ABSTRACT
BACKGROUND: Osteoclast plays an important role in maintaining the balance between bone anabolism and bone catabolism. The abnormality of osteoclast is closely related to osteolytic bone diseases such as osteoporosis, rheumatoid arthritis and tumor bone metastasis. PURPOSE: We aim to search for natural compound that may suppress osteoclast formation and function. STUDY DESIGN: In this study, we assessed the impact of Dauricine (Dau) on the formation and function of osteoclasts in vitro, as well as its potential in preventing bone loss in an ovariectomy mouse model in vivo. METHODS: Multiple in vitro experiments were carried out, including osteoclastogenesis, podosomal belt formation, bone resorption assay, RNA-sequencing, real-time quantitative PCR, ROS level detection, surface plasmon resonance assay, luciferase assay and western blot. To verify the effect in vivo, an ovariectomized mouse model (OVX model) was constructed, and bone parameters were measured using micro-CT and histology. Furthermore, metabolomics analysis was performed on blood serum samples from the OVX model. RESULTS: In vitro experiments demonstrated that Dau inhibits RANKL-induced osteoclastogenesis, podosomal belt formation, and bone resorption function. RNA-sequencing results revealed that Dau significantly suppresses genes related to osteoclast. Functional enrichment analysis indicated that Dau's inhibition of osteoclasts may be associated with NF-κB signaling pathway and reactive oxygen metabolism pathway. Molecular docking, surface plasmon resonance assay and western blot analysis further confirmed that Dau inhibits RANKL-induced osteoclastogenesis by modulating the ROS/NF-κB/NFATc1 pathway. Moreover, administration of Dau to OVX-induced mice validated its efficacy in treating bone loss disease. CONCLUSION: Dau prevents OVX-induced bone loss by inhibiting osteoclast activity and bone resorption, potentially offering a new approach for preventing and treating metabolic bone diseases such as osteoporosis. This study provides innovative insights into the inhibitory effects of Dau in an in vivo OVX model and elucidates the underlying mechanism.
Subject(s)
Benzylisoquinolines , NF-kappa B , NFATC Transcription Factors , Osteoclasts , Osteogenesis , Ovariectomy , RANK Ligand , Reactive Oxygen Species , Animals , Benzylisoquinolines/pharmacology , Female , RANK Ligand/metabolism , Mice , NF-kappa B/metabolism , Reactive Oxygen Species/metabolism , Osteogenesis/drug effects , Osteoclasts/drug effects , NFATC Transcription Factors/metabolism , Disease Models, Animal , Bone Resorption/drug therapy , Mice, Inbred C57BL , RAW 264.7 Cells , Osteoporosis/drug therapy , Osteoporosis/prevention & control , Humans , TetrahydroisoquinolinesABSTRACT
One of the effective therapeutic strategies to treat rheumatoid arthritis (RA)-related bone resorption is to target excessive activation of osteoclasts. We discovered that 6-O-angeloylplenolin (6-OAP), a pseudoguaianolide from Euphorbia thymifolia Linn widely used for the treatment of RA in traditional Chinese medicine, could inhibit RANKL-induced osteoclastogenesis and bone resorption in both RAW264.7 cells and BMMs from 1 µM and protect a collagen-induced arthritis (CIA) mouse model from bone destruction in vivo. The severity of arthritis and bone erosion observed in paw joints and the femurs of the CIA model were attenuated by 6-OAP administered at both dosages (1 or 5 mg/kg, i.g.). BMD, Tb.N and BV/TV were also improved by 6-OAP treatment. Histological analysis and TRAP staining of femurs further confirmed the protective effects of 6-OAP on bone erosion, which is mainly due to reduced osteoclasts. Molecular docking indicated that c-Src might be a target of 6-OAP and phosphorylation of c-Src was suppressed by 6-OAP treatment. CETSA and SPR assay further confirmed the potential interaction between 6-OAP and c-Src. Three signaling molecules downstream of c-Src that are vital to the differentiation and function of osteoclasts, NF-κB, c-Fos and NFATc1, were also suppressed by 6-OAP in vitro. In summary, the results demonstrated that the function of c-Src was disrupted by 6-OAP, which led to the suppression of downstream signaling vital to osteoclast differentiation and function. In conclusion, 6-OAP has the potential to be further developed for the treatment of RA-related bone erosion.
