Untreated OVX Rats Research Articles

Blackcurrant extract promotes differentiation of MC3T3‑E1 pre‑osteoblasts.

Osteoporosis risk increases in menopausal individuals owing to the decrease in estrogen secretion. Blackcurrant extract (BCE) ameliorates osteoporosis; however, the underlying mechanisms are unclear. Furthermore, although BCE has phytoestrogenic activity, its effects on osteoblasts are unknown. In the present study, we investigated BCE-mediated attenuation of osteoporosis using mouse MC3T3-E1 pre-osteoblasts, with a focus on osteogenesis. After treating MC3T3-E1 cells with BCE for 48 h, cell proliferation was assessed using Cell Counting Kit-8. Levels of osteoblast differentiation markers, namely alkaline phosphatase activity and total collagen content in the cells, were evaluated after 3 and 14 days of BCE treatment, respectively. The expression of genes encoding osteoblast differentiation markers, including collagen type I (Col-I), alkaline phosphatase (Alp), bone γ-carboxyglutamate protein (Bglap), and runt-related transcription factor 2 (Runx2), was evaluated using reverse transcription-quantitative polymerase chain reaction. Mineralization of the cells was evaluated using Alizarin Red staining. Femoral tissues of ovariectomized (OVX) rats with or without 3% BCE were stained using ALP to evaluate osteogenic differentiation in femoral tissue. After treating MC3T3-E1 cells with BCE, cell proliferation had increased. BCE treatment increased Alp activity and total collagen content. Moreover, the expression of Col-I, Alp, Bglap, and Runx2 increased in BCE-treated cells. Furthermore, when MC3T3-E1 cells were treated with BCE for 21 days, the levels of calcified nodules increased. Alp staining intensity was stronger in the epiphyses on femoral tissue of OVX rats treated with 3% BCE than in those of untreated OVX rats. The results suggest that BCE may promote osteogenesis by inducing osteoblast differentiation.

Estrogenic and anti-amnesic potential of Millettia griffoniana Baill. (Fabaceae) ethanolic extract on scopolamine-induced memory impairment in ovariectomized Wistar rats.

Dementias including Alzheimer disease (AD) are three times higher in menopausal women than in men. Phytoestrogens, a group of plant-derived compounds are known to alleviate menopausal complaints including dementia. Millettia griffoniana Baill is a phytoestrogen-rich plant used to treat menopausal complaints and dementia. Evaluating the estrogenic and neuroprotective potential of Millettia griffoniana on ovariectomized (OVX) rats. The in vitro safety of M. griffoniana ethanolic extract was assayed by MTT in human mammary epithelial (HMEC) and mouse neuronal (HT-22) cells and its lethal dose 50 (LD50) was estimated following OECD 423 guidelines. For estrogenicity, in vitro the well known E-screen assay on MCF-7cells was performed and in vivo four groups of OVX rats were treated either with 75, 150 and 300mg/kg M. griffoniana extract doses or estradiol (1mg/kg BW) for three days; and changes in uterine and vagina were analyzed. Then, for neuroprotective effect, Alzheimer-type dementia induction was achieved by scopolamine (1.5mg/kg B.W., i.p.) injection four days/week and M. griffoniana extract as well as piracetam (standard) were administered daily for 2 weeks to evaluate the extract's neuroprotective potential. The endpoints were the assessment of learning and working memory, oxidative stress state (SOD, CAT, and MDA) in brain, acetylcholine esterase (AChE) activity and the histopathological changes in hippocampus. No toxic effect was observed when incubating mammary (HMEC) and neuronal (HT-22) cells with M. griffoniana ethanol extract for 24h and its LD50 was found >2000mg/kg. The extract also exhibited both in vitro and in vivo estrogenic activities, displayed by a significant (p<0.01) increment in MCF-7cells population in vitro and an increase in the epithelium height of the vagina and the wet weight of the uterus mainly with the 150mg/kg BW extract dose compared to untreated OVX rats. The extract also reversed scopolamine-induced memory impairment in rat by improving learning, working and reference memory. This was associated with an increment in CAT and SOD expression, alongside a decrement in MDA content and AChE activity in hippocampus. Further, the extract reduced neuronal cell loss in hippocampal structures (CA1, CA3 and dentate gyrus). High Performance Liquid Chromatography coupled with Mass Spectrometry (HPLC-MS) spectra, revealed the presence of numerous phytoestrogens in M. griffoniana extract. M. griffoniana ethanolic extract has estrogenic, anticholinesterase and antioxidant activities that could account for its anti-amnesic effects. These findings therefore sheds light on why this plant is commonly used in the therapy of menopausal complaints and dementia.

