Effect of Tart Cherry Polyphenols on Osteoclast Differentiation and Activity.

Bone is maintained by an intricate balance between bone formation and bone resorption. The presence of inflammation can contribute to an imbalance in bone homeostasis by enhancing differentiation and activity of osteoclasts, the cells that participate in the breakdown of bone. Polyphenols such as flavonoids found in plant-derived foods have been shown to have anti-inflammatory effects in various tissues. Tart cherries are a rich source of such polyphenolic compounds. Using mouse macrophage cells (RAW 264.7), we examined whether tart cherry polyphenols could dose dependently inhibit the proliferation and activity of receptor activator of nuclear factor kappa-B ligand (RANKL) differentiated osteoclasts under inflammatory conditions. Tartrate-resistant acid phosphatase (TRAP) activity and staining of TRAP-positive multinucleated cells, used as indicators of osteoclast differentiation and activity, tended to decrease with tart cherry polyphenols treatment. Osteoprotegerin expression by osteoclasts was decreased in a similar manner. A significant increase in nitrite concentration was observed with the lower doses of tart cherry polyphenols of 50 and 100 µg/mL (P < .05). However, higher doses of tart cherry polyphenols (200 and 300 µg/mL) reduced nitrite concentrations below that of the control that received no tart cherry polyphenols treatment (P < .05). Western blot analyses showed that protein expression of cyclooxygenase-2 (COX-2) followed a similar trend, although results were not statistically significant. On the other hand, tart cherry polyphenols treatments dose dependently increased inducible nitric oxide synthase protein expression, with statistical significance noted at doses of 200 and 300 µg/mL. Overall, our findings suggest that the polyphenols associated with tart cherries potentially inhibit osteoclast differentiation and activity, which may be beneficial to bone health.

Anti-Inflammatory Action of Blueberry Polyphenols in HIG-82 Rabbit Synoviocytes.

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease resulting in joint destruction and disability in the adult population. The etiology of RA is not well understood and presently there is no known cure for this disease. The accumulation and proliferation of fibroblast-like synoviocytes may be involved in cartilage destruction. Both in vitro and in vivo studies support an anti-inflammatory role of dietary polyphenols, the bioactive constituents found in fruits and vegetables. The objective of this study was to investigate the anti-inflammatory role of blueberry polyphenols (BBPs) using rabbit synoviocytes stimulated with tumor necrosis factor alpha (TNFα). Rabbit synoviocytes (HIG-82) were treated with varying doses of BBPs and stimulated with TNFα. Stimulation of rabbit synoviocytes with the proinflammatory cytokine TNFα increased cell proliferation by ∼19% compared with the nonstimulated control. Cell proliferation was significantly decreased in a dose-dependent manner by the treatment with BBPs. Post-TNFα stimulation, cells treated with BBPs resulted in decreases in interleukin 1 beta and nuclear factor kappa B (NFκB) concentration. Reverse transcription-polymerase chain reaction analysis showed that matrix metalloproteinase 3 increased fivefold in the control TNFα-stimulated group, but was decreased by threefold in the blueberry treatment group. These results suggest that downregulation of proinflammatory cytokines and transcription factor NFκB by naturally occurring bioactives such as BBPs may be a potential therapeutic strategy for reducing inflammation associated with RA.

Effect of blueberry polyphenols on 3T3-F442A preadipocyte differentiation.

Today obesity is an epidemic, and its prevalence has increased significantly over the last few decades. To avoid excessive accumulation of fat, optimum energy intake along with regular exercise is mandatory. Polyphenols present in green tea, grape seeds, orange, and grapefruit combat adipogenesis at the molecular level and also induce lipolysis. However, very little is known regarding the role of blueberry polyphenols on adipocyte differentiation. Hence we tested the dose-dependent effects of blueberry polyphenols on mouse 3T3-F442A preadipocyte differentiation and lipolysis. 3T3-F442A preadipocytes were incubated with three doses of blueberry polyphenols (150, 200, and 250 µg/mL [BB-150, BB-200, and BB-250, respectively]), and intracellular lipid content, cell proliferation, and lipolysis were assayed. Blueberry polyphenols suppressed adipocyte differentiation determined by Oil Red-O staining and AdipoRed assay. Intracellular lipid content in control (11,385.51±1,169.6 relative fluorescence units) was significantly higher (P<.05) than with the three doses of blueberry polyphenols (8336.86±503.57, 4235.67±323.17, and 3027.97±346.61, respectively). This corresponds to a reduction of 27%, 63%, and 74%, respectively. Cell proliferation was observed to be significantly higher in the control (0.744±0.035 optical density units) than with BB-150 (0.517±0.031), BB-200 (0.491±0.023), and BB-250 (0.455±0.012). However, when tested for lipolysis, there was no significant difference observed among the groups. We conclude that blueberry polyphenols may play an effective role in inhibiting adipogenesis and cell proliferation.

Combination of genistin and fructooligosaccharides prevents bone loss in ovarian hormone deficiency.

