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Background: The differential responses of the myokine irisin, in combination with changes in markers and regulators of bone remodeling to high-intensity interval exercise of high and low impact, were examined in 18 young adult females (22.5 ± 2.7 years). Methods: Participants performed two high-intensity interval exercise trials in random order: running on a treadmill and cycling on a cycle ergometer. Trials consisted of eight 1 min running or cycling intervals at ≥ 90% of maximal heart rate, separated by 1 min passive recovery intervals. Blood samples were collected at rest (pre-exercise) and 5 min, 1 h, and 24 h following each exercise trial. Irisin, osteocalcin, sclerostin, osteoprotegerin (OPG), receptor activator nuclear factor kappa-β ligand (RANKL), and parathyroid hormone (PTH) were analyzed in serum, with post-exercise concentrations being corrected for exercise-induced changes in plasma volume. Results: Irisin was elevated 24 h post-exercise compared to its resting values in both trials (20%, p < 0.05) and was higher after cycling compared to running (exercise mode effect, p < 0.05) with no interaction. Osteocalcin, sclerostin, PTH, and RANKL increased from pre- to 5 min post-exercise (18%, 37%, 83%, and 33%, respectively, p < 0.05), returning to baseline levels in 1 h, with no trial or interaction effects. OPG showed a time effect (p < 0.05), reflecting an overall increase at 5 min and 1 h post-exercise, which was not significant after the Bonferroni adjustment. Conclusions: In young adult females, high-intensity interval exercise induced an immediate response in markers and regulators of bone remodeling and a later response in irisin concentrations, which was independent of the gravitational impact.

Implant-related bone infections are a major complication in orthopedic surgery that lead to inflammation and bone destruction. Bacterial biofilm formation on the implant is discussed to polarize the immune response towards tolerance and to facilitate bacterial persistence. In addition to their role in the early immune response, macrophages are osteoclast precursor cells. Therefore, macrophages can link inflammation and RANKL-mediated osteoclastogenic bone destruction. We investigated the influence of Staphylococcus aureus (SA) and epidermidis (SE) biofilm formation on immune function and osteoclastogenesis using RAW264.7 cells and conditioned media (CM) of planktonic and biofilm cultures in the presence and absence of the osteoclastogenic transcription factor RANKL. Analysis of immune cell activation, metabolic activity and osteoclast formation revealed that a planktonic environment causes a pro-inflammatory response. This was also partially induced by biofilm CM. Simultaneous stimulation with CM and RANKL suppressed osteoclast formation in favor of a long-term immune activation. While the early macrophage response towards CM was dominated by glycolysis, the CM and RANKL approach shifted metabolism towards increased mitochondrial biomass and activity. This was most evident in biofilm CM. We further showed that planktonic CM effects are mediated through activation of TLR signaling and induction of IFN-β production. In biofilm CM, high lactate levels seem to significantly contribute to the modulation of macrophages. Our results can contribute to find targets for therapeutic intervention that restore an effective pro-inflammatory immune response, which could help to control implant-related bone infections. Importance The number of endoprosthetic and bone reconstructive surgeries is rising and accordingly incidences of implant-related bone infections increase. Biofilm formation on the implant surface protects bacteria against most antibiotics and the host’s immune response. Thus, novel therapeutic strategies for prevention or treatment of biofilm formation are needed. Furthermore, bone infections are associated with osteoclast-mediated bone destruction, which promotes infection progression and ultimately leads to implant loosening. We investigated the effect of staphylococci-mediated biofilm formation on macrophage immune activation and osteoclastogenic differentiation, respectively. In planktonic and biofilm environments, immune cell activation dominated over osteoclastogenesis. The effects of planktonic environments were mainly mediated through activation of TLR signaling and resulted in a pronounced pro-inflammatory and IFN-β macrophage immune response. In biofilm environments, high lactate levels impaired osteoclast formation but also prevented an effective long-term immune activation. Therefore, targeting biofilm-derived metabolites but also IFN-β signaling might be promising novel treatment strategies.

Aseptic loosening of the prosthesis caused by wear-particle-induced osteolysis is a long-term complication and one of the most common reasons for the failure of joint implants. The primary cause of aseptic loosening of the prosthesis is overactive bone resorption caused by wear-particle-activated osteoclasts in both direct and indirect ways. Therefore, drugs that can inhibit differentiation and bone resorption of osteoclasts need investigation as a potential therapeutic strategy to prevent and treat peri-prosthetic osteolysis and thereby prolong the service life of the prosthesis. This study has verified the potential inhibitory effect of LY450139 on inflammatory osteolysis induced by titanium particles in a mice skull model. In addition, we found that LY450139 inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis, bone resorption, and podosomal actin belt formation in a dose-dependent manner without evidence of cytotoxicity in vitro. In addition, LY450139 significantly decreased the expression of osteoclast-specific markers, including TRAP, CTSK, V-ATPase d2, CTR, DC-STAMP, NFATc1, and the downstream target gene Hes1 in Notch signaling pathway. Further investigation of the molecular mechanism demonstrated that LY450139 inhibited the formation of osteoclasts via inhibition of the NF-κB and Notch signaling pathways. In summary, LY450139 inhibited the formation of RANKL-mediated osteoclasts via NF-κB and Notch signaling and inhibited Ti particle-induced inflammatory osteolysis in vivo. LY450139 is a potential targeted drug for the treatment of peri-prosthetic osteolysis and other osteolytic disease associated with overactive osteoclasts.

Osteoporosis is a reduction in skeletal mass due to the decrease of osteogenic ability and the activation of the osteoclastic function. Inhibiting bone resorption and accelerating the new bone formation is a promising strategy to repair the bone defect of osteoporosis. In this study, we first systematically investigated the roles of Chinese medicine Asperosaponin VI (ASP VI) on osteogenic mineralization of BMSCs and osteoclastogenesis of BMMs, and then explored the synergistic effect of ASP VI and BS (BMP-2 immobilized in 2-N, 6-O-sulfated chitosan) on bone formation. The result showed that ASP VI with the concentration lower than 10−4 M contributed to the expression of osteogenic gene and inhibited osteoclastic genes RANKL of BMSCs. Simultaneously, ASP VI significantly reduced the differentiation of mononuclear osteoclasts in the process of osteoclast formation induced by M-CSF and RANKL. Furthermore, by stimulating the SMADs, TGF-β1, VEGFA, and OPG/RANKL signaling pathways, ASBS (ASP VI and BS) substantially enhanced osteogenesis, greatly promoted angiogenesis, and suppressed osteoclastogenesis. The findings provide a new perspective on osteoporosis care and prevention., Graphical abstract Image 1, Highlights • Asperosaponin VI down-regulated the osteoclastic genes RANKL expression. • First combined Asperosaponin VI with rhBMP-2 and 2-N, 6-O-sulfated chitosan. • Show high osteogenic activity and excellent anti-osteoclastogenesis. • Explored mechanism on promoting osteogenesis and inhibiting osteoclastogenesis.