Suppression of high bone remodelling by E'Jiao in ovariectomised rats.

The current prevention options for postmenopausal osteoporosis are very limited. E'Jiao is a collagen-rich traditional Chinese medicine with the potential to prevent osteoporosis but more comprehensive investigations are lacking. This study aimed to investigate the skeletal protective effects of E'Jiao in a rat model of osteoporosis caused by ovariectomy. Female Sprague Dawley rats (n=42) were randomly assigned into baseline, sham, ovariectomised (OVX) control, OVX-treated with low-dose (0.26g/kg), medium dose (0.53g/kg) and high dose E'Jiao (1.06g/kg), as well as calcium carbonate (1% w/v) groups. Daily treatment through oral gavage was initiated 7 days after OVX. The rats were euthanised after eight weeks of treatment. Bone mineral density and content were measured at baseline, 1 and 2 months after treatment. Blood was collected for the measurement of bone remodelling markers. Femur and tibial bones were collected for histomorphometry and biomechanical strength analysis. Untreated OVX rats showed high bone remodelling marked by the increased bone formation and bone resorption markers, as well as increased mineralising surface/bone surface ratio. In addition, osteoclast surface and single-labelled surface were increased while mineral apposition rate was reduced in the untreated OVX rats. These changes were antagonised by E'Jiao at all doses. However, the structural, cellular and biomechanical parameters were not affected by ovariectomy and treatment. In conclusion, E'Jiao prevented high bone remodelling during oestrogen deficiency but a long-term study will be required to establish its effects on structural and biomechanical changes due to oestrogen deficiency.

Normalisation of High Bone Remodelling due to Oestrogen Deficiency by Traditional Chinese Formulation Kang Shuai Lao Pian in Ovariectomised Rats.

Postmenopausal osteoporosis transpires due to excessive osteoclastic bone resorption and insufficient osteoblastic bone formation in the presence of oestrogen insufficiency. Kang Shuai Lao Pian (KSLP) is a red ginseng-based traditional Chinese medicine known for its anti-ageing properties. However, studies on its effect on bone loss are lacking. Thus, the current study examined the skeletal protective effects of KSLP in an ovariectomised rodent bone loss model. Three-month-old female Sprague Dawley rats (n=42) were randomised into baseline, sham and ovariectomised (OVX) groups. The OVX rats were supplemented with low- (KSLP-L; 0.15 g/kg), medium- (KSLP-M; 0.30 g/kg), high-dose KSLP (KSLP-H; 0.45 g/kg) or calcium carbonate (1% w/v). The daily supplementation of KSLP was performed via oral gavage for eight weeks. Gavage stress was stimulated in the ovariectomised control with distilled water. The rats were euthanised at the end of the study. Whole-body and femoral bone mineral content and density scans were performed at baseline and every four weeks. Blood samples were obtained for the determination of bone remodelling markers. Histomorphometry and biomechanical strength testing were performed on femurs and tibias. High bone remodelling typically due to oestrogen deficiency, indicated by the elevated bone formation and resorption markers, osteoclast surface, single-labelled surface and mineralising surface/bone surface ratio, was observed in the untreated OVX rats. Whole-body BMD adjusted to body weight and Young's modulus decreased significantly in the untreated OVX rats. High-dose KSLP supplementation counteracted these degenerative changes. In conclusion, KSLP improves bone health by normalising bone remodelling, thereby preventing bone loss and decreased bone strength caused by oestrogen deficiency. Its anti-osteoporosis effects should be validated in patients with postmenopausal osteoporosis.

Chrysin alleviates alteration of bone-remodeling markers in ovariectomized rats and exhibits estrogen-like activity in silico.

Evidences are beginning to accrue that flavonoids, particularly phytoestrogens, could have beneficial effects against several age-related diseases linked to estrogen deficiency including postmenopausal osteoporosis. In this study, the effect of chrysin on selected bone-remodeling markers in ovariectomized rats and its estrogen-like activity in silico were investigated. The data indicated that administration of chrysin at 50mg/kg and 100mg/kg for 6weeks to OVX rats significantly (p < 0.05) prevented body weight gain and partially reverse uterine weight loss. In addition, treatment of OVX rats significantly (p < 0.01) increased femur dry weight, femur ash weight, bone ash calcium, and phosphorous levels in a dose-dependent manner. However, there was significant (p < 0.001) decline in serum estradiol level in all OVX rats compared to the sham-operated group. Interestingly, administration of chrysin significantly (p < 0.05) reversed the reduction of estradiol induced by ovariectomy compared to untreated OVX rats. Moreover, administration of chrysin to OVX rats significantly (p < 0.05) suppressed excessive elevation of bone-remodeling markers expression compared to untreated OVX rats. Similarly, molecular docking analysis revealed that chrysin interacts with both α and β estrogen receptors with exothermic binding energies of -229.83kcal/Mol and -252.72kcal/Mol, respectively, and also fits perfectly into the active site of both α and β estrogen receptors. This study demonstrated that chrysin exhibits potential antiosteoporotic effects against bone loss in OVX rats through enhanced bone mineral contents and preventing excessive elevation of bone-remodeling markers and bone-resorbing cytokine.

