Tag Archives: animal

Vitamin E Prevents Steroid-Induced Bone Loss in Rabbits

Abstract

Vitamin E prevents steroid-induced osteonecrosis in rabbits.

Prevention of osteonecrosis after corticosteroid administration would be important. We examined the potential of vitamin E (alpha-tocopherol) to reduce the incidence of corticosteroid-induced osteonecrosis in an animal model.
Japanese white rabbits were divided into 2 groups; the control group was fed a normal diet and the experimental group was fed alpha-tocopherol-supplemented diet in which alpha-tocopherol (600 mg/kg diet) was added to the normal diet. To induce osteonecrosis, high-dose methylprednisolone acetate (MPSL) (20 mg/kg body weight) was injected once into the right gluteus medius muscle of all rabbits. 4 weeks after the injection of MPSL, the presence or absence of osteonecrosis of bilateral femurs was examined histopathologically. The tocopherol/cholesterol ratios were calculated. The plasma levels of thiobarbituric acid-reactive substances (TBARS) were measured.
Alpha-tocopherol-supplemented diet reduced the incidence of osteonecrosis, which developed in 14 of 20 rabbits in the control group and 5 of 21 rabbits in the experimental group (p = 0.004). The tocopherol/cholesterol ratio was higher in the experimental group than in the control group after the alpha-tocopherol administration. The plasma TBARS level was lower in the experimental group than in the control group at 4 weeks after the MPSL administration.
Our findings may offer a new approach for the prevention of corticosteroid-induced osteonecrosis.

Kuribayashi M, Fujioka M, Takahashi KA, Arai Y…
Acta Orthop Feb 2010
PMID: 20146637  | PMID: 20170436| Free Full Text

Vitamin E Reverses Nicotine-Induced Bone Resorption in Rats

Abstract

Vitamin E reversed nicotine-induced toxic effects on bone biochemical markers in male rats.

Vitamin E is beneficial in restoring bone histomorphometric parameters in nicotine-treated rats. This study determined the effectiveness of 3 forms of vitamin E in restoring bone metabolism in nicotine-treated rats.
Thirty-five male Sprague-Dawley rats were divided into 5 groups: (1) control (C), (2) nicotine cessation (NC), (3) α-tocopherol (ATF), (4) tocotrienol-enhanced fraction (TEF) and (5) γ-tocotrienol (GTT). Treatment was carried out for 4 months. The control group was administered normal saline and olive oil throughout the treatment period while treatment for groups 2-5 was performed in 2 phases. In the first phase, the groups received nicotine 7 mg/kg intraperitoneally for 2 months. The following 2 months, group 2 received normal saline and olive oil while groups 3-5 received ATF, TEF or GTT, 60 mg/kg orally. Pre-treatment and post-treatment serum was collected for bone biochemical marker measurement using the ELISA method.
Nicotine increased serum bone-resorbing cytokines (interleukin-1 and interleukin-6) and the bone resorption marker pyridinoline (PYD) while reducing the bone formation marker osteocalcin after 2 months of nicotine treatment. The parameters failed to improve after nicotine was stopped for 2 months. Supplementation with the 3 forms of vitamin E improved the parameters, i.e. reduced the cytokines and pyridinoline as well as increased the osteocalcin. In addition, the TEF and GTT groups had a higher level of osteocalcin than the control group.
Nicotine impaired bone metabolism and cessation of nicotine treatment did not reverse the effects. Vitamin E, especially the tocotrienols, restored bone metabolism that was impaired due to nicotine.

Norazlina M, Hermizi H, Faizah O, Nazrun AS…
Arch Med Sci Aug 2010
PMID: 22371792 | Free Full Text

Spirulina Protects Against Osteoporosis in Rats

Abstract

Spirulina protects against rosiglitazone induced osteoporosis in insulin resistance rats.