Postmenopausal osteoporosis is a systemic disease characterized by the loss of bone mass and increased bone fracture risk largely resulting from significantly reduced levels of the hormone estrogen after menopause. Besides the direct negative effects of estrogen-deficiency on bone, indirect effects of altered immune status in postmenopausal women might contribute to ongoing bone destruction, as postmenopausal women often display a chronic low-grade inflammatory phenotype with altered cytokine expression and immune cell profile. In this context, it was previously shown that various immune cells interact with osteoblasts and osteoclasts either via direct cell-cell contact, or more likely via paracrine mechanisms. For example, specific subtypes of T lymphocytes express TNFα, which was shown to increase osteoblast apoptosis and to indirectly stimulate osteoclastogenesis via B cell-produced receptor-activator of NF-κB ligand (RANKL), thereby triggering bone loss during postmenopausal osteoporosis. Th17 cells release interleukin-17 (IL-17), which directs mesenchymal stem cell differentiation towards the osteogenic lineage, but also indirectly increases osteoclast differentiation. B lymphocytes are a major regulator of osteoclast formation via granulocyte colony-stimulating factor secretion and the RANKL/osteoprotegerin system under estrogen-deficient conditions. Macrophages might act differently on bone cells dependent on their polarization profile and their secreted paracrine factors, which might have implications for the development of postmenopausal osteoporosis, because macrophage polarization is altered during disease progression. Likewise, neutrophils play an important role during bone homeostasis, but their over-activation under estrogen-deficient conditions contributes to osteoblast apoptosis via the release of reactive oxygen species and increased osteoclastogenesis via RANKL signaling. Furthermore, mast cells might be involved in the development of postmenopausal osteoporosis, because they store high levels of osteoclastic mediators, including IL-6 and RANKL, in their granules and their numbers are greatly increased in osteoporotic bone. Additionally, bone fracture healing is altered under estrogen-deficient conditions with the increased presence of pro-inflammatory cytokines, including IL-6 and Midkine, which might contribute to healing disturbances. Consequently, in addition to the direct negative influence of estrogen-deficiency on bone, immune cell alterations contribute to the pathogenesis of postmenopausal osteoporosis., publishedVersion

Introduction: Favipiravir and Vitamin C (Vit C) were used together in the treatment of the COVID-19 pandemic. However, the effects of favipiravir on the periodontium are still unknown. Therefore, the aim of this study was to investigate the effects of Favipiravir and Vit C treatment on alveolar bone metabolism. Experimental: Fifty healthy adult male Sprague-Dawley rats (2-3 months old) were randomly divided into five equal groups (n = 10): Control, Favi 20, Favi 100, Favi 20+Vit C, Favi 100+Vit C. Favipiravir (20 mg/kg and 100 mg/kg, i.m.) and Vit C (150 mg/kg/day, oral) were administered to the rats for 14 days. Alveolar bone loss (ABL) and histopathological changes were examined using a light microscope. Immunohistochemistry was used to determine levels of receptor activator of nuclear factor kappa-B ligand (RANKL), caspase-3, bone morphogenic protein 2 (BMP-2) and alkaline phosphaTaşe (ALP) in the bone tissues. Results: Favipiravir increased the levels of RANKL and caspase-3 expression but decreased BMP-2 and ALP levels in a dose-dependent manner. Favi 20+Vit C and Favi 100 +Vit C groups showed decreased RANKL and caspase-3 levels in addition to increased BMP-2 and ALP levels. Conclusion: Favipiravir can cause histopathological damage to the periodontium, but administration of favipiravir combined with Vit C can provide a protective effect against this damage.

Osteoporosis is a systemic metabolic bone disease with an increasing incidence rate. Chinese medicinal herbs have a long history of treating bone diseases. Polysaccharides are an important category of phytochemicals in Chinese medicinal herbs, and their health benefits have increased the interest of the public. Numerous studies have indicated that polysaccharides exhibit anti-osteoporosis effects by balancing bone resorption and bone formation, but the detailed effects and mechanism have not been systematically summarized. We performed a comprehensive review of the literature to consolidate studies for the period 2000–2021 by conducting electronic searches on the PubMed, CNKI, VIP, and Wanfang databases. In total, polysaccharides from 19 kinds of Chinese medicinal herbs in 54 studies have shown bone homeostasis protective properties. In vivo and in vitro experiments have demonstrated that polysaccharides present properties in the treatment of postmenopausal osteoporosis, senile osteoporosis, and glucocorticoid-induced secondary osteoporosis, especially postmenopausal osteoporosis. Moreover, a number of signalling pathways, such as the Wnt/β-catenin signalling pathway, BMP/SMAD/RUNX2 signalling pathway, OPG/RANKL/RANK signalling pathway, apoptosis pathway, and transcription factors, are regulated by polysaccharides and participate in improving bone homeostasis. This review will provide a better understanding of the anti-osteoporotic effects of polysaccharides and the concomitant modulations of signalling pathways.

Mechanical strain regulated osteoclastic differentiation and angiogenesis are crucial for bone modeling and remodeling, and previous data indicate that high-magnitude strain within physiological load regulates osteoclastic differentiation. However, the underlying mechanisms are still not fully understood. In the present study, the RAW264.7 mouse monocyte/macrophage was used as an osteoclast precursor, and the bone marrow-derived macrophages (BMMs) were isolated and cultured in vitro. The above cells were subjected to macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL) for the induction of osteoclast differentiation. Subsequently, the above cells were stretched by differential strain magnitudes to simulate the mechanical stimuli in the physiological conditions, and we found that low-magnitude strain (100 με) increased the expression levels of Acp5, Clcn7, MMP9 and Ctsk to promote osteoclastogenesis, while high-magnitude strain (3000 με) had opposite effects. In addition, we noticed that high-magnitude strain upregulated PTEN to inactivate the PI3K/Akt signaling pathway, and silencing of PTEN abrogated the suppressing effects of high-magnitude strain on osteoclastic differentiation. Next, we screened out that high-magnitude strain downregulated miR-21 to promote PTEN expressions in a competing endogenous RNA (ceRNA)-dependent manner. Finally, upregulation of miR-21 recovered osteoclastic differentiation in RAW264.7 and BMMs cells stimulated with high-magnitude strain. Collectively, our findings suggested that high-magnitude mechanical strain affected osteoclastic differentiation through modulating the miR-21/PTEN/PI3K/Akt signaling cascade, which provided potential strategies for the treatment of bone-related diseases. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00507-x.

Osteoporosis is a systemic skeletal disorder characterized by reduced bone mineral density (BMD) and bone quality and increased bone porosity, which increase the risk of bone fracture. Inflammation, one of the important mechanisms related to aging, is associated with osteoporosis. Treatment with anti-inflammatory agents is effective for alleviating senile osteoporosis. Alginate oligosaccharide (AOS) can prevent and treat diseases related to inflammation, oxidative stress, and immunity. This study evaluates the effect of AOS on osteoporosis and investigates the underlying mechanism. Osteoporosis model was induced by D-galactose (D-gal) (200 mg kg-1 day-1 ) for eight weeks. Three groups were administered via AOS (50, 100, and 150 mg kg-1 day-1 ) for four weeks, while a control group received sterile water (5 ml kg-1 day-1 ) for 8 weeks. The results showed that AOS improved bone density and bone microstructure in D-gal-induced osteoporosis mice. AOS inhibited osteoclast proliferation, probably through the suppression of receptor activator of nuclear factor-kappa B ligand (RANKL)-associated nuclear factor kappa B (NF-κB) and c-Fos signaling pathway. AOS also increased osteoprotegerin (OPG) expression and competitively inhibited the binding between RANK and RANKL in senile osteoporosis. Further, AOS decreased the secretion of serum osteocalcin and reduced bone conversion. Together, these results demonstrate the anti-osteoporosis activity of AOS in mice with osteoporosis.

Ameloblastoma is an odontogenic neoplasm characterized by slow intraosseous growth with progressive jaw resorption. Recent reports have revealed that ameloblastoma harbours an oncogenic BRAFV600E mutation with mitogen-activated protein kinase (MAPK) pathway activation and described cases of ameloblastoma harbouring a BRAFV600E mutation in which patients were successfully treated with a BRAF inhibitor. Therefore, the MAPK pathway may be involved in the development of ameloblastoma; however, the precise mechanism by which it induces ameloblastoma is unclear. The expression of ADP-ribosylation factor (ARF)-like 4c (ARL4C), induced by a combination of the EGF-MAPK pathway and Wnt/β-catenin signalling, has been shown to induce epithelial morphogenesis. It was also reported that the overexpression of ARL4C, due to alterations in the EGF/RAS-MAPK pathway and Wnt/β-catenin signalling, promotes tumourigenesis. However, the roles of ARL4C in ameloblastoma are unknown. We investigated the involvement of ARL4C in the development of ameloblastoma. In immunohistochemical analyses of tissue specimens obtained from 38 ameloblastoma patients, ARL4C was hardly detected in non-tumour regions but tumours frequently showed strong expression of ARL4C, along with the expression of both BRAFV600E and RAF1 (also known as C-RAF). Loss-of-function experiments using inhibitors or siRNAs revealed that ARL4C elevation depended on the RAF1-MEK/ERK pathway in ameloblastoma cells. It was also shown that the RAF1-ARL4C and BRAFV600E-MEK/ERK pathways promoted cell proliferation independently. ARL4C-depleted tumour cells (generated by knockdown or knockout) exhibited decreased proliferation and migration capabilities. Finally, when ameloblastoma cells were co-cultured with mouse bone marrow cells and primary osteoblasts, ameloblastoma cells induced osteoclast formation. ARL4C elevation in ameloblastoma further promoted its formation capabilities through the increased RANKL expression of mouse bone marrow cells and/or primary osteoblasts. These results suggest that the RAF1-MEK/ERK-ARL4C axis, which may function in cooperation with the BRAFV600E-MEK/ERK pathway, promotes ameloblastoma development. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

