Tag Archives: animal

Ferutinin Inhibits Resorption in Ovariectomized Rats

Abstract

Effects of different doses of ferutinin on bone formation/resorption in ovariectomized rats.

This study analyzes the effects of different doses of ferutinin on bone loss caused by estrogen deficiency in ovariectomized rats, in comparison with estradiol benzoate. Thirty female Sprague-Dawley rats were ovariectomized and treated for 30 days from the day after ovariectomy. Static/dynamic histomorphometric analyses were performed on trabecular and cortical bone of lumbar vertebrae and femurs. Very low weight increments were recorded only in all F-OVX groups, with respect to the others. Although the great differences in weight, that could imply a decrease of bone mass in F-OVX groups compared to the control ovariectomized group (C-OVX), trabecular bone in lumbar vertebrae did not show significant differences, suggesting that ferutinin, opposing estrogen deficiency, inhibits bone resorption. Newly formed cortical bone was always low in all F-OVX groups and high in C-OVX, suggesting that it is mainly devoted in answering mechanical demands. In contrast, in distal femoral metaphyses, trabecular bone was reduced and the number of osteoclasts was increased in C-OVX with respect to all other groups, suggesting that it is mainly devoted in answering metabolic demands; moreover, ferutinin dose of 2 mg/kg seemed to be more effective than the lower doses used and estrogens, particularly in those skeletal regions with higher metabolic activity. Our results suggest that the role of ferutinin in preventing osteoporosis caused by estrogen deficiency is expressed in decreasing bone erosion; moreover, in all F-OVX groups bone turnover is very low and seems correlated to the trivial body weight increase, which, in turn, depends on ferutinin treatment.

Cavani F, Ferretti M, Carnevale G, Bertoni L…
J. Bone Miner. Metab. Nov 2012
PMID: 22828874

Vitamin E Does Not Prevent Bone Loss in Rats

Abstract

Vitamin E does not prevent bone loss and induced anxiety in rats with ligature-induced periodontitis.

The purpose of this study was to investigate the effect of vitamin E on alveolar bone loss (ABL) and anxiety in rats with ligature-induced experimental periodontitis (EP).
Wistar rats were subjected to ligature-induced EP and treated with vitamin E (500mg/kg, orally) for 9 days. Then anxiety was tested using the elevated plus-maze (EPM) test. All of the animals were euthanised by cervical dislocation on day 11. ABL was analysed morphometrically and histopathologically. Lipid peroxidation quantification, activity of the enzyme superoxide dismutase and immunohistochemistry to tumour necrosis factor-alpha (TNF-α) and inducible isoform of nitric oxide synthases (iNOS) were also tested.
EP induced a marked inflammatory process and intense ABL. Treatment with vitamin E decreased inflammatory reaction, prevented malondialdehyde formation and reduced the immunoreactivity to iNOS, but did not decrease ABL. Vitamin E had an anxiogenic effect on rats with or without EP.
Vitamin E may have potential to reduce oxidative damage and inflammatory response in EP but does not prevent ABL. Attention should be given to indiscriminate use of vitamin E due to the risk of causing anxiety in patients.

Carvalho Rde S, de Souza CM, Neves JC, Holanda-Pinto SA…
Arch. Oral Biol. Jan 2013
PMID: 22664314

Blueberry May Prevent Collagen and Bone Loss in Rat Cells

Abstract

Blueberry consumption prevents loss of collagen in bone matrix and inhibits senescence pathways in osteoblastic cells.

Ovariectomy (OVX)-induced bone loss has been linked to increased bone turnover and higher bone matrix collagen degradation as the result of osteoclast activation. However, the role of degraded collagen matrix in the fate of resident bone-forming cells is unclear. In this report, we show that OVX-induced bone loss is associated with profound decreases in collagen 1 and Sirt1. This was accompanied by increases in expression and activity of the senescence marker collagenase and expression of p16/p21 in bone. Feeding a diet supplemented with blueberries (BB) to pre-pubertal rats throughout development or only prior to puberty [postnatal day 21 (PND21) to PND34] prevents OVX-induced effects on expression of these molecules at PND68. In order to provide more evidence and gain a better understanding on the association between bone collagen matrix and resident bone cell fate, in vitro studies on the cellular senescence pathway using primary calvarial cells and three cell lines (ST2 cells, OB6, and MLO-Y4) were conducted. We found that senescence was inhibited by collagen in a dose-response manner. Treatment of cells with serum from OVX rats accelerated osteoblastic cell senescence pathways, but serum from BB-fed OVX rats had no effect. In the presence of low collagen or treatment with OVX rat serum, ST2 cells exhibited higher potential to differentiate into adipocytes. Finally, we demonstrated that bone cell senescence is associated with decreased Sirt1 expression and activated p53, p16, and p21. These results suggest that (1) a significant prevention of OVX-induced bone cell senescence from adult rats can occur after only 14 days consumption of a BB-containing diet immediately prior to puberty, and (2) the molecular mechanisms underlying this effect involves, at least in part, prevention of collagen degradation.

