Nitensidine A,a guanidine alkaloid from Pterogyne nitens,induces osteoclastic cell death

Nitensidine A is a guanidine alkaloid isolated from Pterogyne nitens, a common plant in South America. To gain insight into the biological activity of P. nitens-produced compounds, we examined herein their biological effects on osteoclasts, multinucleated giant cells that regulate bone metabolism by resorbing bone. Among four guanidine alkaloids (i.e., galegine, nitensidine A, pterogynidine, and pterogynine), nitensidine A and pterogynine exhibited anti-osteoclastic effects at 10 μM by reducing the number of osteoclasts on the culture plate whereas galegine and pterogynidine did not. The anti-osteoclastic activities of nitensidine A and pterogynine were exerted in a concentration-dependent manner, whereas nitensidine A exhibited an approximate threefold stronger effect than pterogynine (IC₅₀ values: nitensidine A, 0.93 ± 0.024 μM; pterogynine, 2.7 ± 0.40 μM). In the present study, the anti-osteoclastic effects of two synthetic nitensidine A derivatives (nitensidine AT and AU) were also examined to gain insight into the structural features of nitensidine A that exert an anti-osteoclastic effect. The anti-osteoclastic effect of nitensidine A was greatly reduced by substituting the imino nitrogen atom in nitensidine A with sulfur or oxygen. According to the differences in chemical structures and anti-osteoclastic effects of the four guanidine alkaloids and the two synthetic nitensidine A derivatives, it is suggested that the number, binding site, and polymerization degree of isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their anti-osteoclastic effects.     

Curcumin synergizes with resveratrol to stimulate the MAPK signaling pathway in human articular chondrocytes in vitro

The mitogen-activated protein kinase (MAPK) pathway is stimulated in differentiated chondrocytes and is an important signaling cascade for chondrocyte differentiation and survival. Pro-inflammatory cytokines such as interleukin 1β (IL-1β) play important roles in the pathogenesis of osteoarthritis (OA) and rheumatoid arthritis (RA). In this study, we investigated whether curcumin and resveratrol can synergistically inhibit the catabolic effects of IL-1β, specifically the inhibition of the MAPK and subsequent apoptosis in human articular chondrocytes. Chondrocytes were either left untreated or treated with 10 ng/ml IL-1β or 1 μM U0126, a specific inhibitor of MAPK pathway alone for the indicated time periods or pre-treated with 10 μM curcumin, 10 μM resveratrol or 10 μM resveratrol and 10 μM curcumin for 4 h followed by co-treatment with 10 ng/ml IL-1β or 1 μM U0126 and 10 μM resveratrol, 10 μM curcumin or 10 μM resveratrol and 10 μM curcumin for the indicated time periods. Cultures were evaluated by immunoblotting and transmission electron microscopy. Incubation of chondrocytes with IL-1β resulted in induction of apoptosis, downregulation of β1-integrins and the extracellular signal-regulated kinase 1/2 (Erk1/2). Interestingly, U0126 induced apoptosis and blocked the above-mentioned proteins in a similar way to IL-1β. Furthermore, curcumin and resveratrol inhibited IL-1β- or U0126-induced apoptosis and downregulation of β1-integrins and Erk1/2 in human articular chondrocytes. These results suggest that combining these two natural compounds activates MEK/Erk signaling, a pathway that is involved in the maintenance of chondrocyte differentiation and survival.     