SNP rs3755955 (major/minor allele: G/A) located in Iduronidase-Alpha-L- (IDUA) gene was reported to be significant for human bone mineral density (BMD). This follow-up study was to uncover the underlying association mechanism through molecular and cellular functional assays relevant to bone. We tested the effects of single nucleotide polymorphisms (SNP) rs3755955 (defined allele G as wild-type and allele A as variant-type) on osteoblastic and osteoclastic functions, as well as protein phosphorylation in stably transfected human fetal osteoblast (hFOB) cell and mononuclear-macrophage (RAW264.7) cell. In hFOB cells, transfection with variant-type IDUA significantly decreased osteoblastic gene expression (OPN, COL1A1 and RANKL) (p

Periodontal disease is an inflammatory disease caused by periodontopathogenic bacteria, which eventually leads to bone tissue (alveolar bone) destruction as inflammation persists. Periodontal tissues have an immune system against the invasion of these bacteria, however, due to the persistent infection by periodontopathogenic bacteria, the host innate and acquired immunity is impaired, and tissue destruction, including bone tissue destruction, occurs. Osteoclasts are essential for bone destruction. Osteoclast progenitor cells derived from hematopoietic stem cells differentiate into osteoclasts. In addition, bone loss occurs when bone resorption by osteoclasts exceeds bone formation by osteoblasts. In inflammatory bone disease, inflammatory cytokines act on osteoblasts and receptor activator of nuclear factor-κB ligand (RANKL)-producing cells, resulting in osteoclast differentiation and activation. In addition to this mechanism, pathogenic factors of periodontal bacteria and mechanical stress activate osteoclasts and destruct alveolar bone in periodontitis. In this review, we focused on the mechanism of osteoclast activation in periodontitis and provide an overview based on the latest findings.

The possible mechanisms underlying the quantitative and qualitative effects of cinacalcet on bone were explored in a chronic kidney disease-mineral and bone disorder (CKD-MBD) mouse model in relation to the influence of the interactions among the osteoclast (OC) endoplasmic reticulum (ER) stress, autophagy and apoptosis pathways on OC differentiation. Body weight and biochemical parameters improved significantly in the CKD + cinacalcet groups compared to the CKD group. Micro-computed tomography (μCT) revealed both cortical and trabecular parameters deteriorated significantly in the CKD group and were reversed by cinacalcet in a dose-dependent manner. Nanoindentation analysis of bone quality proved that both cortical hardness and elastic modulus improved significantly with high dose cinacalcet treatment. In vitro studies revealed that cinacalcet inhibited receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB (RANK)-induced OC differentiation in a concentration-dependent manner through a close interaction between activation of caspase-related apoptosis, reversal of OC autophagy through the protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and adenosine monophosphate-activated protein kinase (AMPK) pathways, and attenuation of the OC ER stress/CREBH/NFATc1 signaling pathway. Cinacalcet improves both bone quantity and bone quality in CKD mouse model and inhibits OC differentiation through regulation of the interactions among the apoptosis, ER stress, and autophagy pathways within OCs. © 2021 American Society for Bone and Mineral Research (ASBMR).

As part of our continuous program to identify new potential candidates for controlling osteolytic bone diseases from natural products, the alkaloid fraction of barley (Hordeum vulgare var. hexastichon) grass (HVA) significantly inhibited RANKL-induced osteoclast formation and protected mice from LPS-induced bone loss. A phytochemical investigation of HVA afforded nine indole alkaloids, including one new compound [hordeumin A (1)] and eight known analogues (2-9). Of them, four (1, 2, 4, and 5) were anti-osteoclastogenic compounds. Of these four, compound 5 significantly suppressed RANKL-induced osteoclast formation, actin ring formation, and bone resorption in a concentration-dependent manner. It also suppressed the RANKL-induced NF-κB and MAPK signaling pathways and the activation of c-Fos and NFATc1. Compound 5 also reduced the expression levels of osteoclast-specific marker genes, including TRAP, CtsK, DC-STAMP, OSCAR, and MMP9. Our findings suggest that HVA and its alkaloid constituents could be valuable candidates for the prevention and treatment of osteolytic bone diseases.

Cadmium (Cd) contamination in the environment is a major public health concern since it has been linked to osteoporosis and other bone deformities. Linarin is a flavonoid glycoside, and it can promote osteoblastogenesis. This research aimed to investigate the potential role of linarin against Cd-exposed bone deformations in mice model. In our research, male mice were randomly allocated into four groups: control, Cd-exposed, and Cd + linarin (20 and 40mg/kg/bw, respectively). Linarin prevented body weight loss, increased serum calcium (Ca) and phosphorus (P), and bone alkaline phosphatase (BAP) levels in Cd-exposed groups. Furthermore, linarin treatment at 20 and 40mg/kg/bw significantly decreased RANK and OPG, resulting in an increase in RANKL mRNA levels and protein distribution in the bone of Cd-exposed mice. In addition, the bone of Cd-exposed mice administered with linarin showed higher TRAP, NFATc1, MMP9, and RUNX2 mRNA levels and protein distribution. Linarin significantly decreased oxidative stress in Cd-exposed mice bone by decreasing MDA, a lipid peroxidation product. Moreover, linarin protects Cd-exposed mice antioxidant enzymes by increasing bone SOD, CAT, and GPx levels. Besides, linarin suppresses alterations in the inflammatory system, i.e., NF-κB p65/IKKβ, by reducing NF-κB p65, IKKβ, IL-6, and TNF-α in the bone of Cd-exposed animals. This study concluded that linarin has potential to cure osteoporosis in Cd-exposed mice by reducing oxidative stress and inflammation and modulating the RANK/RANKL/OPG pathway.

Objective Receptor activator of NF-κβ ligand (RANKL) is crucial for the development of hepatic insulin resistance and poor glucose uptake; therefore, inhibiting RANKL with Denosumab could improve fasting plasma glucose (FPG) and insulin (FPI). Methods A systematic review was conducted to evaluate the effects of Denosumab on glycemic parameters. PubMed, SCOPUS, EBSCO, and LILACS databases were searched for studies that investigated the effect of Denosumab on FPG, glycated hemoglobin (HbA1c), FPI, and Homeostatic Model Assessment for Insulin Resistance (HOMA1-IR). The pooled standard difference in means (SDM) and 95% confidence intervals (95%CI) were calculated. The results were stratified into (1) Normal Glucose Tolerance (NGT) and (2) Impaired Glucose Tolerance (IGT). Results Six publications (1203 participants) were included. There was a significant association between Denosumab and FPG (SDM = −0.388, 95%CI: −0.705 to −0.070, p = .017) and with HOMA1-IR (SDM = −0.223, 95%CI: −0.388 to −0.058, p = .008), but not for HbA1c and FPI. When stratified by glucose tolerance, the association between Denosumab and FPG, HbA1c, and HOMA1-IR was present for the IGT group. Lastly, Denosumab had a time-dependent effect on HbA1c (slope = −0.037, 95%CI: −0.059 to −0.015, p

Type 2 Diabetes Mellitus is a chronic metabolic disease that causes endothelial damage and is an important risk factor for atherosclerosis. In the present study vitamin D3 supplementation in rats was used to determine the role of Osteoprotegerin (OPG)/Receptor activator kB ligand (RANKL) signalling in endothelial damage and changes in the expression levels of genes involved in this pathway. We hypothesized that vitamin D3 supplementation affects OPG and RANKL activity in the aorta. Diabetes was induced in rats via injections of 40 mg/kg of streptozotocin followed by a high fructose (10%) diet. Group 2 (healthy) and 4 (diabetic) received 170 IU/kg of vitamin D3 weekly for 5 weeks, while Group 1 (healthy) and 2 (diabetic) received sterile saline. The aortas of each group were collected to analyse mRNA expression using the real-time PCR method and also to evaluate magnesium and calcium levels using inductively coupled plasma mass spectrometry. Opg and Il-1b expression levels were significantly associated with both diabetes and vitamin D3 supplementation in the aortas of the study groups (p ≤ 0.05). Opg mRNA expression was also found to correlate with both Icam-1 and Nos3 mRNA expression levels (r = 0.699, p = 0.001 and r = 0.622, p = 0.003, respectively). In addition, when mineral levels in the aortic tissues were compared among all groups, it was found that the interaction of diabetes and vitamin D3 supplementation significantly affected Mg levels and Mg/Ca ratios. It is concluded that vitamin D3 supplementation has a modulatory effect on OPG/RANKL activity in the vessel wall by ameliorating endothelial damage in diabetes. This effect may contribute to the regulation of cytokine-mediated vascular homeostasis and mineral deposition in the aorta; therefore, further comprehensive studies are proposed to demonstrate this relationship.