The study was undertaken to assess the protective effect of Spirulina fusiformis extract against Rosiglitazone induced osteoporosis and pharmacodynamic effects of Rosiglitazone with Spirulina in treating hyperglycemia and hyperlipidemia of insulin resistance rat.
For this aim, 30 Wistar albino rats were equally divided into five groups as control (C), diabetes mellitus (DM), diabetes mellitus+Rosiglitazone (DM+R), diabetes mellitus+Spirulina (DM+S), and diabetes mellitus+Rosiglitazone+Spirulina (DM+R+S). Serum glucose, triglyceride, HDL, LDL and insulin concentrations were estimated by routine standard methods in blood samples collected on 21th day. Integrity of the bone surface was examined by scanning electronic microscopy, and bone strength was measured by micro-hardness test on 45th day.
A significant decrease in total bone mineral density was observed in group DM+R rats (p<0.05). The number and depth of resorptive pits on surface of the bone in Rosiglitazone treated rats improved clearly with Spirulina administration. The intactness and integrity of the bone surface as well as the bone strength improved due to the high content of calcium and phosphorous in Spirulina. Besides, chromium and gamma-linoleic acid in Spirulina helped to decrease the fasting serum glucose, HDL, LDL and triglycerides levels in insulin resistance rats.
These findings suggest that combination therapy of Rosiglitazone with Spirulina reduced the risk of osteoporosis in insulin resistance rats. Additionally, Spirulina complemented the antihyperglycemic and antilipidemic activity of Rosiglitazone.

Gupta S, Hrishikeshvan HJ, Sehajpal PK
Diabetes Res. Clin. Pract. Jan 2010
PMID: 19896232

Black Tea Suppresses Bone Turnover in Rats

Abstract

Evidence for a prospective anti-osteoporosis effect of black tea (Camellia Sinensis) extract in a bilaterally ovariectomized rat model.

The purpose of this study was to examine whether whole aqueous black tea extract (BTE) prevents bone loss induced by ovarian hormone deficiency. Eighteen 95-100 days old female albino rats were randomly assigned to three treatment groups [sham -operated control (sham); bilaterally ovariectomized (ovx) and ovx + aqueous black tea extract (BTE) ] and sacrificed after 28 days. All animals were fed a standard laboratory diet with free access to deionized water except on days of urinary parameter studies when animals were given only calcium free deionized water during the entire 24 h period of urine collection. Body weight study revealed that rats in the ovx group had significantly higher final body weight than rats in the sham group. This higher final body weight was not observed in animals receiving BTE. The ovx group also had significantly higher abdominal fat mass and liver weight and significantly lower uterus, right kidney and left kidney weights than in other two groups. All these organ weight changes in ovx group also were not observed in animals receiving BTE. Results of urinary studies revealed that rats in the ovx group had significantly higher urinary excretion of calcium (Ca), phosphate, creatinine (Cr), calcium to creatinine (Ca:Cr) ratio (P< 0.001) and hydroxyproline (HPr) (P< 0.01) than rats in the sham group. Significant recovery of all these parameters were observed in animals receiving BTE. The ovx group also had significantly higher (P< 0.001) serum alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) activity than rats in the other two groups. These changes could not be seen in animals receiving BTE. Also, identical changes were seen in bone density experiments. Rats in the ovx group had significantly lower densities of the right femur (P<0.001), eighth thoracic rib (P< 0.001), eighth thoracic vertebra (P< 0.05), and fourth lumbar vertebra (P< 0.01) than rats in the sham group; and significant improvement in densities of these bones were seen in animals supplemented with BTE. Animals of ovx group also showed significant decrease in calcium and phosphate level in all these bones which could be regained significantly when these animals were supplemented with BTE. Our findings suggest that aqueous BTE may be effective in preventing bone loss due to ovarian hormone deficiency. Because serum activity of AP, TRAP and urinary loss of bone minerals (Ca and Phosphate) and also the organic components of bone (Cr and HPr) were significantly greater in the ovx group, compared to sham animals and ovx + BTE group. This confirms that ovariectomy enhances and BTE suppresses the rate of bone turnover. The density results of ovx + BTE group are significantly greater than rats in the ovx group, suggesting further that formation exceeded resorption. Detailed studies are underway to clarify the mechanism of this protective effect of BTE on hypogonadal bone loss.

