Mending the bones with natural products

In this issue, Qiu and colleagues [1] demonstrate in vitro and in vivo activity of a triterpene glycoside from black cohosh. The isolated compound acts as a suppressor of osteoclastogenesis, targeting specifically RANKL, a member of the TNF superfamily.     

Receptor activator of NF-kappaB ligand enhances the activity of macrophages as antigen presenting cells

Receptor activator of NFkappaB ligand (RANKL) is known as a key regulator of osteoclastogenesis. However, the fact that fibroblasts and periodontal ligament cells express RANKL in response to bacterial substances, suggests that RANKL may have evolved as a part of the immunity to infection. As RANKL increases the survival and activity of dendritic cells, it may have similar effects on macrophages. To address this issue, we studied the effect of RANKL on various functions of macrophages using mouse bone marrow derived macrophages. RANKL enhanced the survival of macrophages and up-regulated the expression of CD86. RANKL-treated macrophages showed increased allogeneic T cell activation and phagocytic activity compared to control cells. In addition, RANKL increased the expression of TNFalpha, MCP-1, and IL-6 but not of IL-10, IL-12, IFN-gamma, and iNOS. Collectively, RANKL augmented the activity of macrophages especially as antigen presenting cells, suggesting its new role in immune regulation.     

High extracellular Ca2+ alone stimulates osteoclast formation but inhibits in the presence of other osteoclastogenic factors

High ambient Ca2+ at bone resorption sites have been implicated to play an important role in the regulation of bone remodeling. The present study was performed to clarify the mode of high extracellular Ca2+ (Ca2+(e))-induced modulation of osteoclastogenesis and the expression of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG), thereby to define its role in osteoclast formation. Mouse bone marrow cells were cocultured with osteoblastic cells in the absence or presence of osteoclastogenic factors such as 1,25-dihydroxyvitaminD3 (1,25-(OH)2vitD3)and macrophage colony-stimulating factor/soluble RANKL. Ca2+ concentration in media (1.8 mM) was adjusted to 3, 5, 7 or 10 mM. Osteoclast formation was confirmed by the appearance of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells and the expression of osteoclast phenotypic markers (calcitonin receptor, vitronectin receptor, cathepsin K, matrix metalloproteinase-9, carbonic anhydrase 2). High Ca2+(e) alone significantly stimulated osteoclast formation in a dose-dependent manner. However, in the presence of highly osteoclastogenic factors, high Ca2+(e) significantly inhibited osteoclastogenesis. High Ca2+(e) alone continuously up-regulated RANKL expression while only transiently increased OPG expression. However, in the presence of 1,25-(OH)(2)vitD(3), high Ca2+(e) did not change the 1,25-(OH)2vitD3-induced RANKL expression while increased OPG expression. Taken together, these findings suggest that high Ca2+(e) alone increase osteoclastogenesis but inhibit in the presence of other osteoclastogenic factors. In addition, high CaCa2+(e)-induced osteoclastogenesis may be mediated by osteoblasts via up-regulation of RANKL expression. Meanwhile up-regulated OPG might participate in the inhibitory effect of high Ca2+(e) on 1,25-(OH)2vitD3-induced osteoclastogenesis.     

Simvastatin inhibits osteoclast differentiation by scavenging reactive oxygen species

Osteoclasts, together with osteoblasts, control the amount of bone tissue and regulate bone remodeling. Osteoclast differentiation is an important factor related to the pathogenesis of bone-loss related diseases. Reactive oxygen species (ROS) acts as a signal mediator in osteoclast differentiation. Simvastatin, which inhibits 3-hydroxy-3-methylglutaryl coenzyme A, is a hypolipidemic drug which is known to affect bone metabolism and suppresses osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand (RANKL). In this study, we analyzed whether simvastatin can inhibit RANKL-induced osteoclastogenesis through suppression of the subsequently formed ROS and investigated whether simvastatin can inhibit H2O2-induced signaling pathways in osteoclast differentiation. We found that simvastatin decreased expression of tartrate-resistant acid phosphatase (TRAP), a genetic marker of osteoclast differentiation, and inhibited intracellular ROS generation in RAW 264.7 cell lines. ROS generation activated NF-κB, protein kinases B (AKT), mitogen-activated protein kinases signaling pathways such as c-JUN N-terminal kinases, p38 MAP kinases as well as extracellular signal- regulated kinase. Simvastatin was found to suppress these H2O2-induced signaling pathways in osteoclastogenesis. Together, these results indicate that simvastatin acts as an osteoclastogenesis inhibitor through suppression of ROS-mediated signaling pathways. This indicates that simvastatin has potential usefulness for osteoporosis and pathological bone resorption.     

