Ethanol Extract of Radix Asteris Suppresses Osteoclast Differentiation and Alleviates Osteoporosis.

Radix Asteris, the root of Aster tataricus L. f., is historically significant in East Asian medicine for treating respiratory conditions. Yet, its implications on bone health remain uncharted. This research investigated the impact of an aqueous ethanol extract of Radix Asteris (EERA) on osteoclast differentiation and its prospective contribution to osteoporosis management. We discerned that EERA retards osteoclast differentiation by inhibiting receptor activator of nuclear factor kappa-B ligand (RANKL) expression and obstructing RANKL-induced osteoclastogenesis. EERA markedly suppressed RANKL-induced expression of NFATc1, a pivotal osteoclastogenic factor, via modulating early RANK signaling. EERA's therapeutic potential was underscored by its defense against trabecular bone degradation and its counteraction to increased body and perigonadal fat in ovariectomized mice, mirroring postmenopausal physiological changes. In the phytochemical analysis of EERA, we identified several constituents recognized for their roles in regulating bone and fat metabolism. Collectively, our findings emphasize the potential of EERA in osteoclast differentiation modulation and in the management of osteoporosis and associated metabolic changes following estrogen depletion, suggesting its suitability as an alternative therapeutic strategy for postmenopausal osteoporosis intertwined with metabolic imbalances.

Water Extract of Angelica dahurica Inhibits Osteoclast Differentiation and Bone Loss.

Angelica dahurica radix has a long history of traditional use in China and Korea for treating headaches, cold-damp pain and skin diseases. Despite various pharmacological studies on A. dahurica, its impact on bones remains unclear. Hence, this study investigated the inhibitory effect of A. dahurica's radix water extract (WEAD) on osteoclast differentiation. In vitro experiments showed that WEAD effectively suppresses osteoclast differentiation. Treatment of an osteoclast precursor with WEAD significantly suppressed the expression of nuclear factor of activated T-cells 1 (NFATc1), essential transcription factor for osteoclastogenesis, while increasing the expression of negative regulators, interferon regulatory factor 8 (Irf8) and v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MafB). Consistent with the in vitro findings, the oral administration of WEAD (100 and 300 mg/kg/day) to mice subjected to surgical ovariectomy for a duration of six weeks alleviated bone loss, while also mitigating weight gain and liver fat accumulation. In addition, we also identified phytochemicals present in WEAD, known to regulate osteoclastogenesis and/or bone loss. These results suggest the potential use of WEAD for treating various bone disorders caused by excessive bone resorption.

Isolation and Characterization of an Anti-Osteoporotic Compound from Melia toosendan Fructus.

Melia toosendan fructus, traditionally employed in traditional Chinese and Korean herbal medicine, exhibits diverse biological properties encompassing anti-tumor, anti-inflammatory, and anti-viral effects. However, its influence on bone metabolism remains largely unexplored. In this study, we investigated the impact of an ethanolic extract of Melia toosendan fructus (MTE) on osteoclast differentiation and characterized its principal active constituent in osteoclast differentiation and function, as well as its effects on bone protection. Our findings demonstrate that MTE effectively inhibits the differentiation of osteoclast precursors induced by receptor activator of nuclear factor κB ligand (RANKL). Utilizing a bioassay-guided fractionation approach coupled with UHPLC-MS/MS analysis, we isolated and identified the triterpenoid compound toosendanin (TSN) as the active constituent responsible for MTE's anti-osteoclastogenic activity. TSN treatment downregulated the expression of nuclear factor of activated T cells c1, a pivotal osteoclastogenic transcription factor, along with molecules implicated in osteoclast-mediated bone resorption, including tumor necrosis factor receptor-associated factor 6, carbonic anhydrase II, integrin beta-3, and cathepsin K. Furthermore, treatment of mature osteoclasts with TSN impaired actin ring formation, acidification, and resorptive function. Consistent with our in vitro findings, TSN administration mitigated trabecular bone loss and reduced serum levels of the bone resorption marker, C-terminal cross-linked telopeptides of type I collagen, in a mouse bone loss model induced by intraperitoneal injections of RANKL. These results suggest that TSN, as the principal active constituent of MTE with inhibitory effects on osteoclastogenesis, exhibits bone-protective properties by suppressing both osteoclast differentiation and function. These findings imply the potential utility of TSN in the treatment of diseases characterized by excessive bone resorption.