The Impact of Genistein Supplementation on Tendon Functional Properties and Gene Expression in Estrogen-Deficient Rats.

Tendinopathy risk increases with menopause. The phytoestrogen genistein prevents collagen loss during estrogen deficiency (ovariectomy [OVX]). The influence of genistein on tendon function and extracellular matrix (ECM) regulation is not well known. We determined the impact of genistein on tendon function and the expression of several genes important for the regulation of tendon ECM. Eight-week-old rats (n = 42) were divided into three groups: intact, OVX, or OVX-genistein (6 mg/kg/day) for 6 weeks. Tail fascicles were assessed with a Deben tensile stage. Achilles tendon mRNA expression was determined with digital droplet polymerase chain reaction. Compared to intact, fascicle stress tended to be lower in untreated OVX rats (P = .022). Furthermore, fascicle modulus and energy density were greater in genistein-treated rats (P < .05) compared to intact. Neither OVX nor genistein altered expression of Col1a1, Col3a1, Casp3, Casp8, Mmp1a, Mmp2, or Mmp9 (P > .05). Compared to intact, Tnmd and Esr1 expression were greater and Pcna and Timp1 expression were lower in OVX rats (P < .05). Genistein treatment returned Tnmd, Pcna, and Timp1 to levels of intact-vehicle (P < .05), but did not alter Scx or Esr1 (P > .05). Several β-catenin/Wnt signaling-related molecules were not altered by OVX or genistein (P > .05). Our findings demonstrate that genistein improves tendon function in estrogen-deficient rats. The effect of genistein in vivo was predominately on genes related to cell proliferation rather than collagen remodeling.

The Effect of Systemic Delivery of Bisphosphonates on Trabecular and Cortical Bone Mass of Ovariectomized Rats

Background: Bisphosphonates are one of the most important drug families, which inhibit osteoclast-mediated bone loss due to osteoporosis and other disorders. However, the impact of different members of this family of drugs on the various cortical and trabecular bone mineral density wasn’t investigated. Objectives: In this study the therapeutic effects of bisphosphonates in Ovariectomized (OVX) female rats was assessed, for eight weeks, and these effects were compared on trabecular and cortical bones. Methods: Thirty adult female rats (225 + 25 g) were divided to five equal groups [Sham, the untreated OVX rats (negative control), and the OVX rats treated with zoledronic acid, alendronate, and risedronate]. The effectiveness of these treatments after eight weeks was comprehensively evaluated by in vivo and in vitro analysis. Results: Systemic delivery of zoledronic acid, alendronate, and risedronate had statistically significant effects on serum parameters in comparison to the untreated OVX animals. Moreover, administration of zoledronic acid, alendronate, and risedronate increased expression of osteogenic genes (OCN, ALP, and Col1), bone mineral density (BMD), and biomechanical performance of the OVX-induced osteoporotic bones in comparison with the untreated OVX rats (P < 0.05). However, different analyses showed that trabecular BMD significantly increased in zoledronic acid- and risedronate-treated animals in comparison to those of the alendronate-treated group (P < 0.05). Conclusions: It was concluded that zoledronic acid, alendronate, and risedronate sufficiently improved OVX-induced osteopenia. Moreover, it was found out that zoledronic acid and risedronate considerably improved the regeneration of trabecular bones in comparison to the alendronate.