An efficient in vitro shoot regeneration from immature inflorescence and ex vitro rooting of Arnebia hispidissima (Lehm). DC. - A red dye (Alkannin) yielding plant

Arnebia hispidissima, which belongs to the family Boraginaceae, is an important medicinal and dye yielding plant. The alkannin, a red dye, are root-specific secondary metabolites of A. hispidissima. Shoots were regenerated from callus derived from immature inflorescence explants obtained from field grown plants. MS medium containing 4.52 μM 2, 4-D and 3.33 μM BAP was found to be most effective for the proliferation of callus, induced on medium containing 4.52 μM 2, 4-D. Maximum number (43.1 ± 0.25) with average length (5.2 ± 0.23) of shoots regenerated when callus was transferred to MS medium supplemented with 1.11 μM BAP, 1.16 μM Kin and 0.57 μM IAA. About 75.5 % of in vitro regenerated shoots were rooted on half-strength MS medium supplemented with 9.84 μM of IBA and 200 mg l⁻¹ of activated charcoal. In comparison to in vitro, higher percent (90.2 %) of shoots were rooted under ex vitro conditions when treated with IBA (0.98 mM) for 5 min. Plantlets rooted in vitro as well as ex vitro were acclimatized successfully under the green house conditions. Ex vitro rooted plants exhibited higher survival percentage (75 %) as compared to in vitro rooted plantlets (60 %). Present study may be applicable in the large-scale root-specific red dye (alkannin) production via root induction under ex vitro condition.     

In vitro propagation and withaferin A production in Withania ashwagandha,a rare medicinal plant of India

Withania ashwagandha, belonging to the family Solanaceae, is an important medicinal herb of India with restricted geographic distribution. It is a rich source of withaferin A (WA) and other bioactive withanolides. In the present study a rapid in vitro mass propagation protocol of W. ashwagandha was developed from nodal explants. Nodal explants were cultured on MS medium supplemented with various concentrations and combinations of plant growth regulators (PGRs). The highest number of regenerated shoots per ex-plant (33 ± 2.7) and highest WA (13.4 ± 1.15 mg/g of DW) production was obtained on MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). In vitro raised shoots were further rooted on half-strength MS medium containing 2.0 μM Indole-3-butyric acid (IBA) and analyzed for WA production. The rooted plantlets when transferred to poly bags in the greenhouse showed 90 % survival frequency. Levels of WA were higher in the in vitro and ex vitro derived shoot and root tissues as compared to field grown mother plants. In an attempt to further maximize WA production, shoot cultures were further grown in liquid MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). Root cultures were grown on half strength MS liquid medium fortified with 2.0 μM of IBA. WA production in the liquid cultures was significantly higher compared to the static composition of the same media. This protocol, first of its kind in this plant, can be successfully employed for conservation, proliferation and large-scale production of WA. The regenerated plants can also be used in traditional medicine as an alternative to naturally collected plants.     

Micropropagtion of Terminalia bellerica from nodal explants of mature tree and assessment of genetic fidelity using ISSR and RAPD markers

The present study reports an efficient in vitro micropropagation protocol for a medicinally important tree, Terminalia bellerica Roxb. from nodal segments of a 30 years old tree. Nodal segments taken from the mature tree in March-April and cultured on half strength MS medium gave the best shoot bud proliferation response. Combinations of serial transfer technique (ST) and incorporation of antioxidants (AO) [polyvinylpyrrolidone, PVP (50 mg l⁻¹) + ascorbic acid (100 mg l⁻¹) + citric acid (10 mg l⁻¹)] in the culture medium aided to minimize browning and improve explant survival during shoot bud induction. Highest multiplication of shoots was achieved on medium supplemented with 6-benzyladenine (BA, 8.8 μM) and α-naphthalene acetic acid (NAA, 2.6 μM) in addition to antioxidants. Shoot elongation was obtained on MS medium containing BA (4.4 μM) + phloroglucinol (PG, 3.9 μM). Elongated shoots were transferred to half strength MS medium containing indole-3-butyric acid (IBA, 2.5 μM) for root development. The acclimatization of plantlets was carried out under greenhouse conditions. The genetic fidelity of the regenerated plants was checked using inter simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. Comparison of the bands among the regenerants and mother plant confirmed true-to-type clonal plants.     

