Effects of tocopherols and tocotrienols on body composition and bone calcium content in adrenalectomized rats replaced with dexamethasone

Long-term glucocorticoid treatment is associated with severe side effects, such as obesity and osteoporosis. A palm oil-derived vitamin E mixture had been shown previously to be protective against osteoporosis in rats given 120 microg/kg dexamethasone daily for 12 weeks. In this study we determined the effects of two isomers of vitamin E (i.e., palm oil-derived gamma-tocotrienol and the commercially available alpha-tocopherol, 60 mg/kg of body weight/day) on body composition and bone calcium content in adrenalectomized rats replaced with two doses of dexamethasone, 120 microg/kg and 240 microg/kg daily. Treatment period was 8 weeks. gamma-Tocotrienol (60 mg/kg of body weight/day) was found to reduce body fat mass and increase the fourth lumbar vertebra bone calcium content in these rats, while alpha-tocopherol (60 mg/kg of body weight/day) was ineffective. Therefore, in conclusion, palm oil-derived gamma-tocotrienol has the potential to be utilized as a prophylactic agent in prevention of the side effects of long-term glucocorticoid use.     

Effects of ω3- and ω6-Polyunsaturated Fatty Acids on RANKL-Induced Osteoclast Differentiation of RAW264.7 Cells: A Comparative in Vitro Study

Polyunsaturated fatty acids (PUFAs) have been reported to have an anabolic effect on bone in vivo, but comparative studies to identify inhibitors of osteoclast formation amongst ω3- and ω6-PUFAs are still lacking. Here we assessed the effects of the ω3-PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the ω6-PUFAs, arachidonic acid (AA) and γ-linolenic acid (GLA) on a RAW264.7 osteoclast differentiation model. The effects of PUFAs on RANKL-induced osteoclast formation were evaluated by counting tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells. PUFAs significantly inhibited RANKL-induced osteoclast formation in a dose-dependent manner with AA- and DHA-mediated inhibition being the strongest. Furthermore, RANKL-induced mRNA- and protein expression of the key osteoclastogenic genes cathepsin K and TRAP were inhibited by AA and more potently by DHA. Owing to the attenuated osteoclastogenesis by DHA and AA, actin ring formation and bone resorptive activity of these cells as evaluated on bone-mimetic plates were severely compromised. Hence, of the tested PUFAs, AA and DHA were found to be the most effective in inhibiting RANKL-induced osteoclast formation with the latter providing the strongest inhibitory effects. Collectively, the data indicates that these PUFAs may play an important role in regulating bone diseases characterized by excessive osteoclast activity.     

Three-Dimensional Co-Culture System of Human Osteoblasts and Osteoclast Precursors from Osteoporotic Patients as an Innovative Model to Study the Role of Nutrients: Focus on Vitamin K2

Several natural compounds, such as vitamin K2, have been highlighted for their positive effects on bone metabolism. It has been proposed that skeletal disorders, such as osteoporosis, may benefit from vitamin K2-based therapies or its regular intake. However, further studies are needed to better clarify the effects of vitamin K2 in bone disorders. To this aim, we developed in vitro a three-dimensional (3D) cell culture system one step closer to the bone microenvironment based on co-culturing osteoblasts and osteoclasts precursors obtained from bone specimens and peripheral blood of the same osteoporotic patient, respectively. Such a 3-D co-culture system was more informative than the traditional 2-D cell cultures when responsiveness to vitamin K2 was analyzed, paving the way for data interpretation on single patients. Following this approach, the anabolic effects of vitamin K2 on the osteoblast counterpart were found to be correlated with bone turnover markers measured in osteoporotic patients’ sera. Overall, our data suggest that co-cultured osteoblasts and osteoclast precursors from the same osteoporotic patient may be suitable to generate an in vitro 3-D experimental model that potentially reflects the individual’s bone metabolism and may be useful to predict personal responsiveness to nutraceutical or drug molecules designed to positively affect bone health.     