Euonymus alatus (Thunb.) Siebold Prevents Osteoclast Differentiation and Osteoporosis.

Euonymus alatus (Thunb.) Siebold, a traditional medicinal plant, has been used in China and several other Asian countries to address a variety of health concerns. The extensive research conducted on E. alatus is driven by its diverse pharmacological applications. However, its biological effects on osteoclastogenesis and osteoporosis have not been previously studied. In this research, we investigated the impact of an ethanolic extract of E. alatus (EEEA) on osteoclast differentiation and function as well as estrogen deficiency-induced bone loss. We found that EEEA inhibits osteoclast differentiation by downregulating the expression of the receptor activator of nuclear factor-κB ligand (RANKL) in osteoclast-supporting cells and by directly impeding RANKL-mediated signaling pathways for osteoclastogenesis in precursor cells. In addition, EEEA inhibited the bone-resorptive function of mature osteoclasts in vitro. Furthermore, oral administration of EEEA significantly alleviated bone loss in an ovariectomy-induced osteoporosis mouse model. Additionally, we identified phytochemicals in EEEA that have suppressive effects on osteoclast differentiation and bone loss. Collectively, these results suggest that EEEA holds potential as a biotherapeutic candidate for anti-postmenopausal osteoporosis.

Water Extract of Piper longum Linn Ameliorates Ovariectomy-Induced Bone Loss by Inhibiting Osteoclast Differentiation.

Piper longum linn has traditionally been used for the treatment of respiratory and gastrointestinal disorders in India. Although various pharmacological effects of P. longum have been studied, its effects on bone have not been clearly elucidated. Therefore, this study examined the inhibitory effect of the water extract of P. longum Linn (WEPL) on osteoclast differentiation. WEPL directly affected the osteoclast precursors and suppressed osteoclast differentiation in vitro. In addition, the expression levels of c-Fos and nuclear factor of activated T cells 1, a critical transcription factor for osteoclastogenesis, were significantly downregulated by WEPL via the suppression of the receptor activator of nuclear factor (NF)-κB ligand-induced mitogen-activated protein kinase and NF-κB signaling pathways. Consistent with the in vitro results, oral administration of WEPL (100 and 300 mpk) to ovariectomized mice for six weeks relieved the OVX-induced bone loss. We also identified phytochemicals in WEPL that are reported to exert inhibitory effects on osteoclastogenesis and/or bone loss. Collectively, the findings of our study indicate that WEPL has an anti-osteoporotic effect on OVX-induced bone loss by diminishing osteoclast differentiation, suggesting that it may be useful to treat several bone diseases caused by excessive bone resorption.

Ethanolic extract of Pyrrosia lingua (Thunb.) Farw. ameliorates OVX-induced bone loss and RANKL-induced osteoclastogenesis.

Pyrrosia lingua (Thunb.) Farw is a common plant that has been widely used as a traditional herbal medicine in China and Korea to treat patients suffering from pain, vaginal bleeding and urolithiasis. However, the pharmacological effects of P. lingua on bone remain unknown. We investigated the anti-osteoporotic effects of an ethanolic extract of P. lingua (EEPL). We found that EEPL suppressed osteoclast differentiation by directly acting on osteoclast precursor cells. EEPL suppressed the expression of receptor activator of nuclear factor-κB ligand (RANKL)-induced nuclear factor of activated T cells 1, a major transcription factor for osteoclastogenesis, by inhibiting RANKL-induced expression of aryl hydrocarbon receptor/c-Fos, and activation of nuclear factor-κB and mitogen-activated protein kinases. Moreover, administration of EEPL inhibited trabecular bone loss and weight gain in ovariectomized mice. Furthermore, we identified phytochemicals in EEPL that are known to exert anti-osteoclastogenic or anti-osteoporotic effects using ultra-high-performance liquid chromatography-tandem mass-spectrometry analysis. Overall, the results of this study suggest that EEPL is effective therapeutic candidate that can be used to prevent or treat postmenopausal osteoporosis.

