ABSTRACT
Ganoderic Acid A (GAA) has demonstrated beneficial effects in anti-inflammatory and anti-oxidative stress studies. However, it remains unknown whether GAA exerts positive impacts on bone loss induced by lipopolysaccharide (LPS). This study aims to investigate the influence of GAA on bone loss in LPS-treated rats. The study assesses changes in the viability and osteogenic potential of MC3T3-E1 cells, as well as osteoclast differentiation in RAW264.7 cells in the presence of LPS using CCK-8, ALP staining, AR staining, and Tartrate-resistant acid phosphatase (TRAP) staining. In vitro experiments indicate that LPS-induced inhibition of osteoclasts (OC) and Superoxide Dismutase 2 (SOD2) correlates with heightened levels of inflammation and oxidative stress. Furthermore, GAA has displayed the ability to alleviate oxidative stress and inflammation, enhance osteogenic differentiation, and suppress osteoclast differentiation. Animal experiment also proves that GAA notably upregulates SOD2 expression and downregulates TNF-α expression, leading to the restoration of impaired bone metabolism, improved bone strength, and increased bone mineral density. The collective experimental findings strongly suggest that GAA can enhance osteogenic activity in the presence of LPS by reducing inflammation and oxidative stress, hindering osteoclast differentiation, and mitigating bone loss in LPS-treated rat models.
Subject(s)
Cell Differentiation , Heptanoic Acids , Inflammation , Lanosterol , Lipopolysaccharides , Osteoclasts , Osteogenesis , Oxidative Stress , Rats, Sprague-Dawley , Superoxide Dismutase , Animals , Lipopolysaccharides/pharmacology , Oxidative Stress/drug effects , Male , Mice , Rats , RAW 264.7 Cells , Superoxide Dismutase/metabolism , Inflammation/metabolism , Inflammation/drug therapy , Osteoclasts/drug effects , Osteoclasts/metabolism , Cell Differentiation/drug effects , Osteogenesis/drug effects , Lanosterol/analogs & derivatives , Lanosterol/pharmacology , Lanosterol/therapeutic use , Heptanoic Acids/pharmacology , Heptanoic Acids/therapeutic use , Bone Density/drug effects , Tumor Necrosis Factor-alpha/metabolism , Bone Resorption/prevention & control , Bone Resorption/drug therapy , Bone Resorption/metabolismABSTRACT
Short-beak and dwarf syndrome (SBDS) is caused by novel goose parvovirus (NGPV) infection, which leads to farm economic losses. Our research aimed to investigate the potential of administering isolated lactic acid bacteria (LAB) in alleviating SBDS in ducks. Eight wild LAB strains were isolated from duck feces and their biosecurity was investigated in both duck embryo fibroblast (DEF) and live ducks. Moreover, the LAB strains exhibited no detrimental effects on bone metabolism levels and facilitated the tight junction proteins (TJPs) mRNA expression, and contributing to the mitigation of inflammation in healthy ducks. Subsequently, we conducted in vitrol and in vivo experiments to assess the impact of LAB on NGPV infection. The LAB strains significantly reduced the viral load of NGPV and downregulated the mRNA levels of pro-inflammatory factors in DEF. Additionally, LAB treatment alleviated SBDS in NGPV-infected ducks. Furthermore, LAB treatment alleviated intestinal damage, and reduced the inflammatory response, while also mitigating bone resorption in NGPV-infected ducks. In conclusion, the LAB strains isolated from duck feces have favorable biosecurity and alleviate SBDS in ducks, and the mechanism related to LAB improves intestinal barrier integrity, alleviates inflammation, and reduces bone resorption. Our study presents a novel concept for the prevention and treatment of NGPV, thereby establishing a theoretical foundation for the future development of probiotics in the prevention and treatment of NGPV.