Subject(s)
Arthritis, Experimental , Bone Resorption , NF-kappa B , NFATC Transcription Factors , Osteoclasts , Osteogenesis , Animals , Mice , NFATC Transcription Factors/metabolism , RAW 264.7 Cells , Bone Resorption/drug therapy , Bone Resorption/metabolism , Bone Resorption/prevention & control , Arthritis, Experimental/drug therapy , Arthritis, Experimental/pathology , Arthritis, Experimental/metabolism , Arthritis, Experimental/chemically induced , Osteogenesis/drug effects , NF-kappa B/metabolism , Osteoclasts/drug effects , Osteoclasts/metabolism , Male , Signal Transduction/drug effects , CSK Tyrosine-Protein Kinase/metabolism , Molecular Docking Simulation , src-Family Kinases/metabolism , src-Family Kinases/antagonists & inhibitorsABSTRACT
BACKGROUND: The discovering of an osteoclast (OC) coupling active agent, capable of suppressing OC-mediated bone resorption while concurrently stimulating osteoblast (OB)-mediated bone formation, presents a promising strategy to overcome limitations associated with existing antiresorptive agents. However, there is a lack of research on active OC coupling agents. PURPOSE: This study aims to investigate the potential of Jiangu Formula (JGF) in inhibiting OCs while maintaining the OCOB coupling function. METHODS: The anti-osteoporosis efficacy of JGF was evaluated in osteoporosis models induced by ovariectomy in C57BL/6 mouse and SD rats. The effect of JGF on OCs was evaluated by detecting its capacity to inhibit OC differentiation and bone resorption in an in vitro osteoclastogenesis model induced by RANKL. The OCOB coupling activity of JGF was evaluated by measuring the secretion levels of OC-derived coupling factors, OB differentiation activity of MC3T3-E1 interfered with conditioned medium, and the effect of JGF on OC inhibition and OB differentiation in a C3H10T1/2-RAW264.7 co-culture system. The mechanism of JGF was studied by network pharmacology and validated using western blot, immunofluorescence (IF), and ELISA. Following that, the active ingredients of JGF were explored through a chemotype-assembly approach, activity evaluation, and LC-MS/MS analysis. RESULTS: JGF inhibited bone resorption in murine osteoporosis without compromising the OCOB coupling effect on bone formation. In vitro assays showed that JGF preserved the coupling effect of OC on OB differentiation by maintaining the secretion of OC-derived coupling factors. Network analysis predicted STAT3 as a key regulation point for JGF to exert anti-osteoporosis effect. Further validation assays confirmed that JGF upregulated p-STAT3(Ser727) and its regulatory factors IL-2 in RANKL-induced RAW264.7 cells. Moreover, 23 components in JGF with anti-OC activity identified by chemotype-assembly approach and verification experiments. Notably, six compounds, including ophiopogonin D, ginsenoside Re, ginsenoside Rf, ginsenoside Rg3, ginsenoside Ro, and ononin were identified as OC-coupling compounds. CONCLUSION: This study first reported JGF as an agent that suppresses bone loss without affecting bone formation. The potential coupling mechanism of JGF involves the upregulation of STAT3 by its regulators IL-2. Additionally, the chemotype-assembly approach elucidated the activity compounds present in JGF, offering a novel strategy for developing an anti-resorption agent that preserves bone formation.
Subject(s)
Bone Resorption , Cell Differentiation , Drugs, Chinese Herbal , Mice, Inbred C57BL , Osteoblasts , Osteoclasts , Osteoporosis , Rats, Sprague-Dawley , Animals , Osteoclasts/drug effects , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/chemistry , Mice , Osteoporosis/drug therapy , Osteoblasts/drug effects , Female , RAW 264.7 Cells , Cell Differentiation/drug effects , Bone Resorption/drug therapy , Ovariectomy , RANK Ligand , Rats , Osteogenesis/drug effects , Disease Models, Animal , STAT3 Transcription Factor/metabolismABSTRACT
Declining estrogen production in postmenopausal females causes osteoporosis in which the resorption of bone exceeds the increase in bone formation. Although clinical drugs are currently available for the treatment of osteoporosis, sustained medication use is accompanied by serious side effects. Corydalis bungeana Herba, a famous traditional Chinese herb listed in the Chinese Pharmacopoeia Commission, constitutes various traditional Chinese Medicine prescriptions, which date back to thousands of years. One of the primary active components of C. bungeana Turcz. is Corynoline (Cor), a plant isoquinoline alkaloid derived from the Corydalis species, which possesses bone metabolism disease therapeutic potential. The study aimed at exploring the effects as well as mechanisms of Cor on osteoclast formation and bone resorption. TRAcP staining, F-actin belt formation, and pit formation were employed for assessing the osteoclast function. Western blot, qPCR, network pharmacology, and docking analyses were used for analyzing the expression of osteoclast-associated genes and related signaling pathways. The study focused on investigating how Cor affected OVX-induced trabecular bone loss by using a mouse model. Cor could weaken osteoclast formation and function by affecting the biological receptor activators of NF-κB and its ligand at various concentrations. Mechanistically, Cor inhibited the NF-κB activation, and the MAPKs pathway stimulated by RANKL. Besides, Cor enhanced the protein stability of the Nrf2, which effectively abolished the RANKL-stimulated ROS generation. According to an OVX mouse model, Cor functions in restoring bone mass, improving microarchitecture, and reducing the ROS levels in the distal femurs, which corroborated with its in vitro antiosteoclastogenic effect. The present study indicates that Cor may restrain osteoclast formation and bone loss by modulating NF-κB/MAPKs and Nrf2 signaling pathways. Cor was shown to be a potential drug candidate that can be utilized for the treatment of osteoporosis.