Water Extract of Fritillariae thunbergii Bulbus Inhibits RANKL-Mediated Osteoclastogenesis and Ovariectomy-Induced Trabecular Bone Loss.

Fritillariae thunbergii bulbus has been widely used to treat symptoms of coughs and airway congestion in the chest due to pathological colds and damp phlegm in traditional Chinese medicine. Despite its long history of traditional use, its pharmacological activities on osteoclastogenesis and osteoporosis have not been evaluated. This study investigated the effects of the water extract of Fritillariae thunbergii bulbus (WEFT) on osteoclast differentiation in bone marrow-derived macrophage cells and on ovariectomy (OVX)-induced osteoporosis in mice. We found that WEFT significantly inhibited osteoclastogenesis by downregulating the receptor activator of the NF-κB ligand (RANKL) signaling-induced nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expression. In an OVX-induced osteoporosis model, WEFT significantly prevented the OVX-induced trabecular loss of femurs, accompanied by a reduction in fat accumulation in the bone marrow and liver. In addition, WEFT significantly prevented weight gain and gonadal fat gain without recovering uterine atrophy. Using ultrahigh-performance liquid chromatography-tandem mass spectrometry, seven alkaloids (peimisine glucoside, yibeissine, peiminoside, sipeimine-glucoside, peimisine, peimine, and peiminine) were identified in WEFT. The results of this study suggest that WEFT can be a potential pharmacological candidate to reduce menopausal osteoporosis and menopause-related symptoms, such as fat accumulation.

Inhibitory Effect of Rosae Multiflorae Fructus Extracts on the Receptor Activator of NF-κB Ligand-Induced Osteoclastogenesis through Modulation of P38- and Ca2+-Mediated Nuclear Factor of Activated T-Cells Cytoplasmic 1 Expression.

BACKGROUND: Rosae Multiflorae fructus (RMF), known to have anti-inflammatory and antioxidant properties, has been used as a traditional remedy for inflammatory diseases such as arthritis in Eastern Asia. However, its effect on osteoclasts, which play a crucial role in resorptive inflammatory bone diseases, is yet to be elucidated. METHODS: The effect of extract of RMF (RMF-E) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis was examined by tartrate-resistant acid phosphatase (TRAP) staining, real-time polymerase chain reaction and western blot analysis. In addition, RANKL-induced Ca2+-oscillation was also investigated. RESULTS: RMF-E remarkably inhibited TRAP+-osteoclast and resorptive pit formation in a dose-dependent manner. In addition, the expression of c-Fos and nuclear factor of activated T-cells cytoplasmic, known as pivotal transcription factors for osteoclast formation in vitro and in vivo, and that of the osteoclast differentiation markers such as Acp5, Oscar, CtsK, Atp6v0d2, Tm7sf4, and Nfatc1 were significantly decreased by RMF-E treatment during osteoclastogenesis. The inhibitory effect of RMF-E on RANKL-induced osteoclastogenesis was caused by the suppression of p38 mitogen-activated protein kinase activation, and RANKL-induced Ca2+-oscillation removal via inactivation of Bruton's tyrosine kinase (BTK), and subsequently phospholipase C-γ2. CONCLUSIONS: RMF-E negatively regulates osteoclast differentiation and formation. These findings suggest the possibility of RMF-E as a traditional therapeutic agent against osteoclast-related bone disorders such as osteoporosis, rheumatoid arthritis, and periodontitis.

Actin-binding LIM protein 1 regulates receptor activator of NF-κB ligand-mediated osteoclast differentiation and motility.