Subject(s)
Ducks , Inflammation , Lactobacillales , Poultry Diseases , Animals , Ducks/virology , Ducks/microbiology , Poultry Diseases/microbiology , Poultry Diseases/prevention & control , Poultry Diseases/virology , Inflammation/veterinary , Inflammation/prevention & control , Lactobacillales/genetics , Parvoviridae Infections/veterinary , Parvoviridae Infections/prevention & control , Parvoviridae Infections/virology , Parvoviridae Infections/microbiology , Feces/microbiology , Feces/virology , Bone Resorption/prevention & control , Bone Resorption/microbiology , Bone Resorption/veterinary , Intestines/microbiology , Intestines/virology , Probiotics/administration & dosage , Probiotics/pharmacology , Probiotics/therapeutic use , Parvovirus/genetics , Geese/virologyABSTRACT
Background: Osteoporosis is a disease associated with bone resorption, characterized primarily by the excessive activation of osteoclasts. Ginkgetin is a compound purified from natural ginkgo leaves which has various biological properties, including anti-inflammation, antioxidant, and anti-tumor effects. This study investigated the bone-protective effects of ginkgetin in ovariectomized (OVX) mice and explored their potential signaling pathway in inhibiting osteoclastogenesis in a mouse model of osteoporosis. Methods: Biochemical assays were performed to assess the levels of Ca, ALP, and P in the blood. Micro CT scanning was used to evaluate the impact of ginkgetin on bone loss in mice. RT-PCR was employed to detect the expression of osteoclast-related genes (ctsk, c-fos, trap) in their femoral tissue. Hematoxylin and eosin (H&E) staining was utilized to assess the histopathological changes in femoral tissue due to ginkgetin. The TRAP staining was used to evaluate the impact of ginkgetin osteoclast generation in vivo. Western blot analysis was conducted to investigate the effect of ginkgetin on the expression of p-NF-κB p65 and IκBα proteins in mice. Results: Our findings indicate that ginkgetin may increase the serum levels of ALP and P, while decreasing the serum level of Ca in OVX mice. H&E staining and micro CT scanning results suggest that ginkgetin can inhibit bone loss in OVX mice. The TRAP staining results showed ginkgetin suppresses the generation of osteoclasts in OVX mice. RT-PCR results demonstrate that ginkgetin downregulate the expression of osteoclast-related genes (ctsk, c-fos, trap) in the femoral tissue of mice, and this effect is dose-dependent. Western blot analysis results reveal that ginkgetin can inhibit the expression of p-NF-κB p65 and IκBα proteins in mice. Conclusion: Ginkgetin can impact osteoclast formation and activation in OVX mice by inhibiting the NF-κB/IκBα signaling pathway, thereby attenuating bone loss in mice.
Subject(s)
Biflavonoids , NF-kappa B , Osteoclasts , Signal Transduction , Animals , Biflavonoids/pharmacology , Biflavonoids/therapeutic use , Signal Transduction/drug effects , Mice , NF-kappa B/metabolism , Female , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteoporosis/drug therapy , Osteoporosis/metabolism , Osteoporosis/pathology , Ovariectomy , Disease Models, Animal , Bone Resorption/drug therapy , Bone Resorption/metabolism , Bone Resorption/prevention & control , Bone Resorption/pathology , X-Ray Microtomography , NF-KappaB Inhibitor alpha/metabolism , Mice, Inbred C57BLABSTRACT
Inhibition of excessive osteoclastic activity is an efficient therapeutic strategy for many bone diseases induced by increased bone resorption, such as osteoporosis. BMS-582949, a clinical p38α inhibitor, is a promising drug in Phase II studies for treating rheumatoid arthritis. However, its function on bone resorption is largely unknown. In this study, we find that BMS-582949 represses RANKL-induced osteoclast differentiation in a dose-dependent manner. Moreover, BMS-582949 inhibits osteoclastic F-actin ring formation and osteoclast-specific gene expression. Mechanically, BMS-582949 treatment attenuates RANKL-mediated osteoclastogenesis through mitogen-activated protein kinases (MAPKs) and protein kinase B (AKT) signaling pathways without disturbing nuclear factor-κB (NF-κB) signaling. Interestingly, BMS-582949 impairs osteoclastic mitochondrial biogenesis and functions, such as oxidative phosphorylation (OXPHOS). Furthermore, BMS-582949 administration prevents bone loss in ovariectomized mouse mode by inhibiting both bone resorption and bone formation in vivo. Taken together, these findings indicate that BMS-582949 may be a potential and effective drug for the therapy of osteolytic diseases.
