Search

BACKGROUND: Previous studies have demonstrated that icariin has important roles in promoting bone formation and inhibiting bone resorption, but its effects on osteoporosis-mediated bone pain have not been reported. OBJECTIVE: To investigate the possible mechanism of icariin alleviating bone pain in postmenopausal senile osteoporosis. METHODS: (1) Animal experiment: 200 C57BL/6 mice were randomly divided into 4 groups: sham group (n=50), model group (n=50), icariin treatment group (n=50), and carbonic anhydrase II inhibitor (Brinzolamide) treatment group (n=50). Ovariectomy was performed on C57BL/6 mice to establish a postmenopausal osteoporosis model in all groups except the sham group. The icariin group was given icariin on the second day after modeling, and pain behavior tests (Von Frey, Hot Plate, and Tail Flick tests) were performed every 2 weeks for 20 weeks. After sampling, bone mass was detected by microCT, osteoclast activity was detected by hematoxylin-eosin and tartrate-resistant acid phosphatase staining, and neuronal morphology and related ion channel expression were detected by tissue immunofluorescence staining. (2) Cell experiment: Osteoclast precursor cells derived from mouse bone marrow were extracted and induced to differentiate into osteoclasts using the RANKL/M-CSF system in vitro and supplemented with icariin of different concentrations (1 and 10 μmol/L). Tartrateresistant acid phosphatase staining was used to detect osteoclast differentiation, ghost pen cyclic peptide staining was used to detect osteoclast actin ring, bone plate absorption assay was used to detect osteoclast osteophagy function, pH value of the system was detected by pH meter, and expression of osteoclast differentiation-related proteins was detected by western blot. In addition, mouse dorsal root ganglion-derived nerve cells were extracted and treated with icariin. Cell counting kit-8 was used to detect neuronal activity and CGRP staining was used to detect neuronal morphology. RESULTS AND CONCLUSION: Compared with the model group, the icariin treatment group had higher bone mineral density, fewer tartrate-resistant acid phosphatase-positive osteoclasts in bone tissue, decreased neuronal activity, and decreased neuronal transient receptor potential vanilloid subtype 1 channel and carbonic anhydrase II expression. Behavioral results showed that the icariin treatment group was less sensitive to pain than the model group. Icariin inhibited osteoclast differentiation and bone phagocytosis in vitro. Icariin enhanced the activity of dorsal root ganglion neurons and inhibited the expression of calcitonin gene-related peptide and transient receptor potential vanilloid subtype 1 channels in dorsal root ganglia in a relatively non-toxic pH range. To conclude, icariin alleviates bone pain caused by postmenopausal osteoporosis by regulating the acidic microenvironment through its effect on osteoclasts. [ABSTRACT FROM AUTHOR]

Background and Objectives: The alveolar bone remodelling promoted by reasonable mechanical force triggers orthodontic tooth movement (OTM). The generation of osteoclasts is essential in this process. However, the mechanism of mechanical force mediating osteoclast differentiation remains elusive. Small nucleolar RNA host gene 5 (SNHG5), which was reported to mediate the osteogenic differentiation of bone marrow mesenchymal stem cells in our previous study, was downregulated in human periodontal ligament cells (hPDLCs) under mechanical force. At the same time, the RANKL/OPG ratio increased. Based on this, we probed into the role of SNHG5 in osteoclast formation during OTM and the relevant mechanism. Materials and Methods: SNHG5 and the RANKL/OPG ratio under different compressive forces were detected by western blotting (WB) and qRT‐PCR. Impact of overexpression or knockdown of SNHG5 on osteoclast differentiation was detected by qRT‐PCR, WB and transwell experiments. The combination of SNHG5 and C/EBPβ was verified by RNA immunoprecipitation and RNA pull‐down assays. The expression of SNHG5 and osteoclast markers in gingiva were analysed by qRT‐PCR and the paraffin sections of periodontal tissues were used for histological analysis. Results: Compressive force downregulated SNHG5 and upregulated the RANKL/OPG ratio in hPDLCs. Overexpression of SNHG5 inhibited RANKL's expression and osteoclast differentiation. SNHG5 combined with C/EBPβ, a regulator of osteoclast. The expression of SNHG5 in periodontal tissue decreased during OTM. Conclusion: SNHG5 inhibited osteoclast differentiation during OTM, achieved by affecting RANKL secretion, which may provide a new idea to interfere with bone resorption during orthodontic treatment. [ABSTRACT FROM AUTHOR]

\nPlain Language SummaryIn this study, we investigated the inhibitory effects of radiofrequency exposure on RANKL-induced osteoclast differentiation in RAW264.7 cells, along with the underlying mechanisms. RAW264.7 cells were subjected to radiofrequency exposure at three distinct power densities: 50 µW/cm2, 150 µW/cm2, and 450 µW/cm2. The results showed that, among the three dosage levels, exposure to 150 µW/cm2 of radiofrequency radiation significantly reduced the proliferation capacity of RAW264.7 cells. RF exposure at three power densities resulted in significant increases in the level of osteoclast apoptosis and notable decreases in osteoclast differentiation. Notably, the most pronounced effects on apoptosis, differentiation in RAW 264.7 cells were observed at the 150 µW/cm2 power density. These effects were accompanied by concurrent decreases in mRNA and protein levels of osteoclast-specific genes, including RANK, NFATc1, and TRACP. Furthermore, radiofrequency exposure at power density of 150 µW/cm2 induced a significant decrease in cytoplasmic NF-κB protein levels while increasing its nuclear fraction, thereby counteracting the effects of RANKL-induced NF-κB activation. These data suggest that radiofrequency exerts inhibitory properties on RANKL-induced NF-κB transcriptional activity, subsequently indirectly suppressing the expression of downstream NF-κB target genes, such as NFATc1 and TRACP. In conclusion, our study demonstrates that radiofrequency radiation effectively inhibits osteoclast differentiation by modulating the NF-κB signaling pathway. These findings have important implications for potential therapeutic interventions in osteoporosis.Osteoporosis is a common bone disease where bones become weak and brittle, often leading to fractures. It frequently occurs in older adults, especially postmenopausal women, due to low estrogen levels and inadequate calcium intake. This causes increased activity of bone cells called osteoclasts which break down bone tissue, resulting in severe bone loss. Currently, the primary treatment is long-term use of medications like bisphosphonates. However, these drugs can have side effects. The main adverse reactions include fever, vomiting, rash, diarrhea, dizziness, abdominal pain, musculoskeletal pain, headache, allergic-like reactions, indigestion, edema, and ocular symptoms.This study explored using radiofrequency (RF) radiation as a safe, non-invasive alternative therapy for osteoporosis. RF radiation is a type of energy used in communications like cell phones and WiFi. We tested whether exposure to 900MHz RF radiation could inhibit the formation and activity of osteoclasts to prevent excessive bone breakdown.We treated osteoclast precursor cells with RANKL, a protein that stimulates osteoclast formation. Cells were then exposed to RF radiation at various intensities. The results showed that medium-level RF radiation (150 μW/cm2) significantly suppressed RANKL-induced osteoclast differentiation and bone resorption capacity. This effect was like the osteoclast inhibition seen with estrogen treatment.Further analysis revealed that RF radiation blocks the activation of NF-κB, a key signaling molecule that promotes osteoclast formation when RANKL is present. This in turn reduced production of downstream signals like NFATc1 and TRACP which are essential for osteoclast differentiation.In summary, this study demonstrates that medium-intensity RF radiation could potentially prevent excessive osteoclastic bone resorption in osteoporosis patients by interfering with NF-κB signaling cascade. The research highlights RF radiation’s promise as a novel, non-invasive osteoporosis therapy.Osteoporosis is a common bone disease where bones become weak and brittle, often leading to fractures. It frequently occurs in older adults, especially postmenopausal women, due to low estrogen levels and inadequate calcium intake. This causes increased activity of bone cells called osteoclasts which break down bone tissue, resulting in severe bone loss. Currently, the primary treatment is long-term use of medications like bisphosphonates. However, these drugs can have side effects. The main adverse reactions include fever, vomiting, rash, diarrhea, dizziness, abdominal pain, musculoskeletal pain, headache, allergic-like reactions, indigestion, edema, and ocular symptoms.This study explored using radiofrequency (RF) radiation as a safe, non-invasive alternative therapy for osteoporosis. RF radiation is a type of energy used in communications like cell phones and WiFi. We tested whether exposure to 900MHz RF radiation could inhibit the formation and activity of osteoclasts to prevent excessive bone breakdown.We treated osteoclast precursor cells with RANKL, a protein that stimulates osteoclast formation. Cells were then exposed to RF radiation at various intensities. The results showed that medium-level RF radiation (150 μW/cm2) significantly suppressed RANKL-induced osteoclast differentiation and bone resorption capacity. This effect was like the osteoclast inhibition seen with estrogen treatment.Further analysis revealed that RF radiation blocks the activation of NF-κB, a key signaling molecule that promotes osteoclast formation when RANKL is present. This in turn reduced production of downstream signals like NFATc1 and TRACP which are essential for osteoclast differentiation.In summary, this study demonstrates that medium-intensity RF radiation could potentially prevent excessive osteoclastic bone resorption in osteoporosis patients by interfering with NF-κB signaling cascade. The research highlights RF radiation’s promise as a novel, non-invasive osteoporosis therapy. [ABSTRACT FROM AUTHOR]

ZC4H2 encodes a C4H2-type zinc finger protein, mutations of which lead to a spectrum of diseases known as ZC4H2 associated rare disorders (ZARD). In addition to neurological phenotypes, the most typical symptoms of ZARD are multiple joint contractures of varying degrees, accompanied by abnormal development of muscles and bones, and osteoporosis in some cases. The pathogenic mechanisms of such bone related phenotypes, however, remain unclear. Here, we showed that ZC4H2 is expressed in the developing bones in mice. ZC4H2 knockout mice were neonatal-lethal and smaller in size, with reduced calcification of long bones. Upon induced loss of ZC4H2 postnatally, the femoral bones developed an osteoporosis-like phenotype, with reduced bone mineral density, bone-volume fraction, and trabecular bone number. Knockdown of ZC4H2 showed no clear effect on the expression of osteogenic differentiation genes in in vitro models using mesenchymal stem cells. Interestingly, ZC4H2 knockdown significantly enhanced osteoclast differentiation and bone resorption in induced bone marrow-derived macrophages. We further confirmed that the number of osteoclasts in the long bone of ZC4H2 knockout mice was increased, as well as the expression of the serum bone resorption/osteoporosis marker CTX-1. Our study unveils a new role of ZC4H2 in osteoclast differentiation and bone development, providing new clues on the pathology of ZARD. [ABSTRACT FROM AUTHOR]