Osteoporosis is an age‐related disease characterized by low mineral density, compromised bone strength and increased risk of fragility fracture. Most agents for treating osteoporosis focus primarily on anti‐resorption by inhibiting osteoclast activity. Bisphosphonate (BP) is a potent anti‐resorptive agent that has been used clinically for decades and is proven to be effective. However, BP has a variety of side effects and is far from being an ideal anti‐osteoporosis agent. BP selectively binds to calcium crystals, which are subsequently taken up or released by osteoclasts. Based on the action of BP, we previously demonstrated the inhibitory effect of a novel bone‐targeting BP derivative, bisphosphonate‐enoxacin (BE). In the current study, we used bone marrow‐derived osteoclast cultures to further assess the inhibitory effect of BE on osteoclastogenesis and employed reverse transcription PCR and real‐time PCR to examine expression of osteoclast‐specific genes. Additionally, we used bone resorption and F‐actin immunofluorescence assays to evaluate the effect of BE on osteoclast function and investigated the potential mechanisms affecting osteoclast differentiation and function in vitro. Furthermore, an ovariectomized (OVX) rat model was established to evaluate the therapeutic effects of BE on preventing bone loss. Results showed that BE exerted potent inhibitory effects on osteoclast formation and bone resorption by specifically abrogating RANKL‐induced JNK signalling, and that it preserved OVX rat bone mass in vivo without any notable side effects. Collectively, these results indicated that the BP derivative BE may have significant potential as a treatment for osteoporosis and other osteolytic diseases.

Osteosarcoma is a major form of malignant bone tumor that typically occurs in young adults and children. The combination of aggressive surgical strategies and chemotherapy has led to improvements in survival time, although individuals with recurrent or metastatic conditions still have an extremely poor prognosis. This disappointing situation strongly indicates that testing novel, targeted therapeutic agents is imperative to prevent the progression of osteosarcoma and enhance patient survival time. Curcumin, a naturally occurring phenolic compound found in Curcuma longa, has been shown to have a wide variety of anti-tumor, anti-oxidant, and anti-inflammatory activities in many types of cancers including osteosarcoma. Curcumin is a highly pleiotropic molecule that can modulate intracellular signaling pathways to regulate cell proliferation, inflammation, and apoptosis. These signaling pathways include RANK/RANKL, Notch, Wnt/β-catenin, apoptosis, autophagy, JAK/STAT, and HIF-1 pathways. Additionally, curcumin can regulate the expression of various types of microRNAs that are involved in osteosarcoma. Therefore, curcumin may be a potential candidate for the prevention and treatment of osteosarcoma. This comprehensive review not only covers the use of curcumin in the treatment of osteosarcoma and its anti-cancer molecular mechanisms but also reveals the novel delivery strategies and combination therapies with the aim to improve the therapeutic effect of curcumin.

Fracture healing is a complex event with the involvement of many cell systems, cytokines, as well as mRNAs. Herein, we report the interactions among long noncoding RNA X-inactive specific transcript (XIST)/microRNA-135 (miR-135)/cAMP response element-binding protein 1 (CREB1) axis during fracture healing. We observed increased expression of XIST in patients with long-term unhealed fracture by microarray analysis. Subsequently, a mouse model with tibial fracture and a cell model using osteoblast-like MC3T3-E1 cells were generated. The XIST overexpression during fracture healing decreased proliferation and differentiation of MC3T3-E1 cells, while silencing of XIST facilitated MC3T3-E1 cell growth. Furthermore, miR-135 targeted CREB1 and negatively regulated its expression. XIST acted as a sponge for miR-135, thereby upregulating CREB1 and promoting the activity of the TNF-α/RANKL pathway. Transfection of miR-135 inhibitor or CREB1 overexpression blocked the stimulating effects of XIST knockdown on MC3T3-E1 cell growth. Besides, specific inhibitors of the TNF-α/RANKL pathway reversed the repressive role of XIST in cell osteogenic differentiation. All in all, these findings suggest that XIST knockdown induces the differentiation of osteoblast-like cells via regulation of the miR-135/CREB1/TNF-α/RANKL axis. XIST, as a consequence, represents an attractive therapeutic strategy to accelerate fracture healing.

A simple methylenedioxy dibromoindole alkaloid, amakusamine (1), was isolated from a marine sponge of the genus Psammocinia, and its structure was determined from spectroscopic data, time-dependent density-functional theory calculations, and synthesis. Compound 1 inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced formation of multinuclear osteoclasts with an IC50 value of 10.5 μM in RAW264 cells. The structure-activity relationship of 1 was also investigated with synthetic derivatives.

Background/purpose: Local anesthesia (LA) application is a routine dental work in clinic. The aim of present study was to evaluate the extent of biologic effects of LA on periodontal ligaments (PDL) or bone cells (MG63). Materials and methods: Local anesthetics (LAs) at different concentrations were added to PDL and MG 63 cells. The viability of the cells was analyzed using an MTT assay. The inflammatory markers, COX-2, IL-1, IL-6 and TNF-A, of PDL and MG63 cells treated with LAs were analyzed with a Western blot assay. The extract medium of the LA-treated PDL cells was added to the MG63 cells for subsequent culture and to examine the RANKL, ALP, and OPG expression. The data were statistically analyzed with p 0.05). The RANKL expression in the MG63 cells increased as the Septanest and Scandonest concentrations were increased in the PDL extract medium (p

The main metabolic function of bone is the homeostasis of calcium and phosphate. This is achieved through a balance of bone resorption and formation. To gain an understanding of bone turnover it is important to appreciate the different cells, regulatory steroids and hormones involved and how these have their actions. This article summarizes bone cells, normal bone metabolism, bone turnover, regulatory mechanisms and the clinical applications of these. The receptor activator of nuclear factor-kB, receptor activator of nuclear factor-kB ligand and osteoprotegerin (RANK/RANKL/OPG) cell signalling pathway is discussed in detail. Understanding these topics enhances knowledge of the pathological processes and diseases that result from failures of bone metabolism.

OBJECTIVES Notch signaling pathway, known to influence bone resorption in several oral diseases, has not been analyzed in peri-implantitis yet. Therefore, the aims of the present study were to determine the levels of Notch cascade, bone remodeling mediators, and pro-inflammatory cytokines, in conjunction with clinical parameters, in subjects with peri-implant mucositis and peri-implantitis. MATERIAL AND METHODS Clinical parameters: peri-implant probing depth, bleeding on probing, suppuration on probing, and plaque index (PI) were recorded. Samples were collected from 130 participants, divided into peri-implantitis (PI), peri-implant mucositis (PM), and healthy implants (HI) group. Relative expression levels (REL) of Notch 1, Notch 2, Jagged 1, Hes 1, Hey 1, TNF-α, IL-17, IL-1β, IL-6, RANKL, and OPG mRNA were determined by reverse transcriptase-real-time polymerase chain reaction. Quantitation of Notch 1, Il-17, and IL-6 proteins was performed using ELISA assays. RESULTS All clinical parameters were significantly higher in PI compared to HI. Significant decrease of Notch 1, and higher REL of Hey 1, IL-1β, IL-6, and RANKL were found in PI compared to HI. PM showed significant increase of IL-1β REL in comparison with HI. In PI versus PM, significantly higher REL was found for Hey 1, TNF-α, IL-17, IL-1β, IL-6, and RANKL. Additionally, higher protein concentrations of IL-6 and IL-17 were detected in PI versus PM and versus HI group. CONCLUSION The combined effect of Notch 1 down-regulation and elevated expression of some key inflammation modulators might result in osteoclast activity increase and subsequent osteolysis in peri-implantitis.