Subject(s)
Mice, Inbred C57BL , Osteoclasts , Osteogenesis , Ovariectomy , p38 Mitogen-Activated Protein Kinases , Animals , Mice , Ovariectomy/adverse effects , Female , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteogenesis/drug effects , p38 Mitogen-Activated Protein Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Bone Resorption/prevention & control , Bone Resorption/drug therapy , Bone Resorption/metabolism , RAW 264.7 Cells , Protein Kinase Inhibitors/pharmacology , Bone Remodeling/drug effects , RANK Ligand/metabolism , Cell Differentiation/drug effects , Dose-Response Relationship, DrugABSTRACT
Osteoporosis is characterized by bone loss and deterioration in bone microstructure, leading to bone fragility. It is strongly correlated with menopause in women. Previously, we reported that diets supplemented with a kudzu (Pueraria lobata) vine extract suppressed bone resorption in ovariectomized (OVX) mice, a postmenopausal model. The main isoflavone in kudzu is puerarin (daidzein-8-C-glycoside). Puerarin (daidzein-8-C-glycoside), which is main isoflavone of kudzu, probably contributes to the beneficial effect. However, the underlying mechanism is unclear. Therefore, the nutrikinetics of puerarin and the comparison with the suppressive effects of kudzu isoflavones on osteoclast differentiation was examined in this study. We demonstrated that orally administered puerarin was absorbed from the gut and entered the circulation in an intact form. In addition, puerarin accumulated in RAW264.7 pre-osteoclast cells in a time-dependent manner. Tartrate-resistant acid phosphatase activity was decreased by puerarin treatment in a concentration-dependent manner in RAW264.7 cells stimulated with the receptor activator of nuclear factor kappa-B ligand. Ovariectomy-induced elevated bone resorption was suppressed, and the fragile bone strength was improved by puerarin ingestion in the diet. These findings suggested that orally administered puerarin was localized in bone tissue and suppressed bone resorption and osteoclastogenesis in ovariectomized mice.
Subject(s)
Cell Differentiation , Femur , Isoflavones , Osteoclasts , Ovariectomy , Pueraria , Animals , Isoflavones/pharmacology , Isoflavones/administration & dosage , Osteoclasts/drug effects , Female , Mice , Femur/drug effects , Femur/metabolism , Pueraria/chemistry , Cell Differentiation/drug effects , RAW 264.7 Cells , Bone Resorption/prevention & control , Plant Extracts/pharmacology , Plant Extracts/administration & dosage , Osteoporosis/prevention & control , Osteoporosis/drug therapy , Tartrate-Resistant Acid Phosphatase/metabolismABSTRACT
PURPOSE: To evaluate the effect of interleukin-6 (IL-6) inhibitor (tocilizumab) on bacterial infection-associated bone resorption around implants during osseointegration in rabbits. MATERIALS AND METHODS: At total of 24 male, 9-monthold New Zealand white rabbits were included, and their two mandibular anterior teeth were extracted. Three months after extraction, 24 one-piece Dentium implants (Ø 2.5 mm, intraosseous length of 12 mm) were inserted in the anterior mandible, and the rabbits were divided into four groups (n = 6 per group). Different treatment methods were used in each group: blank control group (BC); only silk ligation (negative control [NC]); silk ligation and injection with minocycline hydrochloride ointment (positive control [PC]); and silk ligation and injection with tocilizumab at 8 mg/kg via the auricle vein (experimental [EP]). Eight weeks later, the animals were sacrificed, and samples were collected and then analyzed using microcomputed tomography (microCT) scanning, immunohistochemical analysis, and histologic analysis. RESULTS: From the microCT measurement, the ratio of the bone volume to the total volume (BV/TV) in the EP group was 67.00% ± 2.72%, which was higher than that in the other three groups (58.85% ± 2.43% in the BC group, 55.72% ± 2.48% in the PC group, and 36.52% ± 3.02% in the NC group). From immunohistochemical analysis, the expression of IL-6 was found to be higher in the NC group than in the BC, PC, and EP groups, but there was no statistical difference between these three groups. Furthermore, the RANKL (receptor activator of nuclear factor-κB ligand) expression was the lowest in the EP group, followed by the BC group, the PC group, and the NC group, which had the highest expression; there was no difference between the NC and PC groups. Upon histologic analysis, significant new bone was found on the implant surfaces in the EP group, sparse and less new bone could be seen in the BC and PC groups, and the most serious bone resorption occurred in the NC group. CONCLUSIONS: Tocilizumab, an inhibitor of IL-6, has a certain effect in preventing bone loss around implants caused by bacterial infection during the osseointegration period.
