Animal Model Of Postmenopausal Osteoporosis Research Articles

Using broadly targeted plant metabolomics technology combined with network pharmacology to explore the mechanism of action of the Yishen Gushu formula in the treatment of postmenopausal osteoporosis in vivo

Ethnopharmacological relevanceYishen Gushu Formula (YSGSF) is composed of Epimedium, prepared Rehmannia, Drynaria, Eucommia, Dodder, ginseng, Astragalus, Ligusticum wallichii, Aucklandia and Panax notoginseng. It can improve bone mineral density by regulating bone metabolism. However, the mechanism of YSGSF in the treatment of Postmenopausal osteoporosis (PMOP) remains unclear. Aim of the studyThe compounds, targets, and molecular mechanisms of YSGSF in the treatment of PMOP were investigated using broad-spectrum target metabolomics from plants, combined with network pharmacology and animal studies, leading to a discussion on a novel approach to understanding YSGSF's action in PMOP treatment. Materials and methodsUsing ultra-performance liquid chromatography coupled with triple quadrupole-linear ion trap tandem mass spectrometry (UPLC-QTRAP-MS/MS) within a comprehensive targeted metabolomics framework, the active constituents of YSGSF were identified. This, alongside network pharmacology and molecular docking, facilitated the identification of critical signaling pathways and targets pertinent to YSGSF's therapeutic effect on PMOP. Subsequently, an animal model for PMOP was developed. Following intervention grouping, rats' weight changes were recorded; serum bone metabolic factors were assessed via ELISA; bone microstructure was examined using HE staining and Micro-CT; and key signaling pathway proteins and genes were analyzed through immunohistochemistry to validate YSGSF's potential mechanism in PMOP treatment. ResultsA total of 84 main active components of YSGSF were identified. The key signaling pathways affected by YSGSF in the treatment of PMOP were the TNF and IL-7 signaling pathways, closely related to TNF-α, IL-1β, c-jun and other protein targets. The results of animal experiments showed that YSGSF could downregulate the expression of TNF-a, IL-1β and c-Jun proinflammatory factors by regulating the TNF and IL-7 signaling pathways and regulate the inflammatory response, osteocyte differentiation and apoptosis to control the development of PMOP. ConclusionYSGSF activates the TNF-α and IL-7 signaling pathways in PMOP rats, reducing TNF-α and IL-1β levels, the c-Jun inflammatory response, and osteocyte differentiation and apoptosis, thus playing a significant role in treating PMOP.

Effect of probiotics on postmenopausal bone health: a preclinical meta-analysis.

Postmenopausal osteoporosis is a major concern for women worldwide due to increased risk of fractures and diminished bone quality. Recent research on gut microbiota has suggested that probiotics can combat various diseases, including postmenopausal bone loss. Although several preclinical studies have explored the potential of probiotics in improving postmenopausal bone loss, the results have been inconsistent and the mechanism of action remains unclear. To address this, a meta-analysis was conducted to determine the effect of probiotics on animal models of postmenopausal osteoporosis. The bone parameters studied were bone mineral density (BMD), bone volume fractions (BV/TV), and hallmarks of bone formation and resorption. Pooled analysis showed that probiotic treatment significantly improves BMD and BV/TV of the ovariectomised animals. Probiotics, while not statistically significant, exhibited a tendency towards enhancing bone formation and reducing bone resorption. Next, we compared the effects of Lactobacillus sp. and Bifidobacterium sp. on osteoporotic bone. Both probiotics improved BMD and BV/TV compared with control, but Lactobacillus sp. had a larger effect size. In conclusion, our findings suggest that probiotics have the potential to improve bone health and prevent postmenopausal osteoporosis. However, further studies are required to investigate the effect of probiotics on postmenopausal bone health in humans.