Purpose Emerging evidence suggests that the progesterone-mediated receptor activator of nuclear factor κB (RANK)/soluble RANK ligand (sRANKL)/osteoprotegerin (OPG) pathway plays an important role in mammary carcinogenesis and is hyperactivated in germline (g)BRCA1/2 mutation carriers. We analyzed the effects of a 3-month intensive lifestyle intervention within the LIBRE-1 study on the serum levels of OPG and sRANKL and hypothesized that the intervention program provides a beneficial impact on the biomarkers by increasing OPG and reducing sRANKL serum concentrations. Methods Serum levels of OPG and sRANKL of 49 gBRCA1/2 mutation carriers were quantified using enzyme-linked immunosorbent assays. We used previously collected blood samples from participants of the prospective LIBRE-1 study, who were randomized into an intervention group (IG), increasing physical activity and adherence to the Mediterranean diet (MedD) through supervised sessions from study entry to the first study visit after 3 months and a usual-care control group (CG). Differences in biomarker levels before and after the 3-month intervention were tested within and between study groups. Results The lifestyle intervention resulted in a significant increase in OPG for participants in both the IG (q = 0.022) and CG (q = 0.002). sRANKL decreased significantly in the IG (q = 0.0464) and seemed to decrease in the CG (q = 0.5584). An increase in the intake of Omega-3 polyunsaturated fatty acids was significantly associated with an increase in OPG (r = 0.579, q = 0.045). Baseline serum levels of sRANKL were a strong predictor for the change of sRANKL in the course of the intervention (ß-estimate = − 0.70; q = 0.0018). Baseline physical fitness (assessed as VO2peak) might predict the change of OPG in the course of the intervention program (ß-estimate = 0.133 pg/ml/ml/min/kg; p = 0.0319; q = 0.2871). Conclusion Findings from this pilot study seem to confirm our hypothesis by showing an increase in OPG and decrease in sRANKL over a 3-month lifestyle intervention and suggest that increased physical activity and adherence to the MedD are potent modulators of the biomarkers OPG and potentially sRANKL.

Objective This study was aimed to determine the levels of salivary receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) and its association with periodontal status among periodontitis patients. Materials and Methods A cross-sectional study was designed and performed at the Dental Clinic, Hospital Universiti Sains Malaysia (HUSM). Random sampling was employed to identify 88 participants into three groups: 30 mild periodontitis, 30 moderate to severe periodontitis, and 28 healthy (nonperiodontitis) patients. Periodontal parameters: periodontal pocket depth (PPD), clinical attachment level (CAL), plaque score (PS), and gingival bleeding index (GBI) were recorded. In total, 4 mL of unstimulated whole saliva was collected to determine the levels of salivary RANKL and OPG proteins by using ELISA technique. Data were analyzed by using SPSS software version 24.0. Results Mean values for PPD (5.3 ± 0.5) and CAL (5.6 ± 0.5) were observed higher for moderate to severe periodontitis as compared with values (4.4 ± 0.2) (4.5 ± 0.2) in mild periodontitis patients. The mean salivary RANKL and OPG was 0.23 ± 0.07 ng/mL and 1.78 ± 0.70 ng/mL respectively in moderate to severe periodontitis. Only salivary RANKL levels were significantly and positively correlated with all the clinical periodontal parameters. Conclusion The levels of salivary RANKL were higher as opposed to lower OPG levels in periodontitis patients in contrast to healthy (nonperiodontitis) patients. RANKL levels were significantly associated with the periodontal parameters. Therefore, we can conclude that RANKL can potentially aid as an adjunctive diagnostic protein in evaluating periodontal disease.

The negative impact of rheumatoid arthritis (RA) on bone mineral density is well characterized. Notably, articular bone erosion is a central feature of RA, leading to joint damage and disabilities. In addition, the axial and appendicular skeleton can be affected, which secondly manifests in bone fracture. The main trigger of RA-associated bone loss is excessive bone degradation by osteoclasts and impaired bone formation by osteoblasts. In particular, the inflammatory status, reflected by high level of proinflammatory cytokines, receptor activator of nuclear factor κB ligand (RANKL), and autoantibodies induces the formation of bone-resorbing osteoclasts. Today, antirheumatic therapy effectively hampers synovial inflammation and bone erosion. However, current medication is unable to repair established bone lesions. This review outlines the knowledge gained about the pathophysiology of rheumatoid arthritis and the molecular mechanisms that promote osteoclast-mediated bone erosion and inhibit osteoblast-related bone formation, pointing out possible new intervention for inflammatory bone disease.

Objectives: This study aimed to evaluate the levels of TNF-alpha, PGE., RANKL, RANK, OPG, the markers of periimplant bone toss in peri-implant crevicutar fluid obtained around standard and extra short implants. Moreover, the levels of putative oral pathogens were investigated in the submucosal biofilm samples. Material and Methods: The implants were divided into two groups according to their Lengths: standard (>= 8 mm) and extra short (4 mm). A total of 60 implants were researched in 30 patients. The probing depth (PD), clinical attachment level (CAL), presence of bleeding on probing (BOP), 3-year survival rate (CSR), and bone toss (BL) were measured. Results: No statistically significant difference was found in the values of PD, CAL, BOP, CSR, and BL between the groups (P> 0.05). Total amounts of PGE,, RANKL, RANK, OPG, and RANKL/OPG were not statistically significantly different between the groups (P> 0.05). The abundance of F. nucleatum, T. forsythia, P. intermedia, P. gingivalis, S. oralis and T. denticola was compared between the groups and the results were not statistically significant (P> 0.05). Conclusion: The results of this study suggested that PGE(2), TNF-alpha, RANKL, RANK, OPG, and RANKL/OPG in PICF, as well as microbiological parameters in submucosal biofilms, were similar between standard (>= 8 mm) and extra short (4 mm) implants. Therefore, the implant length does not seem to influence the bone loss, levels of osteoimmunological and microbiological markers in the peri-implant tissues and survival rates.

Objective: This study was to determine OPG and RANKL expression after hydroxyapatite (HA) scaffold from crab shells (Portunus pelagicus) application in tooth socket of Cavia cobaya. Methods: This study was a post-test only control group design. Twenty four Cavia cobaya was divided into 4 groups. The lower left incisor was extracted and given a combination of HA and gelatin scaffold. Experimental animals were sacrificed on the 7th and 14th day. The amount of OPG and RANKL expression was calculated under a light microscope at 1000x magnification. The statistical analysis was done by One Way ANOVA Test and Tukey HSD. Results: Compared to other groups, the lowest and the highest level of OPG and RANKL were in P14 group. Conclusion: HA scaffold from crab shells (Portunus pelagicus) can increase OPG expression and decrease RANKL expression in the process of regenerating alveolar bone after tooth extraction.

Osteoporosis is a polygenic disease associated with low bone mineral density and deterioration of bone miniscule architecture and increased chance of bone fractures. However, several signaling pathways regulate bone mineral density including parathyroid hormone (PTH), Core-binding factor α-1 (CBFA1), Wnt/β-catenin, the receptor activator of the nuclear factor kappa-B (NF-κB) ligand (RANKL), myostatin, estrogen, and osteogenic exercise signaling pathways. These signaling pathways occur at protein level that depends not only on messenger RNA transcriptional regulation but also on a number of translational and posttranslational controls. Moreover, proteomic alterations in bone tissue due to a disease may occur in several ways that are unpredictable from either genome or transcriptome analysis. Decades of genome and transcriptome analyses have identified few causative genes; nonetheless, the majority of osteoporosis susceptibility genes remain unknown. It appears that a deeper view of bone proteome alterations will influence bone health and disease. This article highlights the efficacy of proteomics as an emerging tool for the discovery of bone mineral density molecular pathways.

