Midlife women, bone health, vegetables, herbs and fruit study. The Scarborough Fair study protocol

BACKGROUND: Bone loss is accelerated in middle aged women but increased fruit/vegetable intake positively affects bone health by provision of micronutrients essential for bone formation, buffer precursors which reduce acid load and phytochemicals affecting inflammation and oxidative stress. Animal studies demonstrated bone resorption inhibiting properties of specific vegetables, fruit and herbs a decade ago. Objective: To increase fruit/vegetable intake in post menopausal women to 9 servings/day using a food specific approach to significantly reduce dietary acid load and include specific vegetables, fruit and herbs with bone resorbing inhibiting properties to assess effect on bone turnover, metabolic and inflammatory markers. METHODS/DESIGN: The Scarborough Fair Study is a randomised active comparator controlled multi centre trial. It aimed to increase fruit and vegetable intake in 100 post menopausal women from ≤ 5 servings/day to ≥ 9 servings/day for 3 months. The women in the dietary intervention were randomly assigned to one of the two arms of the study. Both groups consumed ≥ 9 servings/day of fruit/vegetables and selected herbs but the diet of each group emphasised different fruit/vegetables/herbs with one group (B) selecting from a range of vegetables, fruit and culinary herbs with bone resorbing inhibiting properties. 50 women formed a negative control group (Group C usual diet). Primary outcome variables were plasma bone markers assessed at baseline, 6 weeks and 12 weeks. Secondary outcome variables were plasma inflammation and metabolic markers and urinary electrolytes (calcium, magnesium, potassium and sodium) assessed at baseline and 12 weeks. Dietary intake and urine pH change also were outcome variables. The dietary change was calculated with 3 day diet diaries and a 24 hour recall. Intervention participants kept a twice weekly record of fruit, vegetable and herb intake and urine pH. DISCUSSION: This study will provide information on midlife women’s bone health and how a dietary intervention increasing fruit and vegetable/herb intake affects bone, inflammatory and metabolic markers and urinary electrolyte excretion. It assesses changes in nutrient intake, estimated dietary acid load and sodium: potassium ratios. The study also explores whether specific fruit/vegetables and herbs with bone resorbing properties has an effect on bone markers. TRIAL REGISTRATION: ACTRN 1261100076394

Geraniol attenuates osteoclast differentiation by suppressingNF-kB activity and expression of osteoclastogenic genes

Osteoporotic patients have lower bone mass due to increased bone resorption by osteoclasts. The aim of this study was to investigate the cytotoxic and antiosteoclastogenic effects of geraniol, a natural monoterpene on human CD14+ monocytes (ex vivo) and murine RAW264.7 macrophages (in vitro) using alamar blue and tartrate resistant acid phosphatase staining respectively. The anti-osteoclastogenic activity of geraniol was further explored by analyzing its effects on actin ring formation and bone resorptive function of osteoclasts. Geraniol significantly (p < 0.001) inhibited osteoclast formation in CD14+ monocytes and RAW264.7 macrophages without cytotoxicity. Moreover, reduced osteoclastogenesis in these cells led to an arrest in actin ring formation and diminished bone resorption. Analysis of underlying molecular mechanisms revealed that geraniol alleviated NF-kB activity, an indispensable upstream modulator of osteoclast formation. Furthermore, expression of key osteoclastogenic genes such as dendritic cell-specific transmembrane protein (DC-STAMP) involved in cell-cell fusion and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), a master transcription factor essential for osteoclast differentiation was downregulated by geraniol. These observations indicate that inhibition of osteoclast differentiation is presumably one of the pharmacological properties of geraniol.Grants from the University of Pretoria Vice Chancellor’s Postdoctoral Research Fellowship; RESCOM, University of Pretoria and the University of Pretoria’s Strategic Institutional Research Theme in Food, Nutrition and Well-being.http://link.springer.com/journal/442017-12-31hb2016Food ScienceHuman NutritionPhysiolog

Factors associated with bone mineral density and bone resorption markers in postmenopausal hiv-infected women on antiretroviral therapy: A prospective cohort study

