Monthly Archives: September 2013

Isoflavones More Effective Than Pure Genistein in Ovariectomized Rats

Abstract

Comparison of the bone protective effects of an isoflavone-rich diet with dietary and subcutaneous administrations of genistein in ovariectomized rats.

Administration of the isoflavone genistein (GEN) has been described to result in bone protection but also to induce uterotrophic responses. To compare bone protective effects of GEN with an isoflavone-rich diet (IRD) and to further elucidate molecular mechanisms involved in bone-protection, ovariectomized rats (OVX) received either a diet low in isoflavone content (IDD) enriched with GEN (42 mg kg(-1)b.wtd(-1)) (GEN(d)), an IRD (14 mg kg(-1)b.wtd(-1) GEN, 14 mg kg(-1)b.wtd(-1) daidzein) or were treated subcutaneously (s.c.) with GEN (10 mg kg(-1)b.wtd(-1)) (GEN(sc)) for 12 weeks. Intact (SHAM), vehicle treated OVX animals and those substituted with 17beta-estradiol (2microg kg(-1)b.wtd(-1)) (E(2)), served as controls. OVX-induced bone loss could be antagonized in E(2), GEN(sc), GEN(d) and IRD groups. Uterine wet weight (UWW) was only stimulated in E(2) and GEN(sc) animals. Serum biomarkers of bone-formation (osteocalcin, osteopontin) and bone-resorption (telopeptides of collagen type I, pyridinoline cross-links) were elevated in OVX compared to SHAM and E(2) animals. Feeding IRD stimulated bone-formation and inhibited bone-resorption, whereas s.c. or dietary administration of GEN only resulted in a stimulation of bone-formation. The results of the present study indicate that in contrast to s.c. administration, dietary intake of GEN resulted in bone protection without stimulation of UWW. Dietary intake of isoflavones by an IRD also did not result in a stimulation of UWW, yet IRD appeared to be more effective in bone protection than administration of pure GEN.

Hertrampf T, Schleipen B, Offermanns C, Velders M…
Toxicol. Lett. Feb 2009
PMID: 19063953

Review: Isoflavone Optimal Intake is 50-90mg

Abstract

Investigating the optimal soy protein and isoflavone intakes for women: a perspective.

Traditional soyfoods have been consumed for centuries throughout much of East Asia and, recently, these foods have also become popular in the West. Soyfoods and specific soybean components, such as the protein and isoflavones, have attracted attention for their possible health benefits. Isoflavones are classified as phytoestrogens and have been postulated to be natural alternatives to hormone therapy for menopausal women. To provide guidance on optimal soy intake, this article evaluates Asian soy consumption and both clinical and Asian epidemiologic studies that examined the relationship between soy intake and a variety of health outcomes. On the basis of these data and the standard principles of dietary practice the author suggests that optimal soy protein and isoflavone intakes are 15-20 g/day and 50-90 mg/day, respectively. In addition, an intake of 25 g/day soy protein can be specifically used as the recommendation for cholesterol reduction.

Messina M
Womens Health (Lond Engl) Jul 2008
PMID: 19072500

Genistein May Work by Suppressing Inflammation in Ovariectomized Rats

Abstract

The phytoestrogen genistein reduces bone loss in short-term ovariectomized rats.

The incidence of fractures and of osteoporosis differs between Oriental and Western Caucasian women. This may depend, at least in part, on nutritional factors, including dissimilarities in dietary intake of phytoestrogens. To investigate this possibility, 2-month-old female rats were ovariectomized (OVX) or sham-operated (SHAM), fed a casein-based diet, injected daily with subcutaneous genistein (GEN), the most abundant and best characterized phytoestrogen, or vehicle (Veh) and killed 21 days after surgery. As expected, ovariectomy resulted in loss of bone mineral density (BMD) and in uterine atrophy. However, administration of 5 micrograms GEN per gram body weight (b.w.) ameliorated the ovariectomy-induced loss of BMD (189 +/- 2 mg/cm2 in OVX and 192 +/- 2 in OVX with 5 micrograms GEN/g b.w. per day; p < 0.05). One microgram GEN per gram body weight did not affect the BMD loss and the effect of the 5 micrograms and 25 micrograms GEN per gram body weight were statistically not different. A trend toward reduced uterine atrophy (21% reduction) was noted with the 25 micrograms GEN dose, but not with the 1 microgram and 5 micrograms doses. A separate experiment with 2 x 2 factorial design was conducted to elucidate the mechanism by which GEN ameliorates ovariectomy-induced bone loss. In this experiment, histomorphometry demonstrated a dramatic reduction in trabecular bone volume after ovariectomy (7.6 +/- 0.7% of total bone volume in SHAM-Veh vs 3.3 +/- 0.2% in OVX-Veh; p < 0.01) and less bone loss in OVX rats injected with 5 micrograms GEN per gram per day (3.3 +/- 0.2% of total bone volume in OVX-Veh vs 5.2 +/- 0.4% in OVX-GEN; p < 0.01). Administration of GEN was associated with higher bone formation rate per tissue volume and with a trend toward a higher number of osteoblasts per bone perimeter. The parameters of bone resorption were not affected by GEN. The concentration of serum osteocalcin and the urinary excretion of deoxypyridinoline provided corroborating results. Since production of proinflammatory cytokines is intimately involved in the pathogenesis of postmenopausal osteoporosis, the effect of GEN on lipopolysaccharide-induced in vitro production of Tumor necrosis factor-alpha (TNF alpha) was tested in monocytic cells from the same four rat groups. Production of TNF alpha was markedly elevated in OVX-Veh as compared with the SHAM-Veh rats, but this was blocked by GEN in the OVX rats. This study shows that GEN reduces both trabecular and compact bone loss after ovariectomy and that this protective effect differs from that of estrogen, since it depends on stimulation of bone formation rather than on suppression of bone resorption. Lack of action of GEN on uterine atrophy supports the possibility that this GEN dose affects target tissues via non-estrogenic mechanisms. Modulation of cytokine production may be involved in the effect of GEN on bone.

Fanti P, Monier-Faugere MC, Geng Z, Schmidt J…
Osteoporos Int 1998
PMID: 9797913

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