Inflammation-induced osteoclast proliferation is a crucial contributor to impaired bone metabolism. Kurarinone (KR), a flavonoid extracted from the Radix Sophorae Flavescentis, exhibits notable anti-inflammatory properties. Nevertheless, the precise influence of KR on osteoclast formation remains unclear. This study's objective was to assess the impact of KR on osteoclast activity in vitro and unravel its underlying mechanism. Initially, a target network for KR-osteoclastogenesis-osteoporosis was constructed using network pharmacology. Subsequently, the intersecting targets were identified through the Venny platform and a PPI network was created using Cytoscape 3.9.1. Key targets within the network were identified employing topological algorithms. GO enrichment and KEGG pathway analysis were then performed on these targets to explore their specific functions and pathways. Additionally, molecular docking of potential core targets of KR was conducted, and the results were validated through cell experiments. A total of 83 target genes overlapped between KR and osteoclastogenesis-osteoporosis targets. Enrichment analysis revealed their role in inflammatory response, protein tyrosine kinase activity, osteoclast differentiation, and MAPK and NF-κB signaling pathways. PPI analysis and molecular docking demonstrate that key targets MAPK14 and MAPK8 exhibit more stable binding with KR compared to other proteins. In vitro experiments demonstrate that KR effectively inhibits osteoclast differentiation and bone resorption without cellular toxicity. It suppresses key osteoclast genes (NFATc1, c-Fos, TRAP, MMP9, Ctsk, Atp6v2), hinders IκB-α degradation, and inhibits ERK and JNK phosphorylation, while not affecting p38 phosphorylation. The results indicate that KR may inhibit osteoclast maturation and bone resorption by blocking NF-κB and MAPK signaling pathways, suggesting its potential as a natural therapeutic agent for osteoporosis. [ABSTRACT FROM AUTHOR]

The construction of a chemical library based on natural products is a promising method for the synthesis of natural product‐like compounds. In this study, we synthesized a terpenoid alkaloid‐like compound library based on the humulene skeleton. Our strategy, which enables access to diverse ring systems such as 11‐membered monocyclic, oxabicyclic, and medium‐sized aza ring‐containing scaffolds, involves the introduction of a nitrogen atom, an intermolecular C−O bond formation via Lewis acid‐mediated epoxide‐opening transannulation, and a ring‐reconstruction strategy based on olefin metathesis. A cheminformatics analysis based on their structural and physicochemical properties revealed that the synthesized compounds have high three‐dimensionality and high natural product likeness scores but with structural novelty. The usefulness of the terpenoid alkaloid‐like compound library for drug discovery and the accessibility to structure‐activity relationship studies were validated by performing an assay for osteoclast‐specific tartrate‐resistant acid phosphatase activity, resulting in the identification of a seed compound for bone‐resorptive diseases such as osteoporosis. [ABSTRACT FROM AUTHOR]

RANKL (Receptor Activator of Nuclear Factor Kappa-B Ligand) is a cytokine that plays a crucial role in bone remodeling by regulating differentiation and activation of osteoclast, which breaks down old bone tissue. In this study, we attempted to produce human-derived RANKL using plant expression systems. The RANKL expressed in Nicotiana benthamiana leaves was designed to accumulate within the endoplasmic reticulum lumen. The purified RANKL from N. benthamiana induced the differentiation of bone marrow-derived macrophages to mature osteoclast, albeit modestly. In conclusion, human-derived RANKL could be produced using a plant expression system, but it requires further improvement. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder characterized by joint destruction due to synovial hypertrophy and the infiltration of inflammatory cells. Despite substantial progress in RA treatment, challenges persist, including suboptimal treatment responses and adverse effects associated with current therapies. This study investigates the anti-rheumatic capabilities of the newly identified multi-protein kinase inhibitor, KMU-11342, aiming to develop innovative agents targeting RA. In this study, we synthesized the novel multi-protein kinase inhibitor KMU-11342, based on indolin-2-one. We assessed its cardiac electrophysiological safety using the Langendorff system in rat hearts and evaluated its toxicity in zebrafish in vivo. Additionally, we examined the anti-rheumatic effects of KMU-11342 on human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS), THP-1 cells, and osteoclastogenesis in RAW264.7 cells. KMU-11342 demonstrated the ability to inhibit LPS-induced chemokine inhibition and the upregulation of pro-inflammatory cytokines, cyclooxygenase-2, inducible nitric oxide synthase, p-IKKα/β, p-NF-κB p65, and the nuclear translocation of NF-κB p65 in RA-FLS. It effectively suppressed the upregulation of NLR family pyrin domain containing 3 (NLRP3) and caspase-1 cleavage. Furthermore, KMU-11342 hindered the activation of osteoclast differentiation factors such as RANKL-induced TRAP, cathepsin K, NFATc-1, and c-Fos in RAW264.7 cells. KMU-11342 mitigates LPS-mediated inflammatory responses in THP-1 cells by inhibiting the activation of NLRP3 inflammasome. Notably, KMU-11342 exhibited minimal cytotoxicity in vivo and electrophysiological cardiotoxicity ex vivo. Consequently, KMU-11342 holds promise for development as a therapeutic agent in RA treatment. [ABSTRACT FROM AUTHOR]

Periodontitis, the second most common oral disease, is primarily initiated by inflammatory responses and osteoclast differentiation, in which the MAPK signaling pathway and mitochondrial function play important roles. 3‐methyl‐1H‐indol‐1‐yl dimethylcarbamodithioate (3o), a hybrid of indole and dithiocarbamate, was first synthesized by our group. It has shown anti‐inflammatory activity against lipopolysaccharide‐induced acute lung injury. However, it is not known if 3o can exert effects in periodontitis. In vitro study: LPS‐induced macrophage inflammation initiation and a receptor activator of nuclear factor κB ligand‐stimulated osteoclast differentiation model were established. Cell viability, inflammatory cytokines, osteoclast differentiation, the MAPK signaling pathway, and mitochondrial function before and after treatment with 3o were investigated. In vivo study: Alveolar bone resorption, inflammatory cytokine expression, osteoclast differentiation, and the underlying mechanisms were assessed in mice with periodontitis. Inflammatory cytokine expression and osteoclast differentiation appeared downregulated after 3o treatment. 3o inhibited the MAPK signaling pathway and restored mitochondrial function, including mitochondrial reactive oxygen species, mitochondrial membrane potential, and ATP production. Meanwhile, 3o reduced inflammation activation and bone resorption in mice with periodontitis, reflected by the decreased expression of inflammatory cytokines and osteoclasts, implying that 3o inhibited the MAPK signaling pathway and the mitochondrial oxidative DNA damage marker 8‐OHdG. These results highlight the protective role of 3o in periodontitis in mice and reveal an important strategy for preventing periodontitis. [ABSTRACT FROM AUTHOR]

Objective To observe the expression profile of epidermal growth factor receptor (EGFR) during the differentiation of osteoclasts, and to explore EGFR-related signaling pathways and key genes during the process. Methods Bone marrow derived macrophages were extracted from 24 healthy SPF-grade male C57BL/6 mice (6-8 weeks old, weighing 19~21 g), and then treated with M-CSF and RANKL co-stimulation to establish a model of mouse osteoclast differentiation. TRAP staining and RT-qPCR were used to detect the differentiation status of osteoclasts and the expression level of EGFR. Bioinformatics were employed to systematically identify EGFR-related genes during osteoclast differentiation, and then RT-qPCR and EGFR activation and inhibition models were applied to validate the genes. Results In vitro osteoclast differentiation model showed the EGFR expression level was in a continuous increase during the differentiation of mouse osteoclasts (P < 0.01). RNA-seq data indicated that EGFR expression was significantly associated with multiple signaling pathways, including MAPK pathway (P < 0.05). Weighted correlation network analysis (WGCNA) and protein-protein interaction (PPI) analysis identified co-expression and correlations of EGFR with ATP binding cassette subfamily C member 1 (ABCC1) and cell division cycle 37 (CDC37) (P < 0.01). The osteoclast differentiation model showed significant increases in the expression levels of ABCC1 (P < 0.05) and CDC37 (P < 0.01) during differentiation, and activation of EGFR further enhanced the levels (P < 0.01) while EGFR inhibition decreased them (P < 0.05). Conclusion Activation of EGFR signal induces osteoclast differentiation and up-regulates the expression levels of ABCC1 and CDC37.

Diabetic osteoporosis (DO) presents significant clinical challenges. This study aimed to investigate the potential of magnetic nanoparticle-enhanced extracellular vesicles (GMNPE-EVs) derived from bone marrow mesenchymal stem cells (BMSCs) to deliver miR-15b-5p, thereby targeting and downregulating glial fibrillary acidic protein (GFAP) expression in rat DO models. Data was sourced from DO-related RNA-seq datasets combined with GEO and GeneCards databases. Rat primary BMSCs, bone marrow-derived macrophages (BMMs), and osteoclasts were isolated and cultured. EVs were separated, and GMNPE targeting EVs were synthesized. Bioinformatic analysis revealed a high GFAP expression in DO-related RNA-seq and GSE26168 datasets for disease models. Experimental results confirmed elevated GFAP in rat DO bone tissues, promoting osteoclast differentiation. miR-15b-5p was identified as a GFAP inhibitor, but was significantly downregulated in DO and enriched in BMSC-derived EVs. In vitro experiments showed that GMNPE-EVs could transfer miR-15b-5p to osteoclasts, downregulating GFAP and inhibiting osteoclast differentiation. In vivo tests confirmed the therapeutic potential of this approach in alleviating rat DO. Collectively, GMNPE-EVs can effectively deliver miR-15b-5p to osteoclasts, downregulating GFAP expression, and hence, offering a therapeutic strategy for rat DO. [ABSTRACT FROM AUTHOR]