Subject(s)
Berberine Alkaloids , Bone Resorption , Osteoporosis , Female , Humans , Osteogenesis , NF-kappa B/genetics , NF-kappa B/metabolism , Reactive Oxygen Species/metabolism , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Signal Transduction , Osteoclasts , Bone Resorption/drug therapy , Bone Resorption/genetics , Bone Resorption/metabolism , Osteoporosis/drug therapy , Osteoporosis/genetics , Osteoporosis/metabolism , RANK Ligand/genetics , RANK Ligand/metabolism , Cell DifferentiationABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Osteoporosis (OP) is a metabolic disorder characterized by disrupted osteoclastic bone resorption and osteoblastic bone formation. Curculigo orchioides Gaertn has a long history of application in traditional Chinese and Indian medicine for treating OP. Orcinol gentiobioside (OGB) is a principal active constituent derived from Curculigo orchioides Gaertn and has been shown to have anti-OP activity. However, the therapeutic efficacy and mechanism of OGB in modulating osteoclastic bone resorption remain undefined. AIM OF THE STUDY: To evaluate the effect of OGB on the formation, differentiation and function of osteoclasts derived from bone marrow macrophages (BMMs), and further elucidate the underlying action mechanism of OGB in OP. MATERIALS AND METHODS: Osteoclasts derived from BMMs were utilized to evaluate the effect of OGB on osteoclast formation, differentiation and bone resorption. Tartrate-resistant acid phosphatase (TRAP) staining and activity assays were conducted to denote the activity of osteoclasts. Osteoclast-related genes and proteins were determined by RT-PCR and Western blotting assays. The formation of the F-actin ring was observed by confocal laser microscopy, and bone resorption pits were observed by inverted microscopy. The target of OGB in osteoclasts was predicted by using molecular docking and further verified by Cellular Thermal Shift Assay (CETSA) and reversal effects of the target activator. The apoptosis of osteoclasts was analyzed by flow cytometry, and autophagic flux in osteoclasts was determined by confocal laser microscopy. RESULTS: OGB inhibited osteoclast formation and differentiation, osteoclast-related genes and proteins expression, F-actin ring formation, and bone resorption activity. Molecular docking and CETSA analysis demonstrated that OGB exhibited good affinity for c-Jun N-terminal Kinase 1 (JNK1). In addition, OGB induced apoptosis and inhibited autophagy in osteoclasts, and the JNK agonist anisomycin reversed the increase in apoptosis and inhibition of autophagy induced by OGB in osteoclasts. CONCLUSION: OGB inhibited osteoclastogenesis by promoting apoptosis and diminishing autophagy via JNK1 signaling.
Subject(s)
Bone Resorption , Osteogenesis , Resorcinols , Humans , Actins/metabolism , Molecular Docking Simulation , Cells, Cultured , Osteoclasts , Bone Resorption/drug therapy , Bone Resorption/metabolism , Apoptosis , Autophagy , RANK Ligand/pharmacology , RANK Ligand/metabolism , Cell DifferentiationABSTRACT
Cyathulae Radix, a traditional Chinese medicine and a common vegetable, boasts a history spanning millennia. It enhances bone density, boosts metabolism, and effectively alleviates osteoporosis-induced pain. Despite its historical use, the molecular mechanisms behind Cyathulae Radix's impact on osteoporosis remain unexplored. In this study, we investigated the effects and mechanisms of Cyathulae Radix ethanol extract (CEE) in inhibiting osteoporosis and osteoclastogenesis. Eight-week-old female mice underwent ovariectomy and were treated with CEE for eight weeks. Micro-computed tomography (micro-CT) assessed histomorphometric parameters, bone tissue staining observed distal femur histomorphology, and three-point bending tests evaluated tibia mechanical properties. Enzyme-linked immunosorbent assay (ELISA) measured serum estradiol (E2), receptor activator for nuclear factor B ligand (RANKL), and osteoprotegerin (OPG) levels. Osteoclastogenesis-related markers were analyzed via Western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, CEE effects on RANKL-induced osteoclast formation and bone resorption were investigated in vitro using tartrate-resistant acid phosphatase (TRAP) staining, qRT-PCR, and WB assay. Compared with the ovariectomy (OVX) group, CEE treatment enhanced trabecular bone density, maximal load-bearing capacity, and various histomorphometric parameters. Serum E2 and OPG levels significantly increased, while Receptor activator of nuclear factor-κB (RANK) decreased in the CEE group. CEE downregulated matrix metallopeptidase 9 (MMP-9), Cathepsin K (CTSK), and TRAP gene and protein expression. In bone marrow macrophages (BMMs), CEE reduced mature osteoclasts, bone resorption pit areas, and MMP-9, CTSK, and TRAP expression during osteoclast differentiation. Compared with DMSO treatment, CEE markedly inhibited RANK, TNF receptor associated factor 6 (TRAF6), Proto-oncogene c-Fos (c-Fos), Nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expressions, and Extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), NF-kappa B-p65 (p65) phosphorylation in osteoclasts. In conclusion, CEE significantly inhibits OVX-induced osteoporosis and RANKL-induced osteoclastogenesis, potentially through modulating the Estrogen Receptor (ER)/RANK/NFATc1 signaling pathway.
Subject(s)
Bone Resorption , Osteoporosis , Female , Mice , Animals , Humans , Osteoclasts/metabolism , X-Ray Microtomography , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Bone Resorption/drug therapy , Bone Resorption/genetics , Bone Resorption/metabolism , Osteoporosis/drug therapy , RANK Ligand/metabolism , RANK Ligand/pharmacology , Cell Differentiation , NF-kappa B/genetics , NF-kappa B/metabolism , OvariectomyABSTRACT
Forsythia suspensa tea is a popular traditional Chinese medicine decoction for its healthy and therapeutic benefits. However, its effects in bone metabolism were not clear. In recent study, we uncovered anti-osteoclastogenesis property of Phillygenin (Phi), a compound abundant in Forsythia suspensa leaves, and aimed to investigate the effect and mechanism of Phi on bone metabolism in vivo and in vitro. Lipopolysaccharides-induced murine calvaria osteolysis and ovariectomy-induced bone loss animal models were used to identify the bone-protective effect of Phi in vivo and micro-CT, pQCT, and TRAP staining were applied. We used CCK8, TUNEL, BrdU, and TRAP staining to evaluate the efficacy of Phi on the proliferation and formation of OCs in primary mBMMs. RNA sequence, activity-based protein profiling, molecular docking, G-LISA, and WB were used to inspect the target and underlying mechanism of Phi's actions in mBMMs. We found Phi significantly inhibited bone resorption in vivo and inhibited mBMMs osteoclastogenesis in vitro. Ras homolog gene family member A (RhoA) was identified as the direct target of Phi. It counteracted the effects of RhoA activator and acted as a RhoA inhibitor. By targeting RhoA, Phi modulated Rho-associated coiled-coil containing protein kinase 1 (ROCK1) activity and regulated its downstream NF-κB/NFATc1/c-fos pathway. Furthermore, Phi depressed the disassembling of F-actin ring through cofilin and myosin1a. Our findings provided Phi as a potential option for treating bone loss diseases by targeting RhoA and highlighted the importance of F. suspensa as a preventive approach in bone disorders.