Zinc deficiency or excess within the physiological range increases genome instability and cytotoxicity, respectively, in human oral keratinocyte cells

Zinc (Zn) is an essential component of Zn-finger proteins and acts as a cofactor for enzymes required for cellular metabolism and in the maintenance of DNA integrity. The study investigated the genotoxic and cytotoxic effects of Zn deficiency or excess in a primary human oral keratinocyte cell line and determined the optimal concentration of two Zn compounds (Zn Sulphate (ZnSO₄) and Zn Carnosine (ZnC)) to minimise DNA damage. Zn-deficient medium (0 μM) was produced using Chelex treatment, and the two Zn compounds ZnSO₄ and ZnC were tested at concentrations of 0.0, 0.4, 4.0, 16.0, 32.0 and 100.0 μM. Cell viability was decreased in Zn-depleted cells (0 μM) as well as at 32 μM and 100 μM for both Zn compounds (P < 0.0001) as measured via the MTT assay. DNA strand breaks, as measured by the comet assay, were found to be increased in Zn-depleted cells compared with the other treatment groups (P < 0.05). The Cytokinesis Block Micronucleus Cytome assay showed a significant increase in the frequency of both apoptotic and necrotic cells under Zn-deficient conditions (P < 0.05). Furthermore, elevated frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) were observed at 0 and 0.4 μM Zn, whereas these biomarkers were minimised for both Zn compounds at 4 and 16 μM Zn (P < 0.05), suggesting these concentrations are optimal to maintain genome stability. Expression of PARP, p53 and OGG1 measured by western blotting was increased in Zn-depleted cells indicating that DNA repair mechanisms are activated. These results suggest that maintaining Zn concentrations within the range of 4–16 μM is essential for DNA damage prevention in cultured human oral keratinocytes.     

Resveratrol Treatment Delays Growth Plate Fusion and Improves Bone Growth in Female Rabbits

Trans-resveratrol (RES), naturally produced by many plants, has a structure similar to synthetic estrogen diethylstilbestrol, but any effect on bone growth has not yet been clarified. Pre-pubertal ovary-intact New Zealand white rabbits received daily oral administration of either vehicle (control) or RES (200 mg/kg) until growth plate fusion occurred. Bone growth and growth plate size were longitudinally monitored by X-ray imaging, while at the endpoint, bone length was assessed by a digital caliper. In addition, pubertal ovariectomized (OVX) rabbits were treated with vehicle, RES or estradiol cypionate (positive control) for 7 or 10 weeks and fetal rat metatarsal bones were cultured in vitro with RES (0.03 µM–50 µM) and followed for up to 19 days. In ovary-intact rabbits, sixteen-week treatment with RES increased tibiae and vertebrae bone growth and subsequently improved final length. In OVX rabbits, RES delayed fusion of the distal tibia, distal femur and proximal tibia epiphyses and femur length and vertebral bone growth increased when compared with controls. Histomorphometrical analysis showed that RES-treated OVX rabbits had a wider distal femur growth plate, enlarged resting zone, increased number/size of hypertrophic chondrocytes, increased height of the hypertrophic zone, and suppressed chondrocyte expression of VEGF and laminin. In cultured fetal rat metatarsal bones, RES stimulated growth at 0.3 µM while at higher concentrations (10 μM and 50 μM) growth was inhibited. We conclude that RES has the potential to improve longitudinal bone growth. The effect was associated with a delay of growth plate fusion resulting in increased final length. These effects were accompanied by a profound suppression of VEGF and laminin expression suggesting that impairment of growth plate vascularization might be an underlying mechanism.     