维生素E同系化合物抑制人肝癌细胞HepG2增殖的作用

通过探讨α 生育酚 (α T)、γ 生育酚 (γ T)、δ 生育酚 (δ T)及维生素E琥珀酸酯 (VES)抑制肝癌细胞生长的生物学效应 ,为维生素E(VE)防治肝癌提供理论依据 ,在体外培养的人肝癌细胞 (HepG2 )培养液中分别加入 1 2 5、2 5 0、50 0、1 0 0 0和 2 0 0mg L的VE同系化合物 ,采用细胞计数法和噻唑蓝比色法 (MTT)比较VE同系化合物抑制HepG2细胞增殖的效应 ;采用流式细胞仪检测四种VE同系化合物对HepG2细胞周期的影响。结果显示在 1 2 5和 2 5 0mg L浓度下 ,4种VE同系化合物均未显示出对HepG2细胞生长的抑制效应 ;在50、1 0 0和 2 0 0mg L浓度下 ,δ 生育酚、VES处理的HepG2生长速度明显减缓、细胞存活率显著降低 ,δ 生育酚处理的HepG2细胞生长轻度减缓 ,而γ 生育酚处理的HepG2细胞未见明显改变。VE同系化合物抑制HepG2细胞增殖的效应为δ 生育酚 >VES >γ 生育酚 >α 生育酚 ;细胞周期结果显示HepG2细胞增殖阻断于G1 期。提示VE同系化合物抑制肝癌细胞生长作用不同 ,δ 生育酚和VES在体外显示出较强的抗肝癌细胞增殖效应     

Flavonoid intake and bone health

Flavonoids, found in a wide diversity of plant foods from fruits and vegetables, herbs and spices, essential oils, and beverages, have the most potential of dietary components for promotion of bone health beyond calcium and vitamin D. Recent epidemiological studies show flavonoid consumption to have a stronger association with bone than general fruit and vegetable consumption. Bioactive flavonoids are being assessed for properties beyond their chemical antioxidant capacity, including anti-inflammatory actions. Some have been reported to enhance bone formation and to inhibit bone resorption through their action on cell signaling pathways that influence osteoblast and osteoclast differentiation. Future research is needed to determine which of the flavonoids and their metabolites are most effective and at what dose, as well as the mechanism of modulating cellular events, in order to set priorities for clinical trials.     

Effect of Vitamin E Supplement on Bone Turnover Markers in Postmenopausal Osteopenic Women: A Double-Blind,Randomized, Placebo-Controlled Trial

Vitamin E is a strong anti-oxidative stress agent that affects the bone remodeling process. This study evaluates the effect of mixed-tocopherol supplements on bone remodeling in postmenopausal osteopenic women. A double-blinded, randomized, placebo-controlled trial study was designed to measure the effect of mixed-tocopherol on the bone turnover marker after 12 weeks of supplementation. All 52 osteopenic postmenopausal women were enrolled and allocated into two groups. The intervention group received mixed-tocopherol 400 IU/day, while the control group received placebo tablets. Fifty-two participants completed 12 weeks of follow-up. Under an intention-to-treat analysis, vitamin E produced a significant difference in the mean bone resorption marker (serum C-terminal telopeptide of type I collagen (CTX)) compared with the placebo group (−0.003 ± 0.09 and 0.121 ± 0.15, respectively (p < 0.001)). In the placebo group, the CTX had increased by 35.3% at 12 weeks of supplementation versus baseline (p < 0.001), while, in the vitamin E group, there was no significant change of bone resorption marker (p < 0.898). In conclusion, vitamin E (mixed-tocopherol) supplementation in postmenopausal osteopenic women may have a preventive effect on bone loss through anti-resorptive activity.     

Effect of vitamin E on linoleic acid-mediated induction of peroxisomal enzymes in cultured porcine endothelial cells