Effect of dietary soy isoflavones on bone loss in ovariectomized rats

Purpose : To determine the effect of dietary soy isoflavone supplementation on bone loss in ovariectomized (OVX) rats. Methods : Forty-eight rats were assigned randomly to groups of OVX rats receiving soy isoflavones (20, 30, or 40 mg/kg of body weight daily), untreated OVX rats, or untreated intact rats. After 8 weeks, bone mineral density (BMD), mineral (Ca, P, Mn, Mg, and Zn) concentrations, and the expression of osteoblast-related genes were measured in femur tissue samples. Results : Eight weeks after OVX, there was a significant decrease in body weight, serum levels of osteocalcin, alkaline phosphatase, and oestradiol, BMD and mineral elements, as well as the expressions of Ctnnb1, Runx2, and Sp7 (all p < 0.05). These decreases were accompanied by reduced maximum load capacity of lumbar vertebrae. Daily supplementation with soy isoflavones dosedependently ameliorated these effects (all p < 0.05). Western blotting revealed that these effects were likely due to the reversal of the OVX-induced decrease in Notch1 proteins in bone and muscle. Conclusion : Soy isoflavone treatment represents a potential therapy for preventing postmenopausal bone loss by stimulating the Notch signalling. Keywords : Mineral elements, Alkaline phosphatase, Isoflavones, Bone loss, Notch pathway

Comparative impact of systemic delivery of atorvastatin, simvastatin, and lovastatin on bone mineral density of the ovariectomized rats.

In addition to lipid-lowering properties, statins have been suggested to affect bone turnover by increasing the osteoblastic bone formation and blocking the osteoclastogenesis. However, there are many controversial reports regarding the beneficial effect of statins on osteoporosis. In this study, we investigated the therapeutic effects of the most important lipophilic statins administered orally for 60 days to the ovariectomized (OVX) female Sprague-Dawley rats and compared the effects on different harvested trabecular and compact bones. Thirty female rats were divided into five equal groups including the normal rats, untreated OVX rats (negative control), and the OVX rats treated with atorvastatin (20 mg/kg/day), simvastatin (25 mg/kg/day), and lovastatin (20 mg/kg/day). The osteoporotic animals were treated daily for 60 days and euthanized at the end of experiments. The effectiveness of these treatments was evaluated by biomechanical testing, histopathologic, histomorphometric, micro-CT scan, real-time PCR, and serum biochemical analysis. Moreover, the hepatotoxicity and rhabdomyolysis related with these treatments were assessed by biochemistry analysis and histopathological evaluation. The results and statistical analysis showed that systemic delivery of simvastatin and lovastatin significantly increased serum calcium level, expression of osteogenic genes, bone mineral density (BMD), and biomechanical properties in comparison to the untreated OVX rats, especially in trabecular bones (P < 0.05). The results of different analysis also indicated that there was no statistical difference between the atorvastatin-treated animals and the negative control. Among all treatments, only atorvastatin showed an evident hepatotoxicity and myopathy. It was concluded that the lovastatin and simvastatin efficiently ameliorated the OVX-induced osteoporosis. Moreover, the simvastatin-treated animals showed more resemblance to the normal group in terms of BMD, expression of osteogenic genes, serum biochemical parameters, histomorphometric findings, and biomechanical performance with no significant side-effects.

Effects of low-intensity non-coherent light therapy on the inflammatory process in the calcaneal tendon of ovariectomized rats.

The aim of this experimental study was to investigate the effects of low-intensity light-emitting diode (LED) phototherapy on the inflammatory process in the calcaneal tendon of ovariectomized rats (OVX) through the involvement of the inflammatory mediators interleukin (IL)-6, IL-10, and tumor necrosis factor-alpha (TNF-α). Thirty-five female Wistar rats were divided into 4 groups: 3 groups of OVX rats totaling 30 rats (untreated OVX rats [OVX injury group], treated OVX rats [OVX LED group], and control OVX rats; subgroups existed based on the sampling times, which were 3, 7, and 14days) and 1 group of non-OVX rats (not OVX; n = 5). Tendon injury was induced by trauma using a 208-g mass placed at 20cm from the right tendon of each animal with energy of 0.70J. The animals were treated 12h after tendonitis with LED therapy and every 48h thereafter until euthanasia (at 3, 7, or 14days). The tendons were dissected and stored in liquid nitrogen at -196°C, thawed only at the time of immunoenzymatic testing (ELISA). Groups treated with LED showed a decrease in the number of pro-inflammatory cells, IL-6, and TNF-α (p <0.05), and an increase in IL-10 (p < 0.05) when compared to the not OVX group (p < 0.05). It was concluded that low-intensity LED treatment using the parameters and wavelength of 945nm in the time periods studied reduced the release of IL-6 and TNF-α and increased the release of IL-10, thereby improving the inflammatory response in OVX rats.