Actin-binding LIM protein 1 (ABLIM1), a member of the LIM-domain protein family, mediates interactions between actin filaments and cytoplasmic targets. However, the role of ABLIM1 in osteoclast and bone metabolism has not been reported. In the present study, we investigated the role of ABLIM1 in the receptor activator of NF-κB ligand (RANKL)- mediated osteoclastogenesis. ABLIM1 expression was induced by RANKL treatment and knockdown of ABLIM1 by retrovirus infection containing Ablim1-specific short hairpin RNA (shAblim1) decreased mature osteoclast formation and bone resorption activity in a RANKL-dose dependent manner. Coincident with the downregulated expression of osteoclast differentiation marker genes, the expression levels of c-Fos and the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), critical transcription factors of osteoclastogenesis, were also decreased in shAblim1-infected osteoclasts during RANKLmediated osteoclast differentiation. In addition, the motility of preosteoclast was reduced by ABLIM1 knockdown via modulation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/Rac1 signaling pathway, suggesting another regulatory mechanism of ABLIM1 in osteoclast formation. These data demonstrated that ABLIM1 is a positive regulator of RANKLmediated osteoclast formation via the modulation of the differentiation and PI3K/Akt/Rac1-dependent motility. [BMB Reports 2018; 51(7): 356-361].

Pueraria lobate Inhibits RANKL-Mediated Osteoclastogenesis Via Downregulation of CREB/PGC1ß/c-Fos/NFATc1 Signaling.

Puerariae radix, the dried root of Pueraria lobate Ohwi, is known to prevent bone loss in ovariectomized mice; however, the precise molecular mechanisms are not understood. In this study, we investigated the effects and underlying mechanisms of action of Puerariae radix extract (PRE) on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. PRE dose-dependently inhibited osteoclast differentiation and formation, decreased the bone-resorbing activity of osteoclasts, and downregulated the expression of osteoclast differentiation marker genes. The expression of osteoclastogenic factors produced by PRE-treated osteoblasts such as RANKL, macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG) was comparable to that of untreated (control) cells. However, the formation of osteoclasts via bone marrow cell and calvaria-derived osteoblast co-cultures was suppressed by PRE treatment. Therefore, the inhibitory effects of PRE on osteoclastogenesis clearly targeted osteoclasts, but not osteoblasts. PRE treatment considerably reduced RANKL-induced mitogen-activated protein kinases (MAPKs) activity, especially c-Jun N-terminal kinase, in osteoclast precursor cells. In addition, PRE markedly suppressed cAMP response element-binding protein (CREB) activation and the induction of peroxisome proliferator-activated receptor gamma coactivator 1ß (PGC1ß), which stimulate osteoclastogenesis - an effect that was not observed for puerarin and 17-ß estradiol. Finally, PRE treatment significantly repressed the expression of c-Fos and the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), which is a master transcription factor for osteoclastogenesis in vitro and in vivo. Overall, these results strongly suggest that PRE is an effective inhibitor of RANKL-induced osteoclastogenesis and may be a potent therapeutic agent for bone-related diseases such as osteoporosis, rheumatoid arthritis, and periodontitis.

Sophorae Flos extract inhibits RANKL-induced osteoclast differentiation by suppressing the NF-κB/NFATc1 pathway in mouse bone marrow cells.

BACKGROUND: Sophorae Flos (SF) is a composite of flowers and buds of Styphnolobium japonicum (L.) Schott and has been used in traditional Korean and Chinese medicine for the treatment of hemostasis and inflammation. Previous studies reported that SF possesses anti-obesity properties, as well as anti-allergic, anti-proliferative, and anti-inflammatory activities. However, the effect of SF in bone resorption has not been studies. In this study, we examined the potential of SF extract (SFE) to inhibit receptor activator of NF-κB ligand (RANKL) -induced osteoclast differentiation in cultured mouse-derived bone marrow macrophages (BMMs). METHODS: BMMs, that act as osteoclast precursors, were cultured with M-CSF (50 ng/ml) and RANKL (100 ng/ml) for 4 days to generate osteoclasts. Osteoclast differentiation was measured by tartrate-resistant acidic phosphatase (TRAP) staining and the TRAP solution assay. Osteoclast differentiation marker genes were analyzed by the quantitative real-time polymerase chain reaction analysis. RANKLs signaling pathways were confirmed through western blotting. RESULTS: SFE significantly decreased osteoclast differentiation in a dose-dependent manner. SFE inhibited RANKL-induced osteoclastogenesis by suppressing NF-κB activation. By contrast, SFE did not affect phospholipase C gamma 2 or subsequent cAMP response element binding activation. SFE inhibited the RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1). CONCLUSIONS: SFE attenuated the RANKL-mediated induction of NF-κB through inhibition of IκBα phosphorylation, which contributed to inhibiting of RANKL-induced osteoclast differentiation through downregulation of NFATc1.