Das AS, Mukherjee M, Mitra C
Asia Pac J Clin Nutr 2004
PMID: 15228990 | Free Full Text

Green Tea Prevents Bone Loss in Rats

Abstract

Protective effect of green tea polyphenols on bone loss in middle-aged female rats.

Recent studies have suggested that green tea polyphenols (GTP) are promising agents for preventing bone loss in women. Findings that GTP supplementation resulted in increased urinary GTP concentrations and bone mass via an increase of antioxidant capacity and/or a decrease of oxidative stress damage suggest a significant role of GTP in bone health of women.
Recent studies suggested that green tea polyphenols (GTP) are promising agents for preventing bone loss in women. However, the mechanism related to the possible protective role of GTP in bone loss is not well understood.
This study evaluated bioavailability, mechanisms, bone mass, and safety of GTP in preventing bone loss in middle-aged rats without (sham, SH) and with ovariectomy (OVX).

A 16-week study of 2 (SH vs. OVX) x 3 (no GTP, 0.1% GTP, and 0.5% GTP in drinking water) factorial design using 14-month-old female rats (n = 10/group) was performed. An additional 10 rats in baseline group were euthanized at the beginning of study to provide baseline parameters.
There was no difference in femur bone mineral density between baseline and the SH+0.5% GTP group. Ovariectomy resulted in lower values for liver glutathione peroxidase activity, serum estradiol, and bone mineral density. GTP supplementation resulted in increased urinary epigallocatechin and epicatechin concentrations, liver glutathione peroxidase activity and femur bone mineral density, decreased urinary 8-hydroxy-2′-deoxyguanosine and urinary calcium levels, but no effect on serum estradiol and blood chemistry levels.
We conclude that a bone-protective role of GTP may contribute to an increase of antioxidant capacity and/or a decrease of oxidative stress damage.

Shen CL, Wang P, Guerrieri J, Yeh JK…
Osteoporos Int Jul 2008
PMID: 18084689

EGCG Suppresses Osteoclasts and Arthritis in Mice

Abstract

(-)-Epigallocatechin-3-gallate suppresses osteoclast differentiation and ameliorates experimental arthritis in mice.

To verify the effects of (-)-epigallocatechin-3-gallate (EGCG) on osteoclast differentiation and on experimental arthritis in mice.
Human osteoclasts were differentiated from peripheral blood monocytes. The effects of EGCG were examined by tartrate-resistant acid phosphatase (TRAP) staining, bone resorption assay, Western blotting, and quantitative real-time polymerase chain reaction.

Arthritis was induced in mice by injecting a cocktail of monoclonal antibodies against collagen. EGCG (20 microg/gm body weight) was administered intraperitoneally every day from day 0 through the end of the experiments (day 15). The effects of EGCG were determined by assessments of joint swelling, histologic changes, and TRAP staining on day 15.
EGCG reduced the generation of TRAP-positive multinucleated cells, bone resorption activity, and osteoclast-specific gene expression without affecting cell viability. EGCG down-regulated expression of nuclear factor of activated T cells c1 (NF-ATc1), but not of NF-kappaB, c-Fos, and c-Jun, suggesting that down-regulation of NF-ATc1 is one of the molecular bases of EGCG action. Additionally, EGCG treatment ameliorated clinical symptoms and reduced histologic scores in arthritic mice (P < 0.05). The in vivo effect of EGCG on osteoclast differentiation was not clear in this model, probably because EGCG suppressed the inflammation itself.
EGCG suppressed osteoclast differentiation and ameliorated experimental arthritis in mice over the short term. It remains to be established whether EGCG is useful for the prevention and treatment of osteoporosis and rheumatoid arthritis.

Morinobu A, Biao W, Tanaka S, Horiuchi M…
Arthritis Rheum. Jul 2008
PMID: 18576345 | Free Full Text

EGCG Inhibits Osteoclasts in Mice

Abstract

Epigallocatechin-3-gallate inhibits osteoclastogenesis by down-regulating c-Fos expression and suppressing the nuclear factor-kappaB signal.