Effect of pumpkin enriched with calcium lactate on iron status in an animal model of postmenopausal osteoporosis

Abstract The homeostasis of calcium (Ca) and iron (Fe) is disturbed during menopause. The present study aimed to determine the effects of Ca-enriched pumpkin on Fe status in ovariectomized rats. A total of 48 female Wistar rats were divided into six groups. One group was fed a standard diet (C), while the other five groups were ovariectomized and fed a standard diet (OVX), a calcium lactate diet (CaL_OVX), calcium lactate-enriched pumpkin (PCaL_OVX), calcium lactate and alendronate (CaL_OVX_B), and calcium lactate-enriched pumpkin and alendronate (PCaL_OVX_B), respectively. The nutritional intervention lasted 12 weeks and rats were euthanized. Tissue samples were collected, and the iron content in the samples was assessed. A comparison of all groups showed a reduction in iron concentrations in femurs, liver, hair, spleen, and kidneys in the ovariectomized groups than in the control group. The PCaL_OVX_B group had a significantly higher blood hemoglobin concentration than the control group. Moreover, spleen and liver Fe concentrations were the highest in PCaL_OVX and PCaL_OVX_B rats among the treated groups and were comparable with the control group. These results indicate that ovariectomy decreases Fe status in rats. Calcium lactate-enriched pumpkin with and without alendronate can increase Fe concentration in liver and spleen in ovariectomized rats.

Protective Effects of the Postbiotic Lactobacillus plantarum MD35 on Bone Loss in an Ovariectomized Mice Model

Postmenopausal osteoporosis is caused by estrogen deficiency, which impairs bone homeostasis, resulting in increased osteoclastic resorption without a corresponding increase in osteoblastic activity. Postbiotics have several therapeutic properties, including anti-obesity, anti-diabetic, anti-inflammatory, and anti-osteoporotic effects. However, the beneficial effects of the postbiotic MD35 of Lactobacillus plantarum on bone have not been studied. In this study, we demonstrated that the postbiotic L. plantarum MD35, isolated from young radish water kimchi, influences osteoclast differentiation in mouse bone marrow-derived macrophage (BMM) culture. In addition, it was effective protecting against estrogen deficiency-induced bone loss in ovariectomized (OVX) mice, an animal model of postmenopausal osteoporosis. In BMM cells, postbiotic MD35 inhibited the receptor activator of nuclear factor-kappa B of NF-κB ligand (RANKL)-induced osteoclast differentiation by attenuating the phosphorylation of extracellular signal-related kinase, significantly suppressing the resorption activity and down-regulating the expression of RANKL-mediated osteoclast-related genes. In the animal model, the oral administration of postbiotic MD35 remarkably improved OVX-induced trabecular bone loss and alleviated the destruction of femoral plate growth. Therefore, postbiotic MD35 could be a potential therapeutic candidate for postmenopausal osteoporosis by suppressing osteoclastogenesis through the regulation of osteoclast-related molecular mechanisms.

The Evaluation of Xiaozeng Qianggu Tablets for Treating Postmenopausal Osteoporosis via up-Regulated Autophagy.

Objective Postmenopausal osteoporosis (PMOP) is a common age-associated disease in the life course. Clinically, Xiaozeng Qianggu Tablets (XQT) have a potent therapeutic effect on the PMOP. However, the bioactive components and the mechanism of XQT underlying the PMOP treatment were unclear and it should be explored to discover the scientific connotation in traditional medical practice. Methods The components in XQT were identified by UPLC-Q-TOF/MS. The animal model of PMOP was established by surgical ovariectomy in the female Sprague-Dawley rats. After treatment of XQT, the therapeutic effect was assessed by the determination of bone metabolism biomarkers in serum and histopathological examination. The effect of XQT on the autophagy and bone micro-situation were tested using western blot, RT-qPCR, and transmission electron microscope. Results There were 27 compounds identified in XQT, including catalpol, monotropein, verbascoside, cryptochlorogenic acid, 5,7-dihydroxychromone 7-rutinoside, biorobin, and so on. The bone metabolism markers (alkaline phosphatase, bone alkaline phosphatase, procollagen type I intact N-terminal propeptide, cross-linked carboxy-terminal telopeptide of type I collagen, and tartrate-resistant acid phosphatase) were significantly increased in the PMOP rats and reversed by XQT administration. Moreover, the width of bone trabeculae and the ratio of the area of calcium deposition to bone trabeculae were also improved after treating the middle dose of XQT. Meanwhile, the bone micro-structure was improved by XQT. The mRNA and protein expression of unc-51 like kinase 1, beclin-1, and microtubule-associated protein 1B-light chain 3 in PMOP rats were down-regulated and up-regulated by XQT administration. Conclusions The compounds in XQT, including catalpol, monotropein, verbascoside cryptochlorogenic acid, and so on, were valuable for further pharmacy evaluation. The pathological changes and bone micro-structure were improved by XQT, and the down-regulated autophagy level was also restored, which suggested a potent effect of XQT on treating PMOP, corresponding to its clinic use.

