory impact on osteoclast differentiation and function. Additionally, employing an in vivo mouse model, we demonstrated that PIN effectively prevented osteoclast-induced bone loss resultant from estrogen deficiency. Conclusion: Our findings highlight the potent inhibitory effects of PIN on osteoclastogenesis, bone resorption, and RANKL-induced signaling pathways, thereby establishing PIN as a promising therapeutic candidate for the prevention and management of osteolytic bone diseases. The translational potential of this article: PIN serves as a promising therapeutic agent for the prevention and management of osteolytic bone diseases and holds promise for future clinical applications in addressing conditions characterized by excessive bone resorption. PIN is a natural compound found in various sources, including damiana, honey, fingerroot, and propolis. Its widespread availability and potential for therapeutic use make it an attractive candidate for further investigation and development as a clinical intervention.

Abstract Background Estrogen deficiency-mediated hyperactive osteoclast represents the leading role during the onset of postmenopausal osteoporosis. The activation of a series of signaling cascades triggered by RANKL-RANK interaction is crucial mechanism underlying osteoclastogenesis. Vorinostat (SAHA) is a broad-spectrum pan-histone deacetylase inhibitor (HDACi) and its effect on osteoporosis remains elusive. Methods The effects of SAHA on osteoclast maturation and bone resorptive activity were evaluated using in vitro osteoclastogenesis assay. To investigate the effect of SAHA on the osteoclast gene networks during osteoclast differentiation, we performed high-throughput transcriptome sequencing. Molecular docking and the assessment of RANKL-induced signaling cascades were conducted to confirm the underlying regulatory mechanism of SAHA on the action of RANKL-activated osteoclasts. Finally, we took advantage of a mouse model of estrogen-deficient osteoporosis to explore the clinical potential of SAHA. Results We showed here that SAHA suppressed RANKL-induced osteoclast differentiation concentration-dependently and disrupted osteoclastic bone resorption in vitro. Mechanistically, SAHA specifically bound to the predicted binding site of RANKL and blunt the interaction between RANKL and RANK. Then, by interfering with downstream NF-κB and MAPK signaling pathway activation, SAHA negatively regulated the activity of NFATc1, thus resulting in a significant reduction of osteoclast-specific gene transcripts and functional osteoclast-related protein expression. Moreover, we found a significant anti-osteoporotic role of SAHA in ovariectomized mice, which was probably realized through the inhibition of osteoclast formation and hyperactivation. Conclusion These data reveal a high affinity between SAHA and RANKL, which results in blockade of RANKL-RANK interaction and thereby interferes with RANKL-induced signaling cascades and osteoclastic bone resorption, supporting a novel strategy for SAHA application as a promising therapeutic agent for osteoporosis.

Aims: The present study investigated receptor activator of nuclear factor kappa-Β ligand (RANKL), osteoprotegerin (OPG), and Runt-related transcription factor 2 (RUNX2) gene expressions in giant cell tumour of bone (GCTB) patients in relationship with tumour recurrence. We also aimed to investigate the influence of CpG methylation on the transcriptional levels of RANKL and OPG. Methods: A total of 32 GCTB tissue samples were analyzed, and the expression of RANKL, OPG, and RUNX2 was evaluated by quantitative polymerase chain reaction (qPCR). The methylation status of RANKL and OPG was also evaluated by quantitative methylation-specific polymerase chain reaction (qMSP). Results: We found that RANKL and RUNX2 gene expression was upregulated more in recurrent than in non-recurrent GCTB tissues, while OPG gene expression was downregulated more in recurrent than in non-recurrent GCTB tissues. Additionally, we proved that changes in DNA methylation contribute to upregulating the expression of RANKL and downregulating the expression of OPG, which are critical for bone homeostasis and GCTB development. Conclusion: Our results suggest that the overexpression of RANKL/RUNX2 and the lower expression of OPG are associated with recurrence in GCTB patients. Cite this article: Bone Joint Res 2024;13(2):84–91.

The number of middle-aged and elderly orthodontic patients is increasing due to changes in age composition. It is important to investigate the detailed mechanisms of bone remodeling in orthodontic tooth movement (OTM) in the elderly. However, there are few reports on the mechanism of tooth movement in the elderly. The purpose of the present study was to analyze OTM and osteoclastogenesis in aged mice and to elucidate the mechanism. It has been reported that tumor necrosis factor (TNF)-α plays an important role in osteoclast formation and OTM. First, 8-week-old and 78-week-old male C57BL/6J mice were subcutaneously injected with TNF-α into the calvaiae, and micro-CT, tartrate-resistant acid phosphatase (TRAP) staining, and real-time PCR were performed to evaluate osteoclast formation and bone resorption. Furthermore, osteoclastogenesis by TNF-α and receptor activator of nuclear factor-kappa B ligand (RANKL) using bone marrow cells was evaluated in vitro. Finally, a nickel-titanium closed-coil spring was attached, mesial movement of the maxillary left first molar was performed, and tooth movement distance and osteoclast formation were evaluated. Compared to 8-week-old mice, 78-week-old mice had decreased TNF-α-induced bone resorption, osteoclastogenesis, and TRAP and cathepsin K expression in the calvariae. In vitro osteoclast formation also decreased in 78-week-old mice. Furthermore, tooth movement distance and osteoclastogenesis were reduced. OTM decreased in aged mice, which was shown to be caused by a decrease in osteoclastogenesis. Therefore, it was suggested that it is necessary to keep in mind that tooth movement may be suppressed when treating elderly patients. [ABSTRACT FROM AUTHOR]