Abstract 2724: NMU induction of mammary tumors in ovariectomized rats administered peroral estrogen and progesterone

Abstract Mammary tumors induced by carcinogens such as N-nitroso-N-methylurea (NMU) are thought to be estrogen dependent since ovariectomy (OVX) or treatment with tamoxifen or aromatase inhibitors inhibits mammary tumor development. However, whether NMU-induced tumor formation can be restored in OVX animals with estradiol (E2) and/or progesterone (P4) administration is unclear. Common methods of hormone administration such as pellet implants, injections or gavage feeding can lead to supraphysiological levels of hormone or unwarranted stress for use in long term tumor studies. To circumvent these problems, we administered hormones daily in peanut butter to OVX Sprague-Dawley rats. Short-term (10 day) pilot studies were conducted to determine the dose of E2 that would decrease body weights and increase uterine weights of OVX animals relative to sham controls. An initial pilot using 150 μg/kg E2 for 10 days led to decreased body weight gain in OVX animals but was unable to restore uterine weights. A second pilot analyzed a dose of 300 μg/kg or 600 μg/kg E2. Body weights were repressed in both groups relative to OVX, but only the 600 μg/kg dose was able to restore uterine weights. Therefore the 600 μg/kg dose was used to test whether hormones fed in peanut butter could drive tumor development. Following surgery on PND 40, rats received daily treatments in peanut butter throughout the study. Rats that were OVX were divided into four treatment groups (n = 8): 1) E2; 2) P4 (15 mg/kg); 3) E2 + P4; and 4) vehicle, with a fifth group given vehicle after sham surgery (n = 8) as a positive control. On PND 50, all animals received a single I.P. injection of 50 mg/kg NMU to induce mammary tumors. Rats were weighed and palpated biweekly. Blood was collected via tail vein at 4, 8 and 12 weeks following NMU administration 2 to 4 hours after hormone feeding. Analysis of serum E2 indicated that levels were elevated and not supraphysiological. Body weights of E2 groups with or without P4 were similar to sham body weights while the P4 group had body weights similar to those of untreated OVX rats. Mammary tumors were first observed between 6 and 7 weeks post NMU-injection in sham, E2 + P4 and E2 animals. At 18 weeks, 62.5, 37.5, and 25% of rats presented with tumors and total tumors per group were 8, 3 and 3 in sham, E2 + P4 and E2 groups, respectively. One tumor was observed in an OVX + vehicle rat at 8.5 weeks while no tumors were observed in the P4 group. Collectively, these studies indicate that administering hormones perorally in peanut butter restores body weights and uterine weights of OVX animals and allows for tumor formation in response to NMU. The lower tumor incidence in OVX animals receiving hormones suggests that additional factors may be necessary to fully restore NMU-induced tumors. Citation Format: Hillary Stires, Mariana D. Saboya, Samantha P. Globerman, Wendie S. Cohick. NMU induction of mammary tumors in ovariectomized rats administered peroral estrogen and progesterone. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2724. doi:10.1158/1538-7445.AM2015-2724

Comparing the effects of 17β-oestradiol and the selective oestrogen receptor modulators, raloxifene and tamoxifen, on prepulse inhibition in female rats

BackgroundEvidence suggests that oestrogen plays a protective role against the development and severity of schizophrenia. However, while oestrogen may be beneficial as a treatment in schizophrenia, its chronic use is associated with side-effects. Selective oestrogen receptor modulators (SERMs) may provide an alternative, however little is known about the mechanism underlying their effects in schizophrenia. MethodsWe investigated the effect of raloxifene and tamoxifen on dopaminergic-induced disruptions of prepulse inhibition (PPI). PPI measures sensorimotor gating and PPI disruptions are considered an endophenotype for schizophrenia. Adult female Sprague–Dawley rats were either intact, ovariectomized (OVX), OVX and 17β-oestradiol-treated, OVX and raloxifene-treated (low or high dose), or OVX and tamoxifen-treated (low or high dose). ResultsThe dopamine D1/D2 receptor agonist, apomorphine (0, 0.1, 0.3 and 1mg/kg), caused the expected dose-dependent disruption in PPI in intact and OVX rats. This PPI disruption was prevented in OVX rats treated with 17β-oestradiol, a high dose of raloxifene or a high dose of tamoxifen. In untreated OVX rats, average PPI was 55% after saline and 34% after 1mg/kg apomorphine treatment, a reduction of 21%. However, oestradiol-treated and raloxifene-treated OVX rats showed only a 7% PPI reduction, and tamoxifen-treated OVX rats had a 4% PPI reduction caused by apomorphine treatment. Startle amplitude was not different between the groups. ConclusionThe SERMs, raloxifene and tamoxifen, can prevent dopamine D1/D2 receptor-mediated disruptions of sensorimotor gating, similar to oestradiol. These data lend support for the use of SERMs as a treatment for schizophrenia.