Subject(s)
Bone Diseases, Metabolic , Bone Resorption , Lignans , Osteolysis , Animals , Female , Mice , Bone Resorption/drug therapy , Bone Resorption/prevention & control , Cell Differentiation , Lignans/pharmacology , Molecular Docking Simulation , NF-kappa B/metabolism , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/pharmacology , Osteoclasts , Osteogenesis , Osteolysis/chemically inducedABSTRACT
Tujia ethnomedicine Xuetong (the stems of Kadsura heteroclita) have been widely used in folk for rheumatoid arthritis (RA), which can alleviate rheumatic pain through liquor soaking in folk. In this study, we aimed to evaluate the pharmacological effects and underlying mechanism of Xuetongsu (a key chemical component of Xuetong) on bone destruction. In our previous study, it was found that Xuetong extract can reduce adjuvant arthritic rats paw swelling and inhibit inflammatory factors in serum. Furthermore, Xuetongsu has been demonstrated to inhibit the proliferation of fibroblast-like synoviocytes, but its potential to inhibit bone destruction has not been explored. To address this, we employed the STRING database to predict protein interactions and utilized Autodock software to simulate the binding of Xuetongsu to target proteins. In this study, administration of Xuetongsu significantly alleviated paw swelling and bone destruction in C57BL/6 mice with collagen-induced arthritis (CIA). Mechanistic studies have indicated that Xuetongsu promotes apoptosis of mature osteoclasts in joint tissues by activating Caspase-3 and Bax, while inhibiting Bcl-2. Additionally, Xuetongsu inhibits osteoclast differentiation by suppressing RANKL, RANK, P-NF-κB, and NFATc1, and reduces bone resorption activity by inhibiting MMP-9, CTSK, and TRAP. Importantly, Xuetongsu exhibits good biocompatibility in major organs of mice. In summary, Xuetongsu has the potential to treat bone destruction by promoting apoptosis of mature osteoclasts, inhibiting osteoclast differentiation, and reducing bone resorption. This study reveals the pharmacological effects of Xuetongsu and its mechanism of action, which may contribute to the development of novel approaches for treating RA.
Subject(s)
Arthritis, Experimental , Arthritis, Rheumatoid , Bone Resorption , Mice , Rats , Animals , Osteoclasts/metabolism , Arthritis, Experimental/drug therapy , Mice, Inbred C57BL , Bone Resorption/drug therapy , RANK Ligand/metabolism , Cell DifferentiationABSTRACT
BACKGROUND: Yunnan Baiyao (YNBY), a traditional Chinese medicine, is renowned for its anti-inflammatory properties. Recent studies have suggested that YNBY plays a significant role in inhibiting osteoclast differentiation and autophagy, which are essential processes in inflammation and bone resorption associated with periodontitis. However, the precise relationship between autophagy and the mechanism by which YNBY inhibits osteoclastogenesis remains unexplored.The primary objective of this study was to investigate the inhibitory effects of YNBY on the process of osteoclastogenesis and its potential in preventing inflammatory bone loss. METHODS: The animals were subjected to sacrifice at intervals of 2, 4, and 6 weeks postintervention whilst under deep anaesthesia, and specimens were subsequently collected. The specimens were subjected to hematoxylin and eosin (HE) staining, in addition to tartrate-resistant acid phosphatase (TRAP) staining and subsequently imaged employing a digital scanner. The confirmation of osteoclast (OC) differentiation and autophagic flux was achieved through various techniques, including western blotting, transmission electron microscopy (TEM), TRAP staining, pit formation assay, and immunofluorescence. RESULTS: The microcomputed tomography images provided evidence of the effective inhibition of alveolar bone absorption at 2, 4, and 6 weeks following YNBY treatment. Additionally, the histomorphometric evaluations of tissue segments stained with HE and TRAP, which involved measuring the distance between the alveolar bone crest (ABC) and cementoenamel junction (CEJ) and quantifying TRAP-positive OCs, yielded comparable results to those obtained through computed tomography analysis. YNBY treatment resulted in a decrease in the CEJ-ABC distance and inhibition of OC differentiation. Furthermore, in vitro studies showed that the autophagy modulators rapamycin (RAP) and 3-methyladenine (3-MA) significantly affected OC differentiation and function. YNBY attenuated the impact of RAP on the differentiation of OCs, autophagy-related factor activation, and bone resorption. CONCLUSIONS: We hypothesise that YNBY suppresses the differentiation of OC and bone resorption by blocking autophagy. This study reveals that targeting autophagy might be a new alternative treatment methodology for periodontitis treatment.