Effects of (R)-ketamine on reduced bone mineral density in ovariectomized mice: A role of gut microbiota

Depression is a high risk for osteoporosis, suggesting an association between depression and low bone mineral density (BMD). We reported that the novel antidepressant (R)-ketamine could ameliorate the reduced BMD in the ovariectomized (OVX) mice which is an animal model of postmenopausal osteoporosis. Given the role of gut microbiota in depression and bone homeostasis, we examined whether gut microbiota plays a role in the beneficial effects of (R)-ketamine in the reduced BMD of OVX mice. OVX or sham was operated for female mice. Subsequently, saline (10 ml/kg/day, twice weekly) or (R)-ketamine (10 mg/kg/day, twice weekly) was administered intraperitoneally into OVX or sham mice for the six weeks. The reduction of cortical BMD and total BMD in the OVX mice was significantly ameliorated after subsequent repeated intermittent administration of (R)-ketamine. Furthermore, there were significant changes in the α- and β-diversity between OVX + saline group and OVX + (R)-ketamine group. There were correlations between several OTUs and cortical (or total) BMD. There were also positive correlations between the genera Turicibacter and cortical (or total) BMD. Moreover, there were correlations between several metabolites in blood and cortical (or total) BMD. These data suggest that (R)-ketamine may ameliorate the reduced cortical BMD and total BMD in OVX mice through anti-inflammatory actions via gut microbiota. Therefore, it is likely that (R)-ketamine would be a therapeutic drug for depressed patients with low BMD or patients with osteoporosis.This article is part of the Special Issue on ‘Ketamine and its Metabolites’.

Moderate Static Magnetic Fields Prevent Bone Architectural Deterioration and Strength Reduction in Ovariectomized Mice

Substantial studies have revealed the capacity of moderate-intensity static magnetic fields (SMFs) on promoting osteogenesis and inhibiting bone resorption. However, it is unknown whether moderate SMF can resist the deterioration of bone microarchitecture and strength induced by the reduction of estrogen in animal model for postmenopausal osteoporosis. Herein, the efficiency of moderate SMF of 0.2~0.4 T and SMF of 0.6 T on bone mineral density (BMD) and bone mineral content (BMC), bone microarchitecture, bone strength, and histological bone turnover was systematically investigated in ovariectomized (OVX) mice. OVX mice and sham mice were subjected to SMF exposure continuously for four weeks. Dual-energy X-ray absorptiometry (DEXA) data showed SMF prevented the reduction of BMD and BMC in OVX mice. Micro-computed tomography (Micro-CT) results revealed that SMF ameliorated the deterioration of trabecular and cortical bone microarchitecture. Three-point bending test showed that SMF mitigated OVX-induced reduction in femoral mechanical properties, including stiffness, ultimate stress, and elasticity modulus. Moreover, bone histochemical analysis demonstrated that SMF decreased osteoclast formation in cancellous bone and cortical bone, while SMF increased osteoblast formation in trabecular bone. Together, these results demonstrate that moderate SMF alleviated menopause-induced bone loss effectively, and imply that moderate SMF might become a potential biophysical treatment modality for postmenopausal osteoporosis.

Therapeutic potential of annatto tocotrienol with self-emulsifying drug delivery system in a rat model of postmenopausal bone loss