To assess the serum and salivary levels of biomarkers related to bone metabolism in cirrhotic patients as well as the evidence of osteoporotic changes on panoramic radiographs. Thirty-eight cirrhotic patients underwent anamnesis and physical examination. Specimens of blood and saliva were collected for evaluation by using Luminex™ xMAP technology to quantify RANKL, OPG, IL-1β, IL-6 and TNF-α. Panoramic radiographs were evaluated based on the mandibular cortical index (MCI) and the resulting data were compared to the expression of biomarkers in serum and saliva. Descriptive data analysis was performed and the Mann–Whitney’s test and Spearman’s correlation were used. Most of the sample consisted of males (68.4%) who had cirrhosis mostly resulting from alcoholism (28.9%). Median concentration values of RANKL (74.44 pg/mL), IL-1 β (45.91 pg/mL), IL-6 (67.69 pg/mL) and TNF-α (5.97 pg/mL) in saliva were higher than those observed in serum. In 72.7% of the panoramic radiographs, MCI was found to be suggestive of osteoporotic changes. No statistically significant correlation was observed between salivary and serum expressions of biomarkers or between biomarkers and MCI. RANKL, OPG, IL-1β, IL-6 and TNF-α are expressed differently in serum and saliva and the concentration of these biomarkers is not related to MCI. This study contributes to the study of the mechanisms of osteoporosis in cirrhotic individuals.

Osteocytes are extremely sensitive to mechanical loading and govern bone remodeling process. Advanced glycation end products (AGEs) have the capacity to induce osteocyte apoptosis. In order to investigate the effects of AGEs on the mechanosensitivity of osteocytes, the osteocytic-like cells (MLO-Y4) were treated with low (50 μg/ml) and high (400 μg/ml) concentrations of AGEs for 1day and exposed to 15 dyne/cm2 of fluid shear stress. Then the F-actin cytoskeleton, prostaglandin E2(PGE2), Nitric oxide (NO), the Wnt/β-catenin signaling pathway activity mRNA expressions were detected for osteocytes mechanical response changes; osteocalcin (OCN) and receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) were detected for the regulation on bone remodeling function of osteocytes. The results showed that AGEs accumulation inhibited the sense of osteocytes to external mechincal loading, promoted shear-induced NO and PGE2 release, suppressed the mechanosensitivity of Wnt/β-catenin signaling pathway, and furthermore promoted OCN and RANKL/OPG mRNA expressions. These indicated AGEs had an adverse impact on the mechanosensitivity of osteocytes, and led to a negative effect on their regulation of bone remodeling process under mechanical stimulation. This work provides a new perspective to interpret the alteration mechanism of osteocytes mechanosensitivity and provides a novel clue for exploring the mechanism of osteoporosis.

Osteoclastogenesis is induced by receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), and can be suppressed by osteoprotegerin (OPG). Beclin1 has a dual role in osteoclastogenesis. However, the role of Beclin1-mediated autophagy during OPG-induced inhibition of osteoclastogenesis remains unclear. Here, we found that Beclin1 and matrix metalloproteinase 9 (MMP-9) expression were increased during osteoclastogenesis. OPG (20, 40, and 80 ng/mL) decreased Src and MMP-9 expression, but augmented Beclin1 expression and fluorescence intensity. Similarly, treatment with the autophagy activator rapamycin increased Beclin1 expression during OPG-induced inhibition of osteoclastogenesis. Further, Beclin1 knockdown restored osteoclast numbers by reducing autophagy during OPG-induced inhibition of osteoclastogenesis. These results indicate that Beclin1 has a positive role during OPG-induced inhibition of osteoclastogenesis by regulating autophagy, which might provide a potential basis for osteoclastogenesis.

Aging is associated with excessive bone loss that is not counteracted with the development of new bone. However, the mechanisms underlying age-related bone loss are not completely clear. Myeloid-derived suppressor cells (MDSCs) are a population of heterogenous immature myeloid cells with immunosuppressive functions that are known to stimulate tumor-induced bone lysis. In this study, we investigated the association of MDSCs and age-related bone loss in mice. Our results shown that aging increased the accumulation of MDSCs in the bone marrow and spleen, while in the meantime potentiated the osteoclastogenic activity of the CD11b(+)Ly6C(hi)Ly6G(+) monocytic subpopulation of MDSCs. In addition, CD11b(+)Ly6C(hi)Ly6G(+) MDSCs from old mice exhibited increased expression of c-fms compared to young mice, and were more sensitive to RANKL-induced osteoclast gene expression. On the other hand, old mice showed elevated production of IL-6 and receptor activator of nuclear factor kappa-B ligand (RANKL) in the circulation. Furthermore, IL-6 and RANKL were able to induce the proliferation of CD11b(+)Ly6C(hi)Ly6G(+) MDSCs and up-regulate c-fms expression. Moreover, CD11b(+)Ly6C(hi)Ly6G(+) MDSCs obtained from old mice showed increased antigen-specific T cell suppressive function, pStat3 expression, and cytokine production in response to inflammatory stimulation, compared to those cells obtained from young mice. Our findings suggest that CD11b(+)Ly6C(hi)Ly6G(+) MDSCs are a source of osteoclast precursors that together with the presence of persistent, low-grade inflammation, contribute to age-associated bone loss in mice.

Fifteen new isopimarane-type diterpenes, taichunins E-S (1-15), and a new 20-nor-isopimarane, taichunin T (16), together with four known compounds were isolated from Aspergillus taichungensis (IBT 19404). The structures of these new compounds were determined by NMR and mass spectroscopy, and their absolute configurations were analyzed by NOESY and TDDFT calculations of ECD spectra. Taichunins G, K, and N (3, 7, and 10) completely inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced formation of multinuclear osteoclasts in RAW264 cells at 5 μM, with 3 showing 92% inhibition at a concentration of 0.2 μM.

Circulating inflammatory factors affect osteoblast and osteoclast formation and activity in osteoporosis. Estrogen affects the migration of Th17 cells via the C-C chemokine receptor type 6 (CCR6) and C-C chemokine ligand 20 (CCL20) signaling pathways to modulate bone metabolism; however, it is unclear whether and how CCR6 modulates bone homeostasis. In the present study, CCR6 knockout (CCR6-/-) mice were selected to investigate the effects of CCR6 in the regulation of homeostasis of osteoblasts and osteoclasts. Primary osteoblasts were isolated from the calvarium of newborn CCR6-/- or wild-type mice, followed by osteoblastic differentiation culture in vitro. CCR6 deletion reduced osteoblast activity in terms of alkaline phosphatase (ALP) activity and inhibited osteoblast mineralization according to the results of Alizarin Red S staining, whereas it did not affect the proliferation of osteoblasts. CCR6 deletion inhibited Osterix mRNA expression in osteoblasts during the late stage of mineralization in vitro, while it did not affect mRNA expression levels of runt-related transcription factor 2 (Runx2) and Collagen-1. The ratio of osteoprotegerin (OPG) /receptor activator of nuclear factor κ-Β ligand (RANKL) mRNA level in osteoblasts was decreased by CCR6 deficiency in the culture treated with 1,25(OH)2D3/PGE2, while there was no effect observed in the normal culture environment. The results provide novel insights, such as that CCR6 deletion suppresses osteoblast differentiation by downregulating the expression levels of the transcription factor Osterix, and indirectly promotes osteoclast production by increasing transcription of RANKL. This may be one of the mechanisms via which CCR6 deletion regulates bone metabolism.

The receptor activator of nuclear factor-kappa B (RANK) and its ligand RANKL are members of the tumor necrosis factor (TNF) super-family of cytokines with a role in progestogen-associated malignancies in breast. Basic and clinical data support the participation of the cytokine pathway in metastatic disease and as poor prognosis markers. The value of RANK/RANKL as prognostic indicators in endometrial and ovarian tumors, as well as the data suggesting a potential role of RANK/RANKL in hormone dependent tumorigenesis in the endometrium, have been described. The D-CARE study could not confirm benefit in the modulation of RANKL in breast cancer.