0042 : Chronic increase in blood flow in rat mesenteric resistance arteries improved endothelium (NO)-mediated dilation: essential role of estrogens

Resistance arteries (RA) have a key role in the control of local blood flow. They undergo outward expansive remodeling in response to a chronic increase in blood flow as seen in post-ischemic collateral arteries growth. This remodeling is associated with improved endothelium-dependent dilation. We hypothesized that estrogens play a role in flow-mediated improvement of endothelium-dependent dilation. Local increase in blood flow in one mesenteric artery was obtained local surgery in three-month old ovariectomized female rats treated or not with 17-b-estradiol (E2). Changes in arterial function and structure were measured after 2 weeks. After 2 weeks, arterial diameter increased in high flow (HF) compared to normal flow (NF) arteries in ovariectomized rats treated with E2, not in untreated rats. Acetylcholine-mediated relaxation was higher in HF than in NF arteries in control and OVX rats treated with E2. In untreated rats, the relaxation was lower in HF than in NF vessels. eNOS expression level was higher in HF than in NF vessels in E2-treated rats only. Acetylcholine-mediated relaxation was fully dependent on the production of NO in E2-treated rats (total inhibition by the NO-synthesis blocker L-NAME). In untreated OVX rats L-NAME blocked only partly the relaxation. Endothelium-independent relaxation (sodium nitroprusside) was not affect by OVX or by E2. Serotoninand phenylephrine-mediated contraction was higher in HF than in NF arteries in both treated and untreated OVX rats. Thus, we demonstrated the essential role of endogenous E2 in flowmediated improvement of endothelium (NO)-mediated dilation in mesenteric resistance arteries.

Effects of sequential osteoporosis treatments on trabecular bone in adult rats with low bone mass.

We used an osteopenic adult ovariectomized (OVX) rat model to evaluate various sequential treatments for osteoporosis, using FDA-approved agents with complementary tissue-level mechanisms of action. Sequential treatment for 3 months each with alendronate (Aln), followed by PTH, followed by resumption of Aln, created the highest trabecular bone mass, best microarchitecture, and highest bone strength. Individual agents used to treat human osteoporosis reduce fracture risk by ∼ 50-60%. As agents that act with complementary mechanisms are available, sequential therapies that mix antiresorptive and anabolic agents could improve fracture risk reduction, when compared with monotherapies. We evaluated bone mass, bone microarchitecture, and bone strength in adult OVX, osteopenic rats, during different sequences of vehicle (Veh), parathyroid hormone (PTH), Aln, or raloxifene (Ral) in three 90-day treatment periods, over 9 months. Differences among groups were evaluated. The interrelationships of bone mass and microarchitecture endpoints and their relationship to bone strength were studied. Estrogen deficiency caused bone loss. OVX rats treated with Aln monotherapy had significantly better bone mass, microarchitecture, and bone strength than untreated OVX rats. Rats treated with an Aln drug holiday had bone mass and microarchitecture similar to the Aln monotherapy group but with significantly lower bone strength. PTH-treated rats had markedly higher bone endpoints, but all were lost after PTH withdrawal without follow-up treatment. Rats treated with PTH followed by Aln had better bone endpoints than those treated with Aln monotherapy, PTH monotherapy, or an Aln holiday. Rats treated initially with Aln or Ral, then switched to PTH, also had better bone endpoints, than monotherapy treatment. Rats treated with Aln, then PTH, and returned to Aln had the highest values for all endpoints. Our data indicate that antiresorptive therapy can be coupled with an anabolic agent, to produce and maintain better bone mass, microarchitecture, and strength than can be achieved with any monotherapy.

Effect of Urocortin on strength and microarchitecture of osteopenic rat femur

As yet there is no evidence of the potential antiosteoporotic effect of Urocortin-1 (UCN), a corticotropin releasing factor related peptide, in vivo. In this study, and for the first time, we investigated the effect of UCN in a rat osteopenia model. Sixty female Sprague-Dawley rats were divided into 5 groups: (1) sham-operated, (2) untreated ovariectomized (OVX) rats, (3) and (4) OVX animals treated for 5 weeks with daily subcutaneous low-dose UCN (3 μg/kg of BW) or high-dose UCN (30 μg/kg of BW) 8 weeks after ovariectomy, and (5) OVX rats treated with daily estrogen (0.2 mg/kg of BW p.o) 8 weeks after ovariectomy for 5 weeks (E). After sacrifice, the femurs were reserved for biomechanical, histomorphometric and ash testing. In the biomechanical test, the high-dose UCN rats showed significantly improved mechanical stiffness (341.6 N/mm) compared with the untreated OVX animals (275.9 N/mm). In the histomorphometric evaluation, the high-dose UCN rats demonstrated an improved trabecular microarchitecture especially and significantly at the distal femur (distal femur Tb.Ar = 41.4% and N.Nd/mm(2) = 26.8, proximal femur Tb.Ar = 71.8% and N.Nd/mm(2) = 28.7) compared with untreated OVX rats (distal femur Tb.Ar = 23.3% and N.Nd/mm(2) = 11.7, proximal femur Tb.Ar = 60.2% and N.Nd/mm(2) = 25.2). Our results show that short-term treatment with UCN seems to have a positive effect on the metaphyseal bone structure and strength of the femur in ovariectomized rats.