The study aimed to determine factors associated with changes in bone mineral density (BMD) and bone resorption markers over two years in black postmenopausal women living with human immunodeficiency virus (HIV) on antiretroviral therapy (ART). Women (n = 120) aged > 45 years were recruited from Potchefstroom, South Africa. Total lumbar spine and left femoral neck (LFN) BMD were measured with dual energy X-ray absorptiometry. Fasting serum C-Telopeptide of Type I collagen (CTx), vitamin D and parathyroid hormone were measured. Vitamin D insufficiency levels increased from 23% at baseline to 39% at follow up. In mixed linear models serum CTx showed no change from baseline to end (p = 0.363, effect size = 0.09). Total and LFN BMD increased significantly over two years, but effect sizes were small. No significant change in spine BMD over time was detected (p = 0.19, effect size = 0.02). Age was significantly positively associated with CTx over time, and negatively with total and LFN BMD. Physical activity (PA) was positively associated with LFN BMD (p = 0.008). Despite a decrease in serum vitamin D, BMD and CTx showed small or no changes over 2 years. Future studies should investigate PA interventions to maintain BMD in women living with HIV

Study Protocol – Metabolic syndrome, vitamin D and bone status in South Asian women living in Auckland, New Zealand: A randomised, placebo-controlled, double-blind vitamin D intervention

<p>Abstract</p> <p>Background</p> <p>The identification of the vitamin D receptor in the endocrine pancreas suggests a role for vitamin D in insulin secretion. There is also some limited evidence that vitamin D influences insulin resistance, and thus the early stages of the development of type 2 diabetes.</p> <p>Methods</p> <p>Eighty-four women of South Asian origin, living in Auckland, New Zealand, were randomised to receive either a supplement (4000IU 25(OH)D₃per day) or a placebo for 6 months. At baseline, all participants were vitamin D deficient (serum 25(OH)D₃<50 nmol/L), insulin resistant (HOMA-IR > 1.93) and/or hyperinsulinaemic, hyperglycemic or had clinical signs of dislipidaemia. Changes in HOMA-IR, lipids, parathyroid hormone, calcium and bone markers were monitored at 3 months and 6 months.</p> <p>Discussion</p> <p>This randomised, controlled trial will be the first to investigate the effect of vitamin D supplementation on insulin resistance in non-diabetic subjects. It will subsequently contribute to the growing body of evidence about the role of vitamin D in metabolic syndrome.Registered clinical.</p> <p>Trial registration</p> <p>Registered clinical trial – Registration No. ACTRN12607000642482</p

Inhibitory effects of eugenol on RANKL-induced osteoclast formation via attenuation of NF-kappa B and MAPK pathways

Bone loss diseases are often associated with increased receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation. Compounds that can attenuate RANKL-mediated osteoclast formation are of great biomedical interest. Eugenol, a phenolic constituent of clove oil possesses medicinal properties; however, its anti-osteoclastogenic potential is unexplored hitherto. Here, we found that eugenol dose-dependently inhibited the RANKL-induced multinucleated osteoclast formation and TRAP activity in RAW264.7 macrophages. The underlying molecular mechanisms included the attenuation of RANKL-mediated degradation of IκBα and subsequent activation of NF-κB pathway. Furthermore, increase in phosphorylation and activation of RANKL-induced mitogen-activated protein kinase pathways (MAPK) was perturbed by eugenol. RANKL-induced expression of osteoclast-specific marker genes such as TRAP, cathepsin K (CtsK) and matrix metalloproteinase-9 (MMP-9) was remarkably downregulated by eugenol. These findings provide the first line of evidence that eugenol mediated attenuation of RANKL-induced NF-κB and MAPK pathways could synergistically contribute to the inhibition of osteoclast formation. Eugenol could be developed as therapeutic agent against diseases with excessive osteoclast activity.The Vice Chancellor’s Postdoctoral Research Fellowship and Institute for Food, Nutrition and Well-being, University of Pretoria.http://informahealthcare.com/journal/cts2016-06-30hb201

Arachidonic acid and Docosahexaenoic acid suppress osteoclast formation and activity in human CD14+ monocytes, in vitro