The role of monocytes in postmenopausal osteoporosis is widely recognized; however, the mechanisms underlying monocyte reprogramming remain unknown. In this study, single-cell RNA sequencing (scRNA-seq) was conducted on CD14+ bone marrow monocytes obtained from 3 postmenopausal women with normal BMD and 3 women with postmenopausal osteoporosis (PMOP). Monocle2 was used to classify the monocytes into 7 distinct clusters. The proportion of cluster 1 significantly decreased in PMOP patients, while the proportion of cluster 7 increased. Further analysis via GSEA, transcription factor activity analysis, and sc-metabolic analysis revealed significant differences between clusters 1 and 7. Cluster 7 exhibited upregulated pathways associated with inflammation, immunity, and osteoclast differentiation, whereas cluster 1 demonstrated the opposite results. Monocle2, TSCAN, VECTOR, and scVelo data indicated that cluster 1 represented the initial subset and that cluster 7 represents one of the terminal subsets. BayesPrism and ssGSEA were employed to analyze the bulk transcriptome data obtained from the GEO database. The observed alterations in the proportions of 1 and 7 were validated and found to have diagnostic significance. CD16 serves as the marker gene for cluster 7, thus leading to an increased proportion of CD16+ monocytes in women with PMOP. Flow cytometry was used to assess the consistency of outcomes with those of the bioinformatic analysis. Subsequently, an additional scRNA-seq analysis was conducted on bone marrow mononuclear cells obtained from 3 patients with PMOP and 3 postmenopausal women with normal BMD. The differential proportions of cluster 1 and cluster 7 were once again confirmed, with the pathological effect of cluster 7 may attribute to cell–cell communication. The scRNA-seq findings suggest that an imbalance in monocyte subsets is a characteristic feature of PMOP. These findings elucidate the limitations of utilizing bulk transcriptome data for detecting alterations in monocytes, which may influence novel research inquiries. Lay Summary: Monocytes are a type of white blood cell that plays a role in postmenopausal osteoporosis (PMOP), a condition where bones become weak and brittle after menopause. However, how monocytes change in this condition is not fully understood. In this study, single-cell RNA sequencing was used to analyze bone marrow monocytes from postmenopausal women with normal bone density and those with osteoporosis. Two distinct types of monocytes were identified, which were called clusters 1 and 7. In women with PMOP, there was a decrease in cluster 1 monocytes and an increase in cluster 7 monocytes. This change was validated in external datasets and in peripheral blood. Further analysis showed that cluster 7 monocytes positively correlated with inflammation, immunity, and osteoclast differentiation (a process that leads to bone resorption). Cluster 1 monocytes were found to be the initial subset, while cluster 7 monocytes were one of the terminal subsets. Overall, this study suggests that an imbalance in monocyte subsets is a characteristic feature of postmenopausal osteoporosis. These findings have important implications for understanding the role of monocytes in bone health. Graphical Abstract [ABSTRACT FROM AUTHOR]

Osteoporosis is characterized by an imbalance between osteoclast-mediated bone resorption and osteoblast-related bone formation, particularly increased osteoclastogenesis. However, the mechanisms by which epigenetic factors regulate osteoclast precursor differentiation during osteoclastogenesis remain poorly understood. Here, we show that the specific knockout of the chromatin remodeling factor Arid1a in bone marrow–derived macrophages (BMDMs) results in increased bone mass. The loss of Arid1a in BMDM inhibits cell–cell fusion and maturation of osteoclast precursors, thereby suppressing osteoclast differentiation. Mechanistically, Arid1a increases the chromatin access in the gene promoter region of sialic acid–binding Ig-like lectin 15 (Siglec15) by transcription factor Jun/Fos, which results in the upregulation of Siglec15 and promotion of osteoclast differentiation. However, the loss of Arid1a reprograms the chromatin structure to restrict Siglec15 expression in osteoclast precursors, thereby inhibiting BMDM differentiation into mature osteoclasts. Deleting Arid1a after ovariectomy (a model for postmenopausal bone loss) alleviated bone loss and maintained bone mass. In summary, epigenetic reprogramming mediated by Arid1a loss suppresses osteoclast differentiation and may serve as a promising therapeutic strategy for treating bone loss diseases. Lay Summary: Osteoporosis is a common disease, usually diagnosed by decreased bone density and increased fragility. The people with osteoporosis has higher risk of fractures. Nearly one-third of the aged people will suffer from osteoporosis-related fractures and even lose their lives because of this. Therefore, there is an urgent need for early intervention and effective treatment options for osteoporosis in the aging population. Bone tissue is a highly dynamic tissue that undergoes continuous remodeling throughout an individual's entire life. The balance of remodeling depends on the bone formation mediated by osteoblasts and bone resorption by osteoclasts. When this balance is disrupted, osteoporosis occurs. Thus, the aim of our research is to explore the behind mechanism of this imbalance. Here, we demonstrate that the loss of Arid1a, a chromatin remodeler, leads to chromatin reprogramming that restricts access to promoters by transcription factors such as Jun/Fos, thereby suppressing osteoclast activation and bone resorption. Our findings offer insights into the epigenetic mechanisms underlying osteoporosis and suggest potential strategies for its prevention and treatment. Graphical Abstract [ABSTRACT FROM AUTHOR]

YTHDC1 has been confirmed to mediate osteoporosis (OP) progression by regulating osteogenic differentiation. However, whether YTHDC1 mediates osteoclast differentiation and its molecular mechanism remains unclear. Quantitative real-time polymerase chain reaction and Western blot analysis were performed to detect the levels of YTHDC1, PTPN6, NFATc1, TRAP, RUNX2, alkaline phosphatase, and HUR. YTHDC1 knockout mice was constructed by CRISPR/Cas9 system, and the OP mice model was established by ovariectomy. Hematoxylin and eosin staining and micro-computed tomography were used to evaluate bone formation and bone mass. Mouse primary bone marrow macrophage cells were isolated and induced into osteoclasts. TRAP-positive cells were detected using TRAP staining. MeRIP-qPCR, RIP-qPCR assay, RNA affinity isolation assay, and co-immunoprecipitation assay were used to confirm the interactions among YTHDC1, PTPN6, and HUR. YTHDC1 expression was reduced and positively correlated with lumbar bone mineral density in OP patients. In the ovariectomy model of YTHDC1 knockout mice, bone formation was reduced, bone histomorphology was changed, and osteoclastic-related factor (NFATc1 and TRAP) levels were enhanced. Overexpression YTHDC1 inhibited osteoclast differentiation. YTHDC1 increased PTPN6 messenger RNA stability in an m6A-dependent manner. Moreover, YTHDC1 interacted with HUR to positively regulate PTPN6 expression. PTPN6 knockdown promoted osteoclast differentiation, and this effect was reversed by overexpressing HUR or YTHDC1. YTHDC1 was involved in regulating OP progression through inhibiting osteoclast differentiation by enhancing PTPN6 messenger RNA stability in an m6A-HUR–dependent manner. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study was to explore the potential role of zinc‐finger homeodomain transcription factor (TCF8) in osteoclastogenesis and inflammation during periodontitis. Materials and Methods: Rats with periodontitis were induced via Porphyromonas gingivalis‐lipopolysaccharide (Pg‐LPS) injection. The recombinant lentivirus delivering short hairpin RNA (shRNA) against TCF8 was used to downregulate TCF8 in vivo. Alveolar bone loss in rats was determined by micro‐computed tomography (Micro‐CT). Typical pathological changes, periodontal tissue inflammation, and osteoclastogenesis were evaluated via histological analyses. The RAW264.7‐derived osteoclasts were induced by RANKL stimulation. TCF8 downregulation in vitro was achieved by lentivirus infection. The osteoclast differentiation and inflammatory signaling in RANKL‐induced cells were measured via immunofluorescence methods and molecular biology approaches. Results: Porphyromonas gingivalis‐lipopolysaccharide induced rats exhibited overexpressed TCF8 in their periodontal tissues, while TCF8 knockdown attenuated the bone loss, tissue inflammation, and osteoclastogenesis in LPS‐induced rats. Besides, TCF8 silencing inhibited RANKL‐induced osteoclast differentiation in RAW264.7 cells, as evidenced by the reduced numbers of TRAP‐positive osteoclasts, less formation of F‐actin rings, and downregulated expressions of osteoclast‐specific markers. It also exerted an inhibitory effect on the NF‐κB signaling in RANKL‐induced cells via blocking NF‐κB p65 phosphorylation and nuclear translocation. Conclusions: TCF8 silencing inhibited alveolar bone loss, osteoclast differentiation, and inflammation in periodontitis. [ABSTRACT FROM AUTHOR]

Abstract Psoriatic arthritis mutilans (PAM) is the rarest and most severe form of psoriatic arthritis, characterized by erosions of the small joints and osteolysis leading to joint disruption. Despite its severity, the underlying mechanisms are unknown, and no susceptibility genes have hitherto been identified. We aimed to investigate the genetic basis of PAM by performing massive parallel sequencing in sixty-one patients from the PAM Nordic cohort. We found rare variants in the NADPH oxidase 4 (NOX4) in four patients. In silico predictions show that the identified variants are potentially damaging. NOXs are the only enzymes producing reactive oxygen species (ROS). NOX4 is specifically involved in the differentiation of osteoclasts, the cells implicated in bone resorption. Functional follow-up studies using cell culture, zebrafish models, and measurement of ROS in patients uncovered that these NOX4 variants increase ROS levels both in vitro and in vivo. We propose NOX4 as the first candidate susceptibility gene for PAM. Our study links high levels of ROS caused by NOX4 variants to the development of PAM, offering a potential therapeutic target.

Abstract Objective Osteoporosis is the imbalance in bone homeostasis between osteoblasts and osteoclasts. In this study, we investigated the effects of the circ_0029463/miR-134-5p/Rab27a axis on RANKL-induced osteoclast differentiation. Methods RT-qPCR and western blotting were used to detect the expression of circ_0029463, miR-134-5p, and Rab27a in tissues from patients with osteoporosis and in RANKL-induced osteoclasts. Osteoclast differentiation was verified by TRAP staining. Osteoclast biomarkers, including NFATc1, TRAP, and CTSK, were measured. The target and regulatory relationships between circ_0029463, miR-134-5p, and the Rab27a axis were verified using RIP, dual-luciferase reporter gene, and RNA pull-down assays. Results Elevated expression of circ_0029463 and Rab27a and decreased miR-134-5p expression were observed in the tissues of patients with osteoporosis, and a similar expression pattern was observed in RANKL-induced osteoclasts. Suppression of circ_0029463 expression or miR-134-5p overexpression curbed RANKL-induced osteoclast differentiation, whereas such an effect was abolished by Rab27 overexpression. circ_0029463 sponges miR-134-5p to induce Rab27a expression. Conclusion circ_0029463 sponges miR-134-5p to abolish its suppressive effect of miR-134-5p on Rab27a expression, thereby promoting osteoclast differentiation.

The pathogenic effects of type 2 diabetes on bone tissue are gaining attention, but the cellular and molecular mechanisms underlying osteoimmunology are still unclear in diabetes-related bone diseases. We delineated the single-cell transcriptome of bone marrow cells from both wide type and type 2 diabetes mice, which provided the first detailed global profile of bone marrow cells and revealed a distinct bone immune microenvironment at the genetic level under type 2 diabetic condition. It was observed that osteoclast activity was inhibited due to a dysregulated cytokine network, which ultimately led to decreased osteoclast formation and differentiation. In type 2 diabetes mice, a specific Cd36+ cluster (cluster 18, monocytes/macrophages 2) was identified as the precursor of osteoclasts with diminished differentiation potential. AP-1 was demonstrated to be the key transcription factor in the underlying mechanism.