Morin is a natural compound isolated from moraceae family members and has been reported to possess a range of pharmacological activities. However, the effects of morin on bone-associated disorders and the potential mechanism remain unknown. In this study, we investigated the anti-osteoclastogenic effect of morin in vitro and the potential therapeutic effects on ovariectomy (OVX)-induced osteoporosis in vivo. In vitro, by using a bone marrow macrophage-derived osteoclast culture system, we determined that morin attenuated receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclast formation via the inhibition of the mitogen-activated protein kinase (MAPK), NF-κB and calcium pathways. In addition, the subsequent expression of nuclear factor of activated T cells c1 (NFATc1) and c-fos was significantly suppressed by morin. In addition, NFATc1 downregulation led to the reduced expression of osteoclastogenesis-related marker genes, such as V-ATPase-d2 and Integrin β3. In vivo, results provided that morin could effectively attenuate OVX-induced bone loss in C57BL/6 mice. In conclusion, our results demonstrated that morin suppressed RANKL-induced osteoclastogenesis via the NF-κB, MAPK and calcium pathways, in addition, its function of preventing OVX-induced bone loss in vivo, which suggested that morin may be a potential therapeutic agent for postmenopausal osteoporosis treatment.

1. Phosphorus (P) is a necessary nutrient for egg production and bone quality in poultry diets. To investigate the effects of low dietary available P (avP) on keel bone, 180 laying hens were fed either a control (C, 0.3% avP) or low phosphorus (LP, 0.15% avP) diet from 20-36 weeks of age (WOA). Each diet was replicated in six cages with 15 birds per cage. Keel samples were collected at 24, 28, 32, and 36 WOA to measure indicators.2. The incidence of keel bone damage in the LP group was higher than C group and increased with age throughout the experiment period. Keel bone length from laying hens in the LP group was shorter than C group (P < 0.05) at 32 and 36 WOA.3. The mRNA expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and ratio of RANKL to osteoprotegerin (OPG) were upregulated (P < 0.05), and that of sclerostin and OPG was downregulated (P < 0.05) in the LP group in comparison to hens in the C group. Meanwhile, mRNA expression of the integrin-binding sialoprotein was increased at 24 and 28 WOA (P < 0.05), and decreased at 32 and 38 WOA (P < 0.05) in the LP group.4. Laying hens in LP group had increased trabecular separation and bone surface fraction (P < 0.05), decreased bone volume, bone volume fraction, trabecular number and thickness, and bone mineral density (P < 0.05) at 32 WOA. The LP-fed hens had increased K, Ti, Mn, Fe, Zn, Se, Sr and Pb bone concentrations (P < 0.05), and decreased P and TI bone concentrations (P < 0.05) at 36 WOA.5. Feeding hens a P-deficient diet with 0.15% avP and 3.37% Ca during the laying period impaired keel bone quality, which could be related to the osteoporosis.

Adiponectin (AD) highly expressed in synovial tissue correlates closely with progressive bone erosion in rheumatoid arthritis (RA). However, it remains unknown whether AD receptor mediates the pathogenesis of RA. This study aimed to investigate the effects of adiponectin receptor 1 (AdipoR1) signaling on synovial inflammation and joint damage in collagen-induced arthritis.AD and AdipoR1 expression in synovial tissue of RA and osteoarthritis (OA) patients were tested by PCR and western blotting. The frequency of AdipoR1 on RA synovial fibroblasts (RASFs) was examined by flow cytometry after stimulation with AD, IL-6, or TNF-α. AdipoR1 was knocked down in human RASF cell line (MH7A) and CIA mice joints using lentiviral particles carrying the AdipoR1 short hairpin RNA (shAdipoR1). Both the proliferation and apoptosis of MH7A and the secretion of inflammatory factors from MH7A were examined in vitro. The therapeutic effect of local AdipoR1 inhibition on CIA mice was assessed in vivo.Both the expression of AD and AdipoR1 were significantly higher in RA synovial tissue. AdipoR1 on RASFs was upregulated by AD. Silencing AdipoR1 remarkably reduced lipopolysaccharides-induced proliferation and promoted the apoptosis of MH7A. Moreover, AdipoR1 knockdown inhibited the release of inflammatory factors in vitro. In CIA mice, local AdipoR1 inhibition effectively decreased joint inflammation and alleviated bone destruction via suppressing RANKL and RANKL/OPG ratio in vivo.AdipoR1 signaling participates in the process of synovial inflammation and joint damage in CIA. Local blockade of AdipoR1 might be a new target for the clinical treatment of RA. Key points • Local AdipoR1 inhibition decreased joint inflammation and alleviated bone destruction in CIA.

Slc1a5 (ASCT2) encodes a small neutral amino-acid exchanger and is the most well-studied glutamine transporter in cancer cells. To investigate the role of Slc1a5 in osteoclastogenesis, we developed Slc1a5-deficient mice by using a conventional gene-targeting approach. The Slc1a5–/– mice showed no obvious abnormalities in growth. Glutamine uptake was assessed in Slc1a5+/+ and Slc1a5–/– bone marrow cells stimulated with RANKL. The rate of glutamine uptake in Slc1a5–/– bone marrow cells was reduced to 70% of that of cells from Slc1a5+/+ bone marrow. To confirm the involvement of Slc1a5 in osteoclast formation, bone marrow cells derived from Slc1a5+/+ or Slc1a5–/– mice were stimulated with RANKL and macrophage colony-stimulating factor and stained with tartrate-resistant acid phosphatase. The bone resorption activity and actin ring formation of stimulated cells were measured. The formation of multinucleated osteoclasts in bone marrow cells isolated from Slc1a5–/– mice was severely impaired compared with those from Slc1a5+/+ mice. RANKL-induced expression of ERK, NFκB, p70S6K, and NFATc1 was suppressed in Slc1a5–/– osteoclasts. These results show that Slc1a5 plays an important role in osteoclast formation.

Bone absorption is necessary for the maintenance of bone homeostasis. An osteoclast (OC) is a monocyte–macrophage lineage cell that absorbs bone tissue. Extracellular signal-regulated kinases (ERKs) are known to play important roles in regulating OC growth and differentiation. In this study, we examined specific downstream signal pathways affected by ERK inhibition during OC differentiation. Our results showed that the ERK inhibitors PD98059 and U0126 increased receptor activator of NF-κB ligand (RANKL)-induced OC differentiation in RAW 264.7 cells, implying a negative role in OC differentiation. This is supported by the effect of ERK2-specific small interfering RNA on increasing OC differentiation. In contrast to our findings regarding the RAW 264.7 cells, the ERK inhibitors attenuated the differentiation of bone marrow-derived cells into OCs. The ERK inhibitors significantly increased the phosphorylation of adenosine 5′-monophosphate-activated protein kinase (AMPK) but not the activation of p38 MAPK, Lyn, and mTOR. In addition, while the ERK inhibition increased the expression of the RANKL receptor RANK, it decreased the expression of negative mediators of OC differentiation, such as interferon regulatory factor-8, B-cell lymphoma 6, and interferon-γ. These dichotomous effects of ERK inhibition suggest that while ERKs may play positive roles in bone marrow-derived cells, ERKs may also play negative regulatory roles in RAW 264.7 cells. These data provide important information for drug development utilizing ERK inhibitors in OC-related disease treatment.

The bone microenvironment is a complex tissue in which heterogeneous cell populations of hematopoietic and mesenchymal origin interact with environmental cues to maintain tissue integrity. Both cellular and matrix components are subject to physiologic challenges and can dynamically respond by modifying cell/matrix interactions. When either component is impaired, the physiologic balance is lost. Here, we review the current state of knowledge of how glycosaminoglycans – organic components of the bone extracellular matrix – influence the bone micromilieu. We point out how they interact with mediators of distinct signaling pathways such as the RANKL/OPG axis, BMP and WNT signaling, and affect the activity of bone remodeling cells within the endosteal niche summarizing their potential for therapeutic intervention.