A disulfide bond replacement strategy enables the efficient design of artificial therapeutic peptides

We demonstrate that disulfide bond replacement is an efficient strategy for engineering therapeutic peptides. In previous work, short peptide fragments, known as WP9QY, with sequence homology with the predicted ligand contact surface of the receptor activator of NF-κB (RANK) were crosslinked through intramolecular disulfide bonds to inhibit RANK ligand (RANKL)-induced signaling, osteoclastogenesis, bone resorption in vitro, and bone loss in vivo. We report that replacement of the disulfide bond of WP9QY with an amine cross-linkage results in a significant improvement in enzymatic stability, with only a slight loss of bone resorption-blocking activity in vitro. Furthermore, the WP9QY derivative inhibits bone loss significantly in vivo, whereas the native form of WP9QY was not effective under the same conditions.     

β-Boswellic Acid Inhibits RANKL-Induced Osteoclast Differentiation and Function by Attenuating NF-κB and Btk-PLCγ2 Signaling Pathways

Osteoporosis is a systemic metabolic bone disorder that is caused by an imbalance in the functions of osteoclasts and osteoblasts and is characterized by excessive bone resorption by osteoclasts. Targeting osteoclast differentiation and bone resorption is considered a good fundamental solution for overcoming bone diseases. β-boswellic acid (βBA) is a natural compound found in Boswellia serrata, which is an active ingredient with anti-inflammatory, anti-rheumatic, and anti-cancer effects. Here, we explored the anti-resorptive effect of βBA on osteoclastogenesis. βBA significantly inhibited the formation of tartrate-resistant acid phosphatase-positive osteoclasts induced by receptor activator of nuclear factor-B ligand (RANKL) and suppressed bone resorption without any cytotoxicity. Interestingly, βBA significantly inhibited the phosphorylation of IκB, Btk, and PLCγ2 and the degradation of IκB. Additionally, βBA strongly inhibited the mRNA and protein expression of c-Fos and NFATc1 induced by RANKL and subsequently attenuated the expression of osteoclast marker genes, such as OC-STAMP, DC-STAMP, β3-integrin, MMP9, ATP6v0d2, and CtsK. These results suggest that βBA is a potential therapeutic candidate for the treatment of excessive osteoclast-induced bone diseases such as osteoporosis.     

Osteoprotegerin is present on the membrane of osteoclasts isolated from mouse long bones

Osteoprotegerin (OPG), a member of the tumor necrosis factor receptor superfamily, is known to inhibit osteoclastogenesis by acting as a soluble decoy receptor for the receptor activator of NF-kappaB ligand (RANKL). We report the presence of OPG on the membrane of osteoclasts and the possibility of the direct action of OPG on them. Highly pure osteoclast precursors were isolated from mouse long bones and induced to differentiate into mature osteoclasts by M-CSF and soluble RANKL (sRANKL). The presence of OPG on the membrane of these cells was confirmed by western blotting and immunostaining. Furthermore, sRANKL was found to be bound to the OPG on the osteoclast precursors. These results suggest that OPG might have a new role during the differentiation of osteoclasts beyond its role as a soluble decoy receptor. The mechanism of the existence of OPG on osteoclast precursors remains to be found.     