Background: Patients with multiple myeloma exhibit malignant osteolytic bone disease due to excessive osteoclast formation and function. We recently identified that osteoclastogenic stimulator selenoprotein W (SELENOW) is upregulated via ERK signaling and downregulated via p38 signaling during receptor activator of nuclear factor (NF)-κΒ ligand (RANKL)-induced osteoclast differentiation. In the intrinsic physiological process, RANKL-induced downregulation of SELENOW maintains proper osteoclast differentiation; in contrast, forced overexpression of SELENOW leads to overactive osteoclast formation and function. Methods and results: We observed that SELENOW is highly expressed in multiple myeloma-derived peripheral blood mononuclear cells (PBMCs) and mature osteoclasts when compared to healthy controls. Also, the level of tumor necrosis factor alpha (TNFα), a pathological osteoclastogenic factor, is increased in the PBMCs and serum of patients with multiple myeloma. ERK activation by TNFα was more marked and sustained than that by RANKL, allowing SELENOW upregulation. Excessive expression of SELENOW in osteoclast progenitors and mature osteoclasts derived from multiple myeloma facilitated efficient nuclear translocation of osteoclastogenic transcription factors NF-κB and NFATc1, which are favorable for osteoclast formation. Conclusion: Our findings suggest a possibility that feedforward signaling of osteoclastogenic SELENOW by TNFα derived from multiple myeloma induces overactive osteoclast differentiation, leading to bone loss during multiple myeloma. [ABSTRACT FROM AUTHOR]

Orally administered "tortoiseshell and deer antler gelatin" is a common traditional medicine for patients with osteoporosis or osteoarthritis. From the pepsin-digested gelatin, we previously isolated and identified the osteoblast-stimulating pentapeptide, TSKYR. Its trypsin digestion products include the dipeptide YR, enhancing calcium ion uptake, and tripeptide TSK, resulting in remarkable 30- and 50-fold increases in mineralized nodule area and density in human osteoblast cells. These peptides were chemically synthesized in this study. The composition of deer antler preparations comprises not only proteins and peptides but also a significant quantity of metal ion salts. By analyzing osteoblast growth in the presence of peptide YR and various metal ions, we observed a synergistic effect of calcium and strontium on the effects of YR. Those peptides could also stimulate the growth of C2C12 skeletal muscle cells and human chondrocytes, increasing collagen and glycosaminoglycan content in a three-dimensional environment. The maintenance of bone homeostasis relies on a balance between osteoclasts and osteoblasts. Deer antler peptides were observed to inhibit osteoclast differentiation, as evidenced by ROS generation, tartrate-resistant acid phosphatase (TRACP) activity assays, and gene expression in RAW264.7 cells. In summary, our findings provide a deep understanding of the efficacy of this folk medicine. [ABSTRACT FROM AUTHOR]

Periodontitis (PD) is an inflammatory disease with alveolar bone destruction by osteoclasts (OCs). In PD, both inflammation and OC activation are significantly influenced by periodontal ligament fibroblasts (PDL-Fib). Yet, whether PDL-Fib has heterogeneity and whether distinct PDL-Fib subsets have specific functions have not been investigated. In this study, we discovered the complexity of PDL-Fib in PD, utilizing single-cell RNA sequencing data from human PD patients. We identified distinct subpopulations of PDL-Fib: one expressing interleukin-1 beta (IL-1ß) and another expressing the receptor activator of nuclear factor-kappa B ligand (RANKL), both crucial in OC differentiation and bone resorption. In periodontal tissues of mice with PD, active IL-1ß, cleaved caspase 1, and nucleotide-binding oligomerization domain-like receptor 3 (NLPR3) were significantly elevated, implicating the NLRP3 inflammasome in IL-1ß production. Upon stimulation of PDL-Fib with LPS from Porphyromonas gingivalis (pg), the most well-characterized periodontal bacteria, a more rapid increase in IL-1ß, followed by RANKL induction, was observed. IL-1ß and tumor necrosis factor alpha (TNF-a), another LPS-responsive cytokine, effectively increased RANKL in PDL-Fib, suggesting an indirect effect of pgLPS through IL-1ß and TNF-a on RANKL induction. Immunohistological analyses of mouse periodontal tissues also showed markedly elevated levels of IL-1ß and RANKL upon PD induction and displayed separate locations of IL-1ß-expressing PDL-Fib and RANKL-expressing PDL-Fib in PD. The heterogenic feature of fibroblasts expressing IL-1ß and RANKL was also mirrored in our combined cross-tissue single-cell RNA sequencing datasets analysis. In summary, our study elucidates the heterogeneity of PDL-Fib, highlighting distinct functional groups for producing RANKL and IL-1ß, which collectively promote OC generation and bone destruction in PD. [ABSTRACT FROM AUTHOR]

Psoriatic arthritis mutilans (PAM) is the rarest and most severe form of psoriatic arthritis, characterized by erosions of the small joints and osteolysis leading to joint disruption. Despite its severity, the underlying mechanisms are unknown, and no susceptibility genes have hitherto been identified. We aimed to investigate the genetic basis of PAM by performing massive parallel sequencing in sixty-one patients from the PAM Nordic cohort. We found rare variants in the NADPH oxidase 4 (NOX4) in four patients. In silico predictions show that the identified variants are potentially damaging. NOXs are the only enzymes producing reactive oxygen species (ROS). NOX4 is specifically involved in the differentiation of osteoclasts, the cells implicated in bone resorption. Functional follow-up studies using cell culture, zebrafish models, and measurement of ROS in patients uncovered that these NOX4 variants increase ROS levels both in vitro and in vivo. We propose NOX4 as the first candidate susceptibility gene for PAM. Our study links high levels of ROS caused by NOX4 variants to the development of PAM, offering a potential therapeutic target. Synopsis: Genomic analysis of Psoriatic Arthritis Mutilans (PAM) patients from the Nordic PAM cohort uncovered potentially damaging rare variants in the NOX4 gene. NOX4 belongs to a family of enzymes generating reactive oxygen species (ROS); NOX4 generates mainly hydrogen peroxide (H2O2). Three NOX4 rare variants (p.V369F, p.Y512IfsX20, and p.Y512C) affecting NOX4 functional domains were identified in four PAM patients. Enhanced differentiation and higher generation of ROS activity are observed in patient-derived osteoclasts, the cells responsible for bone resorption. NOX4 rare variants show increased generation of ROS in both zebrafish model and HEK293 cells. Increased levels of ROS, particularly H2O2, present promising therapeutic opportunities for addressing PAM. Genomic analysis of Psoriatic Arthritis Mutilans (PAM) patients from the Nordic PAM cohort uncovered potentially damaging rare variants in the NOX4 gene. NOX4 belongs to a family of enzymes generating reactive oxygen species (ROS); NOX4 generates mainly hydrogen peroxide (H2O2). [ABSTRACT FROM AUTHOR]

It has been known that periodontal ligament‐associated protein‐1 (PLAP‐1/Asporin) not only inhibits cartilage formation in osteoarthritis, but it also influences the healing of skull defect. However, the effect and mechanism of PLAP‐1/Asporin on the mutual regulation of osteoclasts and osteoblasts in periodontitis are not clear. In this study, we utilized a PLAP‐1/Asporin gene knockout (KO) mouse model to research this unknown issue. We cultured mouse bone marrow mesenchymal stem cells with Porphyromonas gingivalis lipopolysaccharide (P.g. LPS) for osteogenic induction in vitro. The molecular mechanism of PLAP‐1/Asporin in the regulation of osteoblasts was detected by immunoprecipitation, immunofluorescence, and inhibitors of signaling pathways. The results showed that the KO of PLAP‐1/Asporin promoted osteogenic differentiation through transforming growth factor beta 1 (TGF‐β1)/Smad3 in inflammatory environments. We further found the KO of PLAP‐1/Asporin inhibited osteoclast differentiation and promoted osteogenic differentiation through the TGF‐β1/Smad signaling pathway in an inflammatory coculture system. The experimental periodontitis model was established by silk ligation and the alveolar bone formation in PLAP‐1/Asporin KO mice was promoted through TGF‐β1/Smad3 signaling pathway. The subcutaneous osteogenesis model in nude mice also confirmed that the KO of PLAP‐1/Asporin promoted bone formation by the histochemical staining. In conclusion, PLAP‐1/Asporin regulated the differentiation of osteoclasts and osteoblasts through TGF‐β1/Smad signaling pathway. The results of this study lay a theoretical foundation for the further study of the pathological mechanism underlying alveolar bone resorption, and the prevention and treatment of periodontitis. [ABSTRACT FROM AUTHOR]

Objective: Osteoporosis is the imbalance in bone homeostasis between osteoblasts and osteoclasts. In this study, we investigated the effects of the circ_0029463/miR-134-5p/Rab27a axis on RANKL-induced osteoclast differentiation. Methods: RT-qPCR and western blotting were used to detect the expression of circ_0029463, miR-134-5p, and Rab27a in tissues from patients with osteoporosis and in RANKL-induced osteoclasts. Osteoclast differentiation was verified by TRAP staining. Osteoclast biomarkers, including NFATc1, TRAP, and CTSK, were measured. The target and regulatory relationships between circ_0029463, miR-134-5p, and the Rab27a axis were verified using RIP, dual-luciferase reporter gene, and RNA pull-down assays. Results: Elevated expression of circ_0029463 and Rab27a and decreased miR-134-5p expression were observed in the tissues of patients with osteoporosis, and a similar expression pattern was observed in RANKL-induced osteoclasts. Suppression of circ_0029463 expression or miR-134-5p overexpression curbed RANKL-induced osteoclast differentiation, whereas such an effect was abolished by Rab27 overexpression. circ_0029463 sponges miR-134-5p to induce Rab27a expression. Conclusion: circ_0029463 sponges miR-134-5p to abolish its suppressive effect of miR-134-5p on Rab27a expression, thereby promoting osteoclast differentiation. [ABSTRACT FROM AUTHOR]