Effect of ormeloxifene on ovariectomy-induced bone resorption, osteoclast differentiation and apoptosis and TGF beta-3 expression

Effect of ormeloxifene, a multifunctional selective estrogen receptor modulator, on prevention of ovariectomy-induced bone resorption in retired breeder female rats, osteoclastogenesis using bone marrow cells from adult Balb/c mice cultured in presence of M-CSF and RANKL, osteoclast apoptosis using terminal deoxynucleotidyl transferase fragment end labeling and TGF beta-3 expression were investigated. Raloxifene, a benzothiophene reported to mimic effects of estrogen in bone, and estradiol were used for comparison. Ormeloxifene (10⁻⁶ and 10⁻⁸ M) significantly inhibited osteoclastogenesis (P < 0.001 versus vehicle control) as evidenced by lower number of TRAP-positive osteoclasts in bone marrow cultures and caused apoptosis of osteoclasts. The effect was almost equivalent to that observed in presence of estradiol-17 beta, except that significant number of cells undergoing apoptosis was evident even at 10⁻⁹ M concentration of estradiol-17 beta (P < 0.001). Raloxifene, though inhibited osteoclastogenesis at much lower concentrations (10⁻⁸ to 10⁻¹² M; P < 0.001), failed to cause apoptosis of osteoclasts at any of the concentrations used. While ormeloxifene, raloxifene and ethynylestradiol significantly prevented ovariectomy-induced bone loss in vivo in retired breeder female rats, prevention of ovariectomy-induced decrease in BMD and trabecular network of proximal tibia, calcium and phosphorus levels in femur and tibia and prevention of ovariectomy-induced down-regulation of TGF beta-3 expression in lumbar vertebrae was of lower order in raloxifene- than ormeloxifene- or ethynylestradiol-supplemented females. Both the SERMs, however, produced considerable estrogenic effects at the uterine level as evidenced by increase in weight, total and endometrial area and luminal epithelial cell height; the effect being generally greater in raloxifene- than ormeloxifene-treated rats. Findings demonstrate that inhibition of estrogen-deficiency osteoporosis by ormeloxifene, as in case of estradiol, was mediated via inhibition of osteoclastogenesis, apoptosis of osteoclasts and up-regulation of TGF beta-3 expression. Raloxifene, though effective in inhibiting osteoclastogenesis in vitro at much lower concentrations, was not only less potent in preventing ovariectomy-induced bone loss in retired breeder female rats in vivo but also appeared to have a different mechanism of action than ormeloxifene and estradiol.     

Evaluation of foam cell formation in cultured macrophages: an improved method with Oil Red O staining and DiI-oxLDL uptake

Macrophage-derived foam cell formation elicited by oxidized low-density lipoprotein (oxLDL) is the hallmark of early atherogenesis. Detection of foam cell formation is conventionally practiced by Oil Red O (ORO) staining of lipid-laden macrophages. Other methods include 1,1′-dioctadecyl-3,3,3′3′-tetra-methylindocyanide percholorate (DiI)-labeled oxLDL (DiI-oxLDL) uptake and Nile Red staining. The purpose of the present study is to report an optimized method for assessing foam cell formation in cultured macrophages by ORO staining and DiI-oxLDL uptake. After incubation with oxLDL (50 μg/ml) for 24 h, the macrophages were fixed, stained with ORO for just 1 min, pronounced lipid droplets were clearly observed in more than 90% of the macrophages. To test the in vivo applicability of this method, lesions (or foam cells) of cryosections of aortic sinus or primary mouse peritoneal macrophages from ApoE deficient mice fed a high cholesterol diet were successfully stained. In another set of experiments, treatment of macrophages with DiI-oxLDL (10 μg/ml) for 4 h resulted in significant increase in oxLDL uptake in macrophages as demonstrated by confocol microscopy and flow cytometry. We conclude that the optimized ORO staining and fluorescent labeled oxLDL uptake techniques are very useful for assessing intracellular lipid accumulation in macrophages that are simpler and more rapid than currently used methods.     