The inhibitory effect of beta-lapachone on RANKL-induced osteoclastogenesis.

ß-lapachone (ß-L) is a substrate of reduced nicotinamide adenine dinucleotide (NADH): quinone oxidoreductase 1 (NQO1). NQO1 reduces quinones to hydroquinones using NADH as an electron donor and consequently increases the intracellular NAD+/NADH ratio. The activation of NQO1 by ß-L has beneficial effects on several metabolic syndromes, such as obesity, hypertension, and renal injury. However, the effect of ß-L on bone metabolism remains unclear. Here, we show that ß-L might be a potent inhibitor of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. ß-L inhibited osteoclast formation in a dose-dependent manner and also reduced the expression of osteoclast differentiation marker genes, such as tartrate-resistant acid phosphatase (Acp5 or TRAP), cathepsin K (CtsK), the d2 isoform of vacuolar ATPase V0 domain (Atp6v0d2), osteoclast-associated receptor (Oscar), and dendritic cell-specific transmembrane protein (Dc-stamp). ß-L treatment of RANKL-induced osteoclastogenesis significantly increased the cellular NAD+/NADH ratio and resulted in the activation of 5' AMP-activated protein kinase (AMPK), a negative regulator of osteoclast differentiation. In addition, ß-L treatment led to significant suppression of the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and peroxisome proliferator-activated receptor gamma coactivator 1ß (PGC1ß), which can stimulate osteoclastogenesis. ß-L treatment downregulated c-Fos and nuclear factor of activated T-cells 1 (NFATc1), which are master transcription factors for osteoclastogenesis. Taken together, the results demonstrated that ß-L inhibits RANKL-induced osteoclastogenesis and could be considered a potent inhibitor of RANKL-mediated bone diseases, such as postmenopausal osteoporosis, rheumatoid arthritis, and periodontitis.

Lysosomal Ca2+ Signaling is Essential for Osteoclastogenesis and Bone Remodeling.

Lysosomal Ca2+ emerges as a critical component of receptor-evoked Ca2+ signaling and plays a crucial role in many lysosomal and physiological functions. Lysosomal Ca2+ release is mediated by the transient receptor potential (TRP) family member TRPML1, mutations that cause the lysosomal storage disease mucolipidosis type 4. Lysosomes play a key role in osteoclast function. However, nothing is known about the role of lysosomal Ca2+ signaling in osteoclastogenesis and bone metabolism. In this study, we addressed this knowledge gap by studying the role of lysosomal Ca2+ signaling in osteoclastogenesis, osteoclast and osteoblast functions, and bone homeostasis in vivo. We manipulated lysosomal Ca2+ signaling by acute knockdown of TRPML1, deletion of TRPML1 in mice, pharmacological inhibition of lysosomal Ca2+ influx, and depletion of lysosomal Ca2+ storage using the TRPML agonist ML-SA1. We found that knockdown and deletion of TRPML1, although it did not have an apparent effect on osteoblast differentiation and bone formation, markedly attenuated osteoclast function, RANKL-induced cytosolic Ca2+ oscillations, inhibited activation of NFATc1 and osteoclastogenesis-controlling genes, suppressed the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), and markedly reduced the differentiation of bone marrow-derived macrophages into osteoclasts. Moreover, deletion of TRPML1 resulted in enlarged lysosomes, inhibition of lysosomal secretion, and attenuated the resorptive activity of mature osteoclasts. Notably, depletion of lysosomal Ca2+ with ML-SA1 similarly abrogated RANKL-induced Ca2+ oscillations and MNC formation. Deletion of TRPML1 in mice reduced the TRAP-positive bone surfaces and impaired bone remodeling, resulting in prominent osteopetrosis. These findings demonstrate the essential role of lysosomal Ca2+ signaling in osteoclast differentiation and mature osteoclast function, which play key roles in bone homeostasis. © 2016 American Society for Bone and Mineral Research.