Subject(s)
Antibodies, Monoclonal, Humanized , Interleukin-6 , Osseointegration , Animals , Rabbits , Male , Pilot Projects , Interleukin-6/analysis , Antibodies, Monoclonal, Humanized/therapeutic use , Antibodies, Monoclonal, Humanized/pharmacology , Osseointegration/drug effects , X-Ray Microtomography , Dental Implants , Bone Resorption/prevention & control , Dental Implantation, Endosseous/methodsABSTRACT
Osteoporosis is a systemic bone disease characterized by decreased bone mass that is tightly regulated by the coordinated actions of osteoclasts and osteoblasts. Apoptosis as a precise programmed cell death involves a cascade of gene expression events which are mechanistically linked to the regulation of bone metabolism. Nevertheless, the critical biomolecules involved in regulating cell apoptosis in osteoporosis remain unknown. To gain a deeper insight into the relationship between apoptosis and osteoporosis, this study integrated the sequencing results of human samples and using a machine learning workflow to overcome the limitations of a single study. Among all immune cell populations, we assessed the apoptotic level and portrayed the distinct subtypes and lineage differentiation of monocytic cells in osteoporotic tissues. Osteoclasts expressed a higher level of Spermidine/spermine-N1-Acetyltransferase1 (SAT1) during osteoclastogenesis which prevented osteoclasts apoptosis and facilitate osteoporosis progression. In addition, Berenil, one potent SAT1 inhibitor, increased osteoclast apoptosis and reversed the bone loss in the femurs of a murine ovariectomy model. In summary, Berenil promotes osteoclast apoptosis, inhibits the bone resorption and improves the abnormal bone structure in vitro and in vivo models by targeting SAT1, demonstrating its potential as a precise therapeutic strategy for clinical osteoporosis treatment.
Subject(s)
Acetyltransferases , Apoptosis , Osteoclasts , Osteoporosis , Apoptosis/drug effects , Animals , Osteoclasts/metabolism , Osteoclasts/pathology , Osteoclasts/drug effects , Osteoporosis/pathology , Osteoporosis/prevention & control , Osteoporosis/metabolism , Humans , Female , Mice , Acetyltransferases/metabolism , Acetyltransferases/genetics , Mice, Inbred C57BL , Bone Resorption/metabolism , Bone Resorption/pathology , Bone Resorption/prevention & control , Ovariectomy , Osteogenesis/drug effects , Cell Differentiation , Disease Models, AnimalABSTRACT
Osteoporosis is a highly prevalent bone metabolic disease in menopause due to estrogen deficiency. Hyperoside is a main compound in Semen cuscutae. Our team previously reported that Semen cuscutae has anti osteoporosis effect on ovariectomized mice by inhibiting bone resorption of osteoclasts. However, it is still unclear whether hyperoside affects osteoclast differentiation and bone resorption, and whether its anti-osteoporosis effect is related to an estrogen-like effect. This study investigates the potential mechanism of hyperoside's anti-osteoporotic effect by examining its impact on osteoclast differentiation and its relationship with the estrogen receptor. DXA, Micro-CT, TRAP staining, HE, and ELISA were used to assess the impact of hyperoside on OVX-induced osteoporosis. The effect of hyperoside on octeoclast differentiation was evaluated using TRAP activity assay, TRAP staining, F-actin staining. The activation of the estrogen receptor by hyperoside and its relationship with osteoclast differentiation were detected using dual-luciferase reporter assay and estrogen receptor antagonists. Our findings revealed that hyperoside (20-80 mg/kg) protect against OVX-induced osteoporosis, including increasing BMD and BMC and improving bone microstructure. Hyperoside inhibited osteoclast differentiation in a concentration dependent manner, whereas estrogen receptor α antagonists reversed its inhibitory effect osteoclast differentiation. Western blot results suggested that hyperoside inhibited TRAP, RANKL, c-Fos and ITG ß3 protein expression in osteoclast or femoral bone marrow of ovariectomized mice. Our findings suggest that hyperoside inhibits osteoclast differentiation and protects OVX-induced osteoporosis through the ERα/ITGß3 signaling pathway.