Zhang J, Lazarenko OP, Blackburn ML, Badger TM…
Age (Dordr) Jun 2013
PMID: 22555620

Melatonin Implants Influence Bone Repair in Rabbits

Abstract

Melatonin promotes angiogenesis during repair of bone defects: a radiological and histomorphometric study in rabbit tibiae.

The pineal gland hormone, melatonin, is an immunomodulator and neuroendocrine hormone; it also stimulates monocyte, cytokine and fibroblast proliferations, which influence angiogenesis. The aim of this study was to investigate the effects of melatonin on angiogenesis during bone defect repair by means of radiological and histomorphometric evaluations of bone response to melatonin implants.
Twenty New Zealand rabbits weighing 3,900-4,500 g were used. Twenty melatonin implants were inserted in the proximal metaphyseal area of the animals’ right tibia and 20 control areas were located in the left proximal metaphyseal area. Following implantation, the animals were sacrificed in groups of five, after 1, 2, 3 and 4 weeks, respectively. Anteroposterior and lateral radiographs were taken, and radiographic thermal imaging analysis was performed for all groups at different time stages following implant insertion. Samples were sectioned at 5 μm and stained using Hematoxylin-Eosin and Masson’s trichrome, supplementing radiographic findings with histomorphometric analysis.
After 4 weeks, radiological images showed complete repair of the bone defects. No healed or residual bone alterations attributable to the presence of the melatonin implant were observed. Histomorphometric analysis at 4 weeks showed the presence of a higher density newly formed bone. There were statistically significant differences in the length of cortical formation between the melatonin group and the control group during the first weeks of the study; there were also statistically significant differences in the number of vessels observed in the melatonin groups at the first two study stages.
Melatonin may have potential beneficial effects on bone defect repair.

Ramírez-Fernández MP, Calvo-Guirado JL, de-Val JE, Delgado-Ruiz RA…
Clin Oral Investig Jan 2013
PMID: 22323056

Bread Crust Does Not Negatively Affect Calcium in Rats

Abstract

Effects of dietary bread crust Maillard reaction products on calcium and bone metabolism in rats.

Maillard reaction products (MRP) consumption has been related with the development of bone degenerative disorders, probably linked to changes in calcium metabolism. We aimed to investigate the effects of MRP intake from bread crust on calcium balance and its distribution, and bone metabolism. During 88 days, rats were fed control diet or diets containing bread crust as source of MRP, or its soluble high molecular weight, soluble low molecular weight or insoluble fractions (bread crust, HMW, LMW and insoluble diets, respectively). In the final week, a calcium balance was performed, then animals were sacrified and some organs removed to analyse calcium levels. A second balance was carried out throughout the experimental period to calculate global calcium retention. Biochemical parameters and bone metabolism markers were measured in serum or urine. Global calcium bioavailability was unmodified by consumption of bread crust or its isolate fractions, corroborating the previously described low affinity of MRP to bind calcium. Despite this, a higher calcium concentration was found in femur due to smaller bones having a lower relative density. The isolate consumption of the fractions altered some bone markers, reflecting a situation of increased bone resorption or higher turnover; this did not take place in the animals fed the bread crust diet. Thus, the bread crust intake does not affect negatively calcium bioavailability and bone metabolism.

Roncero-Ramos I, Delgado-Andrade C, Haro A, Ruiz-Roca B…
Amino Acids Jun 2013
PMID: 22109787

Icariin Inhibits Osteoclasts In Vitro

Abstract

Icariin inhibits the osteoclast formation induced by RANKL and macrophage-colony stimulating factor in mouse bone marrow culture.

Icariin is a prenylated flavonol glycoside contained in the herb Epimedium, which has long been used to improve bone fracture healing or prevent osteoporosis because of the belief that the herb has bone-strengthening action. We have previously demonstrated that icariin enhances the osteogenic differentiation of rat bone marrow stromal cells, and partially explained the bone-strengthening mechanism of the herb. In the present study, the effect of icariin on osteoclastogenesis and bone resorption activity was investigated in mouse bone marrow culture. It was found that icariin dose-dependently inhibited the growth and differentiation of hemopoietic cells from which osteoclasts were formed. Far less TRAP+ multinuclear cells appeared in the 10 microM icariin group than in the control. The bone resorption pits formed in the 10 microM icariin group was also significantly less than that of the control. RT-PCR analysis showed that the gene expression of TRAP, RANK and CTR was obviously lower than that of the control. It can be concluded that icariin has the ability to inhibit the formation and bone resorption activity of osteoclasts, which suggests that icariin should be the effective component for the bone-strengthening action of herb Epimedium.