Objective To explore regulation effects of immunomodulatory molecule IL-33 on RANKL/c-Fos/NFATc1 signal axis in rats with postmenopausal osteoporosis (PMOP) and intervention mechanism of Tenghuang Jiangu capsule (TJC). Methods The rat's PMOP model was performed by bilateralovariectomy method. The rats were divided into sham group, PMOP model group, positive control group (0.09 mg/kg), TJC high, middle and low dose group (0.36, 0.18, 0.09 g/kg). These groups were intragastric administration once a day for 8 weeks. The curative effect of TJC was evaluated including body mass after last administration, BMD, and bone micro architecture. And the levevls of immune regulatory factors IL-33, IL-1, and IL-31in rat serum was checked by ELISA, the expressions of OPG, RANKL, RANK, c-Fos, NFATc1 in rat thigh-bone was determined by qPCR and Western blotting. Results Compared with the sham group, the body mass, BMAT, the levels of IL-1, IL-31, the expression of TRANKL, RANK, c-Fos, NFATc1 in the PMOP model group have a significant increase trend, the content of BMD. the level of IL-33 and the expression of OPG appeared a significant decrease trend (P<0.01). Compared with the PMOP model group, the body mass appeared significantly decrease trend in the TJC high dose group, and the level of IL-33 has a increase trend (P<0.01). BMAT, the expression of RANK appeared significantly decrease trend in the TJC middle dose group and the TJC high dose group, the expression of OPG appeared significantly increase trend ( P < 0 or P < 0.01 ) . The levels of IL-1, IL-31, the expression of TRANKL, RANK, c-Fos, NFATc1 have a significant decrease trend in the TJC low dose group, the TJC middle dose group and the TJC high dose group, the BMD has a significant increase trend ( P < 0.05 or P < 0.01 ) . Conclusion TJC can significantly raise the concentration of IL-33 in PMOP model rats, decrease the concentration ofIL-1, IL-31, and inhibit the expression of osteoclast differentiation marker NFATc1. Its mechanism may be closely related to the inhibition of RANKL/c-Fos/ NFATc1 bone metabolic pathway. [ABSTRACT FROM AUTHOR]

Background: Periodontal ligament‐associated protein‐1 (PLAP‐1), an important target molecule of osteoarthritis research, may affect alveolar bone resorption. The aim of our study was to comprehensively and systematically detect the effect of PLAP‐1 on alveolar bone resorption and the underlying mechanism in PLAP‐1 knockout mouse models. Methods: We used a PLAP‐1 knockout (C57BL/6N‐Plap‐1−/−) mouse model to investigate the effect of PLAP‐1 on osteoclast differentiation and the underlying mechanism by adding Porphyromonas gingivalis lipopolysaccharide to stimulate bone marrow‐derived macrophages. The effect of PLAP‐1 on alveolar bone resorption and the underlying mechanism were studied using a ligature periodontitis model, with microcomputed tomography imaging, immunochemistry, and immunofluorescence. Results: The in vitro analysis results demonstrated that PLAP‐1 knockout significantly inhibited osteoclast differentiation under both normal and inflammatory conditions. Bioinformatic analysis, immunofluorescence, and co‐immunoprecipitation showed colocalization and interaction between PLAP‐1 and transforming growth factor beta 1 (TGF‐β1). The phosphorylation of Smad1 was reduced in the PLAP‐1 knockout cells compared with that in the cells from wild‐type mice. The in vivo analysis results demonstrated that PLAP‐1 knockout decreased bone resorption and the levels of osteoclast differentiation markers in experimental periodontitis compared with those in wild‐type mice. Immunofluorescence staining confirmed colocalization of PLAP‐1 and TGF‐β1 in the experimental periodontitis model. The phosphorylation level of Smad1 was significantly reduced in PLAP‐1 knockout mice compared with that in wild‐type mice. Conclusions: This study revealed that the knockout of PLAP‐1 inhibits osteoclast differentiation and decreases alveolar bone resorption through the TGF‐β1/Smad1 signaling pathway, which could serve as an innovative target for the prevention and treatment of periodontitis. [ABSTRACT FROM AUTHOR]

Background: Osteoporosis is a debilitating condition characterized by reduced bone density and microstructure, leading to increased susceptibility to fractures and increased mortality, particularly among older individuals. Despite the availability of drugs for osteoporosis treatment, the need for targeted and innovative agents with fewer adverse effects persists. Trifolirhizin, a natural pterostalin derived from the root of Sophora flavescens, has been previously studied for its effects on certain anticancer and antiinflammatory. The impact of trifolirhizin on the formation and function of osteoclasts remain unclear. Purpose: Herein, the possible roles of trifolirhizin the formation and function of osteoclasts and the underlying mechanism were explored. Methods: Bone marrow-derived macrophages (BMMs) were employed to evaluate the roles of trifolirhizin on steoclastogenesis, bone absorption and the underlying mechanism in vitro. Bone loss model was established by ovariectomy(OVX) in mice in vivo. Results: Trifolirhizin repressed osteoclastogenesis, bone resorption induced by receptor activator of nuclear factor kappa B ligand (RANKL) in vitro. Mechanistically, trifolirhizin inhibits RANKL-induced MAPK signal transduction and NFATc1 expression. Moreover, trifolirhizin inhibited osteoclast marker gene expression, including NFATc1, CTSK, MMP9, DC-STAMP, ACP5, and V-ATPase-D2. Additionally, trifolirhizin was found to protect against ovariectomy(OVX)-induced bone loss in mice. Conclusion: Trifolirhizin can effectively inhibit osteoclast production and bone resorption activity. The results of our study provide evidence for trifolirhizin as a potential drug for the prevention and treatment of osteoporosis and other osteolytic diseases.

Abstract Background Previous research has revealed that the 18 glycoside hydrolase gene family (GH18) member Chitinase 3-like 1 (Chi3l1) can regulate osteoclast differentiation and bone resorption. However, its downstream receptors and molecular mechanisms during osteoclastogenesis have yet to be elucidated. Methods Initially, we conducted a comprehensive investigation to evaluate the effects of recombinant Chi3l1 protein or Chi3l1 siRNA on osteoclast differentiation and the RANKL-induced MAPK/AKT signaling pathways. Moreover, we used immunofluorescence and immunoprecipitation assays to identify IL13Rα2 as the downstream receptor of Chi3l1. Subsequently, we investigated the impact of IL13Rα2 recombinant protein or IL13Rα2-siRNA on osteoclast differentiation and the associated signaling pathways. Finally, we performed in vivo experiments to examine the effect of recombinant IL13Rα2 protein in an LPS-induced mouse model of cranial osteolysis. Results Our findings highlight that the administration of recombinant Chi3l1 protein increased the formation of osteoclasts and bolstered the expression of several osteoclast-specific genes (TRAP, NFATC1, CTR, CTSK, V-ATPase d2, and Dc-STAMP). Additionally, Chi3l1 significantly promoted the RANKL-induced MAPK (ERK/P38/JNK) and AKT pathway activation, whereas Chi3l1 silencing inhibited this process. Next, using immunofluorescence and co-immunoprecipitation assays, we identified IL13Rα2 as the binding partner of Chi3l1 during osteoclastogenesis. IL13Rα2 recombinant protein or IL13Rα2-siRNA also inhibited osteoclast differentiation, and IL13Rα2-siRNA attenuated the RANKL-induced activation of the MAPK (ERK/P38/JNK) and AKT pathways, similar to the effects observed upon silencing of Chi3l1. Moreover, the promoting effect of recombinant Chi3l1 protein on osteoclastogenesis and the activation of the MAPK and AKT pathways was reversed by IL13Rα2 siRNA. Finally, recombinant LI13Rα2 protein significantly attenuated the LPS-induced cranial osteolysis and the number of osteoclasts in vivo. Conclusions Our findings suggested that IL13Rα2 served as a crucial receptor for Chi3l1, enhancing RANKL-induced MAPK and AKT activation to promote osteoclast differentiation. These findings provide valuable insights into the molecular mechanisms of Chi3l1 in osteoclastogenesis, with potential therapeutic implications for osteoclast-related diseases. Video Abstract

Abstract Background Osteoclast-mediated bone resorption cause bone loss in several bone diseases. Exosomes have been reported to regulate osteoclast differentiation. M2-polarized macrophages exhibit anti-inflammatory activity. This study aimed to explore the effect of exosomes from M2 polarized macrophages (M2-exos) on osteoclastogenesis and molecular mechanisms. Methods M2-exos were isolated from IL-4-induced Raw264.7 cells (M2 macrophages) and used to treat osteoclasts (RANKL-induced Raw264.7 cells). Osteoclast differentiation was visualized using tartrate resistant acid phosphatase staining. Quantitative real-time PCR (qPCR) was conducted to measure the levels of osteoclastogenesis-related genes. The underlying mechanisms of M2-exos were evaluated using qPCR and western blotting. Results M2-exos suppressed osteoclast differentiation induced by RANKL. Additionally, CSF2 was highly expressed in M2 macrophages, and knockdown of CSF2 further enhanced the effects of M2-exos on osteoclast differentiation. Moreover, CSF2 positively regulated TNF-α signaling, which inhibition promoted differentiation of M2-exo-treated osteoclasts. Conclusion M2-exos inhibited RANKL-induced osteoclast differentiation by downregulating the CSF2 expression through inactivating the TNF-α signaling, suggesting the potential application of exosomes in bone disease therapy.

Objective Mechanisms involved in developing intervertebral disc degeneration (IDD) are poorly understood, thus making developing effective therapies difficult. This study aimed to suggest a possible molecular mechanism, based on transcriptome sequencing‐identified transforming growth factor (TGF‐β), underlying the effects on bone homeostasis in IDD. Methods A mouse model for IDD was established. Transcriptome sequencing of nucleus pulposus tissue from mice (n = 3) identified differentially expressed mRNAs and key genes impacting bone homeostasis. A protein–protein interaction network pinpointed core genes. GO and KEGG analysis revealed gene functions. Expression levels of TGF‐β1, tartrate‐resistant acid phosphatase (TRAP), and cathepsin K (CTSK) were measured. Micro‐CT evaluated vertebral structures and vascular imaging. Western Blot measured expression levels of Vegf, Opn, MMP3, and MMP13. Safranin O‐Fast Green and TRAP staining were performed on intervertebral discs and endplates. Results Transcriptomic analysis found 1790 differentially expressed mRNAs in IDD mice. Twenty‐eight genes related to bone homeostasis in IDD were identified. TGF‐β1 was confirmed as the core gene. GO and KEGG showed TGF‐β1 regulates osteoclast markers like CTSK and TRAP through pathways including NF‐κB and MAPK. Experimental validation revealed lower TGF‐β1 expression in IDD mice than controls, and increased TRAP and CTSK expression. Micro‐CT showed decreased bone mass and intervertebral disc space in IDD mice. Vascular imaging showed increased vascular volume in IDD cartilaginous endplates. Western blot displayed increased VEGF and OPN levels, but decreased MMP3 and MMP13 in IDD mice. Safranin O‐fast green staining revealed severe IDD degeneration. However, TGF‐β1 injection improved bone parameters in IDD mice. In vitro experiments confirmed TGF‐β1 inhibits bone marrow macrophages differentiation into osteoclasts. Conclusion From our data, we conclude that TGF‐β1 repressed osteoclast differentiation and aberrant bone‐associated angiogenesis in cartilage endplates (EPs) to alleviate IDD, which may be instrumental for the therapeutic targeting of IDD.