Tocotrienol has been shown to prevent bone loss in animal models of postmenopausal osteoporosis, but the low oral bioavailability might limit its use. A self-emulsifying drug delivery system (SEDDS) could increase the bioavailability of tocotrienol. However, evidence of this system in improving the skeletal effects of tocotrienol is scanty. This study aims to evaluate the therapeutic efficacy of annatto tocotrienol with SEDDS in a rat model of postmenopausal bone loss. Ten-month-old female Sprague Dawley rats were randomized into six groups. The baseline group was euthanatized at the onset of the study. Four other groups underwent ovariectomy to induce estrogen deficiency. The sham underwent similar surgery procedure, but their ovaries were retained. Eight weeks after surgery, the ovariectomized rats received one of the four different regimens orally daily: (a) SEDDS, (b) annatto tocotrienol [60 mg/kg body weight (b.w.)] without SEDDS, (c) annatto-tocotrienol (60 mg/kg b.w.) with SEDDS, (d) raloxifene (1 mg/kg b.w.). After eight weeks of treatment, blood was collected for the measurement of delta-tocotrienol level and oxidative stress markers. The rats were euthanized and their bones were harvested for the evaluation of the bone microstructure, calcium content and strength. Circulating delta-tocotrienol level was significantly higher in rats receiving annatto tocotrienol with SEDDS compared to the group receiving unformulated annatto-tocotrienol (p < 0.05). Treatment with unformulated or SEDDS-formulated annatto tocotrienol improved cortical bone thickness, preserved bone calcium content, increased bone biomechanical strength and increased antioxidant enzyme activities compared with the ovariectomized group (p < 0.05). Only SEDDS-formulated annatto tocotrienol improved trabecular microstructure, bone stiffness and lowered malondialdehyde level (p < 0.05 vs the ovariectomized group). The improvement caused by annatto tocotrienol was comparable to raloxifene. In conclusion, SEDDS improves the bioavailability and skeletal therapeutic effects of annatto tocotrienol in a rat model of postmenopausal bone loss. This formulation should be tested in a human clinical trial to validate its efficacy.

Trilobatin ameliorates bone loss via suppression of osteoclast cell differentiation and bone resorptive function in vitro and in vivo

AimDue to on-going safety concerns or lack of efficacy of currently used medications for the treatment of osteoporosis (OP), identifying new therapeutic agents is an important part of research. In the present study, potential anti-osteoporotic activity of a natural flavonoid glycoside, trilobatin (phloretin 4-O-glucoside, Tri) was evaluated. Material and methodsOsteoclastic cells were established by treating the RAW264.7 macrophage cells with RANKL and ovariectomized (OVX) C57BL/6 female mice were used as an animal model of postmenopausal OP. Actin ring formation, expression levels of osteoclastogenic marker genes and bone resorptive proteins were measured by RT-PCR, western blot, or fluorometric assays. Bone mineral density (BMD) was determined by pDEXA densitometric measurement and serum osteoprotegerin (OPG) and RANKL were measured by ELISA. Key findingTri (5–20 μM) significantly inhibited osteoclast formation and actin ring formation in RANKL-induced osteoclasts. Tri attenuated expression of osteoclastogenic genes (MMP-9 and cathepsin K), bone resorptive proteins (CA II and integrin β3), and osteoclastogenic signalling proteins (TRAF6, p-Pyk2, c-Cbl, and c-Src). Oral administration of Tri to OVX mice augmented BMD and serum OPG/RANKL ratio. Interestingly, while Tri and phloretin aglycone (Phl) showed similar levels of in vitro anti-osteoclastogenic activity, Tri more potently ameliorated bone loss than Phl in OVX mice. SignificanceThis study demonstrated that Tri inhibits osteoclastic cell differentiation and bone resorption by down-regulating the expression of osteoclastogenic marker genes and signalling proteins, bone resorptive proteins, and by augmenting serum OPG/RANKL ratio, suggesting that Tri can be a novel anti-osteoporotic compound for treating senile and postmenopausal OP.

MicroRNA-197-3p Inhibits the Osteogenic Differentiation in Osteoporosis by Down-Regulating KLF 10.

BackgroundStudies have shown that microRNA (miRNA) regulates gene expression of osteoporosis (OS). It is known that miR-197-3p is abnormally expressed in osteoporosis. This study is to investigate the mechanism of miR-197-3p in regulating osteoblast differentiation.MethodsRats were ovariectomized to establish an animal model of postmenopausal osteoporosis. The expression of miR-197-3p and KLF10 was detected in ovariectomized rat models. Primary osteoblasts and MC3T-E1 cells were divided into the control group, miR-197-3p inhibitor group, NC inhibitor group and miR-197-3p inhibitor + si-KLF10 group. The expression of miR-197-3p and Kruppel-like factor 10 (KLF10) was detected by qRT-PCR and Western blot. The relationship between miR-197-3p and KLF10 was analyzed by bioinformatics and luciferase reporter assay. Cell viability was evaluated by MTT assay. The ALP activity measurement and mineralization analysis were performed.ResultsThe expression of miR-197-3p was significantly raised in ovariectomized osteoporosis rats. During the differentiation of osteoblasts, the expression of miR-197-3p was significantly decreased, while the expression of KLF10 was significantly raised in primary osteoblasts and MC3E3T1 cells. The expression of RUNX2, ALP, OCN and OSX in miR-197-3p inhibitor group and MC3T3-E1 group was significantly raised, and the cell survival rate and mineralized nodule were raised as well. KLF10 may be the downstream target gene of miR-197-3p. After co-transfection of miR-197-3p inhibitor and si-klf10, ALP, Runx2, OCN and OSX mRNA, cell survival rate and mineralized nodule were significantly decreased in primary osteoblasts and MC3T3-E1 cells.ConclusionMiR-197-3p Inhibition promoted osteoblast differentiation and reduced OS by up-regulating KLF10.

