Lactoferrin Improves Bone Density and Strength In Vivo, and Inhibits Osteoclasts and Stimulates Osteoblasts In Vitro in Ovariectomized Mice

Abstract

Oral bovine lactoferrin improves bone status of ovariectomized mice.

The aim of the present study was to evaluate the effect of dietary lactoferrin on bone metabolism in vivo using a postmenopausal animal model. We investigated whether bovine lactoferrin (bLF) ingestion could prevent bone loss in ovariectomized mice. Twelve-week-old female C3H mice either ovariectomized or sham operated were fed for 27 wk with the control diet (AIN-93M with 140 g of total milk protein as a protein source per kg of diet). Four groups of ovariectomized mice received diets including different concentrations of bLF (1, 5, 10, or 20 g of total milk protein were replaced by bLF). Ovariectomy induced a decreased uterine weight and a smaller gain of bone mineral density. Immunoreactive bLF was detected in the peripheral blood, and its concentration was related to the amount of bLF ingestion. bLF supplementation to the diet improved bone mineral density (BMD) and femoral failure load in a dose-dependent manner. We confirmed the direct effects of bLF in vitro using established and primary cultures of murine bone cells. Addition of bLF to the culture medium at a concentration of between 1 and 1,000 microg/ml stimulated both cell growth and differentiation of osteoblastic MC3T3 cells while inhibiting the growth of preosteoclastic RAW 267.4 cells. In primary culture of mixed bone cells, an enhanced osteoblast differentiation was associated with an inhibition of osteoclast differentiation at lower bLF concentrations (1-10 microg/ml). In conclusion, these findings suggest that dietary lactoferrin supplementation can have a beneficial effect on postmenopausal bone loss by modulating bone formation and resorption.

Blais A, Malet A, Mikogami T, Martin-Rouas C…
Am. J. Physiol. Endocrinol. Metab. Jun 2009
PMID: 19336659 | Free Full Text

Honokiol Onhibits Osteoclasts In Vitro

Abstract

Honokiol inhibits osteoclast differentiation and function in vitro.

Honokiol, a neolignan, is a physiologically active component of kouboku (Magnolia obovata), a herb used in traditional Chinese medicine. This study investigated the effects of honokiol on the differentiation and function of osteoclasts induced by receptor activator of nuclear factor-kappaB ligand (RANKL). Honokiol markedly inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity and the formation of TRAP-positive multinucleated cells in both bone marrow-derived monocytes and RAW264 cells. In experiments to elucidate its mechanism of action, honokiol was found to suppress RANKL-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). The RANKL-induced expressions of c-Fos and nuclear factor of activated T cells-c1 (NFATc1), which are crucial transcriptional factors for osteoclastogenesis, were also reduced by treatment with honokiol. Furthermore, honokiol induced disruption of the actin rings in mature osteoclasts (mOCs) without affecting the cell viability and suppressed osteoclastic pit formation on dentin slices. Taken together, these results suggest that honokiol inhibits osteoclast differentiation by suppressing the activation of MAPKs (p38 MAPK, ERK and JNK), decreasing the expressions of c-Fos and NFATc1, and attenuates bone resorption by disrupting the actin rings in mOCs. Therefore, honokiol could prove useful for the treatment of bone diseases associated with excessive bone resorption.

Hasegawa S, Yonezawa T, Ahn JY, Cha BY…
Biol. Pharm. Bull. 2010
PMID: 20190414 | Free Full Text

CAPE (From Propolis) Inhibits Resorption in Human Cells

Abstract

Caffeic acid phenethyl ester, an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of RANKL-induced NF-kappaB and NFAT activity.

Receptor activator NF-kappaB ligand (RANKL)-activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF-kappaB inhibitor from honeybee propolis has been shown to have anti-tumor and anti-inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL-induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 microM) dose dependently inhibited RANKL-induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL-induced NF-kappaB and NFAT activation, concomitant with delayed IkappaBalpha degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF-kappaB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases.

Ang ES, Pavlos NJ, Chai LY, Qi M…
J. Cell. Physiol. Dec 2009
PMID: 19681045

CAPE (From Propolis) Inhibits Osteoclasts

Abstract

Caffeic acid phenethyl ester inhibits osteoclastogenesis by suppressing NF kappaB and downregulating NFATc1 and c-Fos.