Improper mechanical stress may induce side effects during orthodontic treatment. If the roots and alveolar bones are extensively resorbed following excess mechanical stress, unplanned tooth mobility and inflammation can occur. Although multiple factors are believed to contribute to the development of side effects, the cause is still unknown. Sonic hedgehog (Shh), one of the hedgehog signals significantly associated with cell growth and cancer development, promotes osteoclast formation in the jawbone. Shh may be associated with root and bone resorptions during orthodontic treatment. In this study, we investigated the relationships between Shh, RANKL, and IL-6 in human periodontal ligament (hPDL) cells exposed to improper mechanical force. Weights were placed on hPDL cells and human gingival fibroblasts (HGFs) for an optimal orthodontic force group (1.0 g/cm2) and a heavy orthodontic force group (4.0 g/cm2). A group with no orthodontic force was used as a control group. Real-time PCR, SDS-PAGE, and Western blotting were performed to examine the effects of orthodontic forces on the expression of Shh, RANKL, and IL-6 at 2, 4, 6, 8, 12, and 24 h after the addition of pressure. The protein expression of Shh was not clearly induced by orthodontic forces of 1.0 and 4.0 g/cm2 compared with the control in HGFs and hPDL cells. In contrast, RANKL and IL-6 gene and protein expression was significantly induced by 1.0 and 4.0 g/cm2 in hPDL cells for forces lasting 6~24 h. However, neither protein was expressed in HGFs. RANKL and IL-6 expressions in response to orthodontic forces and in the control were clearly inhibited by Shh inhibitor RU-SKI 43. Shh did not directly link to RANKL and IL-6 for root and bone resorptions by orthodontic force but was associated with cell activities to be finally guided by the production of cytokines in hPDL cells. [ABSTRACT FROM AUTHOR]

Eosinophilic asthma is the most prevalent and well-defined phenotype of asthma. Despite a majority of patients responding to corticosteroid therapy and T2 biologics, there remains a subset that have recurrent asthma exacerbations, highlighting a need for additional therapies to fully ameliorate airway eosinophilia. Group 2 innate lymphoid cells (ILC2) are considered key players in the pathogenesis of eosinophilic asthma through the production of copious amounts of type 2 cytokines, namely IL-5 and IL-13. ILC2 numbers are increased in the airways of asthmatics and with the greatest numbers of activated ILC2 detected in sputa from severe prednisone-dependent asthma with uncontrolled eosinophilia. Although epithelial-derived cytokines are important mediators of ILC2 activation, emerging evidence suggests that additional pathways stimulate ILC2 function. The tumor necrosis factor super family (TNFSF) and its receptors (TNFRSF) promote ILC2 activity. In this review, we discuss evidence supporting a relationship between ILC2 and TNFSF/TNFRSF axis in eosinophilic asthma and the role of this relationship in severe asthma with airway autoimmune responses. [ABSTRACT FROM AUTHOR]

Glucose-insulinotropic polypeptide (GIP) is an incretin hormone that induces insulin secretion and decreases blood glucose levels. In addition, it has been reported to suppress osteoclast formation. Native GIP is rapidly degraded by dipeptidyl peptidase-4 (DPP-4). (D-Ala2)GIP is a newly developed GIP analog that demonstrates enhanced resistance to DPP-4. This study aimed to evaluate the influence of (D-Ala2)GIP on osteoclast formation and bone resorption during lipopolysaccharide (LPS)-induced inflammation in vivo and in vitro. In vivo, mice received supracalvarial injections of LPS with or without (D-Ala2)GIP for 5 days. Osteoclast formation and bone resorption were evaluated, and TNF-α and RANKL expression were measured. In vitro, the influence of (D-Ala2)GIP on RANKL- and TNF-α-induced osteoclastogenesis, LPS-triggered TNF-α expression in macrophages, and RANKL expression in osteoblasts were examined. Compared to the LPS-only group, calvariae co-administered LPS and (D-Ala2)GIP led to less osteoclast formation, lower bone resorption, and decreased TNF-α and RANKL expression. (D-Ala2)GIP inhibited osteoclastogenesis induced by RANKL and TNF-α and downregulated TNF-α expression in macrophages and RANKL expression in osteoblasts in vitro. Furthermore, (D-Ala2)GIP suppressed the MAPK signaling pathway. The results suggest that (D-Ala2)GIP dampened LPS-triggered osteoclast formation and bone resorption in vivo by reducing TNF-α and RANKL expression and directly inhibiting osteoclastogenesis. [ABSTRACT FROM AUTHOR]