Linoleic acid decreases endothelial barrier function in culture. We hypothesize that the mechanism may involve induction of peroxisomes, with subsequent generation of hydrogen peroxide, and that vitamin E may protect against barrier function loss by preventing the induction of peroxisomal enzymes. To investigate this hypothesis, we exposed cultured endothelial cells to 0 or 90 mumols/L linoleic acid [18:2(n-6)], with or without 25 mumols/L supplemental vitamin E, for 5 d. The induction of peroxisomes by linoleic acid exposure was determined by measuring cellular peroxisomal beta-oxidation and catalase activity. Vitamin E alone had no effect on beta-oxidation or catalase activity, whereas linoleic acid exposure significantly increased both compared with control values. Vitamin E supplementation prevented induction of peroxisomal beta-oxidation and catalase activity by 18:2. In contrast, cell enrichment with vitamin E had no effect on 18:2-induced accumulation of cytoplasmic lipid-like droplets. These results confirm our hypothesis that the protective effects of vitamin E against fatty acid-mediated endothelial cell injury may be due in part to the ability of vitamin E to prevent the induction of peroxisomal beta-oxidation enzymes and thus the formation of excess hydrogen peroxide.     

Comparative activities of daidzein metabolites,equol and <Emphasis Type="Italic">O</Emphasis>-desmethylangolensin,on bone mineral density and lipid metabolism in ovariectomized mice and in osteoclast cell cultures

Daidzein, a major isoflavone predominantly found in soybean, is mainly metabolized to equol and O-desmethylangolensin (O-DMA) by the human gut microflora. Equol exhibits a stronger estrogenic activity than daidzein, however, only approximately 30% of the population has been identified as equol-producers and there are too few direct evidences of the effects of the other major metabolite, O-DMA on estrogen-deficient status. The purpose of this study is therefore, to compare the effect of both O-DMA and equol on bone and lipid metabolism in vivo and in vitro. For the in vivo study, 8-week-old female mice were assigned to five groups as follows: sham-operated (sham), ovariectomized (OVX), OVX + 0.5 mg/day O-DMA (OVX + O-DMA), OVX + 0.5 mg/day equol (OVX + Eq), and OVX + 0.03 microg/day 17beta-estradiol (OVX + E2) administration. Three weeks after the intervention, O-DMA and equol did not affect uterine atrophy in OVX mice. The bone mineral density (BMD) of the femur was lower in the OVX group than in the sham group. The administration of equol but not O-DMA, maintained BMD through the intervention. Values of whole body fat mass and plasma lipids were lower in the equol and O-DMA treated OVX mice than those in OVX mice. In the in vitro study, equol significantly inhibited the osteoclast formation induced by 1alpha,25(OH)(2)D(3) in a dose-dependent manner in a co-culture system of mouse bone-marrow cells with primary osteoblastic cells. However, O-DMA slightly inhibited osteoclast formation, and the effect was not dose dependent. These results suggest that the effects of O-DMA on bone and lipid metabolism in OVX mice and osteoclast cell cultures are weaker than those of equol.     

Lycopene I--effect on osteoclasts: lycopene inhibits basal and parathyroid hormone-stimulated osteoclast formation and mineral resorption mediated by reactive oxygen species in rat bone marrow cultures

Osteoclasts have been shown to produce reactive oxygen species (ROS) that can stimulate bone resorption. We explored the hypothesis that lycopene, the antioxidant carotenoid from tomatoes, can inhibit mineral resorption by inhibiting osteoclast formation and the production of ROS. Cells from bone marrow prepared from rat femur were plated into 16-well calcium phosphate-coated Osteologic Multi-test Slides and cultured in alpha-minimal essential medium supplemented with dexamethasone, beta-glycerophosphate, and ascorbic acid. The cells were treated with varying doses of lycopene in the absence or presence of parathyroid hormone (PTH) at the start of culture and at each medium change (i.e., every 48 hours). On day 8, mineral resorption pits were quantitated. Similar, parallel experiments were carried out in 12-well plastic dishes to assess tartrate-resistant acid phosphatase (TRAP) activity. Results showed that lycopene inhibited TRAP + formation of multinucleated cells in both vehicle- and PTH-treated cultures. Osteoclasts reduced nitroblue tetrazolium (NBT) to purple-colored formazan, indicating the presence of ROS in these cells. The formazan-staining cells were decreased by treatment with 10(-5) M lycopene, indicating that lycopene inhibited the formation of ROS-secreting osteoclasts. In conclusion, we have shown that lycopene inhibits basal and PTH-stimulated osteoclastic mineral resorption and formation of TRAP + multinucleated osteoclasts, as well as the ROS produced by osteoclasts. These findings are novel and may be important in the pathogenesis, treatment, and prevention of osteoporosis.     