Longitudinal Changes of Circulating miRNAs During Bisphosphonate and Teriparatide Treatment in an Animal Model of Postmenopausal Osteoporosis.

ABSTRACTMicroRNAs regulate bone homeostasis, and circulating microRNAs have been proposed as novel bone biomarkers. The effect of anti‐osteoporotic treatment on circulating microRNAs has not been described in detail. Therefore, we performed a comprehensive analysis of microRNA serum levels in ovariectomized (OVX) and sham‐operated (SHAM) rats over 12 weeks of antiresorptive or osteoanabolic treatment. Forty‐two Sprague Dawley rats underwent SHAM surgery (n = 10) or ovariectomy (n = 32). After 8 weeks, OVX rats were randomized to antiresorptive treatment with zoledronate (n = 11), osteoanabolic treatment with teriparatide (n = 11), or vehicle treatment (n = 10). Serum samples were collected at weeks 8, 12, 16, and 20 after surgery. A total of 91 microRNAs were analyzed by RT‐qPCR in serum samples collected at week 20. Based on the results, 29 microRNAs were selected for longitudinal analysis at all four study time points. Changes in bone mineral density and microstructure were followed up by in vivo micro‐CT and ex vivo nano‐CT. Ovariectomy resulted in the loss of trabecular bone, which was reversed by osteoanabolic and antiresorptive treatment. Differential expression analysis identified 11 circulating miRNAs that were significantly regulated after treatment. For example, miR‐107 and miR‐31‐5p increased in vehicle‐treated OVX animals, whereas they decreased during teriparatide treatment. Additional miRNAs were identified that showed significant correlations to bone microstructure or bone miRNA expression, including miR‐203a‐3p, which exhibited a significant negative correlation to vertebral and tibial trabecular bone volume fraction (%). Longitudinal analysis confirmed eight microRNAs with significant changes in serum over time that were prevented by teriparatide and zoledronate treatment (miR‐34a‐5p, miR‐31‐5p, miR‐30d‐3p, miR‐378a‐5p) or teriparatide treatment only (miR‐375‐3p, miR‐183‐5p, miR‐203a‐3p, miR‐203b‐3p). Gene target network analysis identified WNT and Notch signaling as the main signaling pathways controlled by these miRNAs. Thus, ovariectomy results in time‐dependent deregulation of circulating miRNAs compared with SHAM animals. Anti‐osteoporotic treatments can rescue this effect, showing that bone‐related miRNAs might act as novel biomarkers for treatment monitoring. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Post-menopausal Osteoporosis and Probiotics.

Postmenopausal osteoporosis (PMO) is characterized by low bone mass and structural deterioration of bone tissue with increased risk of fracture in postmenopausal women. It is due to the deficiency of estrogen production after menopause, which causes the imbalance in the bone remodeling process where resorption/formation skewed more towards resoption, which leads to bone loss. It causes high morbidity and severe health complication among the affected women. The current PMO therapy has many unwanted side effects and even increases the possibility of tumorigenesis. Therefore, an alternative therapy that is safe and effective is required. Probiotics are dietary supplements consisting of beneficial microbes and when administered in an adequate amount, confer a health benefit to the host. Recent scientific evidences suggested the link between the intestinal microbiota and bone health. This review discusses the process of bone remodeling and the role of intestinal microbiota on the bone metabolism of the host. Further, it summarizes the recent studies of probiotic on an animal model of PMO and also in post postmenopausal women.