Osteoprotegerin (OPG) is a glycoprotein involved in the regulation of bone remodelling. OPG regulates osteoclast activity by blocking the interaction between the receptor activator of nuclear factor kappa B (RANK) and its ligand (RANKL). More and more studies confirm the relationship between OPG and cardiovascular diseases. Numerous studies have confirmed that a high plasma concentration of OPG and a low concentration of tumour necrosis factor–related apoptosis inducing ligand (TRAIL) together with a high OPG/TRAIL ratio are predictors of poor prognosis in patients with myocardial infarction. A high plasma OPG concentration and a high ratio of OPG/TRAIL in the acute myocardial infarction are a prognostic indicator of adverse left ventricular remodelling and of the development of heart failure. Ever more data indicates the participation of OPG in the regulation of the function of vascular endothelial cells and the initiation of the atherosclerotic process in the arteries. Additionally, it has been shown that TRAIL has a protective effect on blood vessels and exerts an anti-atherosclerotic effect. The mechanisms of action of both OPG and TRAIL within the cells of the vascular wall are complex and remain largely unclear. However, these mechanisms of action as well as their interaction in the local vascular environment are of great interest to researchers. This article presents the current state of knowledge on the mechanisms of action of OPG and TRAIL in the circulatory system and their role in cardiovascular diseases. Understanding these mechanisms may allow their use as a therapeutic target in cardiovascular diseases in the future.

ATP-citrate lyase (ACLY), generating most of the nucleocytosolic acetyl coenzyme A (acetyl-CoA) for histone acetylation, links cell metabolism to epigenetic regulation. Recent investigations demonstrated that ACLY activated by metabolic reprogramming played an essential role in both M1 and M2 macrophage activation via histone acetylation. Previous studies also revealed that histone methylation and acetylation were critical for transcriptional regulation of osteoclast-specific genes. Considering that osteoclast differentiation also undergoes metabolic reprogramming and the activity of ACLY is always Akt-dependent, we inferred that receptor activator of NF-κB (RANK) activation might enhance the activity of ACLY through downstream pathways and ACLY might play a role in osteoclast formation. In the current study, we found that ACLY was gradually activated during RANK ligand (RANKL)-induced osteoclast differentiation from bone marrow-derived macrophages (BMMs). Both ACLY knock-down and small molecular ACLY inhibitor BMS-303141 significantly decreased nucleocytosolic acetyl-CoA in BMMs and osteoclasts and suppressed osteoclast formation in vitro. BMS-303141 also suppressed osteoclast formation in vivo and prevents ovariectomy (OVX)-induced bone loss. Further investigations showed that RANKL triggered ACLY translocation into nucleus, consistent with increasing histone H3 acetylation, which was correlated to ACLY. The H3 lysine residues influenced by ACLY were in accordance with GCN5 targets. Using GCN5 knock-down and overexpression, we showed that ACLY and GCN5 functioned in the same pathway for histone H3 acetylation. Analysis of pathways downstream of RANK activation revealed that ACLY was Akt-dependent and predominately affected Akt pathway. With the help of RNA-sequencing, we discovered Rac1 as a downstream regulator of ACLY, which was involved in shACLY-mediated suppression of osteoclast differentiation, cytoskeleton organization, and signal transduction and was transcriptionally regulated by ACLY via histone H3 acetylation. To summarize, our results proved that inhibition of ATP-citrate lyase led to suppression of osteoclast differentiation and function via regulation of histone acetylation. Rac1 could be a downstream regulator of ACLY. © 2021 American Society for Bone and Mineral Research (ASBMR).

The sesquiterpene lactone artemisinin from Artemisia annua L. is well established for malaria therapy, but its bioactivity spectrum is much broader. In this review, we give a comprehensive and timely overview of the literature regarding the immunosuppressive activity of artemisinin-type compounds toward inflammatory and autoimmune diseases. Numerous receptor-coupled signaling pathways are inhibited by artemisinins, including the receptors for interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), β3-integrin, or RANKL, toll-like receptors and growth factor receptors. Among the receptor-coupled signal transducers are extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), AKT serine/threonine kinase (AKT), mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) kinase (MEK), phospholipase C γ1 (PLCγ), and others. All these receptors and signal transduction molecules are known to contribute to the inhibition of the transcription factor nuclear factor κ B (NF-κB). Artemisinins may inhibit NF-κB by silencing these upstream pathways and/or by direct binding to NF-κB. Numerous NF-κB-regulated downstream genes are downregulated by artemisinin and its derivatives, for example, cytokines, chemokines, and immune receptors, which regulate immune cell differentiation, apoptosis genes, proliferation-regulating genes, signal transducers, and genes involved in antioxidant stress response. In addition to the prominent role of NF-κB, other transcription factors are also inhibited by artemisinins (mammalian target of rapamycin [mTOR], activating protein 1 [AP1]/FBJ murine osteosarcoma viral oncogene homologue [FOS]/JUN oncogenic transcription factor [JUN]), hypoxia-induced factor 1α (HIF-1α), nuclear factor of activated T cells c1 (NF-ATC1), Signal transducers and activators of transcription (STAT), NF E2-related factor-2 (NRF-2), retinoic-acid-receptor-related orphan nuclear receptor γ (ROR-γt), and forkhead box P-3 (FOXP-3). Many in vivo experiments in disease-relevant animal models demonstrate therapeutic efficacy of artemisinin-type drugs against rheumatic diseases (rheumatoid arthritis, osteoarthritis, lupus erythematosus, arthrosis, and gout), lung diseases (asthma, acute lung injury, and pulmonary fibrosis), neurological diseases (autoimmune encephalitis, Alzheimer's disease, and myasthenia gravis), skin diseases (dermatitis, rosacea, and psoriasis), inflammatory bowel disease, and other inflammatory and autoimmune diseases. Randomized clinical trials should be conducted in the future to translate the plethora of preclinical results into clinical practice.

Background/aim Side effects of zolendronic acid (ZA) and RANKL inhibitors (RANKL-I) include impaired wound healing and osteonecrosis of the jaw. Platelet rich fibrin (PRF) enhances wound healing and bone remodelling in vivo and in vitro. However, the topical use PRF in the surgical treatment of patients with medicament-related osteonecrosis of the jaw is relatively new and not thoroughly investigated. Furthermore, the potential attenuation of the PRF effect following antiresorptive treatment remains unclear. Therefore, we investigated the concentration of growth factors within the PRF in healthy volunteers and in patients with antiresorptive treatment. Patients and methods Blood samples from healthy volunteers and patients were used to produce PRF. The levels of EGF, VEGF, PDGF-BB, TGF-β1, BMP-2, and CD31 in the PRF was investigated by ELISA. Results ZA treatment induced a significant decrease in EGF and TGF-β1 levels, whereas RANKL-I caused lower TGF-β1 levels. Conclusion Reduced EGF levels in PRF after ZA treatment may explain the delayed wound healing and question the positive effect of PRF in these patients. PRF use in patients undergoing RANKL-I treatment seems to be more justified.

The physiological balance between bone resorption and bone formation is now known to be mediated by a cascade of events parallel to the classic osteoblast-osteoclast interaction. Thus, osteoimmunology now encompasses the role played by other cell types, such as cytokines, lymphocytes and chemokines, in immunological responses and how they help modulate bone metabolism. All these factors have an impact on the RANK/RANKL/OPG pathway, which is the major pathway for the maturation and resorption activity of osteoclast precursor cells, responsible for osteoporosis development. Recently, immunoporosis has emerged as a new research area in osteoimmunology dedicated to the immune system’s role in osteoporosis. The first part of this review presents theoretical concepts on the factors involved in the skeletal system and osteoimmunology. Secondly, existing treatments and novel therapeutic approaches to treat osteoporosis are summarized. These were selected from to the most recent studies published on PubMed containing the term osteoporosis. All data relate to the results of in vitro and in vivo studies on the osteoimmunological system of humans, mice and rats. Treatments for osteoporosis can be classified into two categories. They either target osteoclastogenesis inhibition (denosumab, bisphosphonates), or they aim to restore the number and function of osteoblasts (romozumab, abaloparatide). Even novel therapies, such as resolvins, gene therapy, and mesenchymal stem cell transplantation, fall within this classification system. This review presents alternative pathways in the pathophysiology of osteoporosis, along with some recent therapeutic breakthroughs to restore bone homeostasis.