We have reported that soy isoflavones are capable of preventing loss of bone mineral density (BMD) in rats due to ovariectomy. The intestinal microflora is important in rendering soy isoflavones bioavailability by facilitating their conversion to equol. Hence, substances that can modulate the intestinal microflora could affect the bioavailability of isoflavones. The purpose of this study was to examine whether combination of genistin and fructooligosaccharides (FOS), a prebiotic, can enhance the effects of soy isoflavones on bone in ovariectomized (OVX) female rats. Forty-eight 90-day-old female Sprague-Dawley rats were either sham-operated (Sham; one group) or Ovx (three groups) and were placed on dietary treatment for 50 days. The Sham and one Ovx group received a control diet, and the remaining Ovx groups received genistin-rich isoflavones diet (Ovx+G) or genistin-rich isoflavones and FOS diet (Ovx+G+FOS). After 50 days, blood and bone specimens were collected for analysis. The genistin-rich isoflavones diet was able to significantly increase the whole-body, right femur, and fourth lumbar BMD by 1.6%, 1.48%, and 1.3%, respectively in comparison with the Ovx control. The combination of genistin-rich isoflavones diet and 5% FOS further increased whole-body, right femur, and fourth lumbar BMD more compared to the genistin-rich isoflavones diet. Our findings suggest that although a genistin-rich isoflavones diet can increase the BMD in rats with Ovx-induced bone loss, combination of genistin-rich isoflavones and FOS had greater effect in preventing bone loss in this rat model.

Effect of vitamin E on lipid parameters in ovariectomized rats.

The risk of cardiovascular disease drastically increases at the onset of menopause, in part, because of rise in blood cholesterol and unfavorable changes in lipid profile. This study was designed to investigate the dose-dependent effects of vitamin E supplementation on lipid parameters in ovariectomized (ovx) rats. Sixty 12-month-old female Sprague-Dawley rats were either sham-operated (sham; one group) or ovx (four groups). All rats were maintained on a semipurified caseinbased diet (AIN-93M; 75 IU vitamin E/kg of diet) for a period of 120 days. Thereafter, ovx rats were placed on one of four doses of vitamin E treatment (75, 300, 525, or 750 IU vitamin E/kg of diet), while the sham group was continued on 75 IU vitamin E/kg of diet for 100 days. Ovariectomy tended to increase (by 24%, P = 0.1) serum non?high-density lipoprotein (HDL) cholesterol and decrease (by 14%, P = 0.1) HDL cholesterol. Vitamin E did not have any significant effects on serum lipid parameters. Liver total lipids were notably increased (P < .001) in ovx animals, and supplementation with vitamin E at 525 IU/kg of diet was able to significantly reduce liver total lipids by 13%. Additionally, ovariectomy caused an increase in serum glucose and liver C18:1 fatty acid concentrations along with decreases in C18:0, C20:4, and C22:6 fatty acid concentrations. These alterations on liver fatty acid profiles were unaffected by vitamin E. The findings of this study suggest that vitamin E supplementation moderately improves lipid parameters in ovarian hormone-deficient rats.

Ipriflavone modulates IGF-I but is unable to restore bone in rats.

Previously it has been reported that ipriflavone can prevent bone loss in ovarian hormone deficient rats. The present study evaluated whether ipriflavone was able to restore bone mass in osteopenic ovariectomized rats. Seventy-two, 90 day-old Sprague-Dawley rats were divided into six groups (sham two groups; ovariectomized four groups). Thirty-five days from the date of surgery, one sham and one ovx group were killed to verify the occurrence of bone loss. The remaining four groups were sham, ovx, ovx + ipriflavone (100 mg[sol ]kg body weight per day), or ovx + 17beta-estradiol (10 microg[sol ]kg body weight daily) for a period of 65 days. Ipriflavone was ineffective in restoring bone density and unlike estrogen did not prevent bone resorption as evidenced by increased (p < 0.05) urinary excretion of hydroxyproline and serum tartrate-resistant acid phosphatase activity. Ipriflavone increased (p < 0.05) the expression of IGF-I in the femur. These observations suggest that higher doses of ipriflavone or longer-term studies may be necessary to restore bone mass.

Soy protein has a greater effect on bone in postmenopausal women not on hormone replacement therapy, as evidenced by reducing bone resorption and urinary calcium excretion.