Vitamin D Interactions with Soy Isoflavones on Bone after Menopause: A Review

Vitamin D is known to increase Ca absorption in adults. However, the threshold vitamin D status to benefit Ca absorption is lower than the target vitamin D status for higher bone mineral density and lower fracture risk, pointing to another pathway for vitamin D to benefit bone. One possibility is by affecting osteoblast and osteoclasts directly. Vitamin D-related bone metabolism may also be affected by soy isoflavones, which selectively bind to the estrogen receptor β and may reduce bone loss in postmenopausal women. We discuss a possible synergistic effect of soy isoflavones and vitamin D on bone by affecting osteoblast and osteoclast formation and activity in postmenopausal women.     

Anti-Osteoporosis Effects of the Eleutherococcus senticosus,Achyranthes japonica,and Atractylodes japonica Mixed Extract Fermented with Nuruk

Vigeo is a mixture of fermented extracts of Eleutherococcus senticosus Maxim (ESM), Achyranthes japonica (Miq.) Nakai (AJN), and Atractylodes japonica Koidzumi (AJK) manufactured using the traditional Korean nuruk fermentation method. Although the bioactive effects of ESM, AJN, and AJK have already been reported, the pharmacological effects of Vigeo have not been proven. Therefore, in this study, we investigated whether Vigeo had inhivitory effects on lipopolysaccharide (LPS)-induced inflammatory bone loss in vivo and receptor activator of nuclear factor-B ligand (RANKL)-induced osteoclastogenesis and the related mechanism in vitro. Vigeo administration conferred effective protection against bone loss induced by excessive inflammatory response and activity of osteoclasts in LPS-induced inflammatory osteoporosis mouse model. In addition, Vigeo significantly suppressed the formation of tartrate-resistant acid phosphatase-positive osteoclasts induced by RANKL and inhibited F-actin formation and bone resorbing activity without any cytotoxicity. Moreover, Vigeo significantly inhibited RANKL-induced phosphorylation of p38, ERK, JNK, IκB, and AKT and degradation of IkB. Additionally, Vigeo strongly inhibited the mRNA and protein expression of c-FOS and NFATc1 and subsequently attenuated the expression of osteoclast specific marker genes induced by RANKL. We demonstrated for the first time the anti-osteoporosis effect of Vigeo, suggesting that it could be a potential therapeutic candidate for the treatment of osteoclast-mediated inflammatory bone diseases.     

Vitamin E and macrophage cyclooxygenase regulation in the aged

Aging is associated with increased evidence of cardiovascular disease (CVD). Atherosclerosis, a major cause of CVD, is an inflammatory process whose development is influenced by several proinflammatory mediators. Products of arachidonic acid metabolism, in particular, prostaglandin (PG) E(2) and thromboxane (TX) A(2), play an important role in the development of atherosclerosis. We showed previously that the aged have higher PGE(2) production compared with their young counterparts. This age-associated increase in PGE(2) production is mainly a consequence of increased cyclooxygenase (COX) activity. We demonstrated further that increased COX activity in old mice is due to the increased expression of mRNA and protein for the inducible form of COX, COX-2. Vitamin E has been shown to reduce PGE(2) production and risk of CVD. In aged mice, we showed that a vitamin E-induced decrease in PGE(2) production is due to decreased COX activity. However, vitamin E had no effect on COX mRNA and protein levels, indicating a post-translational regulation of COX by vitamin E. Further experiments indicated that vitamin E decreases COX activity through reducing formation of peroxynitrite, a hydroperoxide shown to be involved in the activation of COX-2. Other homologues of tocopherols were also effective in inhibiting COX activity, but their degree of inhibition varied. The varied potency to inhibit COX activity was not explained totally by differences in their antioxidant capacity. Vitamin E-induced inhibition of COX activity might contribute to its effect of reducing CVD risk.     