Subject(s)
Bone Resorption , Drugs, Chinese Herbal , Periodontitis , Animals , Humans , Osteoclasts , X-Ray Microtomography , China , Bone Resorption/drug therapy , Bone Resorption/prevention & control , Autophagy , Periodontitis/drug therapy , Periodontitis/prevention & control , Sirolimus/pharmacologyABSTRACT
BACKGROUND: Catalpol, a major active component of the Chinese herb Rehmannia glutinosa, possesses various pharmacological benefits, including anti-inflammatory, antidiabetic, and antitumor properties. Recent studies have reported that catalpol can attenuate bone loss and enhance bone formation. Nevertheless, the molecular mechanisms underlying its effects on osteoporosis pathogenesis remain unclear. PURPOSE: We investigated whether catalpol had a protective effect against postmenopausal osteoporosis (PMOP) and explored its exact mechanism of action. METHODS: Seventy-two rats were randomly divided into six groups: sham, model, low-dose catalpol (5 mg/kg/day), medium-dose catalpol (10 mg/kg/day), high-dose catalpol (20 mg/kg/day), and positive control (alendronate, 2.5 mg/kg). In this experiment, a ovariectomy was performed to establish a female rat model of PMOP. After 12 weeks of gavage, micro-computed tomography (micro-CT) and histochemical staining were performed to evaluate bone mass, bone microstructure and histological parameters. Furthermore, RAW 264.7 cells were induced by RANKL to form mature osteoclasts to investigate the effect of catalpol on osteoclast differentiation and apoptosis in vitro. Additionally, the osteoclast apoptosis-related proteins of Sirt6, ERα, FasL, NFATc1, cleaved-caspase 8, cleaved-caspase 3, and Bax were assessed using western blotting. The expressions of NFATc1, Ctsk, Oscar, and Trap were quantified using RT-qPCR. The apoptotic rate of the osteoclasts was determined using flow cytometry. Sirt6 knockdown was performed using siRNA gene silencing in experiments to investigate its role in catalpol-mediated osteoclast apoptosis. The deacetylation of ERα in osteoclasts was tested via co-immunoprecipitation. RESULTS: Catalpol (10 and 20 mg/kg) and alendronate (2.5 mg/kg) could significantly improve bone mineral density (BMD) and microstructure and decrease osteoclast density in ovariectomized (OVX) rats. In addition, catalpol (10 and 20 mg/kg) upregulated the expression of Sirt6, ERα, FasL, cleaved-caspase 8, cleaved-caspase 3, Bax, and downregulated the expression of NFATc1, Ctsk, Oscar, Trap both in vivo and in vitro. Catalpol also promoted ERα deacetylation and stabilized ERα protein to enhance the expression of FasL. In addition, Sirt6 knockdown by siRNA prevented ERα deacetylation and eliminated catalpol-mediated osteoclast apoptosis. CONCLUSIONS: The present study demonstrated that catalpol prevents estrogen deficiency-induced osteoporosis by promoting osteoclast apoptosis via the Sirt6-ERα-FasL axis. These findings revealed a novel molecular mechanism underpinning the impact of catalpol in the progression of osteoporosis and provided novel insights into the treatment of osteoporosis.
Subject(s)
Bone Resorption , Iridoid Glucosides , Osteoporosis, Postmenopausal , Osteoporosis , Humans , Rats , Female , Animals , Osteoclasts , Caspase 3/metabolism , Caspase 8/metabolism , Alendronate/metabolism , Alendronate/pharmacology , Alendronate/therapeutic use , Estrogen Receptor alpha/metabolism , X-Ray Microtomography , bcl-2-Associated X Protein/metabolism , Osteoporosis/prevention & control , Osteogenesis , Osteoporosis, Postmenopausal/drug therapy , Osteoporosis, Postmenopausal/metabolism , Transcription Factors/metabolism , Apoptosis , RNA, Small Interfering/pharmacology , Ovariectomy , Cell Differentiation , RANK Ligand/metabolism , Bone Resorption/drug therapyABSTRACT
Anethum graveolens L., known as European dill, is a versatile herb widely used in both traditional medicine and culinary practices. Despite its long-standing history, the potential impact of the water extract of A. graveolens seeds (WEAG) on bone health remains unexplored. In this study, we investigated the influence of WEAG on osteoclast differentiation and assessed its potential as an anti-osteoporotic agent. WEAG hindered osteoclast differentiation through the suppression of receptor activator of nuclear factor-κB ligand (RANKL) expression in osteoclast-supporting cells and by directly targeting osteoclast precursor cells. WEAG significantly reduced the expression of key osteoclastogenic transcription factors, namely c-Fos and NFATc1, typically induced by RANKL in osteoclast precursors. This reduction was attributed to the suppression of both MAPKs and NF-κB pathways in response to RANKL. In vivo experiments further revealed that WEAG administration effectively reduces trabecular bone loss and weight gain triggered by ovariectomy, mimicking postmenopausal osteoporosis. Furthermore, our comprehensive phytochemical analysis of WEAG identified a range of phytochemical constituents, associated with bone health and weight regulation. Notably, we discovered a specific compound, isorhamnetin-3-O-glucuronide, within WEAG that exhibits anti-osteoclastogenic potential. Overall, this research elucidated the beneficial effects and mechanistic basis of WEAG on osteoclast differentiation and bone loss, indicating its potential as a viable alternative to address bone loss in conditions like postmenopause.