Subject(s)
Cell Differentiation , Estrogen Receptor alpha , Osteoclasts , Osteoporosis , Ovariectomy , Quercetin , Signal Transduction , Animals , Ovariectomy/adverse effects , Female , Signal Transduction/drug effects , Mice , Estrogen Receptor alpha/metabolism , Quercetin/pharmacology , Quercetin/analogs & derivatives , Quercetin/therapeutic use , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteoporosis/drug therapy , Osteoporosis/metabolism , Osteoporosis/pathology , Cell Differentiation/drug effects , Mice, Inbred C57BL , Bone Density/drug effects , Bone Resorption/drug therapy , Bone Resorption/metabolism , Bone Resorption/prevention & controlABSTRACT
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
As bone-resorbing cells rich in mitochondria, osteoclasts require high iron uptake to promote mitochondrial biogenesis and maintain a high-energy metabolic state for active bone resorption. Given that abnormal osteoclast formation and activation leads to imbalanced bone remodeling and osteolytic bone loss, osteoclasts may be crucial targets for treating osteolytic diseases such as periodontitis. Isobavachin (IBA), a natural flavonoid compound, has been confirmed to be an inhibitor of receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation from bone marrow-derived macrophages (BMMs). However, its effects on periodontitis-induced bone loss and the potential mechanism of its anti-osteoclastogenesis effect remain unclear. Our study demonstrated that IBA suppressed RANKL-induced osteoclastogenesis in BMMs and RAW264.7 cells and inhibited osteoclast-mediated bone resorption in vitro. Transcriptomic analysis indicated that iron homeostasis and reactive oxygen species (ROS) metabolic process were enriched among the differentially expressed genes following IBA treatment. IBA exerted its anti-osteoclastogenesis effect by inhibiting iron accumulation in osteoclasts. Mechanistically, IBA attenuated iron accumulation in RANKL-induced osteoclasts by inhibiting the mitogen-activated protein kinase (MAPK) pathway to upregulate ferroportin1 (Fpn1) expression and promote Fpn1-mediated intracellular iron efflux. We also found that IBA inhibited mitochondrial biogenesis and function, and reduced RANKL-induced ROS generation in osteoclasts. Furthermore, IBA attenuated periodontitis-induced bone loss by reducing osteoclastogenesis in vivo. Overall, these results suggest that IBA may serve as a promising therapeutic strategy for bone diseases characterized by osteoclastic bone resorption.
Subject(s)
Iron , Mice, Inbred C57BL , Mitochondria , Organelle Biogenesis , Osteoclasts , Periodontitis , Animals , Mice , Iron/metabolism , RAW 264.7 Cells , Periodontitis/drug therapy , Periodontitis/metabolism , Osteoclasts/drug effects , Osteoclasts/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Osteogenesis/drug effects , Male , Bone Resorption/metabolism , Bone Resorption/drug therapy , Bone Resorption/prevention & control , Bone Resorption/etiology , Alveolar Bone Loss/metabolism , Alveolar Bone Loss/drug therapy , Alveolar Bone Loss/prevention & control , Alveolar Bone Loss/etiology , Alveolar Bone Loss/pathologyABSTRACT
Osteoporosis is particularly prevalent among postmenopausal women and the elderly. In the present study, we investigated the effect of the novel small molecule E0924G (N-(4-methoxy-pyridine-2-yl)-5-methylfuran-2-formamide) on osteoporosis. E0924G significantly increased the protein expression levels of osteoprotegerin (OPG) and runt-related transcription factor 2 (RUNX2), and thus significantly promoted osteogenesis in MC3T3-E1 cells. E0924G also significantly decreased osteoclast differentiation and inhibited bone resorption and F-actin ring formation in receptor activator of NF-κB ligand (RANKL)-induced osteoclasts from RAW264.7 macrophages. Importantly, oral administration of E0924G in both ovariectomized (OVX) rats and SAMP6 senile mice significantly increased bone mineral density and decreased bone loss compared to OVX controls or SAMR1 mice. Further mechanistic studies showed that E0924G could bind to and then activate peroxisome proliferator-activated receptor delta (PPARδ), and the pro-osteoblast effect and the inhibition of osteoclast differentiation induced by E0924G were significantly abolished when PPARδ was knocked down or inhibited. In conclusion, these data strongly suggest that E0924G has the potential to prevent OVX-induced and age-related osteoporosis by dual regulation of bone formation and bone resorption through activation of the PPARδ signaling pathway.