Water Extract of Agastache rugosa Prevents Ovariectomy-Induced Bone Loss by Inhibiting Osteoclastogenesis

Estrogen deficiency in postmenopausal women causes homeostatic imbalance of bone, resulting in bone loss and osteoporosis. Agastache rugosa, a plant belonging to the Lamiaceae family, is an aromatic herb, and the leaves of this herb are widely used as food ingredients. Extracts of A. rugosa have various bioactivities including anti-HIV integration, anti-inflammatory, and anti-atherogenic properties. However, the beneficial effect of A. rugosa on bone has not been studied. Therefore, we investigated the effects of water extract of A. rugosa (WEAR) on osteoclast differentiation and estrogen deficiency-induced bone loss in ovariectomized (OVX) mice as an animal model for postmenopausal osteoporosis. The oral administration of WEAR remarkably improved OVX-induced trabecular bone loss and fat accumulation in the bone marrow. WEAR suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation in osteoclast precursor cells, subsequently inhibiting resorption activity on a bone mimetic surface. WEAR inhibited the expression of cellular oncogene fos (c-Fos) and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), key osteoclastogenic transcription factors, by decreasing RANKL-induced activation of mitogen-activated protein kinases (MAPKs), and nuclear factor-κB (NF-κB) pathways. We also identified seventeen phytochemicals present in WEAR, including five phenols and twelve flavonoids, and found eleven bioactive constituents that have anti-osteoclastogenic effects. Collectively, these results suggest that WEAR could be used to treat and prevent postmenopausal osteoporosis by suppressing osteoclastogenesis.

Three-dimensional microstructures of the intracortical canals in the animal model of osteoporosis.

Osteoporosis is a major disease in aged women, increasing the risk for fractures accompanied by changes in the microarchitecture. The aim of this study was to investigate the three-dimensional (3D) histomorphology of femur diaphysis in the animal model for postmenopausal osteoporosis. The cortical bone of femur diaphysis of the rat was serially sectioned at a thickness of 5 mm and evaluated age-associated changes of the intracortical (osteonal) canal networks three-dimensionally. Cortical microstructures of 10-month old rats were not affected by ovariectomy. Intracortical canal networks were radial toward endosteal aspect and frequently interconnected across the neighboring canals with short arciform and irregular canals reminiscent for resorption spaces in ovarectomized 16-month old rats, contrary to intact canals in 16-month old control rat. Increased proportion of the periosteal circumference lamella and deformed endosteal regions with rare cortical canals hampered reconstructive histomorphology in ovarectomized rats of 26 month age. We have shown that 3D reconstruction of rat femur of the aged model over 16-month old is suitable methods that evaluate and microstructural change of the intracortical canals and cortical bone porosity by estrogen depletion.

MicroRNA levels in bone and blood change during bisphosphonate and teriparatide therapy in an animal model of postmenopausal osteoporosis.

MicroRNAs control the activity of a variety of genes that are pivotal to bone metabolism. Therefore, the clinical utility of miRNAs as biomarkers and drug targets for bone diseases certainly merits further investigation. This study describes the use of an animal model of postmenopausal osteoporosis to generate a comprehensive dataset on miRNA regulation in bone tissue and peripheral blood during bone loss and specifically anti-resorptive and osteo-anabolic treatment. Forty-two Sprague-Dawley rats were randomized to SHAM surgery (n=10) or ovariectomy (OVX, n=32). Eight weeks after surgery, OVX animals were further randomized to anti-resorptive treatment with zoledronate (n=11), osteo-anabolic treatment with teriparatide (n=11), or vehicle treatment (n=10). After 12 weeks of treatment, bone and serum samples were used for microRNA analysis using next-generation sequencing (NGS), mRNA levels using RT-qPCR, and bone microarchitecture analysis using nanoCT. Ovariectomy resulted in loss of trabecular bone, which was fully rescued using osteo-anabolic treatment, and partially rescued using anti-resorptive treatment. NGS revealed that both, anti-resorptive and anabolic treatment had a significant impact on miRNA levels in bone tissue and serum: out of 426 detected miRNAs, 46 miRNAs were regulated by teriparatide treatment an d 10 by zoledronate treatment (p-adj.<0.1). Interestingly, teriparatide and zoledronate treatment were able to revert miRNA changes in tissue and serum of untreated OVX animals, such as the up-regulation of miR-203a-3p, a known osteo-inhibitory miRNA. We confirmed previously established mechanisms of miR-203a by analyzing its direct target Dlx5 in femoral head. Our data reveal a significant effect of ovariectomy-induced bone loss, as well as the two major types of anti-osteoporotic treatment on miRNA transcription in femoral head tissue. These changes are associated with altered activity of target genes relevant to bone formation, such as Dlx5. The observed effects of bone loss and treatment response on miRNA levels in bone are also reflected in the peripheral blood, suggesting the possibility of minimally-invasive monitoring of bone-derived miRNAs using liquid biopsies.