Subject(s)
Anethum graveolens , Bone Resorption , Humans , Female , Anethum graveolens/metabolism , Cell Differentiation , NFATC Transcription Factors/metabolism , Osteoclasts , Osteogenesis , NF-kappa B/metabolism , Phytochemicals/pharmacology , RANK Ligand/metabolism , Bone Resorption/drug therapy , Bone Resorption/prevention & control , Bone Resorption/metabolism , OvariectomyABSTRACT
Current clinical approaches to osteoporosis primarily target osteoclast biology, overlooking the synergistic role of bone cells, immune cells, cytokines, and inorganic components in creating an abnormal osteoporotic microenvironment. Here, metal-polyDNA nanoparticles (Ca-polyCpG MDNs) composed of Ca2+ and ultralong single-stranded CpG sequences were developed to reconstruct the osteoporotic microenvironment and suppress osteoporosis. Ca-polyCpG MDNs can neutralize osteoclast-secreted hydrogen ions, provide calcium repletion, promote remineralization, and repair bone defects. Besides, the immune-adjuvant polyCpG in MDNs could induce the secretion of osteoclastogenesis inhibitor interleukin-12 and reduce the expression of osteoclast function effector protein to inhibit osteoclast differentiation, further reducing osteoclast-mediated bone resorption. PPi4- generated during the rolling circle amplification reaction acts as bisphosphonate analog and enhances bone targeting of Ca-polyCpG MDNs. In ovariectomized mouse and rabbit models, Ca-polyCpG MDNs prevented bone resorption and promoted bone repair by restoring the osteoporotic microenvironment, providing valuable insights into osteoporosis therapy.
Subject(s)
Bone Resorption , Nanoparticles , Osteoporosis , Mice , Animals , Rabbits , Osteoclasts/metabolism , Osteogenesis/genetics , Bone Resorption/drug therapy , Osteoporosis/drug therapy , Osteoporosis/metabolism , Cell DifferentiationABSTRACT
The maintenance of bone is dependent on both osteoclasts, which break down bone, and osteoblasts, which build new bone. Various bone-related disorders, including osteoporosis, can occur as a result of an imbalance between these two cell types. Prolonged use of currently available bone resorption inhibitors may show side effects. Therefore, developing a novel preventive material which effectively inhibits osteoclast differentiation could be beneficial. This study planned to investigate the inhibitory effect of wheat sprout ethanolic extracts (Saegeumgang [SGG] and Arriheuk [ARH]) on the differentiation of osteoclasts induced by RANKL, as well as the mechanisms why fundamental to these effects. The effects of SGG and ARH on bone resorption and osteoclast differentiation were evaluated using RAW 264.7 cells and assessed through TRAP cell count, pit formation, and activity. The expressions of mRNA and protein were accomplished using western blotting, and reverse transcription quantitative polymerase chain reaction analyses were conducted. SGG and ARH were found to suppress osteoclast differentiation in RANKL-stimulated RAW264.7 cells without causing cytotoxic effects. In addition, treatment with SGG and ARH led to a reduction in the number of cells with positive staining for TRAP and TRAP activity. SGG and ARH treatment dose-dependently decreased the pit area in pit formation assays, showing a notable reduction compared to the pit area created by mature osteoclasts. SGG and ARH inhibited osteoclast activity by 84.9% and 95.7% at 200 µg/mL, respectively. In addition, SGG and ARH suppressed the transcriptional activation of various osteoclast-related genes, such as RANK, NFATc1, cathepsin K, c-Fos, TRAP, matrix metallopeptidase-9, dendritic cell-specific transmembrane protein, ATPase H+ transporting v0 subunit d2, and osteoclast-associated receptor in RAW264.7 cells treated with RANKL. SGG and ARH extracts were found to affect the expression of NFATc1 and genes that are specific to osteoclasts during osteoclast differentiation, suggesting their potential use as functional foods or as therapeutic interventions targeting bone health.
Subject(s)
Bone Resorption , Osteoclasts , Triticum/metabolism , Transcription Factors , Bone Resorption/drug therapy , Bone Resorption/metabolism , Signal Transduction , Cell Differentiation , RANK Ligand/metabolismABSTRACT
BACKGROUND: Guizhi Shaoyao Zhimu decoction, a traditional Chinese medicine formula used empirically for the treatment of rheumatoid arthritis (RA), has been shown to alleviate bone destruction in rats with collagen-induced arthritis (CIA). PURPOSE: The aim of this study is to characterize the effects of Guizhi Shaoyao Zhimu granules (GSZGs) on bone destruction in RA and the underlying mechanism. STUDY DESIGN: A CIA arthritis model using DBA/1 mice. The animals were divided into a normal group; CIA model group; low, medium, and high-dose GSZG groups (3, 6, and 9 g/kg/day); and a methotrexate group (1.14 mg/kg/w). In vitro, a cytokine induced osteoclastogenesis model was established. METHODS: After 28 days of treatment, the paw volume was measured, bone destruction was examined by micro-CT, and the generation of osteoclasts in bone tissue was evaluated via tartrate-resistant acid phosphatase (TRAP) staining. Furthermore, the inhibitory effect and underlying mechanism of action of GSZG on RANKL-induced osteoclastogenesis were investigated in vitro. RESULTS: The in vivo analyses demonstrated that the paw volume and degree of bone erosion of mice in the medium- and high-dose GSZG groups were significantly decreased compared to the CIA model group. In addition, GSZG treatment suppressed the excessive generation of osteoclasts in the bone tissue of CIA mice. In vitro, GSZG inhibited RANKL-induced osteoclastogenesis and osteoclast-mediated bone resorption. Specifically, it only inhibited the generation of osteoclast precursors (OCPs); it had no significant effect on the fusion of OCPs or maturation of osteoclasts. Finally, we showed that the inhibitory effect of GSZG on osteoclastogenesis was related to the promotion of PTEN-induced kinase protein 1 (PINK1)/Parkin pathway-mediated mitophagy of osteoclast precursors, which was verified using a PINK1 knockdown small interfering RNA in OCPs. CONCLUSION: These findings indicate that GSZG is a candidate for the treatment of bone destruction in RA and provide a more detailed elucidation of the mechanism of GSZG anti-RA bone erosion, i.e., inhibition of the ROS/NF-κB axis through the PINK1/Parkin-mediated mitochondrial autophagic pathway to inhibit osteoclast precursor production, compared to the published literature.