Osteoclasts are multinuclear cells of myeloid lineage responsible for bone resorption. The anti-inflammatory property of caffeic acid phenethyl ester (CAPE), an active component of the propolis of honeybee hives, has been revealed. Since the regulatory mechanism of differentiation and activation of osteoclasts shares many well-known signaling pathways with that of inflammation, we investigated whether CAPE has any effect on osteoclastogenesis. CAPE potently suppressed osteoclastogenesis in cultures of bone marrow-derived precursor cells with the osteoclast differentiation factor, receptor activator of nuclear factor kappaB ligand (RANKL). While the RANKL-stimulated activation of the ERK, JNK, and p38 MAPK signaling pathways was not affected, the DNA binding and transcription activity of NF kappaB were reduced by CAPE treatment. In addition, CAPE blocked the induction of NFATc1 and c-Fos following RANKL stimulation. Forced expression of c-Fos could reverse the inhibitory effect of CAPE on osteoclastogenesis. Finally, CAPE significantly inhibited the RANKL-induced osteoclast formation in mouse calvariae in vivo. We propose that CAPE might be useful as a therapeutic agent for treatment of bone destructive diseases.

Ha J, Choi HS, Lee Y, Lee ZH…
Int. Immunopharmacol. Jun 2009
PMID: 19285574

Carnitine Slows Bone Growth (and Loss) in Ovariectomized Rats

Abstract

Dietary l-carnitine supplementation improves bone mineral density by suppressing bone turnover in aged ovariectomized rats.

Postmenopausal bone loss is a major public health concern. Although drug therapies are available, women are interested in alternative/adjunct therapies to slow down the bone loss associated with ovarian hormone deficiency. The purpose of this study was to determine whether dietary supplementation of l-carnitine can influence bone density and slow the rate of bone turnover in an aging ovariectomized rat model. Eighteen-month-old Fisher-344 female rats were ovariectomized and assigned to two groups: (1) a control group in which rats were fed ad libitum a carnitine-free (-CN) diet (AIN-93M) and (2) another fed the same diet but supplemented with l-carnitine (+CN). At the end of 8 weeks of feeding, animals were sacrificed and bone specimens were collected for measuring bone mineral content (BMC) and density (BMD) using dual energy X-ray absorptiometry. Femoral microarchitectural properties were assessed by microcomputed tomography. Femoral mRNA levels of selected bone matrix proteins were determined by northern blot analysis. Data showed that tibial BMD was significantly higher in the rat fed the +CN diet than those fed the -CN (control) diet. Dietary carnitine significantly decreased the mRNA level of tartrate-resistant acid phosphatase (TRAP), an indicator of bone resorption by 72.8%, and decreased the mRNA abundance of alkaline phosphatase (ALP) and collagen type-1 (COL), measures of bone formation by 63.6% and 61.2%, respectively. The findings suggest that carnitine supplementation slows bone loss and improves bone microstructural properties by decreasing bone turnover.

Hooshmand S, Balakrishnan A, Clark RM, Owen KQ…
Phytomedicine Aug 2008
PMID: 18539446 | Free Full Text

The part in red above is negative for bone growth. The full text notes:

Our data here showed that carnitine decreased the mRNA levels of TRAP as well as ALP and COL, suggesting that carnitine may suppress bone turnover by decreasing the rates of both bone resorption and formation.

Cinnamon Inhibits Osteoclasts In Vitro

Abstract

Aldehydic components of cinnamon bark extract suppresses RANKL-induced osteoclastogenesis through NFATc1 downregulation.