A Clinical Study of a Standardized Extract of Leaves of Dalbergia sissoo (Roxb ex DC) in Postmenopausal Osteoporosis.

Context:Dalbergia sissoo had shown anti-osteoporotic and fracture-healing activities in animal models of postmenopausal osteoporosis (PMO). Standardized extract of leaves of D. sissoo (SEL-Ds) was clinically evaluated for osteoporosis.Aims:To investigate the anti-osteoporotic activity of D. sissoo in PMO by dual-energy X-ray absorptiometry (DXA), biochemical markers, and effect on clinical profile. Tolerability was assessed by organ function tests and adverse events.Settings and Design:An open-labeled prospective clinical study in ambulant settings was conducted at the menopausal health-care facility of a women's hospital.Materials and Methods:Thirty women (45–69 years) were enrolled for this 1-year study. Evaluations were basally, fortnightly twice, and three monthly four times. SEL-Ds (300 mg) twice daily was administered orally. Calcium (250 mg) and Vitamin D (200 IU) were given twice a day. The efficacy of SEL-Ds was assessed by DXA-scan (spine, femur), by biochemical markers, alkaline phosphatase (ALP), tumor necrosis factor-alpha (TNF-α), and anti-inflammatory marker high-sensitivity C-reactive protein (hs-CRP). Baseline symptom changes and adverse events were carefully recorded.Statistical Analysis:Summary statistics (n, mean, standard deviation, median, and maximum and minimum values) of changes from baseline values and Student's “t-” test for P values were used.Results and Discussion:SEL-Ds was well tolerated at given dose for 1 year. Anti-osteoporotic and anti-inflammatory activities of SEL-Ds were demonstrated by reduction in TNF-α (12.04 ± 2.81–2.35 ± 1.08 pg/ml), ALP (208.75 ± 45.88–154.52 ± 37.25 IU/L), and hs-CRP (6.1 ± 0.77–3.9 ± 0.47 mg/L). BMD-score on DXA-scan also remained unchanged at majority of the bone locations (increased 13/75, unchanged 51/75, and decreased 08/75).Conclusions:D. sissoo has demonstrated anti-osteoporotic and anti-inflammatory activities as indicated by decline in circulating TNF-α along with concurrent reduction in ALP. The nondecline in BMD index in the majority confirms the anti-osteoporotic activity.

An Ovariectomy-Induced Rabbit Osteoporotic Model: A New Perspective.

Study DesignExperimental Animal Model.PurposeThe aim of our study was to validate a pure bilateral ovariectomy (OVX) female New Zealand white rabbit model of postmenopausal osteoporosis utilizing animal-sparing in vivo techniques for evaluating bone mineral density (BMD). We also sought to demonstrate that bilateral OVX in female New Zealand white rabbits can produce diminished BMD in the spinal column and simulate osteoporosis, without the need for adjuvant chemotherapeutic agents (i.e., no additional glucocorticosteroids or other drugs were used for stimulating accelerated BMD loss), which can be assessed by in vivo BMD testing.Overview of LiteratureMultiple animal models of postmenopausal osteoporosis have been described. Rat ovariectomy models have been successful, but are limited by rats' inability to achieve true skeletal maturity and a slight morphology that limits surgical instrumentation. Rabbit models have been described which do not have these limitations, but previous models have relied on adjunctive steroid therapy to achieve osteoporosis and have required animal sacrifice for bone mineral density assessment.MethodsThirty-six skeletally mature female rabbits underwent bilateral OVX. BMD was measured using dual-energy X-ray absorptiometry on the metaphysis of the proximal tibia and distal femur, at baseline and 17 weeks postoperatively.ResultsMean BMD values were significantly reduced by 21.9% (p<0.05) in the proximal tibia and 11.9% (p<0.001) in the distal femur at 17 weeks.ConclusionsThis study is the first to demonstrate a significant bone loss within four months of pure OVX in rabbits using animal-sparing validation techniques. We believe that this OVX model is safe, reproducible, and can be employed to longitudinally evaluate the effect of anti-osteoporosis therapeutics and surgical interventions.

Age-dependent variations of cancellous bone in response to ovariectomy in C57BL/6J mice.