Subject(s)
Bone Resorption , Osteogenesis , Ovariectomy , PPAR delta , Signal Transduction , Animals , Mice , Bone Resorption/drug therapy , Bone Resorption/prevention & control , Bone Resorption/metabolism , Rats , PPAR delta/metabolism , Female , Osteogenesis/drug effects , Signal Transduction/drug effects , Structure-Activity Relationship , Molecular Structure , RAW 264.7 Cells , Osteoporosis/drug therapy , Osteoporosis/prevention & control , Osteoporosis/metabolism , Dose-Response Relationship, Drug , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemistry , Rats, Sprague-Dawley , Osteoclasts/drug effects , Osteoclasts/metabolism , Cell Differentiation/drug effectsABSTRACT
BACKGROUND: Elevated reactive oxygen species levels promote excessive osteoclastogenesis and bone resorption. Puerarin, a natural antioxidant, can prevent bone loss through its antioxidant effects; however, the underlying molecular mechanism remains unclear. This study aimed to explore the effects of puerarin on osteoclast differentiation and bone resorption by regulating the PI3K/AKT/FoxO1 signaling pathway. MATERIALS AND METHODS: An ovariectomized (OVX) rat model of osteoporosis and H2O2-induced oxidative cell model of RAW 264.7 cells were established. The following indices were measured including bone µ-CT scanning and the protein expression levels of FoxO1, p-FoxO1, and catalase were detected using western blotting. RESULTS: Puerarin strongly alleviated oxidative stress-induced bone loss in OVX rats in vivo owing to its antioxidant effects. Puerarin improved the oxidative stress status of cells and inhibited osteoclast formation in vitro. Moreover, the protein expression of FoxO1 and its downstream target, catalase, was upregulated by puerarin. CONCLUSIONS: Puerarin improved the OPG/RANKL ratio, upregulated the protein expression and transcriptional activity of FoxO1, and suppressed the differentiation of RAW264.7 cells into osteoclasts. FoxO1 is a pivotal target of puerarin to confer anti-osteoporosis effects.
Subject(s)
Bone Resorption , Cell Differentiation , Forkhead Box Protein O1 , Isoflavones , Osteoclasts , Osteogenesis , Osteoporosis , Ovariectomy , Oxidative Stress , Signal Transduction , Animals , Isoflavones/pharmacology , Forkhead Box Protein O1/metabolism , Mice , RAW 264.7 Cells , Osteogenesis/drug effects , Osteoclasts/drug effects , Osteoclasts/metabolism , Female , Rats , Bone Resorption/prevention & control , Bone Resorption/drug therapy , Bone Resorption/metabolism , Oxidative Stress/drug effects , Signal Transduction/drug effects , Osteoporosis/drug therapy , Cell Differentiation/drug effects , Antioxidants/pharmacology , Rats, Sprague-Dawley , RANK Ligand/metabolism , Disease Models, Animal , Proto-Oncogene Proteins c-akt/metabolism , Catalase/metabolism , Phosphatidylinositol 3-Kinases/metabolismABSTRACT
Osteoporosis is a common bone disease in aging populations, particularly in postmenopausal women. Anti-resorptive and anabolic drugs have been applied to prevent and cure osteoporosis and are linked with a variety of adverse effects. Antrodia cinnamomea extracts (ACE) are highly renowned for their anticancer, antioxidative, and anti-inflammatory properties. However, whether ACE-enriched anti-osteoporosis functions are largely unknown. In a preclinical animal model, we found that ovariectomy significantly decreased bone volume in the ovariectomized (OVX) rats. Administration of ACE antagonized OVX-induced bone loss. In addition, ACE reversed OVX-reduced biomechanical properties. The serum osteoclast marker also showed improvement in the ACE-treated group. In the cellular model, it was indicated that ACE inhibits RANKL-induced osteoclast formation. Taken together, ACE seems to be a hopeful candidate for the development of novel anti-osteoporosis treatment.