An unbalanced diet can have adverse effects on health. Long chain polyunsaturated fatty acids (LCPUFAs) have been the focus of research owing to their necessity of inclusion in a healthy diet. However, the effects of LCPUFAs on human osteoclast formation and function have not been explored before. A human CD14+ monocyte differentiation model was used to elucidate the effects of an ω-3 LCPUFA, docosahexaenoic acid (DHA), and an ω-6 LCPUFA, arachidonic acid (AA), on osteoclast formation and activity. CD14+ monocytes were isolated from peripheral blood of healthy donors and stimulated with macrophage colony stimulating factor and receptor activator of nuclear factor kappa-B ligand to generate osteoclasts. Data from this study revealed that both the LCPUFAs decreased osteoclast formation potential of CD14+ monocytes in a dose-dependent manner when treated at an early stage of differentiation. Moreover, when exposed at a late stage of osteoclast differentiation AA and DHA impaired the bone resorptive potential of mature osteoclasts without affecting osteoclast numbers. AA and DHA abrogated vitronectin receptor expression in differentiating as well as mature osteoclasts. In contrast, the degree of inhibition for calcitonin receptor expression varied between the LCPUFAs with only AA causing inhibition during osteoclast differentiation. Furthermore, AA and DHA down regulated the expression of key osteoclast-specific genes in differentiating as well as mature osteoclasts. This study demonstrates for the first time that LCPUFAs can modulate osteoclast formation and function in a human primary osteoclast cell line.S1 Fig. Effects of AA and DHA on cell viability. CD14+ monocytes were treated with indicated concentrations of AA and DHA for 48 h and cell viability was measured by alamar blue assay. The results are representative of two independent experiments conducted in triplicate and expressed as percentage cell viability relative to the control.The South African Medical Research Council (MRC), the University of Pretoria School of Medicine Research Committee (RESCOM) and the University of Pretoria Postgraduate Study Abroad Programme.http://www.plosone.orgam201

Piperine alleviates osteoclast formation through the p38/c-Fos/NFATc1 signaling axis

Increased bone fracture is one of the health risk factors in patients with bone loss related disorders such as osteoporosis and breast cancer metastasis to bone. Over activity of osteoclasts leads to uncoupling of bone remodeling favoring bone loss over bone formation. Receptor activator of nuclear factor-κβ ligand (RANKL) triggers the differentiation pathway leading to multinucleated osteoclast formation. Modulation of RANKL or its downstream signaling pathways involved in osteoclast formation is of significant interest in the development of anti-resorptive agents. In this study, we investigated the effects of piperine, an alkaloid present in Piper nigrum L. on osteoclast formation. Piperine inhibited tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclast formation in murine RAW264.7 macrophages and human CD14+ monocytes induced by RANKL and breast cancer cells. Piperine attenuated the p38-mitogen activated protein kinase (MAPK) pathway activation, while the extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) or NF-κβ pathways downstream of RANKL remained unaffected. Concomitantly, expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), the key transcription factors involved in osteoclastogenesis were remarkably inhibited by piperine. Furthermore, piperine disrupted the actin ring structure and bone resorption, a characteristic hallmark of osteoclasts. Collectively, these results suggest that piperine inhibits osteoclast differentiation by suppressing the p38/NFATc1/c-Fos signaling axis.RESCOM, University of Pretoria, the University of Pretoria Vice Chancellor’s Postdoctoral Research Fellowship, the University of Pretoria’s Strategic Institutional Research Theme in Food, Nutrition and Well-being and the Struwig-Germeshuysen Research Trust, South Africa.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1872-80812016-11-30hb201

Ellagic acid inhibits RANKL-induced osteoclast differentiation by suppressing the p38 MAP kinase pathway