Background: The regulation of inflammatory mediators in the degenerating intervertebral disc (IVD) and corresponding ligamentum flavum (LF) is a topic of emerging interest. The study aimed to investigate the expression of a broad array of inflammatory mediators in the degenerated LF and IVD using a dog model of spontaneous degenerative disc disease (DDD) to determine potential treatment targets. Methods: LF and IVD tissues were collected from 22 normal dogs (Pfirrmann grades I and II) and 18 dogs affected by DDD (Pfirrmann grades III and IV). A qPCR gene array was used to investigate the expression of 80 inflammatory genes for LF and IVD tissues, whereafter targets of interest were investigated in additional tissue samples using qPCR, western blot (WB), and immunohistochemistry. Results: Tumor necrosis factor superfamily (TNFSF) signaling was identified as a regulated pathway in DDD, based on the significant regulation (n‐fold ± SD) of various TNFSF members in the degenerated IVD, including nerve growth factor (NGF; −8 ± 10), CD40LG (464 ± 442), CD70 (341 ± 336), TNFSF Ligand 10 (9 ± 8), and RANKL/TNFSF Ligand 11 (85 ± 74). In contrast, TNFSF genes were not significantly affected in the degenerated LF compared to the control LF. Protein expression of NGF (WB) was significantly upregulated in both the degenerated LF (4.4 ± 0.5) and IVD (11.3 ± 5.6) compared to the control group. RANKL immunopositivity was significantly upregulated in advanced stages of degeneration (Thompson grades IV and V) in the nucleus pulposus and annulus fibrosus of the IVD, but not in the LF. Conclusions: DDD involves a significant upregulation of various TNFSF members, with tissue‐specific expression profiles in LF and IVD tissues. The differential involvement of TNFSF members within multiple spinal tissues from the same individual provides new insights into the inflammatory processes involved in DDD and may provide a basis to formulate hypotheses for the determination of potential treatment targets. [ABSTRACT FROM AUTHOR]

Skeletal fragility and high fracture rates are common in CKD. A key component of bone loss in CKD with secondary hyperparathyroidism is high bone turnover and cortical bone deterioration through both cortical porosity and cortical thinning. We hypothesized that RANKL drives high bone resorption within cortical bone leading to the development of cortical porosity in CKD (study 1) and that systemic inhibition of RANKL would mitigate the skeletal phenotype of CKD (study 2). In study 1, we assessed the skeletal properties of male and female Dmp1-cre RANKLfl/fl (cKO) and control genotype (Ranklfl/fl ; Con) mice after 10 wk of adenine-induced CKD (AD; 0.2% dietary adenine). All AD mice regardless of sex or genotype had elevated blood urea nitrogen and high PTH. Con AD mice in both sexes had cortical porosity and lower cortical thickness as well as high osteoclast-covered trabecular surfaces and higher bone formation rate. cKO mice had preserved cortical bone microarchitecture despite high circulating PTH as well as no CKD-induced increases in osteoclasts. In study 2, male mice with established AD CKD were either given a single injection of an anti-RANKL antibody (5 mg/kg) 8 wk post-induction of CKD or subjected to 3×/wk dosing with risedronate (1.2 μg/kg) for 4 wk. Anti-RANKL treatment significantly reduced bone formation rate as well as osteoclast surfaces at both trabecular and cortical pore surfaces; risedronate treatment had little effect on these bone parameters. In conclusion, these studies demonstrate that bone-specific RANKL is critical for the development of high bone formation/high osteoclasts and cortical bone loss in CKD with high PTH. Additionally, systemic anti-RANKL ligand therapy in established CKD may help prevent the propagation of cortical bone loss via suppression of bone turnover. [ABSTRACT FROM AUTHOR]

Infection of ruminants such as cattle with Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease, a disease characterized by chronic inflammation of the small intestine and diarrhoea. Infection with MAP is acquired via the faecal-to-oral route and the pathogen initially invades the epithelial lining of the small intestine. In this study we used an in vitro 3D mouse enteroid model to determine the influence of M cells in infection of the gut epithelia by MAP, in comparison with another bacterial intestinal pathogen of veterinary importance, Salmonella enterica serovar Typhimurium. The differentiation of M cells in the enteroid cultures was induced by stimulation with the cytokine receptor activator of nuclear factor-κB ligand (RANKL), and the effects on MAP and Salmonella uptake and intracellular survival were determined. The presence of M cells in the cultures correlated with increased uptake and intracellular survival of Salmonella, but had no effect on MAP. Interestingly neither pathogen was observed to preferentially accumulate within GP2-positive M cells.