Several major bone diseases are directly attributable to bone loss, including osteoporosis, bone metastasis, and rheumatoid arthritis. The nuclear factor of activated T cell 1 (NFATc1), a transcription factor, has recently been shown to play an essential role in osteoclastogenesis. In this study, we found that of several herbs, Cinnamomum zeylanicum (C. zeylanicum) exhibited the strong inhibitory effects on osteoclastogenesis and that its mechanism of action involves the suppression of NFATc1-mediated signal transduction. C. zeylanicum dose-dependently inhibited osteoclast-like cell formation at concentrations of 12.5-50 microg/ml without affecting cell viability. Resorption pit assays have shown that C. zeylanicum also inhibits the bone-resorbing activity of mature osteoclasts. Treatment with C. zeylanicum inhibited the receptor activator of nuclear factor-kappaB ligand (RANKL)-induced NFATc1 and c-fos expression. Additionally, C. zeylanicum moderately inhibited phosphorylation of IkappaB-alpha, suggesting that the c-fos/NFATc1 pathway, rather than the nuclear factor-kappaB (NF-kappaB) pathway, is the primary target of C. zeylanicum during RANKL-induced osteoclastogenesis. Using an HPLC-DAD system, we identified three major peaks for four characteristic components in the C. zeylanicum extract and identified an unknown peak as 2-methoxycinnamaldehyde via HPLC and a 2D-COSY (1)H NMR study. We identified cinnamaldehyde and 2-methoxycinnamaldehyde as active components reducing osteoclast-like cell formation and inhibiting NFATc1 expression. Notably, in a resorption pit assay, 2-methoxycinnamaldehyde exhibited remarkable inhibition rates of 95% at 2 microM on bone resorption. In summary, this study points to the conclusion that C. zeylanicum inhibits RANKL-induced osteoclastogenesis. This finding raises prospects for the development of a novel approach in the treatment of osteopenic disease.

Tsuji-Naito K
Bioorg. Med. Chem. Oct 2008
PMID: 18823786

CLA Inhibits Osteoclasts in Mouse Cells

Abstract

Conjugated linoleic acid inhibits osteoclast differentiation of RAW264.7 cells by modulating RANKL signaling.

Bone destruction is a pathological hallmark of several chronic inflammatory diseases, including rheumatoid arthritis, periodontitis, and osteoporosis. Inflammation-induced bone loss of this sort results from increased numbers of bone-resorbing osteoclasts. Numerous studies have indicated that conjugated linoleic acid (CLA) positively influences calcium and bone metabolism. Gene-deletion studies have shown that receptor activator of nuclear factor-kappaB ligand (RANKL) is one of the critical mediators of osteoclastogenesis. In this report, we examine the ability of CLA to suppress RANKL signaling and osteoclastogenesis in RAW264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated nuclear factor-kappaB (NF-kappaB), and preexposure of the cells to CLA significantly suppressed RANKL-induced NF-kappaB activation, including phosphorylation of I-kappaBalpha, degradation of I-kappaBalpha, and nuclear translocation of p65. RANKL induced osteoclastogenesis in these monocytic cells, and CLA inhibited RANKL-induced tumor necrosis factor-alpha production and osteoclast differentiation, including osteoclast-specific genes such as tartrate-resistant acid phosphatase, cathepsin K, calcitonin receptor, and matrix metalloproteinase-9 expression and osteoclast-specific transcription factors such as c-Fos, nuclear factor of activated T-cells expression, and bone resorption pit formation. CLA also inhibited RANKL-induced activation of mitogen-activated protein kinase p38 but had little effect on c-Jun N-terminal kinase activation. Collectively, these data demonstrate for the first time that CLA inhibits osteoclastogenesis by modulating RANKL signaling. Thus, CLA may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption by excessive osteoclastogenesis.

Rahman MM, Bhattacharya A, Fernandes G
J. Lipid Res. Aug 2006
PMID: 16702601 | Free Full Text

CLA Isomers Promote Osteoblasts In Vitro

Abstract

Regulation of osteoblast and adipocyte differentiation from human mesenchymal stem cells by conjugated linoleic acid.

Conjugated linoleic acid (CLA) describes a group of isomers of linoleic acid and has variable effects on bone formation and adiposity in vivo and in vitro. The variability may be due to individual effects of the predominant bioactive 9cis,11trans (9,11) and 10trans,12cis (10,12) CLA isomers. Osteoblasts and adipocytes are derived from mesenchymal stem cells (MSCs), and bone loss is accompanied by an increase in marrow adiposity. Osteoblast differentiation from MSCs requires activation of Wnt/beta-catenin signaling by Wnt10b, which inhibits adipocyte differentiation by suppressing CCAAT/enhancer-binding protein (C/EBP) alpha. The objective of this study was to determine if 9,11 and 10,12 CLA affect osteoblast and adipocyte differentiation from MSCs and to determine whether any effects are associated with changes in Wnt10b and C/EBPalpha expression. Osteoblast differentiation was assessed by calcium deposition, alkaline phosphatase (ALP) activity, and the expression of Wnt10b, runx2 and osteocalcin. Adipocyte differentiation was assessed by oil red O staining and C/EBPalpha, PPARgamma and FABP4 expression. Compared to vehicle, 9,11 CLA decreased calcium deposition ( approximately 15%), increased oil red O staining ( approximately 21-28%) and increased FABP4 (AP2) expression ( approximately 58-75%). In contrast, 10,12 CLA increased calcium deposition ( approximately 12-60%), ALP activity ( approximately 2.1-fold) and the expression of Wnt10b ( approximately 60-80%) and osteocalcin ( approximately 90%), but decreased oil red O staining ( approximately 30%) and the expression of C/EBPalpha ( approximately 24-38%) and PPARgamma ( approximately 60%) (P<.05). Thus, our findings demonstrate isomer-specific effects of CLA on MSC differentiation, and suggest that 10,12 CLA may be a useful therapeutic agent to promote osteoblast differentiation from MSCs.