Chen KM, Ge BF, Liu XY, Ma PH…
Pharmazie May 2007
PMID: 17557750

Horny Goat Weed Icariin Metabolites Enhance Osteoblasts and Inhibit Osteoclasts In Vitro

Abstract

Icaritin and its glycosides enhance osteoblastic, but suppress osteoclastic, differentiation and activity in vitro.

Icariin, a principal flavonoid glycoside in Herba Epimedii, is hypothesized to possess beneficial effects on bone mass. Icariin is metabolized to icariside II and then to icaritin in vivo. In the present study, we investigated the in vitro effects of icariin, icariside II and icaritin on both osteoblasts and osteoclasts. After treatment with these compounds at concentrations 10(-5)-10(-8) mol/l, osteoblasts were examined for proliferation, alkaline phosphatase activity, osteocalcin secretion and matrix mineralization, as well as expression levels of bone-related proteins. The formation of osteoclasts was assessed by counting the number of multinucleated TRAP-positive cells. The activity of isolated rat osteoclasts was evaluated by measuring pit area, actin rings and superoxide generation. Icariside II and icaritin increased the mRNA expression of ALP, OC, COL-1 and OPG, but suppressed that of RANKL. In addition, these compounds reduced the number of multinucleated TRAP-positive cells and the osteoclastic resorption area. Also decreases were observed in superoxide generation and actin ring formation that are required for osteoclast survival and bone resorption activity. These findings suggest that icaritin, which was more potent than icariin and icariside II, enhanced the differentiation and proliferation of osteoblasts, and facilitated matrix calcification; meanwhile it inhibited osteoclastic differentiation in both osteoblast-preosteoclast coculture and osteoclast progenitor cell culture, and reduced the motility and bone resorption activity of isolated osteoclasts.

Huang J, Yuan L, Wang X, Zhang TL…
Life Sci. Aug 2007
PMID: 17764702

Icariin, Anemarsaponin BII, and Berberine Benefit Bones

Abstract

Antiosteoporotic chemical constituents from Er-Xian Decoction, a traditional Chinese herbal formula.

Er Xian Decoction (EXD), a traditional Chinese medicine formula, has long been used for the treatment of osteoporosis and menopausal syndrome in China. The present study was designed to investigate the antiosteoporotic constituents of EXD, and evaluate their antiosteoporotic effects in ovariectomized rats.
Osteoblasts in neonatal calvaria cultures and osteoclasts derived from rat marrow cells were used to bioactivity-guided screen the active constituents. The proliferation of osteoblast was assayed by MTT methods. The activity of ALP and TRAP was measured by p- nitrophenyl sodium phosphate assay. The antiosteoporotic effects of icariin (1), anemarsaponin B II (8) and berberine (6) were verified by using OVX rats model. The bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry using the small animal scan mode. The undecalcified longitudinal proximal tibial metaphysical (PTM) sections were cut and stained for the bone histomorphometric analysis.
Bioactivity-guided fractionation has led to the successful isolation of antiosteoporotic constituents, i.e., icariin (1), icariside I (2), baohuoside I (3), mangiferin (4), neomangiferin (5), berberine (6), anemarsaponin B (7), anemarsaponin BII (8), anemarsaponin C (9), anemarrhenasaponin I (10), rubiadin-1-methyl ether (11) and obaculactone (12) from EXD. Further study showed that icariin (1), anemarsaponin BII (8) and berberine (6) increased the BMD in ovariectomized rats, and icariin (1) not only increased the bone formation, but also inhibited bone resorption; anemarsaponin BII (8) mainly increased bone formation and berberine (6) only inhibited the bone resorption in ovariectomized rats.
Our findings demonstrate that multiple ingredients are responsible for antiosteoporotic activity in traditional Chinese medicine formula Er-Xian decoction.

Qin L, Han T, Zhang Q, Cao D…
J Ethnopharmacol Jul 2008
PMID: 18501540

Horny Goat Weed and Icariin may Promote Osteoblasts In Vitro

Abstract

Effects of total flavonoids and flavonol glycosides from Epimedium koreanum Nakai on the proliferation and differentiation of primary osteoblasts.