Subject(s)
Osteoclasts , Osteoporosis , Ovariectomy , Rats, Sprague-Dawley , Animals , Female , Osteoclasts/drug effects , Osteoporosis/prevention & control , Osteoporosis/drug therapy , Osteoporosis/pathology , Mice , Rats , RAW 264.7 Cells , Polyporales/chemistry , Bone Resorption/prevention & control , Bone Resorption/drug therapy , RANK LigandABSTRACT
BACKGROUND AND AIM: Osteoporosis, a systemic metabolic bone disease, is characterized by the decline of bone mass and quality due to excessive osteoclast activity. Currently, drug-targeting osteoclasts show promising therapy for osteoporosis. In this study, we investigated the effect of cichoric acid (CA) on receptor activator of nuclear kappa-B ligand (RANKL)-induced osteoclastogenesis and the bone loss induced by ovariectomy in mice. EXPERIMENTAL PROCEDURE: Molecular docking technologies were employed to examine the interaction between CA and RANKL. CCK8 assay was used to evaluate the cell viability under CA treatment. TRAcP staining, podosome belt staining, and bone resorption assays were used to test the effect of CA on osteoclastogenesis and osteoclast function. Further, an OVX-induced osteoporosis mice model was employed to identify the effect of CA on bone loss using micro-CT scanning and histological examination. To investigate underlying mechanisms, network pharmacology was applied to predict the downstream signaling pathways, which were verified by Western blot and immunofluorescence staining. KEY RESULTS: The molecular docking analysis revealed that CA exhibited a specific binding affinity to RANKL, engaging multiple binding sites. CA inhibited RANKL-induced osteoclastogenesis and bone resorption without cytotoxic effects. Mechanistically, CA suppressed RANKL-induced intracellular reactive oxygen species, nuclear factor-kappa B, and mitogen-activated protein kinase pathways, followed by abrogated nuclear factor activated T-cells 1 activity. Consistent with this finding, CA attenuated post-ovariectomy-induced osteoporosis by ameliorating osteoclastogenesis. CONCLUSIONS AND IMPLICATIONS: CA inhibited osteoclast activity and bone loss by targeting RANKL. CA might represent a promising candidate for treating osteoclast-related diseases, such as osteoporosis.
Subject(s)
Bone Resorption , Caffeic Acids , Osteoporosis , Succinates , Animals , Female , Humans , Mice , Bone Resorption/prevention & control , Cell Differentiation , Mice, Inbred C57BL , Molecular Docking Simulation , NF-kappa B/metabolism , Osteoclasts , Osteogenesis , Osteoporosis/pathology , Ovariectomy/adverse effects , RANK Ligand/metabolismABSTRACT
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
Postmenopausal osteoporosis (PMOP) is a metabolic bone disease that results from overproduction and hyperactivation of osteoclasts caused by insufficient estrogen in women after menopause. Current therapeutic strategies are mainly focused on treating PMOP patients who have already developed severe bone loss or even osteoporotic fractures. Obviously, a better strategy is to prevent PMOP from occurring in the first place. However, such reagents are largely lacking. Piperlongumine (PLM), an amide alkaloid extracted from long pepper Piper longum, exhibits the anti-osteoclastogenic effect in normal bone marrow macrophages (BMMs) and the protective effect against osteolysis induced by titanium particles in mice. This study examined the preventive effect of PLM on PMOP and explored the potential mechanism of this effect using both ovariectomized mice and their primary cells. The result showed that PLM (5 and 10 mg kg-1) administered daily for 6 weeks ameliorated ovariectomy-induced bone loss and osteoclast formation in mice. Further cell experiments showed that PLM directly suppressed osteoclast formation, F-actin ring formation, and osteoclastic resorption pit formation in BMMs derived from osteoporotic mice, but did not obviously affect osteogenic differentiation of bone marrow stromal cells (BMSCs) from these mice. Western blot analysis revealed that PLM attenuated maximal activation of p38 and JNK pathways by RANKL stimulation without affecting acute activation of NF-κB, AKT, and ERK signaling. Furthermore, PLM inhibited expression of key osteoclastogenic transcription factors NFATc1/c-Fos and their target genes (Dcstamp, Atp6v0d2, Acp5, and Oscar). Taken together, our findings suggest that PLM inhibits osteoclast formation and function by suppressing RANKL-induced activation of the p38/JNK-cFos/NFATc1 signaling cascade, thereby preventing ovariectomy-induced osteoporosis in mice. Thus, PLM can potentially be used as an anti-resorption drug or dietary supplement for the prevention of PMOP.