Epigallocatechin-3-gallate (EGCG), the major anti-inflammatory compound in green tea, has been shown to suppress osteoclast differentiation. However, the precise molecular mechanisms underlying the inhibitory action of EGCG in osteoclastogenesis and the effect of EGCG on inflammation-mediated bone destruction remain unclear. In this study, we found that EGCG inhibited osteoclast formation induced by osteoclastogenic factors in bone marrow cell-osteoblast cocultures but did not affect the ratio of receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) to osteoprotegerin induced by osteoclastogenic factors in osteoblasts. We also found that EGCG inhibited osteoclast formation from bone marrow macrophages (BMMs) induced by macrophage colony-stimulating factor plus RANKL in a dose-dependent manner without cytotoxicity. Pretreatment with EGCG significantly inhibited RANKL-induced the gene expression of c-Fos and nuclear factor of activated T-cells (NFATc1), essential transcription factors for osteoclast development. EGCG suppressed RANKL-induced activation of c-Jun N-terminal protein kinase (JNK) pathway, among the three well known mitogen-activated protein kinases and also inhibited RANKL-induced phosphorylation of the NF-kappaB p65 subunit at Ser276 and NF-kappaB transcriptional activity without affecting the degradation of IkappaBalpha and NF-kappaB DNA-binding in BMMs. The inhibitory effect of EGCG on osteoclast formation was somewhat reversed by retroviral c-Fos overexpression, suggesting that c-Fos is a downstream target for antiosteoclastogenic action of EGCG. In addition, EGCG treatment reduced interleukin-1-induced osteoclast formation and bone destruction in mouse calvarial bone in vivo. Taken together, our data suggest that EGCG has an antiosteoclastogenic effect by inhibiting RANKL-induced the activation of JNK/c-Jun and NF-kappaB pathways, thereby suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors.

Lee JH, Jin H, Shim HE, Kim HN…
Mol. Pharmacol. Jan 2010
PMID: 19828731 | Free Full Text

EGCG Improves Bone in Ovariectomized Rats

Abstract

(-)-Epigallocatechin-3-gallate improves bone microarchitecture in ovariectomized rats.

Previously, we reported that (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, increased the osteogenic differentiation of murine bone marrow mesenchymal stem cells by increasing the messenger RNA expression of osteogenesis-related genes, alkaline phosphatase activity, and, eventually, mineralization. The present study further investigated the effects of EGCG on bone microstructure change and possible mechanisms in ovariectomy (OVX)-induced osteopenic rats.
Rats subjected to OVX were administered EGCG systemically for 12 weeks. Proximal tibial bone mineral densities before and after treatment were compared between groups. Changes in the microarchitecture of both the proximal tibia and the third lumbar spine were compared between EGCG-treated and nontreated groups using micro-CT (μCT). Bone histology and immunohistochemistry in the proximal tibia were evaluated.
Results showed that EGCG 3.4 mg/kg/day (estimated peak serum concentration, 10 μmol/L) hampered the decrease in bone mineral density (from 7.97% to 3.96%) and improved the parameters of μCT measurements, including bone volume (from 18% to 27%), trabecular thickness (from 0.17 to 0.22 mm), trabecular number (from 1.13 to 1.37 mm(-1)), and trabecular separation (from 0.91 to 0.69 mm), compared with nontreated ovariectomized rats. Similar improvements in bone volume (from 30% to 49%) and trabecular thickness (from 0.14 to 0.26 mm) were also found in the third lumbar spine. Bone volume in the tibial cortex also increased after EGCG treatment (from 9% to 28%). A higher trabecular number and greater trabecular volume were also seen in histology, further confirming the results of μCT. The immunolocalized bone morphogenetic protein 2 brown-stained area increased from 31% in the OVX group to 53% in the OVX + 10 EGCG group (P < 0.01). Serial biochemistry data revealed no significant systemic toxic effect of EGCG.
Intraperitoneal treatment with EGCG 3.4 mg/kg/day for 3 months can mitigate bone loss and improve bone microarchitecture in ovariectomized rats, and increased expression of bone morphogenetic protein 2 may contribute to this effect.