Tumour necrosis factor‐α promotes BMHSC differentiation by increasing P2X7 receptor in oestrogen‐deficient osteoporosis

The exact mechanism of tumour necrosis factor α (TNF‐α) promoting osteoclast differentiation is not completely clear. A variety of P2 purine receptor subtypes have been confirmed to be widely involved in bone metabolism. Thus, the purpose of this study was to explore whether P2 receptor is involved in the differentiation of osteoclasts. Mouse bone marrow haematopoietic stem cells (BMHSCs) were co‐cultured with TNF‐α to explore the effect of TNF‐α on osteoclast differentiation and bone resorption capacity in vitro, and changes in the P2 receptor were detected at the same time. The P2 receptor was silenced and overexpressed to explore the effect on differentiation of BMHSCs into osteoclasts. In an in vivo experiment, the animal model of PMOP was established in ovariectomized mice, and anti‐TNF‐α intervention was used to detect the ability of BMHCs to differentiate into osteoclasts as well as the expression of the P2 receptor. It was confirmed in vitro that TNF‐α at a concentration of 20 ng/mL up‐regulated the P2X7 receptor of BMHSCs through the PI3k/Akt signalling pathway, promoted BMHSCs to differentiate into a large number of osteoclasts and enhanced bone resorption. In vivo experiments showed that more P2X7 receptor positive osteoclasts were produced in postmenopausal osteoporotic mice. Anti‐TNF‐α could significantly delay the progression of PMOP by inhibiting the production of osteoclasts. Overall, our results revealed a novel function of the P2X7 receptor and suggested that suppressing the P2X7 receptor may be an effective strategy to delay bone formation in oestrogen deficiency‐induced osteoporosis.     

Asprin‐loaded strontium‐containing α‐calcium sulphate hemihydrate/nano‐hydroxyapatite composite promotes regeneration of critical bone defects

Our laboratory originally synthesized strontium(Sr)‐containing α‐calcium sulphate hemihydrate/nano‐hydroxyapatite composite (Sr‐α‐CSH/n‐HA) and demonstrated its ability to repair critical bone defects. This study attempted to incorporate aspirin into it to produce a better bone graft material for critical bone defects. After 5% Sr‐α‐CSH was prepared by coprecipitation and hydrothermal methods, it was mixed with aspirin solution of different concentrations (50 μg/ml, 200 μg/ml, 800 μg/ml and 3200 μg/ml) at a fixed liquid‐solid ratio (0.54 v/w) to obtain aspirin‐loaded Sr‐α‐CSH/n‐HA composite. In vitro experiments were performed on the composite extracts. The tibial defects (3 mm*5 mm) in SD rat model were filled with the composite for 4 weeks and 12 weeks to evaluate its osteogenic capacity in vivo. Our results showed its capability of proliferation, migration and osteogenesis of BMSCs in vitro got improved. In vivo treatment with 800 μg/ml aspirin–loaded Sr‐α‐CSH/n‐HA composite led to significantly more new bone formation in the defects compared with Sr‐α‐CSH/n‐HA composite and significantly promoted the expression of osteogenic‐related genes and inhibited osteoclast activity. In general, our research suggests that aspirin‐loaded Sr‐α‐CSH/n‐HA composite may have a greater capacity of repairing tibial defects in SD rats than simple Sr‐α‐CSH/n‐HA composite.     

Mutagenicity and genotoxicity of dicapthon insecticide

Mutagenic and genotoxic effects of dicapthon were investigated by using the bacterial reverse mutation assay in Salmonella typhimurium TA97, TA98, TA100 and TA102 strains with or without metabolic activation system (S9 mix), and chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) tests in human peripheral blood lymphocytes in vitro. Dicapthon was dissolved in dimethyl sulfoxide for all test systems. 0.1, 1, 10 and 100 μg/plate doses of dicapthon were found to be weakly mutagenic on S. typhimurium TA 98 without S9 mix. The human peripheral lymphocytes were treated with four experimental concentrations of dicapthon (25, 50, 100, and 200 μg/mL) for 24 and 48 h. Dicapthon increased the frequency of SCE only at the 100 μg/mL concentration for the 24 and 48 h applications. Dicapthon also induced abnormal cell frequency, CA/cell ratio and frequency of MN dose dependently for 24 and 48 h. Dicapthon showed a statistically significant cytotoxic effect by decreasing the mitotic index in all concentrations and a cytostatic effect by decreasing nuclear division index in 100 and 200 μg/mL concentrations for both treatment periods when compared with both untreated and solvent controls. These values decreased also in a dose dependent manner.     