In a bioassay-guided drug screening for anti-osteoporosis activity, eight flavonol glycosides were isolated from Epimedium koreanum Nakai, which is traditionally widely used in China for the treatment of impotence and osteoporosis. The effects of total flavonoids and flavonol glycosides on the proliferation and differentiation of rat calvarial osteoblast-like cells were evaluated by the MTT method and measuring the activity of alkaline phosphatase (ALP activity). Total flavonoids (1.2 x10(-2) to 6.0 x10(-7) mg/ml) and flavonol glycosides (2.0 x10(-5) to 1.0 x10(-9) mol/l) exhibited a strong inhibition on the proliferation of primary osteoblasts at most concentrations. However, the total flavonoids and icariin significantly promoted the differentiation of primary osteoblasts. The results suggested that flavonoids from E. koreanum Nakai may improve the development of osteoblasts by promoting the ALP activity; and icariin might be one of the active constituents facilitating the differentiation of osteoblasts.

Zhang DW, Cheng Y, Wang NL, Zhang JC…
Phytomedicine Jan 2008
PMID: 17482445

Resveratrol Enhances Osteogenesis via Runx2 and SIRT1 In Vitro

Abstract

Resveratrol mediated modulation of Sirt-1/Runx2 promotes osteogenic differentiation of mesenchymal stem cells: potential role of Runx2 deacetylation.

Osteogenic repair in response to bone injury is characterized by activation and differentiation of mesenchymal stem cells (MSCs) to osteoblasts. This study determined whether activation of Sirt-1 (a NAD(+)-dependent histone deacetylase) by the phytoestrogen resveratrol affects osteogenic differentiation.
Monolayer and high-density cultures of MSCs and pre-osteoblastic cells were treated with an osteogenic induction medium with/without the Sirt-1 inhibitor nicotinamide or/and resveratrol in a concentration dependent manner.
MSCs and pre-osteoblastic cells differentiated to osteoblasts when exposed to osteogenic-induction medium. The osteogenic response was blocked by nicotinamide, resulting in adipogenic differentiation and expression of the adipose transcription regulator PPAR-γ (peroxisome proliferator-activated receptor). However, in nicotinamide-treated cultures, pre-treatment with resveratrol significantly enhanced osteogenesis by increasing expression of Runx2 (bone specific transcription factor) and decreasing expression of PPAR-γ. Activation of Sirt-1 by resveratrol in MSCs increased its binding to PPAR-γ and repressed PPAR-γ activity by involving its cofactor NCoR (nuclear receptor co-repressor). The modulatory effects of resveratrol on nicotinamide-induced expression of PPAR-γ and its cofactor NCoR were found to be mediated, at least in part, by Sirt-1/Runx2 association and deacetylation of Runx2. Finally, knockdown of Sirt-1 by using antisense oligonucleotides downregulated the expression of Sirt-1 protein and abolished the inhibitory effects of resveratrol, namely nicotinamide-induced Sirt-1 suppression and Runx2 acetylation, suggesting that the acetylated content of Runx2 is related to downregulated Sirt-1 expression.
These data support a critical role for Runx2 acetylation/deacetylation during osteogenic differentiation in MSCs in vitro.

Shakibaei M, Shayan P, Busch F, Aldinger C…
PLoS ONE 2012
PMID: 22539994 | Free Full Text

From the introduction:

Resveratrol is a polyphenolic phytoestrogen (trans-3,5, 4′-trihydroxystilbene) found in the skin of red grapes, red vines, various other fruits, peanuts and root extracts of Polygonum cuspidatum [8]. Resveratrol acts as a mixed agonist/antagonist for the estrogen receptors alpha and beta [9]. Through binding to the estrogen receptor, resveratrol is thought to exert beneficial effects on the cardiovascular system and may reverse osteoporosis by a direct stimulatory effect on bone formation in osteoblastic cells [10]. Many of the biological effects of resveratrol have already been demonstrated in the literature; these include cardiovascular protection [11], anticancer activity [12] and stimulation of proliferation and osteoblastic differentiation in human and mouse MSCs [13], [14]. However, its effects on bone are less studied and are particularly relevant to this investigation.

From the discussion:

Resveratrol’s enhancement of osteogenesis was, at least in part regulated by Runx2 with additional contributions by Sirt-1. Resveratrol increases alkaline phosphatase activity in osteoblastic cells [10] an effect that is blocked by tamoxifen, an estrogen antagonist, suggesting that some of resveratrol’s stimulatory actions may be mediated through the estrogen receptor. Gehm et al. have reported that resveratrol acts as a phytoestrogen (i.e. activating the estrogen receptor) and decreases osteoporosis [43]. Moreover, resveratrol is one of the most potent Sirt-1 activators; through binding to a special binding site it induces a conformational change in Sirt-1, lowering the Km for both the acetylated substrate and NAD, thus resulting in increased enzymatic activity [18]. Sirt-1 facilitates the differentiation of MSCs to osteoblasts by directly regulating factors such as Runx2 and by modulation of nuclear receptor co-repressor NCoR and PPAR-γ.