Subject(s)
Arthritis, Experimental , Arthritis, Rheumatoid , Bone Resorption , Mice , Rats , Animals , Osteoclasts/metabolism , Osteogenesis , Arthritis, Experimental/drug therapy , Arthritis, Experimental/metabolism , Mitophagy , Mice, Inbred DBA , Arthritis, Rheumatoid/drug therapy , Bone Resorption/drug therapy , Bone Resorption/metabolism , Protein Kinases/metabolism , RANK Ligand/pharmacology , RANK Ligand/metabolismABSTRACT
Background: Osteoporosis is a systemic bone disease characterized by decreased bone density and quality, destruction of bone microstructure, and increased bone fragility. Extracellular vesicles are lipid bilayer nanoparticles that participate in intercellular communication. Extracellular vesicles are becoming popular in the study of osteoporosis and the bone cell microenvironment. Extracellular vesicles can transmit cell signals and regulate bone homeostasis. Our previous studies revealed that the Chinese herbal medicine Guilu Erxian Glue promotes type I collagen synthesis and osteoprotegerin secretion by osteoblasts in rats, reverses the imbalance of bone homeostasis, and alleviates osteoporosis. Objective: We investigated how osteoblast-derived extracellular vesicles treated with Guilu Erxian Glue affected osteoclasts in vitro. Methods: We quantified osteoclast differentiation of RAW 264.7 using TRAP staining, cell apoptosis using flow cytometry, extracellular vesicle uptake by fluorescence tracing, bone absorption functions by bone resorption lacuna , and transcription of key genes by quantitative real-time PCR. Results: Fluorescently labeled mouse preosteoblastic MC3T3-E1 cells secreted nanoscale substances less than 1 µm in diameter. Mouse macrophage RAW 264.7 cells adsorbed these nanoparticles and PKH26-labeled extracellular vesicles derived from MC3T3-E1 cells on the cell membrane surface. Extracellular vesicles from MC3T3-E1 cells treated with Guilu Erxian Glue inhibited the differentiation of osteoclasts induced by receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor and reduced the number of lacunae formed by osteoclasts in vitro compared with controls. Extracellular vesicles from MC3T3-E1 cells treated with Guilu Erxian Glue downregulated the relative messenger RNA expression of c-Fos, cathepsin K, nuclear factor of activated T cells 1, and tartrate-resistant acid phosphatase in osteoclasts, which may be part of the mechanism by which they regulate osteoclasts. Conclusions: Our results demonstrate that extracellular vesicles are essential for signal exchange between osteoblasts and osteoclasts. Although we do not know how Guilu Erxian Glue affects the signaling molecules carried by extracellular vesicles, we have shown for the first time, to our knowledge, that Guilu Erxian Glue can inhibit osteoclast differentiation and function via osteoblast-derived extracellular vesicles. Our findings are conducive to providing a new target for the development of osteoporosis drugs.
Subject(s)
Bone Resorption , Drugs, Chinese Herbal , Osteoporosis , Rats , Mice , Animals , Osteoclasts/metabolism , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/therapeutic use , Osteoblasts/metabolism , Bone Resorption/drug therapy , Bone Resorption/metabolism , Osteoporosis/drug therapyABSTRACT
Age-related osteoporosis is associated with increased oxidative stress and cellular senescence. Pyrroloquinoline quinone (PQQ) is a water-soluble vitamin-like compound that has strong antioxidant capacity; however, the effect and underlying mechanism of PQQ on aging-related osteoporosis remain unclear. The purpose of this study was to investigate whether dietary PQQ supplementation can prevent osteoporosis caused by natural aging, and the potential mechanism underlying PQQ antioxidant activity. Here, we found that when 6-month-old or 12-month-old wild-type mice were supplemented with PQQ for 12 months or 6 months, respectively, PQQ could prevent age-related osteoporosis in mice by inhibiting osteoclastic bone resorption and stimulating osteoblastic bone formation. Mechanistically, pharmmapper screening and molecular docking studies revealed that PQQ appears to bind to MCM3 and reduces its ubiquitination-mediated degradation; stabilized MCM3 then competes with Nrf2 for binding to Keap1, thus activating Nrf2-antioxidant response element (ARE) signaling. PQQ-induced Nrf2 activation inhibited bone resorption through increasing stress response capacity and transcriptionally upregulating fibrillin-1 (Fbn1), thus reducing Rankl production in osteoblast-lineage cells and decreasing osteoclast activation; as well, bone formation was stimulated by inhibiting osteoblastic DNA damage and osteocyte senescence. Furthermore, Nrf2 knockout significantly blunted the inhibitory effects of PQQ on oxidative stress, on increased osteoclast activity and on the development of aging-related osteoporosis. This study reveals the underlying mechanism of PQQ's strong antioxidant capacity and provides evidence for PQQ as a potential agent for clinical prevention and treatment of natural aging-induced osteoporosis.