Effects of the Selective Estrogen Receptor Modulator Ospemifene on Bone in Rats

Ospemifene is a non-estrogen agent that exerts tissue-specific estrogen agonistic and weak antagonistic effects (i. e., is a selective estrogen receptor modulator [SERM]). The effects of various once-daily oral doses of ospemifene on bone are examined across 3 studies for 4 or 52 weeks after surgery in the ovariectomized (OVX) rat model of postmenopausal bone loss. Ospemifene treatment reduced the loss of bone mineral content and density observed in untreated OVX rats, significantly increased distal femur bone mineral content at 51 weeks at 25 mg/kg dose compared with untreated OVX rats (p<0.01), and significantly increased trabecular bone mineral density of the distal femur and proximal tibia with 1, 5, or 25 mg/kg doses after 52 weeks. Ospemifene 5 and 25 mg/kg preserved distal femur trabecular structure; trabecular number was significantly increased, whereas trabecular separation and eroded surface values were significantly decreased (all p<0.01). Structural changes associated with ospemifene were accompanied by increased mechanical strength of femurs and 4th lumbar vertebra compared with untreated OVX rats. Ospemifene 10 mg/kg prevented OVX-induced bone loss; trabecular bone volume of distal femurs was increased after 4 weeks. Further, histomorphometric measures revealed decreased bone resorption after 4 weeks of ospemifene treatment, with effects similar to other SERMs (raloxifene and droloxifene). Ospemifene 3 and 10 mg/kg significantly inhibited OVX-induced increases in osteoclast number, and doses ≥0.3 mg/kg dose-dependently reversed the OVX-induced increase in the double-labeled volume:bone volume ratio. These results demonstrate antiresorptive, selective agonist effects of ospemifene on bone that appear similar to raloxifene in this in vivo animal model of estrogen deficiency.

Tualang honey supplement improves memory performance and hippocampal morphology in stressed ovariectomized rats

Recently, our research team has reported that Tualang honey was able to improve immediate memory in postmenopausal women comparable with that of estrogen progestin therapy. Therefore the aim of the present study was to examine the effects of Tualang honey supplement on hippocampal morphology and memory performance in ovariectomized (OVX) rats exposed to social instability stress. Female Sprague-Dawley rats were divided into six groups: (i) sham-operated controls, (ii) stressed sham-operated controls, (iii) OVX rats, (iv) stressed OVX rats, (v) stressed OVX rats treated with 17β-estradiol (E2), and (vi) stressed OVX rats treated with Tualang honey. These rats were subjected to social instability stress procedure followed by novel object recognition (NOR) test. Right brain hemispheres were subjected to Nissl staining. The number and arrangement of pyramidal neurons in regions of CA1, CA2, CA3 and the dentate gyrus (DG) were recorded. Two-way ANOVA analyses showed significant interactions between stress and OVX in both STM and LTM test as well as number of Nissl-positive cells in all hippocampal regions. Both E2 and Tualang honey treatments improved both short-term and long-term memory and enhanced the neuronal proliferation of hippocampal CA2, CA3 and DG regions compared to that of untreated stressed OVX rats.

Impact of estradiol, estrogen receptor subtype specific agonists and genistein on food intake, body weight, and glucose metabolism in leptin resistant ovariectomized Zucker diabetic fatty rats