Chen CH, Kang L, Lin RW, Fu YC…
Menopause Jun 2013
PMID: 23511703

Coffee Delays Bone Repair in Rats

Abstract

Effects of cigarette smoke inhalation and coffee consumption on bone formation and osseous integration of hydroxyapatite implant.

The present study aims to assess the effects of cigarette smoke inhalation and/or coffee consumption on bone formation and osseous integration of a dense hydroxyapatite (DHA) implant in rats. For this study, 20 male rats were divided into four groups (n = 5): CT (control) group, CE (coffee) group, CI (cigarette) group and CC (coffee + cigarette) group. During 16 weeks, animals in the CI group were exposed to cigarette smoke inhalation equivalent to 6 cigarettes per day; specimens in the CE group drank coffee as liquid diet; and rats in the CC group were submitted to both substances. In the 6th week a 5 mm slit in the parietal bone and a 4 mm slit in the tibia were performed on the left side: the former was left open while the latter received a DHA implant. As soon as surgeries were finished, the animals returned to their original protocols and after 10 weeks of exposure they were euthanised (ethically sacrificed) and the mentioned bones collected for histological processing. Data showed that exposure to cigarette smoke inhalation and coffee consumption did not interfere in weight gain and that solid and liquid diet consumption was satisfactory. Rats in the CC group showed a decrease in bone neoformation around the tibial DHA implant (31.8 ± 2.8) as well as in bone formation in the parietal slit (28.6 ± 2.2). On their own, cigarette smoke inhalation or coffee consumption also led to diminished bone neoformation around the implant and delayed the bone repair process in relation to the CT group. However, reduction in the bone repair process was accentuated with exposure to both cigarette smoke inhalation and coffee consumption in this study.

Andrade AR, Sant’Ana DC, Mendes JA, Moreira M…
Braz J Biol Feb 2013
PMID: 23644799 | Free Full Text

Melatonin Increases Bone Density in Blind Mice

Abstract

Effects on bone by the light/dark cycle and chronic treatment with melatonin and/or hormone replacement therapy in intact female mice.

In this study, the effects of the light/dark cycle, hormone replacement therapy (HRT), and nocturnal melatonin supplementation on osteogenic markers and serum melatonin levels were examined in a blind mouse model (MMTV-Neu transgenic mice). Melatonin levels in this mouse strain (FVB/N) with retinal degeneration (rd-/-) fluctuate in a diurnal manner, suggesting that these mice, although blind, still perceive light. Real-time RT-PCR analyses demonstrated that Runx2, Bmp2, Bmp6, Bglap, and Per2 mRNA levels coincide with melatonin levels. The effect of chronic HRT (0.5 mg 17β-estradiol + 50 mg progesterone in 1800 kcal of diet) alone and in combination with melatonin (15 mg/L drinking water) on bone quality and density was also assessed by histomorphometry and microcomputed tomography, respectively. Bone density was significantly increased (P < 0.05) after 1 yr of treatment with the individual therapies, HRT (22% increase) and nocturnal melatonin (20% increase) compared to control. Hormone replacement therapy alone also increased surface bone, decreased trabecular space, and decreased the number of osteoclasts without affecting osteoblast numbers compared to the control group (P < 0.05). Chronic HRT + melatonin therapy did not significantly increase bone density, even though this combination significantly increased Bglap mRNA levels. These data suggest that the endogenous melatonin rhythm modulates markers important to bone physiology. Hormone replacement therapy with or without nocturnal melatonin in cycling mice produces unique effects on bone markers and bone density. The effects of these therapies alone and combined may improve bone health in women in perimenopause and with low nocturnal melatonin levels from too little sleep, too much light, or age.

Witt-Enderby PA, Slater JP, Johnson NA, Bondi CD…
J. Pineal Res. Nov 2012
PMID: 22639972