Subject(s)
Bone Resorption , Osteoporosis , Mice , Animals , Antioxidants/metabolism , PQQ Cofactor/pharmacology , PQQ Cofactor/metabolism , PQQ Cofactor/therapeutic use , NF-E2-Related Factor 2/metabolism , Kelch-Like ECH-Associated Protein 1/metabolism , Up-Regulation , Fibrillin-1/metabolism , Molecular Docking Simulation , Oxidative Stress , Aging , Osteoporosis/drug therapy , Osteoporosis/metabolism , Bone Resorption/drug therapyABSTRACT
BACKGROUND: Osteoporosis is a prevalent bone metabolic disease in menopause, and long-term medication is accompanied by serious side effects. Estrogen deficiency-mediated hyperactivated osteoclasts is the initiating factor for bone loss, which is regulated by nuclear factor-κB (NF-κB) signaling. Safranal (Saf) is a monoterpene aldehyde produced from Saffron (Crocus sativus L.) and possesses multiple biological properties, particularly the anti-inflammatory property. However, Saf's role in osteoporosis remains unknown. PURPOSE: This study aims to validate the role of Saf in osteoporosis and explore the potential mechanism. STUDY DESIGN: The RANKL-exposed mouse BMM (bone marrow monocytes) and the castration-mediated osteoporosis model were applied to explore the effect and mechanism of Saf in vitro and in vivo. METHOD: The effect of Saf on osteoclast formation and function were assessed by TRAcP staining, bone-resorptive experiment, qPCR, immunoblotting and immunofluorescence, etc. Micro-CT, HE, TRAcP and immunohistochemical staining were performed to estimate the effects of Saf administration on OVX-mediated osteoporosis in mice at imaging and histological levels. RESULTS: Saf concentration-dependently inhibited RANKL-mediated osteoclast differentiation without affecting cellular viability. Meanwhile, Saf-mediated anti-osteolytic capacity and Sirt1 upregulation were also found in ovariectomized mice. Mechanistically, Saf interfered with NF-κB signaling by activating Sirt1 to increase p65 deacetylation and inactivating IKK to decrease IκBα degradation. CONCLUSION: Our results support the potential application of Saf as a therapeutic agent for osteoporosis.
Subject(s)
Osteoporosis , Animals , Mice , Mice, Inbred C57BL , Osteoporosis/drug therapy , Osteoporosis/metabolism , Estrogens/deficiency , Estrogens/metabolism , Female , Osteoclasts , Bone Resorption/drug therapy , Bone Resorption/metabolism , Ovariectomy , NF-kappa B/metabolism , AcetylationABSTRACT
Plant-derived extracellular nanovesicles contain RNA and proteins with unique and diverse pharmacological mechanisms. The extracellular nanovesicles encapsulating plant extracts resemble exosomes as they have a round, lipid bilayer morphology. Ginseng is anti-inflammatory, anti-cancer, immunostimulant, and osteogenic/anti-osteoporotic. Here, we confirmed that ginseng-derived extracellular nanovesicles (GDNs) inhibit osteoclast differentiation and elucidated the associated molecular mechanisms. We isolated GDNs by centrifugation with a sucrose gradient. We measured their dynamic light scattering and zeta potentials and examined their morphology by transmission electron microscopy. We used bone marrow-derived macrophages (BMMs) to determine the potential cytotoxicity of GDNs and establish their ability to inhibit osteoclast differentiation. The GDNs treatment maintained high BMM viability and proliferation whilst impeding osteoclastogenesis. Tartrate-resistant acid phosphatase and F-actin staining revealed that GDNs at concentrations >1 µg mL-1 strongly hindered osteoclast differentiation. Moreover, they substantially suppressed the RANKL-induced IκBα, c-JUN n-terminal kinase, and extracellular signal-regulated kinase signaling pathways and the genes regulating osteoclast maturation. The GDNs contained elevated proportions of Rb1 and Rg1 ginsenosides and were more effective than either of them alone or in combination at inhibiting osteoclast differentiation. In vivo bone analysis via microcomputerized tomography, bone volume/total volume ratios, and bone mineral density and bone cavity measurements demonstrated the inhibitory effect of GDNs against osteoclast differentiation in lipopolysaccharide-induced bone resorption mouse models. The results of this work suggest that GDNs are anti-osteoporotic by inhibiting osteoclast differentiation and are, therefore, promising for use in the clinical prevention and treatment of bone loss diseases.
Subject(s)
Bone Resorption , Exosomes , Panax , Animals , Mice , Osteoclasts , Exosomes/metabolism , Bone Resorption/drug therapy , Bone Resorption/metabolism , Ultracentrifugation , Cell DifferentiationABSTRACT
Osteoporosis, an immune disease characterized by bone mass loss and microstructure destruction, is often seen in postmenopausal women. Isoimperatorin (ISO), a bioactive, natural furanocoumarin isolated from many traditional Chinese herbal medicines, has therapeutic effects against various diseases; however, its effect on bone homeostasis remains unclear. In this study, we investigated the effect of ISO on the differentiation and activation of osteoclast and its molecular mechanism in vitro, and evaluated the effect of ISO on bone metabolism by ovariectomized (OVX) rat model. In vitro experiments showed that ISO affected RANKL-induced MAPK, NFAT, NFATc1 trafficking and expression, osteoclast F-actin banding, osteoclast-characteristic gene expression, ROS inhibitory activity, and calcium oscillations, NF-κB signaling pathway. In vivo experiments showed that oral administration of ISO effectively reduced bone loss caused by ovariectomy and retained bone mass.Collectively, ISO inhibits RANK/RANKL binding, thereby reducing the activity of NFATc1, calcium, and ROS and inhibiting osteoclast generation. In addition, ISO protects bone mass by slowing osteoclast production and downregulating NFATc1 gene and protein expression in the bone tissue microenvironment and inhibits OVX-induced bone loss in vivo.