The leptin resistant female Zucker diabetic fatty (ZDF fa/fa) rats are described to be hyperphagic and obese, but compared to its male counterparts they remain euglycaemic. Since estrogens are known to affect glucose metabolism we investigated the long-term effect of estradiol (E2 (4 µg/kg b. wt per day)), ER subtype specific agonists (Alpha (10 µg/kg b. wt per day) and Beta (100 µg/kg b. wt per day)) and genistein (Gen (42 mg/kg b. wt per day)) in ovariectomized ZDF rats. After 4 months food intake, body weight, systemic glucose tolerance, muscular glucose transporter 4 (GLUT4) expression and fiber sizes were determined. Additionally, the impact of E2 and Alpha on glucose uptake in liver, skeletal muscle, and adipose tissue as well as localization of muscular GLUT4 was analyzed. Food intake and body weight were markedly reduced by E2 and Alpha. Glucose tolerance was improved by treatment with E2, Alpha, Beta, and Gen. Glucose absorbance in liver, skeletal muscle and adipose tissue was accelerated in E2 and Alpha treated animals compared to untreated OVX rats (impact of Beta and Gen was not investigated). In the muscle, treatment with Alpha enhanced the expression and translocation of GLUT4 to the cell membrane, whereas Beta substitution resulted in the largest muscle fibers. Administration of Gen suggested affecting both GLUT4 expression and muscle fiber sizes. Thus, our data demonstrates that the ER alpha mediates lower food intake and body weight in leptin resistant female ZDF rats. Activation of ER alpha and beta improves glucose tolerance, but at least in the muscle via different pathways. Further, the ER alpha activation accelerates the glucose absorbance in liver and adipose tissue.

Bovine lactoferrin improves bone mass and microstructure in ovariectomized rats via OPG/RANKL/RANK pathway

Lactoferrin (LF), an 80-kDa iron-binding glycoprotein, is a pleiotropic factor found in colostrum, milk, saliva and epithelial cells of the exocrine glands. The aim of this study was to evaluate the effects of LF on the bones in ovariectomized (Ovx) rats and to identify the pathways that mediate the anabolic action of LF on the bones. Female Sprague-Dawley rats (6-month-old) underwent ovariectomy, and were treated with different doses of LF (10, 100, 1000, and 2000 mg·kg(-1)·d(-1), po) or with 7β-estradiol (0.1 mg·kg(-1), im, each week) as the positive control. By the end of 6 month-treatments, the bone mass and microstructure in the rats were scanned by micro-computed tomography (micro-CT), and the bone metabolism was evaluated with specific markers, and the mRNA levels of osteoprotegerin (OPG) and the receptor-activator of nuclear factor κB ligand (RANKL) in femur were measured using qRT-PCR. LF treatment dose-dependently elevated the bone volume (BV/TV), trabecular thickness (TbTh) and trabecular number (TbN), and reduced the trabecular separation (TbSp) in Ovx rats. Furthermore, higher doses of LF (1000 and 2000 mg·kg(-1)·d(-1)) significantly increased the bone mineral density (BMD) compared with the untreated Ovx rats. The higher doses of LF also significantly increased the serum levels of OC and BALP, and decreased the serum levels of β-CTx and NTX. LF treatment significantly increased the OPG mRNA levels, and suppressed the RANKL mRNA levels, and the RANKL/OPG mRNA ratio in Ovx rats. Oral administration of LF preserves the bone mass and improves the bone microarchitecture. LF enhances bone formation, reduces bone resorption, and decreases bone mass loss, possibly through the regulation of OPG/RANKL/RANK pathway.

Genistein aglycone, a soy‐derived isoflavone, improves skin changes induced by ovariectomy in rats

Ovariectomy accelerates age-related skin changes as adequate oestrogen levels are required to control structural integrity and functional capacity of skin. Genistein, a soy-derived isoflavone, has been tested in anti-ageing cosmetic preparations with interesting results on skin elasticity, photoaging and skin cancer prevention. We investigated the effects of genistein aglycone and compared them with systemic raloxifene hydrochloride and 17-α-ethinyloestradiol on skin changes in aged, ovariectomized (OVX) rats. Six months after ovariectomy, rats were randomly allocated to different groups and treated, daily, with genistein aglycone (1 and 10mg·kg(-1) s.c.), raloxifene hydrochloride (0.05 and 0.5mg·kg(-1) s.c.) or 17-α-ethinyloestradiol (0.003 and 0.03mg·kg(-1) s.c.) for 12 weeks. Controls were untreated OVX and sham OVX rats. At the end of the treatment period, a skin biopsy was carried out and skin samples were assessed for molecular, histological and functional changes. Skin samples of untreated OVX rats showed a decrease in TGF-β1, VEGF, MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 compared with sham OVX rats. All the treatments significantly restored this depressed molecular profile revealed in OVX rats. Genistein aglycone, 1mg·kg(-1) , also significantly increased the thickness of collagen and breaking strength of skin in the OVX rats. Relatively long-term, systemic treatment with genistein aglycone shows comparable efficacy to oestrogen in reversing some molecular, histological and functional changes of the skin associated with ovariectomy in aged rats. This suggests that genistein aglycone might be an effective alternative therapy for the management of age-related skin changes in postmenopausal women.