Bone undergoes continuous remodeling by a coupled action between osteoblasts and osteoclasts. During osteoporosis, osteoclast activity is often elevated leading to increased bone destruction. Hence, osteoclasts are deemed as potential therapeutic targets to alleviate bone loss. Ellagic acid (EA) is a polyphenol reported to possess anticancer, antioxidant and anti-inflammatory properties. However, its effects on osteoclast formation and function have not yet been examined. Here, we explored the effects of EA on RANKL-induced osteoclast differentiation in RAW264.7 murine macrophages (in vitro) and human CD14+monocytes (ex vivo). EA dose-dependently attenuated RANKL-induced TRAP+ osteoclast formation in osteoclast progenitors with maximal inhibition seen at 1 µM concentration without cytotoxicity. Moreover, owing to perturbed osteoclastogenesis, EA disrupted actin ring formation and bone resorptive function of osteoclasts. Analysis of the underlying molecular mechanisms revealed that EA suppressed the phosphorylation and activation of the p38 MAP kinase pathway which subsequently impaired the RANKL-induced differentiation of osteoclast progenitors. Taken together, these novel results indicate that EA alleviates osteoclastogenesis by suppressing the p38 signaling pathway downstream of RANKL and exerts inhibitory effects on bone resorption and actin ring formation.RESCOM, University of Pretoria; the University of Pretoria’s Strategic Institutional Research Theme in Food, Nutrition and Well-being; and in part by the University of Pretoria Vice Chancellor’s Postdoctoral Research Fellowship.http://link.springer.com/journal/122722018-01-31hb2017Human NutritionPhysiolog

Palmitoleic acid inhibits RANKL-induced osteoclastogenesis and bone resorption by suppressing NF- B and MAPK signalling pathways

Osteoclasts are large,multinucleated cells that are responsible for the breakdown or resorption of bone during bone remodelling. Studies have shown that certain fatty acids (FAs) can increase bone formation, reduce bone loss, and influence total bone mass. Palmitoleic acid (PLA) is a 16-carbon, monounsaturated FA that has shown anti-inflammatory properties similar to other FAs. The effects of PLA in bone remain unexplored. Here we investigated the effects of PLA on receptor activator of nuclear factor kappa B (NF- B) ligand (RANKL)-induced osteoclast formation and bone resorption in RAW264.7 murine macrophages. PLA decreased the number of large, multinucleated tartrate resistant acid phosphatase (TRAP) positive osteoclasts and furthermore, suppressed the osteolytic capability of these osteoclasts. This was accompanied by a decrease in expression of resorption markers (Trap, matrix metalloproteinase 9 (Mmp9), cathepsin K (Ctsk)). PLA further decreased the expression of genes involved in the formation and function of osteoclasts. Additionally, PLA inhibited NF- B activity and the activation of mitogen activated protein kinases (MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal–regulated kinase (ERK). Moreover, PLA induced apoptosis in mature osteoclasts. This study reveals that PLA inhibits RANKL-induced osteoclast formation in RAW264.7 murine macrophages through suppression of NF- B and MAPK signalling pathways. This may indicate that PLA has potential as a therapeutic for bone diseases characterized by excessive osteoclast formation.The South African Medical Research Council (SAMRC)—Grantholder M. Coetzee.http://www.mdpi.com/journal/nutrientsam2017Food SciencePhysiolog

Ferulic acid impairs osteoclast fusion and exacerbates survival of mature osteoclasts

Elevated bone loss induced by osteoclasts is a critical and most commonly observed pathological complication during osteolytic diseases such as osteoporosis. Hence, attenuation of osteoclast formation or function is a classical therapeutic approach to regulate bone loss. In this study, we found that ferulic acid (FA), a natural compound potently inhibited osteoclast formation in human CD14+ peripheral blood monocytes (PBMCs) ex vivo with an IC50 of 39 μM.Moreover, due to impaired differentiation of osteoclast progenitors, actin ring formation and bone resorption activity were also perturbed. Investigation of underlying molecular mechanisms revealed that FA inhibited the RANKL-induced expression of dendritic cell-specific transmembrane protein (DC-STAMP), a critical regulator of osteoclast fusion. In addition, expression of matrix metalloproteinase-9 (MMP-9) and cathepsin K (CTSK), the key osteoclast specific lysosomal proteases involved in bone matrix resorption were severely aggravated by FA. A significant reduction in mature osteoclast numbers was detected in the presence of FA accompanied by increased caspase-3 activity and DNA-fragmentation, a characteristic hallmark of apoptosis. Collectively, these results suggested that FA inhibited osteoclast fusion by suppressing the expression of DC-STAMP and induced apoptosis in mature osteoclasts by the caspase-3 pathway.Grants from RESCOM, University of Pretoria; the University of Pretoria’s Strategic Institutional Research Theme in Food, Nutrition and Well-being; and in part by the University of Pretoria Vice Chancellor’s Postdoctoral Research Fellowship.http://link.springer.com/journal/106162017-10-31hb2016Human NutritionPhysiolog