Long noncoding RNAs (lncRNAs) are important regulators of bone metabolism. In this study, lncRNA microarray analysis was used to identify differentially expressed lncRNAs in differentiated osteoclasts. lncRNA-Gm5532 is highly expressed during osteoclast differentiation. lncRNA-Gm5532 knockdown impairs osteoclast formation and bone resorption. Mechanistic experiments show that lncRNA-Gm5532 functions as a competing endogenous RNA (ceRNA) and acts as a sponge for miR-125a-3p, which promotes TNF receptor-associated factor 6 (TRAF6) expression. miR-125a-3p mimics suppress osteoclast differentiation and TAK1/NF-κB/MAPK signaling. The miR-125a-3p inhibitor reverses the negative effects of siGm5532 on osteoclast differentiation. In summary, our study reveals that lncRNA-Gm5532 functions as an activator in osteoclast differentiation by targeting the miR-125a-3p/TRAF6 axis, making it a novel biomarker and potential therapeutic target for osteoporosis.

Background: Previous research has revealed that the 18 glycoside hydrolase gene family (GH18) member Chitinase 3-like 1 (Chi3l1) can regulate osteoclast differentiation and bone resorption. However, its downstream receptors and molecular mechanisms during osteoclastogenesis have yet to be elucidated. Methods: Initially, we conducted a comprehensive investigation to evaluate the effects of recombinant Chi3l1 protein or Chi3l1 siRNA on osteoclast differentiation and the RANKL-induced MAPK/AKT signaling pathways. Moreover, we used immunofluorescence and immunoprecipitation assays to identify IL13Rα2 as the downstream receptor of Chi3l1. Subsequently, we investigated the impact of IL13Rα2 recombinant protein or IL13Rα2-siRNA on osteoclast differentiation and the associated signaling pathways. Finally, we performed in vivo experiments to examine the effect of recombinant IL13Rα2 protein in an LPS-induced mouse model of cranial osteolysis. Results: Our findings highlight that the administration of recombinant Chi3l1 protein increased the formation of osteoclasts and bolstered the expression of several osteoclast-specific genes (TRAP, NFATC1, CTR, CTSK, V-ATPase d2, and Dc-STAMP). Additionally, Chi3l1 significantly promoted the RANKL-induced MAPK (ERK/P38/JNK) and AKT pathway activation, whereas Chi3l1 silencing inhibited this process. Next, using immunofluorescence and co-immunoprecipitation assays, we identified IL13Rα2 as the binding partner of Chi3l1 during osteoclastogenesis. IL13Rα2 recombinant protein or IL13Rα2-siRNA also inhibited osteoclast differentiation, and IL13Rα2-siRNA attenuated the RANKL-induced activation of the MAPK (ERK/P38/JNK) and AKT pathways, similar to the effects observed upon silencing of Chi3l1. Moreover, the promoting effect of recombinant Chi3l1 protein on osteoclastogenesis and the activation of the MAPK and AKT pathways was reversed by IL13Rα2 siRNA. Finally, recombinant LI13Rα2 protein significantly attenuated the LPS-induced cranial osteolysis and the number of osteoclasts in vivo. Conclusions: Our findings suggested that IL13Rα2 served as a crucial receptor for Chi3l1, enhancing RANKL-induced MAPK and AKT activation to promote osteoclast differentiation. These findings provide valuable insights into the molecular mechanisms of Chi3l1 in osteoclastogenesis, with potential therapeutic implications for osteoclast-related diseases. DtV65n_fm6ocbvZ72jE1Sr Video Abstract [ABSTRACT FROM AUTHOR]

Background: Osteoclast-mediated bone resorption cause bone loss in several bone diseases. Exosomes have been reported to regulate osteoclast differentiation. M2-polarized macrophages exhibit anti-inflammatory activity. This study aimed to explore the effect of exosomes from M2 polarized macrophages (M2-exos) on osteoclastogenesis and molecular mechanisms. Methods: M2-exos were isolated from IL-4-induced Raw264.7 cells (M2 macrophages) and used to treat osteoclasts (RANKL-induced Raw264.7 cells). Osteoclast differentiation was visualized using tartrate resistant acid phosphatase staining. Quantitative real-time PCR (qPCR) was conducted to measure the levels of osteoclastogenesis-related genes. The underlying mechanisms of M2-exos were evaluated using qPCR and western blotting. Results: M2-exos suppressed osteoclast differentiation induced by RANKL. Additionally, CSF2 was highly expressed in M2 macrophages, and knockdown of CSF2 further enhanced the effects of M2-exos on osteoclast differentiation. Moreover, CSF2 positively regulated TNF-α signaling, which inhibition promoted differentiation of M2-exo-treated osteoclasts. Conclusion: M2-exos inhibited RANKL-induced osteoclast differentiation by downregulating the CSF2 expression through inactivating the TNF-α signaling, suggesting the potential application of exosomes in bone disease therapy. [ABSTRACT FROM AUTHOR]

Objective: Mechanisms involved in developing intervertebral disc degeneration (IDD) are poorly understood, thus making developing effective therapies difficult. This study aimed to suggest a possible molecular mechanism, based on transcriptome sequencing‐identified transforming growth factor (TGF‐β), underlying the effects on bone homeostasis in IDD. Methods: A mouse model for IDD was established. Transcriptome sequencing of nucleus pulposus tissue from mice (n = 3) identified differentially expressed mRNAs and key genes impacting bone homeostasis. A protein–protein interaction network pinpointed core genes. GO and KEGG analysis revealed gene functions. Expression levels of TGF‐β1, tartrate‐resistant acid phosphatase (TRAP), and cathepsin K (CTSK) were measured. Micro‐CT evaluated vertebral structures and vascular imaging. Western Blot measured expression levels of Vegf, Opn, MMP3, and MMP13. Safranin O‐Fast Green and TRAP staining were performed on intervertebral discs and endplates. Results: Transcriptomic analysis found 1790 differentially expressed mRNAs in IDD mice. Twenty‐eight genes related to bone homeostasis in IDD were identified. TGF‐β1 was confirmed as the core gene. GO and KEGG showed TGF‐β1 regulates osteoclast markers like CTSK and TRAP through pathways including NF‐κB and MAPK. Experimental validation revealed lower TGF‐β1 expression in IDD mice than controls, and increased TRAP and CTSK expression. Micro‐CT showed decreased bone mass and intervertebral disc space in IDD mice. Vascular imaging showed increased vascular volume in IDD cartilaginous endplates. Western blot displayed increased VEGF and OPN levels, but decreased MMP3 and MMP13 in IDD mice. Safranin O‐fast green staining revealed severe IDD degeneration. However, TGF‐β1 injection improved bone parameters in IDD mice. In vitro experiments confirmed TGF‐β1 inhibits bone marrow macrophages differentiation into osteoclasts. Conclusion: From our data, we conclude that TGF‐β1 repressed osteoclast differentiation and aberrant bone‐associated angiogenesis in cartilage endplates (EPs) to alleviate IDD, which may be instrumental for the therapeutic targeting of IDD. [ABSTRACT FROM AUTHOR]

Objective To investigate the underlying mechanism of plastrum testudinis (PT ) in the treatment of osteoporotic fracture ( OF) using network pharmacology and experimental validation . Methods The components of PT and their corresponding targets were obtained with the help of BATMAN database, and relevant targets of OF were obtained by searching Genecards and OMIM databases. Intersection targets were input into the STRING database to obtain protein-protein interaction ( PPI) information. The constructions of PPI network and PT-compound-target-OF network were done with Cytoscape 3. 7. 2, and GO function enrichment and KEGG pathway enrichment analyses were finished with Cytoscape3. 7. 2 and R. Additionally, to verify the reliability of the network pharmacology findings, we also conducted in vivo experiments to verify the effect of PT on improving OF and in vitro experiments, which investigated the phenotypes of human peripheral blood monocytes ( HPBMs) under the intervention of PT. Results Six active compounds and 342 targets of PT were obtained, including 34 targets correlated with OF. There were 802 results gained through GO function enrichment analysis ( P < 0. 05), and 67 results through KEGG pathway enrichment analysis, including 22 relevant signaling pathways ( P < 0.05 ), which were mainly involved in TNF, MARK, estrogen, NF-KB, and other signaling pathways. Notably, our in vivo experiments proved that PT could effectively treat the bone defects in OF model rats. The in vitro experiments showed that PT could inhibit RANKL-induced osteoclast differentiation in HPBMs. Conclusion By regulating inflammatory response, hormone metabolism, and cell cycle, PT may treat OF through numerous compounds, targets and pathways. The inhibition of osteoclast differentiation may play an important role in the mechanism of PT against OF. [ABSTRACT FROM AUTHOR]

Osteoporosis is currently the most prevalent bone disorder worldwide and is characterized by low bone mineral density and an overall increased risk of fractures. To treat osteoporosis, a range of drugs targeting bone homeostasis have emerged in clinical practice, including anti-osteoclast agents such as bisphosphonates and denosumab, bone formation stimulating agents such as teriparatide, and selective oestrogen receptor modulators. However, traditional clinical medicine still faces challenges related to side effects and high costs of these types of treatments. Nanomaterials (particularly gold nanoparticles [AuNPs]), which have unique optical properties and excellent biocompatibility, have gained attention in the field of osteoporosis research. AuNPs have been found to promote osteoblast differentiation, inhibit osteoclast formation, and block the differentiation of adipose-derived stem cells, which thus is believed to be a novel and promising candidate for osteoporosis treatment. This review summarizes the advances and drawbacks of AuNPs in their synthesis and the mechanisms in bone formation and resorption in vitro and in vivo, with a focus on their size, shape, and chemical composition as relevant parameters for the treatment of osteoporosis. Additionally, several important and promising directions for future studies are also discussed, which is of great significance for prevention and treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

Objectives The variable region of most ACPA IgG molecules in the serum of RA patients carries N -glycan (N -glycanV). To analyse the pathogenicity of N -glycanV of ACPAs, we analysed the pathogenicity of a monoclonal ACPA, CCP-Ab1, with or without N -glycanV, which had been isolated from a patient with RA. Methods CCP-Ab1 with no N -glycosylation site in the variable region (CCP-Ab1 N-rev) was generated, and antigen binding, the effect on in vitro differentiation of osteoclasts from bone marrow mononuclear cells of autoimmune arthritis–prone SKG mice (the cell size of TRAP+ cells and bone resorption capacity) and the in vivo effect on the onset or exacerbation of autoimmune arthritis in SKG mice were evaluated in comparison with glycosylated CCP-Ab1. Results Amino acid residues in citrullinated peptide (cfc1), which are essential for binding to CCP-Ab1 N-rev and original CCP-Ab1, were almost identical. The size of TRAP+ cells was significantly larger and osteoclast bone resorption capacity was enhanced in the presence of CCP-Ab1, but not with CCP-Ab1 N-rev. This enhancing activity required the sialic acid of the N -glycan and Fc region of CCP-Ab1. CCP-Ab1, but not CCP-Ab1 N-rev, induced the exacerbation of experimental arthritis in the SKG mouse model. Conclusions These data showed that N -glycanV was required for promoting osteoclast differentiation and bone resorption activity in both in vitro and in vivo assays. The present study demonstrated the important role of N -glycanV in the exacerbation of experimental arthritis by ACPAs. [ABSTRACT FROM AUTHOR]