Caveolin-1 regulates osteoclast differentiation by suppressing cFms degradation

Caveolae are flask-shaped cell-surface membranes, which consist of cholesterol, sphingolipids and caveolin proteins. In a microarray analysis, we found that caveolin-1 (Cav-1) was upregulated by receptor activator of NFκB ligand (RANKL), the osteoclast differentiation factor. Silencing of Cav-1 inhibited osteoclastogenesis and also decreased the activation of mitogen-activated protein kinase and the induction of NFATc1 by RANKL. Cav-1 knockdown suppressed the expression of cFms and RANK, two major receptors for osteoclastogenesis. Interestingly, cFms expression was decreased only at the protein level, not at the messenger RNA (mRNA) level, whereas RANK expression was decreased at both the mRNA and protein levels. Furthermore, Cav-1 deficiency increased the lysosomal degradation of cFms. Taken together, these results demonstrate that Cav-1-dependent cFms stabilization contributes to efficient osteoclastogenesis.     

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.     

Platinum nanoparticles reduce ovariectomy-induced bone loss by decreasing osteoclastogenesis

Platinum nanoparticles (PtNP) exhibit remarkable antioxidant activity. There is growing evidence concerning a positive relationship between oxidative stress and bone loss, suggesting that PtNP could protect against bone loss by modulating oxidative stress. Intragastric administration of PtNP reduced ovariectomy (OVX)- induced bone loss with a decreased level of activity and number of osteoclast (OC) in vivo. PtNP inhibited OC formation by impairing the receptor activator of nuclear factor-κB ligand (RANKL) signaling. This impairment was due to a decreased activation of nuclear factor-κB and a reduced level of nuclear factor in activated T-cells, cytoplasmic 1 (NFAT2). PtNP lowered RANKL-induced long lasting reactive oxygen species as well as intracellular concentrations of Ca(2+) oscillation. Our data clearly highlight the potential of PtNP for the amelioration of bone loss after estrogen deficiency by attenuated OC formation.     

Rutin inhibits osteoclast formation by decreasing reactive oxygen species and TNF-alpha by inhibiting activation of NF-kappaB

Rutin, a glycoside of flavonol, inhibits osteoclast formation induced by receptor activator of NF-kappaB ligand (RANKL) in bone marrow-derived macrophages. It reduces reactive oxygen species produced by RANKL and its inhibitory effect results from reduced levels of TNF-alpha. Rutin also lowers NF-kappaB activation in response to RANKL.     

Deficiency of optineurin enhances osteoclast differentiation by attenuating the NRF2-mediated antioxidant response

Abnormally increased resorption contributes to bone degenerative diseases such as Paget’s disease of bone (PDB) through unclear mechanisms. Recently, the optineurin (OPTN) gene has been implicated in PDB, and global OPTN knockout mice (Optn^−/−) were shown to exhibit increased formation of osteoclasts (osteoclastogenesis). Growing evidence, including our own, has demonstrated that intracellular reactive oxygen species (ROS) stimulated by receptor activator of nuclear factor kappa-B ligand (RANKL) can act as signaling molecules to promote osteoclastogenesis. Here, we report that OPTN interacts with nuclear factor erythroid-derived factor 2-related factor 2 (NRF2), the master regulator of the antioxidant response, defining a pathway through which RANKL-induced ROS could be regulated for osteoclastogenesis. In this study, monocytes from Optn^−/− and wild-type (Optn^+/+) mice were utilized to differentiate into osteoclasts, and both qRT-PCR and tartrate-resistant acid phosphatase (TRAP) staining showed that the Optn^−/− monocytes exhibited enhanced osteoclastogenesis compared to the Optn^+/+ cells. CellROX^® staining, qRT-PCR, and Western blotting indicated that OPTN deficiency reduced the basal expression of Nrf2, inhibited the expression of NRF2-responsive antioxidants, and increased basal and RANKL-induced intracellular ROS levels, leading to enhanced osteoclastogenesis. Coimmunoprecipitation (co-IP) showed direct interaction, and immunofluorescence staining showed perinuclear colocalization of the OPTN-NRF2 granular structures during differentiation. Finally, curcumin and the other NRF2 activators attenuated the hyperactive osteoclastogenesis induced by OPTN deficiency. Collectively, our findings reveal a novel OPTN-mediated mechanism for regulating the NRF2-mediated antioxidant response in osteoclasts and extend the therapeutic potential of OPTN in the aging process resulting from ROS-triggered oxidative stress, which is associated with PDB and many other degenerative diseases.Subject terms: Mechanisms of disease, Stress signalling     