Cytosolic malate dehydrogenase regulates RANKL-mediated osteoclastogenesis via AMPK/c-Fos/NFATc1 signaling.

Cytosolic malate dehydrogenase (malate dehydrogenase 1, MDH1) plays pivotal roles in the malate/aspartate shuttle that might modulate metabolism between the cytosol and mitochondria. In this study, we investigated the role of MDH1 in osteoclast differentiation and formation. MDH1 expression was induced by receptor activator of nuclear factor kappa-B ligand (RANKL) treatment. Knockdown of MDH1 by infection with retrovirus containing MDH1-specific shRNA (shMDH1) reduced mature osteoclast formation and bone resorption activity. Moreover, the expression of marker genes associated with osteoclast differentiation was downregulated by shMDH1 treatment, suggesting a role of MDH1 in osteoclast differentiation. In addition, intracellular ATP production was reduced following the activation of adenosine 5' monophosphate-activated protein kinase (AMPK), a cellular energy sensor and negative regulator of RANKL-induced osteoclast differentiation, in shMDH1-infected osteoclasts compared to control cells. In addition, the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a critical transcription factor of osteoclastogenesis, was decreased with MDH1 knockdown during RANKL-mediated osteoclast differentiation. These findings provide strong evidence that MDH1 plays a critical role in osteoclast differentiation and function via modulation of the intracellular energy status, which might affect AMPK activity and NFATc1 expression.

ADP-Ribosylation Factor 1 Regulates Proliferation, Migration, and Fusion in Early Stage of Osteoclast Differentiation.

Small G-protein adenosine diphosphate (ADP)-ribosylation factors (ARFs) regulate a variety of cellular functions, including actin cytoskeleton remodeling, plasma membrane reorganization, and vesicular transport. Here, we propose the functional roles of ARF1 in multiple stages of osteoclast differentiation. ARF1 was upregulated during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation and transiently activated in an initial stage of their differentiation. Differentiation of ARF1-deficient osteoclast precursors into mature osteoclasts temporarily increased in pre-maturation stage of osteoclasts followed by reduced formation of mature osteoclasts, indicating that ARF1 regulates the osteoclastogenic process. ARF1 deficiency resulted in reduced osteoclast precursor proliferation and migration as well as increasing cell-cell fusion. In addition, ARF1 silencing downregulated c-Jun N-terminal kinase (JNK), Akt, osteopontin, and macrophage colony-stimulating factor (M-CSF)-receptor c-Fms as well as upregulating several fusion-related genes including CD44, CD47, E-cadherin, and meltrin-α. Collectively, we showed that ARF1 stimulated proliferation and migration of osteoclast precursors while suppressing their fusion, suggesting that ARF1 may be a plausible inter-player that mediates the transition to osteoclast fusion at multiple steps during osteoclast differentiation.

Inhibitory Effect of Chrysanthemum zawadskii Herbich var. latilobum Kitamura Extract on RANKL-Induced Osteoclast Differentiation.

Chrysanthemum zawadskii Herbich var. latilobum Kitamura, known as "Gujulcho" in Korea, has been used in traditional medicine to treat various inflammatory diseases, including rheumatoid arthritis. However, these effects have not been tested on osteoclasts, the bone resorbing cells that regulate bone metabolism. Here, we investigated the effects of C. zawadskii Herbich var. latilobum Kitamura ethanol extract (CZE) on osteoclast differentiation induced by treatment with the receptor activator of NF- κ B ligand (RANKL). CZE inhibited osteoclast differentiation and formation in a dose-dependent manner. The inhibitory effect of CZE on osteoclastogenesis was due to the suppression of ERK activation and the ablation of RANKL-stimulated Ca(2+)-oscillation via the inactivation of PLC γ 2, followed by the inhibition of CREB activation. These inhibitory effects of CZE resulted in a significant repression of c-Fos expression and a subsequent reduction of NFATc1, a key transcription factor for osteoclast differentiation, fusion, and activation in vitro and in vivo. These results indicate that CZE negatively regulates osteoclast differentiation and may be a therapeutic candidate for the treatment of various bone diseases, such as postmenopausal osteoporosis, rheumatoid arthritis, and periodontitis.