Platt ID, El-Sohemy A
J. Nutr. Biochem. Dec 2009
PMID: 19019668

CLA Prevents Bone Loss from Corticosteroids in Rats

Abstract

Conjugated linoleic acid prevents growth attenuation induced by corticosteroid administration and increases bone mineral content in young rats.

Corticosteroids are a common therapy in many disease states, despite frequent and potentially serious side effects. Nutritional supplementation with conjugated linoleic acid (CLA) has been shown to increase fat-free mass, whereas supplementation with n-3 and n-6 fatty acids has been shown to increase bone mineral density (BMD). To determine whether CLA can attenuate the side effects of 8 weeks of corticosteroid administration, we randomized twenty-four 5-week-old male Sprague-Dawley rats into 1 of 4 groups: control; control + methylprednisolone (7 mg.kg-1.week-1); CLA diet (1% CLA w/w); or CLA plus methylprednisolone. Body composition, bone mineral content (BMC), and BMD were assessed with dual-energy X-ray absorptiometry at the onset and at the end of the 8-week intervention. The mechanical properties of bone were determined using 3-point femur bending at the end of the intervention. Methylprednisolone resulted in an attenuation of the increase in body mass and lean mass over the 8 weeks (p < 0.05). CLA prevented the methylprednisolone-induced attenuation of body mass and lean mass accumulation. CLA also resulted in a greater increase in BMC (p < 0.05) in the lumbar spine. The energy at failure of the isolated femurs was increased with CLA (p < 0.05). Dietary CLA prevents many of the growth- and bone-related side effects arising from 8 weeks of corticosteroid administration, results in greater increases in BMC and BMD, and can contribute to an improvement in some of the mechanical properties of bone.

Roy BD, Bourgeois J, Rodriguez C, Payne E…
Appl Physiol Nutr Metab Dec 2008
PMID: 19088767

PQQ Inhibits Osteoclasts

Abstract

Inhibition of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation by pyrroloquinoline quinine (PQQ).

The effect of pyrroloquinoline quinine (PQQ) on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation was examined using RAW 264.7 macrophage-like cells. RANKL led to the formation of osteoclasts identified as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in the culture of RAW 264.7 cells. However, PQQ inhibited the appearance of osteoclasts and prevented the decrease of F4/80 macrophage maturation marker on RANKL-stimulated cells, suggesting a preventive action of PQQ on RANKL-induced osteoclast differentiation. PQQ inhibited the activation of nuclear factor of activated T cells (NFATc1), a key transcription factor of osteoclastogenesis, in RANKL-stimulated cells. On the other hand, PQQ did not inhibit the signaling pathway from RANK/RANKL binding to NFATc1 activation, including NF-κB and mitogen-activated protein kinases (MAPKs). PQQ augmented the expression of type I interferon receptor (IFNAR) and enhanced the IFN-β-mediated janus kinase (JAK1) and signal transducer and activator of transcription (STAT1) expression. Moreover, PQQ reduced the expression level of c-Fos leading to the activation of NFATc1. Taken together, PQQ was suggested to prevent RANKL-induced osteoclast formation via the inactivation of NFATc1 by reduced c-Fos expression. The reduced c-Fos expression might be mediated by the enhanced IFN-β signaling due to augmented IFNAR expression.

Odkhuu E, Koide N, Haque A, Tsolmongyn B…
Immunol. Lett. Feb 2012
PMID: 22193059