Ethanol Extract of Amomum tsao-ko Ameliorates Ovariectomy-Induced Trabecular Loss and Fat Accumulation

In Asia, Amomum tsao-ko has long been used as a spice or seasoning in food to stimulate digestion. In the present study, we evaluated the effects of ethanol extract of Amomum tsao-ko (EEAT) on menopausal osteoporosis and obesity. After the administration of EEAT in ovariectomy (OVX) mice models for five weeks, microcomputed tomography and a histological analysis were performed to assess, respectively, the trabecular structure and the fat accumulation in adipose, liver, and bone tissues. We also examined the effects of EEAT on a bone marrow macrophage model of osteoclastogenesis by in vitro stimulation from the receptor activator of nuclear factor-kappa Β ligand (RANKL) through real-time PCR and Western blot analysis. In addition, ultrahigh performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) with authentic standards was applied to characterize the phytochemical profiling of EEAT. We found that EEAT significantly decreased OVX-induced body weight gain and fat accumulation, significantly prevented OVX-induced deterioration of bone mineral density and microstructure of trabecular tissues, and significantly inhibited osteoclast differentiation by downregulating NF-κB/Fos/NFATc1 signaling in osteoclasts. Furthermore, UHPLC–MS/MS identified eight beneficial phytochemicals in EEAT. Collectively, these results suggest that EEAT might be an effective nutraceutical candidate to attenuate menopausal osteoporosis by inhibiting osteoclastogenesis and to prevent obesity by suppressing fat accumulation.     

Osteoclast-Mediated Cell Therapy as an Attempt to Treat Elastin Specific Vascular Calcification

Inflammation and stiffness in the arteries is referred to as vascular calcification. This process is a prevalent yet poorly understood consequence of cardiovascular disease and diabetes mellitus, comorbidities with few treatments clinically available. Because this is an active process similar to bone formation, it is hypothesized that osteoclasts (OCs), bone-resorbing cells in the body, could potentially work to reverse existing calcification by resorbing bone material. The receptor activator of nuclear kappa B-ligand (RANKL) is a molecule responsible for triggering a response in monocytes and macrophages that allows them to differentiate into functional OCs. In this study, OC and RANKL delivery were employed to determine whether calcification could be attenuated. OCs were either delivered via direct injection, collagen/alginate microbeads, or collagen gel application, while RANKL was delivered via injection, through either a porcine subdermal model or aortic injury model. While in vitro results yielded a decrease in calcification using OC therapy, in vivo delivery mechanisms did not provide control or regulation to keep cells localized long enough to induce calcification reduction. However, these results do provide context and direction for the future of OC therapy, revealing necessary steps for this treatment to effectively reduce calcification in vivo. The discrepancy between in vivo and in vitro success for OC therapy points to the need for a more stable and time-controlled delivery mechanism that will allow OCs not only to remain at the site of calcification, but also to be regulated so that they are healthy and functioning normally when introduced to diseased tissue.     

New Generation of Meso and Antiprogestins (SPRMs) into the Osteoporosis Approach

Receptor activator of nuclear factor κB (RANK) and its ligand (RANKL) play key roles in bone metabolism and the immune system. The RANK/RANKL complex has also been shown to be critical in the formation of mammary epithelia cells. The female hormones estradiol and progesterone closely control the action of RANKL with RANK. Blood concentration of these sex hormones in the postmenopausal period leads to an increase in RANK/RANKL signaling and are a major cause of women’s osteoporosis, characterized by altered bone mineralization. Knowledge of the biochemical relationships between hormones and RANK/RANKL signaling provides the opportunity to design novel therapeutic agents to inhibit bone loss, based on the anti-RANKL treatment and inhibition of its interaction with the RANK receptor. The new generation of both anti- and mesoprogestins that inhibit the NF-κB-cyclin D1 axis and blocks the binding of RANKL to RANK can be considered as a potential source of new RANK receptor ligands with anti-RANKL function, which may provide a new perspective into osteoporosis treatment itself as well as limit the osteoporosis rise during breast cancer metastasis to the bone.     