Recent reports suggest that soy protein may reduce the risk of osteoporosis in peri- and postmenopausal women. The objective of this study was to examine whether soy supplementation exerts beneficial effects on serum and urinary biomarkers of bone metabolism in postmenopausal women, regardless of whether or not they are on hormone replacement therapy (HRT). A total of 71 women were randomly assigned to either soy protein (SP) or milk-based protein (MBP), 40 g daily for 3 months, in a double-blind parallel design. Forty-two women completed the study (20 on SP and 22 on MBP). Overall, both protein supplements positively influenced serum IGF-I, known to correlate with bone formation. However, SP had a more pronounced effect on IGF-I than MBP. Urinary deoxypyridinoline (Dpd) excretion, a specific biomarker of bone resorption, was significantly reduced by SP, but not by MBP when all women were included. Furthermore, women on MBP experienced a 33% increase in urinary calcium excretion, whereas SP did not have such an effect. To evaluate whether SP affects women differently on the basis of their HRT status, data from women on HRT (n = 22) and those not on HRT (n = 20) were analyzed separately. The subanalysis of the data indicated that SP had the greatest impact on serum IGF-I (an increase of 97%) in the women not on HRT. The changes in urinary Dpd due to SP were only observed in women not on HRT, indicating that the overall decrease in Dpd occurred with SP in the absence of HRT. These results indicate that soy protein may positively influence bone and calcium homeostasis in postmenopausal women, particularly those not on HRT.

Soy protein supplementation increases serum insulin-like growth factor-I in young and old men but does not affect markers of bone metabolism.

Recent studies suggest that soy protein (SP) protects bone in women; however, its effects on bone metabolism in men have not been investigated. Healthy men (59.2 +/- 17.6 y) were assigned to consume 40 g of either SP or milk-based protein (MP) daily for 3 mo in a double-blind, randomized, controlled, parallel design. Serum insulin-like growth factor-I (IGF-I), which is associated with higher rates of bone formation, was greater (P < 0.01) in men supplemented with SP than in those consuming MP. Serum alkaline phosphatase and bone-specific alkaline phosphatase activities, markers of bone formation, and urinary deoxypyridinoline excretion, a specific marker of bone resorption, were not different between the SP and MP groups. Furthermore, because substantial reductions in bone density occur in men at approximately 65 y of age, data were analyzed separately for men >/=65 y and those <65 y of age. The response to protein supplementation was consistent in the two age groups. The effects of SP on serum IGF-I levels suggest that SP may positively influence bone in men. Longer-duration studies examining the effects of SP or its isoflavones on bone turnover and bone mineral density and content in men are warranted.

Vitamin E improves bone quality in the aged but not in young adult male mice.

It is generally viewed that with advancing age, humans and other animals including mice experience a gradual decline in the rate of bone formation. This, in part, may be due to the rise in oxygen-derived free radical formation. Vitamin E, a strong antioxidant, functions as a free radical scavenger that potentially can suppress bone resorption while stimulating bone formation. Although the effects of vitamin E on immune functions are well documented, there is a paucity of information on its effect on skeletal health in vivo. The purpose of this study was to explore the influence of vitamin E supplementation on bone in young adult and old mice. Six and twenty-four month-old male C57BL/6NIA mice each were divided into two groups and fed a diet containing either adequate (30 mg/kg diet) or high (500 mg/kg diet) levels of vitamin E. Thirty days later, mice were killed and bones were removed for analyses including biomechanical testing using three-point bending and mRNA expressions of insulin-like growth factor-I (IGF-I), osteocalcin, and type 1alpha-collagen using Northern blot. In old but not the young adult mice, high-dose vitamin E enhanced bone quality as evident by improved material and structural bone properties in comparison with adequate. This improved quality was accompanied by increases in bone dry weight, protein, and mRNA transcripts for osteocalcin, type Ialpha-collagen, and IGF-I. These data demonstrate that high-dose vitamin E has pronounced effects on bone quality as well as matrix protein in old mice by augmenting bone matrix protein without reducing bone mineralization as evidenced by unaltered bone density.

Flaxseed improves lipid profile without altering biomarkers of bone metabolism in postmenopausal women.

The risk of cardiovascular disease and osteoporosis drastically increases at the onset of menopause. Phytoestrogens have been suggested to inhibit bone loss and protect the cardiovascular system, in part by improving lipid profiles. The purpose of the present study was to examine the effects of flaxseed, a rich source of the phytoestrogens called lignans, on lipid metabolism and biomarkers of bone turnover in postmenopausal women. Postmenopausal women who were not on hormone replacement therapy were assigned to one of two treatment groups in a double-blind randomized study. Women were asked to consume 40 g of either ground flaxseed or wheat-based comparative control regimen daily for 3 months. In addition, all subjects received 1,000 mg calcium and 400 IU vitamin D daily. Flaxseed supplementation lowered (P < 0.05) both serum total cholesterol and non-high-density lipoprotein cholesterol by 6%, whereas the comparative control regimen had no such effect. Flaxseed regimen reduced serum levels of both low-density- and high-density-lipoprotein cholesterol by 4.7% and triglyceride by 12.8%, albeit not statistically significant. Serum apolipoprotein A-1 and apolipoprotein B concentrations were significantly (P < 0.005) reduced by 6 and 7.5%, respectively, by the flaxseed regimen. Markers of bone formation and resorption were not affected by either of the treatments. The findings of this study indicate that flaxseed supplementation improves lipid profiles but has no effect on biomarkers of bone metabolism in postmenopausal women.