Amid the numerous health concerns faced by the world, osteoporosis a silent epidemic is a major health concern, since it affects millions of people all around the world. Recognizing the limited treatment options for osteoporotic patients, along with their long-term use safety concerns, our research group focused on investigating efficacious, inexpensive and safe alternative therapies for osteoporosis. Investigating the osteogenic potential of CQ, our research group previously reported osteogenic stimulatory effects of CQ (hexane fraction). The present study explores anti-resorptive effects of CQ-H on bones. We utilized RAW264.7 murine macrophage cell line as osteoclast precursors, and resveratrol (RSV) as a standard for its anti-osteoclast activity. Upon RANK-L induction of RAW264.7 cells to differentiate into osteoclasts, we found 10ng/ml CQ-H as most efficacious concentration for inhibiting osteoclastogenesis with 57.8% inhibition of TRAP activity. Whereas, in comparison 20ng/ml RSV showed 49.9% inhibition of TRAP activity. Expression of osteoclast marker genes such as NFATc1, ACP-5, CTSK, MMP-9 and CTR corroborated our results. Distinct downregulation of marker genes was observed in CQ-H and RSV treated differentiated cells compared to the positive control. Moreover, the downregulation was enhanced in CQ-H treated osteoclasts compared to RSV treatment. This study presents in vitro findings on the osteoclast inhibition activity of CQ and provide evidence of anti-osteoporotic therapeutic property of CQ. Further studies on isolation and functional characterization of active compounds of signaling pathways that mediate osteoclastogenesis will contribute further insights on the anti-osteoporotic therapeutic activity of CQ. [ABSTRACT FROM AUTHOR]

Iron overload enhances osteoclastic bone resorption and induces osteoporosis. Excess iron is highly toxic. The modulation of redox and iron homeostasis is critical for osteoclast differentiation under iron-overload condition. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that regulates the cellular defense against oxidative stress and iron overload through the expression of genes involved in anti-oxidative processes and iron metabolism. Our studies demonstrated that NRF2 activation was suppressed during osteoclast differentiation. Under iron-overload condition, NRF2 and its mediated antioxidant and iron metabolism genes were activated by reactive oxygen species (ROS), which enhanced antioxidant capability. NRF2 mediated the upregulation of iron exporter ferroportin 1 (FPN1) and iron storage protein ferritin, contributing to decreased levels of intracellular iron. Nfe2l2 knockout induced oxidative stress and promoted osteoclast differentiation under normal condition, but induced ferroptosis under iron-overload condition. Nfe2l2 knockout alleviated iron overload induced bone loss by inhibiting osteoclast differentiation. Our results suggest that NRF2 activation is essential for osteoclast differentiation by enhancing antioxidant capability and reducing intracellular iron under iron-overload condition. Targeting NRF2 to induce ferroptosis could be a potential therapy for the treatment of iron-overload induced osteoporosis. [ABSTRACT FROM AUTHOR]

Nuclear factor-erythroid 2-related factor 2 (Nrf2) has been shown to be a negative regulator of osteoclast differentiation, but the precise mechanisms have not yet been established. We examined the precise roles of Nrf2 in regulating antioxidants and reactive oxygen species (ROS) levels, especially the cytoplasmic and mitochondrial ROS during osteoclastogenesis in vitro. In the current study, we found that the absence of Nrf2 promotes osteoclast differentiation in bone-marrow-derived macrophages (BMMs) and RAW 264.7 cells. The receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) significantly lowered the levels of Nrf2 and its downstream antioxidant enzymes at mRNA and/or protein levels during osteoclast differentiation in the BMMs of mice and RAW 264.7 mouse leukemic monocytes. Compared to the wild-type cells, Nrf2-deficient cells exhibited heightened sensitivity to both transient RANKL-induced cytoplasmic ROS and prolonged RANKL and M-CSF-induced cytoplasmic and mitochondrial ROS accumulation. Furthermore, exogenous antioxidant agents, including N-acetyl-cysteine (NAC), diphenyleneiodonium chloride (DPI), and mitoquinone mesylate (MitoQ), exhibited substantial capability to suppress the elevation of ROS levels during osteoclast differentiation induced by Nrf2 deficiency, and they consequently inhibited osteoclast differentiation augmented by the lack of Nrf2. The activation of phosphorylated c-FOS resulting from elevated ROS promoted osteoclast differentiation. The inhibition of c-FOS blocked osteoclast differentiation, which was elevated by Nrf2-deficiency. Taken together, these data reveal that Nrf2 effectively decreased the accumulation of intracellular ROS and the phosphorylation of c-FOS during osteoclastic differentiation by regulating antioxidant enzymes and subsequently inhibited RANKL-induced osteoclast differentiation. [ABSTRACT FROM AUTHOR]

Bones serve mechanical and defensive functions, as well as regulating the balance of calcium ions and housing bone marrow.. The qualities of bones do not remain constant. Instead, they fluctuate throughout life, with functions increasing in some situations while deteriorating in others. The synchronization of osteoblast-mediated bone formation and osteoclast-mediated bone resorption is critical for maintaining bone mass and microstructure integrity in a steady state. This equilibrium, however, can be disrupted by a variety of bone pathologies. Excessive osteoclast differentiation can result in osteoporosis, Paget's disease, osteolytic bone metastases, and rheumatoid arthritis, all of which can adversely affect people's health. Osteoclast differentiation is regulated by transcription factors NFATc1, MITF, C/EBPα, PU.1, NF-κB, and c-Fos. The transcriptional activity of osteoclasts is largely influenced by developmental and environmental signals with the involvement of co-factors, RNAs, epigenetics, systemic factors, and the microenvironment. In this paper, we review these themes in regard to transcriptional regulation in osteoclastogenesis. [ABSTRACT FROM AUTHOR]

Osteoporosis is a common bone disorder characterized by reduced bone density and increased risk of fractures. The modulation of bone cell functions, particularly the inhibition of osteoclastic differentiation, plays a crucial role in osteoporosis treatment. Polyphosphoesters (PPEs) have shown the potential in reducing the function of osteoclast cells, but the effect of their chemical structure on osteoclastic differentiation remains largely unexplored. In this study, we evaluated the effect of PPE's chemical structure on the inhibition of osteoclastic differentiation of murine bone marrow mononuclear cells (BMNCs). PPEs containing phosphotriester and phosphodiester units at varying compositions were synthesized. Cytotoxicity testing confirmed the biocompatibility of the copolymers at concentrations below 0.5mg/mL. Isolated from long bones, BMNCs were cultured in a differentiation medium supplemented with different PPE concentrations. Osteoclast formation was assessed through tartrate-resistant acid phosphatase and phalloidin staining. A significant decrease in the size of osteoclast cells formed upon BMNC contact with PPEs was observed, with a more pronounced effect observed at higher PPE concentrations. In addition, an increased composition of phosphodiester units in the PPEs yielded a decreased density of differentiated osteoclasts. Furthermore, real-time PCR analysis of major osteoclastic markers provided gene expression data that correlated with microscopic observations, confirming the effect of phosphodiester units in suppressing osteoclast differentiation of BMNCs from the early stages. These findings highlight the potential of PPEs as polymers are capable of modulating bone cell functions through their chemical structures. [ABSTRACT FROM AUTHOR]

AIM: To investigate the effect of Tenghuang-Jiangu capsule （TJC） on the expression of silent information regulator 1（ SIRT1）/nuclear factor κB（ NF-κB）/nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） signaling pathway in postmenopausal rats with osteoporosis and its molecular mechanism. METHODS: Female Sprague-Dawley rats were randomly divided into sham group, model group ［ovariectomy （OVX） group］, SIRT1 inhibitor （EX-527）+OVX group, positive control estradiol valerate （ESV）+OVX group, and high-, medium- and low-dose TJC groups （TJC-H+OVX, TJC-M+OVX and TJC-M+OVX groups）, with 10 rats in each group. The TJC, ESV or EX-527 intervention after OVX was performed. Double immunofluorescence staining was used to detect SIRT1, NF-κB p65 and NLRP3 proteins, and ELISA was performed to detect inflammatory factors. RT-qPCR and Western blot were used to detect the mRNA and protein expression of SIRT1/NF-κB/NLRP3 signaling pathway-related molecules and osteoclast differentiation markers. RESULTS: Compared with sham group, serum levels of interleukin-1β（ IL-1β）, IL-18, and tumor necrosis factor-α （TNF-α） increased significantly in the OVX model group and in SIRT1 inhibitor EX-527 group （P<0. 01）. Similarly, the fluorescence area of NF-κB p65 and NLRP3 in osteoclast（ P<0. 01）, and the mRNA and protein expression levels of NF-κB p65, NLRP3, and activated T cell c1 nuclear factor cytoplasmic 1 （NFATc1） in the femur also increased significantly （P<0. 01）. However, the fluorescence area of SIRT1 in osteoclast decreased significantly in the OVX model group and in SIRT1 inhibitor EX-527 group（ P<0. 01）. Compared with OVX group, serum IL-1β content and NLRP3 protein expression level in the femur in the TJC-H treatment group decreased significantly. A similar decrease was observed for the serum IL-18 content, NF-κB p65 and NFATc1 protein expression levels in the femur in the TJC-H and TJC-M group rats. Serum TNF-α content, NF-κB p65 and NLRP3 expression fluorescence area in osteoclast, NF-κB p65, NLRP3, and NFATc1 mRNA expression levels in the femur decreased significantly in the TJC-H, TJC-M, and TJC-L groups. In contrast, the fluorescence area of SIRT1 in osteoclast increased significantly （P<0. 05）. Compared with EX-527+OVX group, NLRP3 protein expression level in the TJC-H treatment group decreased significantly. Serum IL-1β content in the TJC-H and TJC-M treatment group decreased significantly. A similar decrease was observed for the serum IL-18 content, NF-κB p65 and NFATc1 protein expression levels in the femur in the TJC-H and TJC-M groups. Serum TNF-α content, NF-κB p65 and NLRP3 expression fluorescence area in osteoclast, NF-κB p65, NLRP3, and NFATc1 mRNA expression levels in the femur decreased significantly in the TJC-H, TJC-M, and TJC-L treatment groups, in contrast, the fluorescence area of SIRT1 in osteoclast increased significantly （P<0. 05）. CONCLUSION: Tenghuang-Jiangu capsule can inhibit osteoclast differentiation in postmenopausal osteoporosis model rats. The mechanism may be related to the inhibition of SIRT1/NF-κB/NLRP3 signaling pathway activation and alleviation of inflammatory response. [ABSTRACT FROM AUTHOR]

Caveolin-1 ( Cavl) is a structural protein component of the Caveolae and a major regulator of a variety of signaling molecules involved in a variety of cellular processes including signal transduction, endocytosis, cholesterol distribution and cell migration, and is a candidate target for the prevention and treatment of many diseases. With further research, the role of Cavl in maintaining bone metabolic balance was also discovered. In this paper, the gene structure and protein structure of Cavl were reviewed, and the regulatory mechanism of Cavl on bone metabolism was respectively summarized from osteogenic and adipogenic differentiation of Mesenchymal Stem Cells ( MSCs) and osteoclastic differentiation of Hematopoietic Stem Cells ( HSCs). [ABSTRACT FROM AUTHOR]

Abstract Background Extracorporeal shock wave therapy (ESWT) has been reported to promote osteoblast differentiation. However, the role of ESWT on osteoclast differentiation is still elusive. Methods This study analyzed the differentiation of osteoclasts in the shock wave group and the control group in vitro, and TRAP staining, RT-PCR, WB assays, and MTT assays were assessed between the two groups. Furthermore, we analyzed the bone formation in these two groups in vivo and micro-CT and trap staining were assessed between the two groups. Results We found that ESWT inhibited osteoclast maturation in vitro and ESW treatment of femur promoted bone formation in vivo. Mechanically, osteoclast differentiation was inhibited as the number of impulses increased and ESWT decreased endogenous levels of NTAFc1 and P65 protein. Conclusions ESWT may be a potential therapy of osteoporosis through NF-κB signaling pathway.