Inhibitory action of bisphosphonates on bone resorption does not involve the regulation of RANKL and OPG expression

The mechanism of inhibitory action of bisphosphonates on bone resorption is not fully elucidated. Osteoclast formation and activity are regulated by osteoblast-derived factors such as the osteoclast differentiating factor, receptor activator of NF-kappaB ligand (RANKL) and the inhibitor, osteoprotegerin (OPG). To investigate in vitro effects of bisphosphonates on mouse osteoblastic cells, we examined the expression levels of RANKL and OPG in the cells treated with alendronate or pamidronate (10(-8) approximately 10(-5) M) alone, or combined with 10 nM of 1,25-(OH)2VitD3 for 24 or 48 h. Various concentrations of alendronate and pamidronate did not change the mRNA expression of RANKL and OPG consistently irrespective of 1,25-(OH)2VitD3 presence. When added into cocultures of mouse osteoblastic cells and bone marrow cells, both alendronate and pamidronate inhibited osteoclast formation and bone resorption but failed to alter the RANKL and OPG mRNA expression. These results indicate that the inhibition of bone resorption by bisphosphonates is not mediated by the regulation of RANKL and OPG expression.     

The Skeletal Effects of Tanshinones: A Review

Background: Osteoporosis results from excessive bone resorption and reduced bone formation, triggered by sex hormone deficiency, oxidative stress and inflammation. Tanshinones are a class of lipophilic phenanthrene compounds found in the roots of Salvia miltiorrhiza with antioxidant and anti-inflammatory activities, which contribute to its anti-osteoporosis effects. This systematic review aims to provide an overview of the skeletal beneficial effects of tanshinones. Methods: A systematic literature search was conducted in January 2021 using Pubmed, Scopus and Web of Science from the inception of these databases. Original studies reporting the effects of tanshinones on bone through cell cultures, animal models and human clinical trials were considered. Results: The literature search found 158 unique articles on this topic, but only 20 articles met the inclusion criteria and were included in this review. The available evidence showed that tanshinones promoted osteoblastogenesis and bone formation while reducing osteoclastogenesis and bone resorption. Conclusions: Tanshinones modulates bone remodelling by inhibiting osteoclastogenesis and osteoblast apoptosis and stimulating osteoblastogenesis. Therefore, it might complement existing strategies to prevent bone loss.     

Alkaloids and saponins in dietary supplements of blue cohosh (Caulophyllum thalictroides)

Preparations of blue cohosh (Caulophyllum thalictroides) have been used traditionally by Native Americans for medicinal purposes. Dietary supplements containing dried roots or extracts of blue cohosh rhizomes are available as dietary supplements. The safety and efficacy of these preparations have not been systematically evaluated. Recent studies indicate that ingestion of specific alkaloids in blue cohosh preparations can produce birth defects and neonatal heart failure. Blue cohosh also contains saponins, which may be responsible for uterine-stimulating effects. We determined the amounts of major alkaloids and saponins in preparations of blue cohosh by high-performance liquid chromatography (HPLC). Alkaloids and saponins were monitored with a photodiode array detector and an evaporative light-scattering detector, respectively. Profiles were compared with those of authenticated blue cohosh root extracts. Identities of the alkaloids and saponins were confirmed by HPLC/mass spectrometry and nuclear magnetic resonance spectrometry. Calculations based on the results of analyses of dietary supplements showed that maximum daily intake of alkaloids and saponins will vary with the form (e.g., root, liquid extract) and doses recommended in product labeling. Intakes may vary from < 1 to 75 mg/day for alkaloids and from about 9 to 420 mg/day for saponins.