Lead exposure inhibits fracture healing and is associated with increased chondrogenesis, delay in cartilage mineralization, and a decrease in osteoprogenitor frequency

Lead exposure continues to be a significant public health problem. In addition to acute toxicity, Pb has an extremely long half-life in bone. Individuals with past exposure develop increased blood Pb levels during periods of high bone turnover or resorption. Pb is known to affect osteoblasts, osteoclasts, and chondrocytes and has been associated with osteoporosis. However, its effects on skeletal repair have not been studied. We exposed C57/B6 mice to various concentrations of Pb acetate in their drinking water to achieve environmentally relevant blood Pb levels, measured by atomic absorption. After exposure for 6 weeks, each mouse underwent closed tibia fracture. Radiographs were followed and histologic analysis was performed at 7, 14, and 21 days. In mice exposed to low Pb concentrations, fracture healing was characterized by a delay in bridging cartilage formation, decreased collagen type II and type X expression at 7 days, a 5-fold increase in cartilage formation at day 14 associated with delayed maturation and calcification, and a persistence of cartilage at day 21. Fibrous nonunions at 21 days were prevalent in mice receiving very high Pb exposures. Pb significantly inhibited ex vivo bone nodule formation but had no effect on osteoclasts isolated from Pb-exposed animals. No significant effects on osteoclast number or activity were observed. We conclude that Pb delays fracture healing at environmentally relevant doses and induces fibrous nonunions at higher doses by inhibiting the progression of endochondral ossification.     

氟对骨形态发生蛋白的直接作用

目的：研究氟对骨形态发生蛋白（ＢＭＰ）活性的直接作用。方法 在纯化的ＢＭＰ中加入氟化物植再植入大鼠皮下,通过比较植入物在不同植入时间的组织学变化,碱性磷酸酶、酸性磷酸酶活性及钙含量来反映ＢＭＰ的生物活性。结果：氟对ＢＭＰ的生物活性有直接的影响,氟能促进ＢＭＰ将更多的成纤维细胞化为成骨细胞,并能增强成骨细胞的功能,加速钙盐沉积,而破骨细胞功能有所下降。结论 氟能直接影响骨形态发生蛋白的活性。     

Growth-inhibitory effects of vitamin E succinate on retrovirus-transformed tumor cells in vitro

Vitamin E succinate inhibited proliferation of C4#1 cells, an established avian retrovirus [reticuloendotheliosis virus (REV)]-transformed immature lymphoid tumor cell line, in a dose-dependent manner. The cytostatic effects of vitamin E succinate were reversible in that treated cells regained their ability to divide after vitamin E succinate removal. Possible mechanism(s) for the antiproliferative actions of vitamin E succinate were investigated. Analyses of C4#1 cell surface membrane antigen profiles and morphology indicated that vitamin E succinate was not inducing differentiation of the tumor cells to a more mature, differentiated, nonproliferative state. Five antioxidants, including a synthetic analogue of vitamin E, Trolox, as well as the active vitamin form, DL-alpha-tocopherol, were incapable of inhibiting C4#1 tumor cell growth, indicating that a mechanism of action other than or in addition to functions as an antioxidant may be operating. Cell cycle analyses suggested that C4#1 tumor cells treated with vitamin E succinate were blocked in the G0G1/early S phases of the cell cycle. Tumor growth arrested by vitamin E succinate did not affect the expression of the REV-encoded oncogene, v-rel, at either the RNA or protein level. These studies demonstrated that vitamin E, in the form of vitamin E succinate, inhibited the growth of retrovirus-transformed tumor cells in vitro and suggested that the antiproliferative effects of vitamin E succinate did not involve antioxidant properties but rather, as yet, unidentified mechanisms leading to cell cycle blockage.     

Suppressive effects of the leaf of Terminalia catappa L. on osteoclast differentiation in vitro and bone weight loss in vivo

Oral administration of Terminalia catappa extract (TCE; 1,000 mg/kg) for 5 wk suppressed bone weight loss and trabecular bone loss in ovariectomized mice. An in vitro experiment showed that TCE (1.3-20 μg/mL) did not increase alkaline phosphatase activity, which would indicate osteoclast formation, in osteoblast-like 3T3-L1 cells. On the other hand, TCE (12.5 μg/mL) markedly decreased the number of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells, which would indicate osteoclast formation, in a co-culture system (bone marrow cells/osteoblastic UAMS-32 cells). A detailed analysis of the stages of osteoclast differentiation revealed that TCE mainly suppressed the differentiation of bone marrow mononuclear cells into osteoclast progenitor cells in the presence of M-CSF and TGF-β. An additional experiment using fractionated TCE revealed that the water-soluble fraction suppressed the bone weight loss in OVX-mice and osteoclast differentiation in vitro. Therefore, the suppressive effects of TCE on bone weight loss in mice might be due to the suppressive effects of highly polar components on the early stage of osteoclast differentiation.     