Jiuqing Lu,1,2,&ast; Nijia Yu,1,&ast; Qian Liu,2,&ast; Yajia Xie,1 Lei Zhen2 1Oral Biomedical Engineering Laboratory, Shanghai Stomatological Hospital, Fudan University, Shanghai, People’s Republic of China; 2Department of Stomatology, Tongji Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Lei Zhen, Department of Stomatology, Tongji Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China, Tel +86 1 582 108 9038, Email zhenleilei123@126.comIntroduction: Periodontitis is a chronic inflammatory disease that causes alveolar bone loss. Diabetes is one of the most important factors contributing to periodontitis. Exosomes derived from mesenchymal stem cells (MSCs-Exo) have been reported to promote bone regeneration. This study aimed to examine the function and mechanism of exosomes derived from periodontal ligament stem cells (PDLSCs-Exo) in regulating periodontal regeneration in diabetic periodontitis.Methods: Exosomes derived from normal-glucose-cultured PDLSCs (NG-PDLSCs-Exo) and high-glucose-preconditioned PDLSCs (HG-PDLSCs-Exo) were used. Their effects on RAW264.7 cells were investigated by TRAP staining and quantitative real time-polymerase chain reaction (qRT-PCR). The role of exosomal miR-31-5p in osteoclast differentiation was tested using qRT-PCR, double luciferase analysis, and Western blotting. We investigated the effects of these two types of PDLSCs-Exo on alveolar bone loss in vivo in mice with experimental periodontitis.Results: PDLSCs-Exo were transferred to RAW264.7, and HG-PDLSCs-Exo inhibited osteoclast formation to a lesser extent than NG-PDLSCs-Exo. Further studies revealed the effect of PDLSCs-Exo on osteoclastogenesis via the miR-31-5p/eNOS signaling pathway. In mice with experimental periodontitis, PDLSCs-Exo reduced alveolar bone destruction and decreased the number of osteoclasts on the alveolar bone surface.Conclusion: Our results suggest that exosomal miR-31-5p derived from PDLSCs regulates alveolar bone regeneration by targeting eNOS.Keywords: diabetes, periodontitis, macrophage, osteoclast differentiation

Abstract Background Osteoporosis, characterized by reduced bone mass and deterioration of bone quality, is a significant health concern for postmenopausal women. Considering that the specific role of circRNAs in osteoporosis and osteoclast differentiation remains poorly understood, this study aims to shed light on their involvement in these processes to enhance our understanding and potentially contribute to improved treatment strategies for osteoporosis. Methods An osteoporotic model was constructed in vivo in ovariectomized mouse. In vitro, we induced osteoclast formation in bone marrow-derived macrophages (BMDMs) using M-CSF + RANKL. To assess osteoporosis in mice, we conducted HE staining. We used MTT and TRAP staining to measure cell viability and osteoclast formation, respectively, and also evaluated their mRNA and protein expression levels. In addition, RNA pull-down, RIP and luciferase reporter assays were performed to investigate interactions, and ChIP assay was used to examine the impact of circZNF367 knockdown on the binding between FUS and CRY2. Results We observed increased expression of CircZNF367, FUS and CRY2 in osteoporotic mice and M-CSF + RANKL-induced BMDMs. Functionally, knocking down circZNF367 inhibited osteoporosis in vivo. Furthermore, interference with circZNF367 suppressed osteoclast proliferation and the expression of TRAP, NFATc1, and c-FOS. Mechanistically, circZNF367 interacted with FUS to maintain CRY2 mRNA stability. Additionally, knocking down CRY2 rescued M-CSF + RANKL-induced osteoclast differentiation in BMDMs promoted by circZNF367 and FUS. Conclusion This study reveals that the circZNF367/FUS axis may accelerate osteoclasts differentiation by upregulating CRY2 in osteoporosis and suggests that targeting circZNF367 may have potential therapeutic effects on osteoporosis.

Background: Abnormal osteoclast and osteoblast differentiation is an essential pathological process in osteoporosis. As an important deubiquitinase enzyme, ubiquitin-specific peptidase 7 (USP7) participates in various disease processes through posttranslational modification. However, the mechanism by which USP7 regulates osteoporosis remains unknown. Herein, we aimed to investigate whether USP7 regulates abnormal osteoclast differentiation in osteoporosis. Methods: The gene expression profiles of blood monocytes were preprocessed to analyze the differential expression of USP genes. CD14+ peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected from osteoporosis patients (OPs) and healthy donors (HDs), and the expression pattern of USP7 during the differentiation of CD14+ PBMCs into osteoclasts was detected by western blotting. The role of USP7 in the osteoclast differentiation of PBMCs treated with USP7 siRNA or exogenous rUSP7 was further investigated by the F-actin assay, TRAP staining and western blotting. Moreover, the interaction between high-mobility group protein 1 (HMGB1) and USP7 was investigated by coimmunoprecipitation, and the regulation of the USP7-HMGB1 axis in osteoclast differentiation was further verified. Osteoporosis in ovariectomized (OVX) mice was then studied using the USP7-specific inhibitor P5091 to identify the role of USP7 in osteoporosis. Results: The bioinformatic analyses and CD14+ PBMCs from osteoporosis patients confirmed that the upregulation of USP7 was associated with osteoporosis. USP7 positively regulates the osteoclast differentiation of CD14+ PBMCs in vitro. Mechanistically, USP7 promoted osteoclast formation by binding to and deubiquitination of HMGB1. In vivo, P5091 effectively attenuates bone loss in OVX mice. Conclusion: We demonstrate that USP7 promotes the differentiation of CD14+ PBMCs into osteoclasts via HMGB1 deubiquitination and that inhibition of USP7 effectively attenuates bone loss in osteoporosis in vivo.The translational potential of this article:The study reveals novel insights into the role of USP7 in the progression of osteoporosis and provides a new therapeutic target for the treatment of osteoporosis.

Abstract Background We have previously reported that extracellular vesicles (EVs) derived from osteoblastic, osteoclastic and mixed prostate cancer cells promote osteoclast differentiation and inhibit osteoblast differentiation via transferring miR-92a-1-5p. In the present study, we focused on engineering miR-92a-1-5p into EVs and determining any therapeutic roles and mechanisms of the engineered EVs. Methods A stable prostate cancer cell line (MDA PCa 2b) overexpressing miR-92a-1-5p was constructed by lentivirus, and EVs were isolated by ultracentrifugation. The overexpression of miR-92a-1-5p in both cells and EVs was tested using qPCR. Osteoclast function was evaluated by Trap staining, mRNA expression of osteoclastic markers ctsk and trap, immunolabeling of CTSK and TRAP and microCT using either in vitro and in vivo assays. Target gene of miR-92a-1-5p was proved by a dual-luciferase reporter assay system. siRNAs were designed and used for transient expression in order to determine the role of downstream genes on osteoclast differentiation. Results Stable overexpression cells of miRNA-92a-5p was associated with EVs upregulating this microRNA, as confirmed by qPCR. Further, miR-92a-1-5p enriched EVs promote osteoclast differentiation in vitro by reducing MAPK1 and FoxO1 expression, associated with increased osteoclast function as shown by TRAP staining and mRNA expression of osteoclast functional genes. siRNA targeting MAPK1 or FoxO1 resulted in similar increase in osteoclast function. In vivo, the miR-92a-1-5p enriched EVs given via i.v. injection promote osteolysis, which was associated with reduction of MAPK1 and FoxO1 expression in bone marrow. Conclusion These experiments suggest that miR-92a-1-5p enriched EVs regulate osteoclast function via reduction of MAPK1 and FoxO1. Graphical Abstract

Inflammatory disorders have been found to induce bone loss through sustained and persistent activation of osteoclast differentiation, leading to heightened bone resorption. The current pharmacological interventions for combating bone loss to harbor adverse effects or contraindications. There is a pressing need to identify drugs with fewer side effects. The effect and underlying mechanism of sulforaphene (LFS) on osteoclast differentiation were illustrated in vitro and in vivo with RANKL-induced Raw264.7 cell line osteoclastogenesis and lipopolysaccharide (LPS)-induced bone erosion model. In this study, LFS has been shown to effectively impede the formation of mature osteoclasts induced from both Raw264.7 cell line and bone marrow macrophages (BMMs), mainly at the early stage. Further mechanistic investigations uncovered that LFS suppressed AKT phosphorylation. SC-79, a potent AKT activator, was found to reverse the inhibitory impact of LFS on osteoclast differentiation. Moreover, transcriptome sequencing analysis revealed that treatment with LFS led to a significant upregulation in the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant-related genes. Then it's validated that LFS could promote NRF2 expression and nuclear translocation, as well as effectively resist oxidative stress. NRF2 knockdown reversed the suppression effect of LFS on osteoclast differentiation. In vivo experiments provide convincing evidence that LFS is protective against LPS-induced inflammatory osteolysis. These well-grounded and promising findings suggest LFS as a promising agent to addressing oxidative-stress related diseases and bone loss disorders. [Display omitted] • Sulforaphene effectively attenuated both Raw264.7 and bone marrow macrophage osteoclastogenesis. • Sulforaphene promoted NRF2 expression and nuclear translocation to alleviate oxidative stress. • Sulforaphene has the ability to protect from LPS-induced bone loss in vivo. [ABSTRACT FROM AUTHOR]