Subject(s)
Alkaloids , Benzodioxoles , Bone Resorption , Osteoporosis, Postmenopausal , Osteoporosis , Humans , Female , Animals , Mice , Osteogenesis , MAP Kinase Signaling System , Osteoclasts , Bone Resorption/drug therapy , Bone Resorption/prevention & control , Osteoporosis/etiology , Osteoporosis/genetics , Cell Differentiation , NF-kappa B/metabolism , Osteoporosis, Postmenopausal/metabolism , Ovariectomy/adverse effects , Alkaloids/metabolism , RANK Ligand/metabolismABSTRACT
Vitamin D is a vital indicator of musculoskeletal health, as it plays an important role through the regulation of bone and mineral metabolism. This meta-analysis was performed to investigate the effects of vitamin D supplementation/fortification on bone turnover markers in women. All human randomised clinical trials reported changes in bone resorption markers (serum C-terminal telopeptide of type-I collagen (sCTX) and urinary type I collagen cross-linked N-telopeptide (uNTX)) or bone formation factors (osteocalcin (OC), bone alkaline phosphatase (BALP) and procollagen type-1 intact N-terminal propeptide (P1NP)) following vitamin D administration in women (aged ≥ 18 years) were considered. Mean differences (MD) and their respective 95 % CI were calculated based on fixed or random effects models according to the heterogeneity status. Subgroup analyses, meta-regression models, sensitivity analysis, risk of bias, publication bias and the quality of the included studies were also evaluated. We found that vitamin D supplementation had considerable effect on sCTX (MD: -0·038, n 22) and OC (MD: -0·610, n 24) with high heterogeneity and uNTX (MD: -8·188, n 6) without heterogeneity. Our results showed that age, sample size, dose, duration, baseline vitamin D level, study region and quality of studies might be sources of heterogeneity in this meta-analysis. Subgroup analysis also revealed significant reductions in P1NP level in dose less than 600 µg/d and larger study sample size (>100 participants). Moreover, no significant change was found in BALP level. Vitamin D supplementation/fortification significantly reduced bone resorption markers in women. However, results were inconsistent for bone formation markers.
Subject(s)
Biomarkers , Bone Remodeling , Dietary Supplements , Vitamin D , Humans , Vitamin D/blood , Vitamin D/administration & dosage , Female , Biomarkers/blood , Bone Remodeling/drug effects , Randomized Controlled Trials as Topic , Bone Resorption/prevention & control , Collagen Type I/blood , Bone and Bones/metabolism , Bone and Bones/drug effects , Osteocalcin/blood , Alkaline Phosphatase/blood , Peptides/blood , Food, FortifiedABSTRACT
Repeated chromatography of the CH2Cl2 and EtOAc soluble fractions from the methanol extract of Belamcanda chinensis root yielded six new sucrosephenylpropanoid esters (1-6) and twenty-one known compounds (7-27). The structures of 1-6 were elucidated using diverse nuclear magnetic resonance (NMR) techniques and high-resolution mass spectrometry (HRMS) data analysis, together with chemical methods. All the twenty-seven isolated compounds were evaluated for their anti-osteoclastogenic activities. Preliminary screening results revealed that compounds 1 and 19 exhibited strong effects against RANKL-induced osteoclast formation in RAW264.7 cells. In addition, the treatment of mouse bone marrow macrophages (BMMs) with compounds 1 and 19 significantly decreased RANKL-induced TRAP-positive multinucleated osteoclast formation in a concentration-dependent manner without affecting cell viability. Further bioassay investigation showed that compounds 1 and 19 inhibited the expression of some osteoclast-specific marker genes and the transcription factor nuclear factor of activated T cells cytoplasmic 1 (NFATc1) in response to RANKL. To the best of our knowledge, this is the first investigation of anti-osteoclastogenic activity for compounds isolated from B. chinensis.
Subject(s)
Bone Resorption , Isoflavones , Animals , Mice , Bone Resorption/drug therapy , Bone Resorption/metabolism , Bone Resorption/prevention & control , Cell Differentiation , NFATC Transcription Factors/drug effects , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Osteoclasts , Osteogenesis/drug effects , Isoflavones/chemistry , Isoflavones/pharmacology , Plant Roots/chemistryABSTRACT
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
Osteoporosis (OP) is a bone remodeling disease characterized by an imbalance between bone resorption and formation. Osteoclasts are the primary therapeutic targets for treating bone destruction. Koumine (KM), the most bioactive component in Gelsemium alkaloids, exhibits antitumor, immunosuppressive, anti-inflammatory, and analgesic properties. However, the effects of bone loss have not been well studied. This study conducted in vitro and in vivo verification experiments on KM. The results showed that KM inhibited bone resorption and tartrate-resistant acid phosphatase positive (TRAP+) osteoclasts development by mature osteoclasts in a dose-dependent manner. Moreover, KM prevented OVX-induced OP in vivo and potentially inhibited ubiquitination, a process closely related to various biological activities, including protein interaction, transcription, and transmembrane signal transduction regulation, especially within the nuclear factor-κB (NF-κB) pathway. Previous studies have demonstrated that several proteins ubiquitination promotes osteoclastogenesis, our study indicated that KM inhibits early NF-κB activation and receptor activator of NF-κB ligand induced ubiquitination, a critical factor in osteoclast differentiation. In conclusion, our research suggests that KM holds potential as an effective therapeutic agent for OP.