The ovariectomized (OVX) mouse model has been widely accepted to be suitable for the study of postmenopausal osteoporosis. However, whether C57BL/6J mice, a commonly used genetic background mouse strain, is an appropriate model for postmenopausal osteoporosis remains controversial. The present study investigated the effect of the OVX model on alterations in bone density and microarchitecture in C57BL/6J female mice of different ages. C57BL/6J mice were divided into 8-, 12- and 16-week-old groups (OVX8, OVX12 and OVX16) from the beginning of OVX. At 8 weeks post-surgery, the mice were anesthetized and micro-computed tomography was used to analyze the bone density and microarchitecture. The results revealed that OVX-induced loss of cancellous bone was greatest in OVX8, moderate in OVX12, and only a weak bone loss was observed in the OVX16 group when compared with the SHAM16 control group. In addition, the effect of genetic backgrounds in response to the OVX model were examined. Several other strains of mice, including inbred (BALB/c) and outbred (ICR and Kunming), were used in the present study, all of which were subjected to OVX at 8 weeks of age. The present findings revealed that the highest rate of bone loss was detected in C57BL/6J female mice. In addition, treatment with estrogen (17β-estradiol, 30 µg/kg five times per week) led to a significant increase in bone density in C57BL/6J mice compared with the other strains of mice. Therefore, these results may provide novel insights into the age- and strain-associated effect of OVX on regulating turnover of bone in female mice. The present findings also suggest 8-week-old C57BL/6J mice as an animal model for postmenopausal osteoporosis and preclinical testing of potential therapies for this disease.

Can we induce osteoporosis in animals comparable to the human situation?

Osteoporosis is a chronic systemic bone disease of growing relevance due to the on-going demographic change. Since the underlying regulatory mechanisms of this critical illness are still not fully understood and treatment options are not satisfactorily resolved, there is still a great need for osteoporosis research in general and animal models in particular.Ovariectomized rodents are standard animal models for postmenopausal osteoporosis and highly attractive due to the possibility to specifically modify their genetic background. However, some aspects can only be addressed in large animal models; such as metaphyseal fracture healing and advancement of orthopedic implants. Among other large animal models sheep in particular have been proven invaluable for osteoporosis research in this context.In conclusion, today we are able to influence the bone metabolism in animals causing a more or less pronounced systemic bone loss and structural deterioration comparable to the situation found in patients suffering from osteoporosis. However, there is no perfect model for osteoporosis, but a variety of models appropriate for answering specific questions. Though, the appropriateness of an animal model is not only defined in regard to the similarity to human physiology and the disease itself, but also in regard to acquisition, housing requirements, handling, costs, and particularly ethical concerns and animal welfare.

Pomegranate and its derivatives can improve bone health through decreased inflammation and oxidative stress in an animal model of postmenopausal osteoporosis

Recently, nutritional and pharmaceutical benefits of pomegranate (PG) have raised a growing scientific interest. Since PG is endowed with anti-inflammatory and antioxidant activities, we hypothesized that it may have beneficial effects on osteoporosis. We used ovariectomized (OVX) mice as a well-described model of postmenopausal osteoporosis to study the influence of PG consumption on bone health. Mice were divided into five groups as following: two control groups sham-operated and ovariectomized (OVX CT) mice fed a standard diet, versus three treated groups OVX mice given a modified diet from the AIN-93G diet, containing 5.7% of PG lyophilized mashed totum (OVX PGt), or 9.6% of PG fresh juice (OVX PGj) or 2.9% of PG lyophilized mashed peel (OVX PGp). As expected, ovariectomy was associated with a decreased femoral bone mineral density (BMD) and impaired bone micro-architecture parameters. Consumption of PGj, PGp, or PGt induced bone-sparing effects in those OVX mice, both on femoral BMD and bone micro-architecture parameters. In addition, PG (whatever the part) up-regulated osteoblast activity and decreased the expression of osteoclast markers, when compared to what was observed in OVX CT animals. Consistent with the data related to bone parameters, PG consumption elicited a lower expression of pro-inflammatory makers and of enzymes involved in ROS generation, whereas the expression of anti-inflammatory markers and anti-oxidant actors was enhanced. These results indicate that all PG parts are effective in preventing the development of bone loss induced by ovariectomy in mice. Such an effect could be partially explained by an improved inflammatory and oxidative status.