Selective uptake of dietary tocotrienols into rat skin

Using a vitamin E mixture extracted from palm oil, the tissue distribution of dietary tocotrienols and tocopherols was examined in rats and mice. Wistar rats (4-wk-old) were fed a diet containing 48.8 mg/kg alpha-tocopherol, 45.8 mg/kg alpha-tocotrienol and 71.4 mg/kg gamma-tocotrienol for 8 wk. Nude mice (BALB/c Slc-nu, 8-wk-old) and hairless mice (SKH1, 8-wk-old) were fed the same diet for 4 wk. alpha-Tocopherol was abundantly retained in the skin, liver, kidney and plasma of rats and mice. alpha-Tocotrienol and gamma-tocotrienol were detected slightly in the liver, kidney and plasma, while substantial amounts of these tocotrienols were detected in the skin of both rats and mice. The present study suggests that the skin is a unique tissue in respect to its ability to discriminate between various vitamin E analogs.     

Mechanism in inhibitory effects of vitamin K2 on osteoclastic bone resorption: in vivo study in osteopetrotic (op/op) mice

Osteoclast deficiency in op/op mice was cured by a single injection of 5 micrograms recombinant human macrophage colony-stimulating factor (M-CSF). On d 5, the osteoclast number reached a maximum value. By d 15, the osteoclast number had decreased to about 70% of the maximum level. Moreover, by d 20, the osteoclast number had decreased to about 30% of its maximum level. On d 5, the osteoclast number of vitamin K2 12 h previously had decreased to about 30% of the M-CSF-only injected mice. Moreover, on d 5, the osteoclast number of the mice receiving a single injection of vitamin K2 24 h previously had decreased to about 15% that of mice injected only with M-CSF. These results indicate that vitamin K2 inhibits in vivo osteoclast formation. On d 20, the osteoclast number of the mice injected with a single dose of vitamin K2 12 or 24 h previously had decreased to 0% compared with those receiving only M-CSF. The present results suggest that the vitamin K2 "causes cell death" to mature osteoclasts and inhibits in vivo osteoclast formation.     

Index of Authors by Abstract Numbers

The role of beta-carotene and vitamin E in modifying the effect of cell differentiating agent has not been studied. This study has investigated the effects of beta-carotene and d-alpha-tocopheryl succinate (alpha-TS) on adenosine 3′,5′-cyclic monophosphate (cAMP) induced differentiated functions in murine neuroblastoma cells (NBP2) in culture.

Prostaglandin E1 (PGE1), a stimulator of adenylate cyclase, and 4-(3-butoxy-4-methoxy-benzyl)-2-imidazolidinone (R020-1724), an inhibitor of cyclic nucleotide phosphodiesterase, were used to induce differentiation in NB cells.

Both beta-carotene and alpha-TS markedly enhanced the level of morphologic differentiation (neurite formation) induced by both PGE1 and R020-1724. However, beta-carotene and alpha-TS by themselves were ineffective. These vitamins increased tyrosine hydroxylase (TH) activity. However, beta-carotene did not significantly affect PGE1- and R020-1724-stimulated rise in TH activity. alpha-TS at a higher concentration inhibited PGE1- and R020-1724-stimulated increase in TH activity. None of the above treatments affected basal choline acetyltransferase (ChAT) activity. beta-carotene and alpha-TS caused a transient increase in cAMP level, and they also enhanced the effect of PGE1 and R020-1724 on cAMP level in a transient manner.

These results suggest that beta-carotene and alpha-TS modify the effects of cAMP stimulating agents on differentiation of NB cells in culture.