Protection against Ovariectomy-Induced Bone Loss by Tranilast

Background

Tranilast (N-(3′,4′-dimethoxycinnamonyl) anthranilic acid) has been shown to be therapeutically effective, exerting anti-inflammatory and anti-oxidative effects via acting on macrophage. We hypothesized that Tranilast may protect against oxidative stress-induced bone loss via action in osteoclasts (OCs) that shares precursors with macrophage.

Methodology and Principal Findings

To elucidate the role of Tranilast, ovariectomy (OVX)-induced bone loss in vivo and OC differentiation in vitro were evaluated by µCT and tartrate-resistant acid phosphatase staining, respectively. Oral administration of Tranilast protected against OVX-induced bone loss with decreased serum level of reactive oxygen species (ROS) in mice. Tranilast inhibited OC formation in vitro. Decreased osteoclastogenesis by Tranilast was due to a defect of receptor activator of nuclear factor-κB ligand (RANKL) signaling, at least partly via decreased activation of nuclear factor-κB and reduced induction and nuclear translocation of nuclear factor of activated T cells, cytoplasmic 1 (or NFAT2). Tranilast also decreased RANKL-induced a long lasting ROS level as well as TGF-β to inhibit osteoclastogenesis. Reduced ROS caused by Tranilast was due to the induction of ROS scavenging enzymes (peroxiredoxin 1, heme oxygenase-1, and glutathione peroxidase 1) as well as impaired ROS generation.

Conclusions/Significance

Our data suggests the therapeutic potential of Tranilast for amelioration of bone loss and oxidative stress due to loss of ovarian function.     

Genotoxicity of drinking water disinfection by-products (bromoform and chloroform) by using both Allium anaphase-telophase and comet tests

Genotoxic effects of bromoform and chloroform, disinfection by-products of the chlorination of drinking water, were examined by using mitotic index (MI), mitotic phase, chromosome aberrations (CAs) and comet assay on root meristematic cells of Allium cepa. Different concentrations of bromoform (25, 50, 75 and 100 μg/mL) and chloroform (25, 50, 100 and 200 μg/mL) were introduced to onion tuber roots. Distilled water was used as a negative control and methyl methansulfonate (MMS-10 μg/mL) as positive control. All obtained data were subjected to statistical analyses by using SPSS 15.0 for Windows software. For comparison purposes, Duncan multiple range tests by using one-way analysis of variance were employed and p < 0.05 was accepted as significant value. Exposure of both chemicals (except 25 μg/mL applications of bromoform) significantly decreased MI. Bromoform and chloroform (except 25 μg/mL applications) increased total CAs in Allium anaphase-telophase test. A significant increase in DNA damage was also observed at all concentrations of both bromoform and chloroform examined by comet assay. The damages were higher than that of positive control especially at 75–100 μg/mL for bromoform and 100–200 μg/mL for chloroform.     

Downregulated fat mass and obesity-associated protein inhibits bone resorption and osteoclastogenesis by nuclear factor-kappa B inactivation

During osteoporosis, fat mass and obesity-associated protein (FTO) promotes the shift of bone marrow mesenchymal stem cells to adipocytes and represses osteoblast activity. However, the role and mechanisms of FTO on osteoclast formation and bone resorption remain unknown. In this study, we investigated the effect of FTO on RAW264.7 cells and bone marrow monocytes (BMMs)-derived osteoclasts in vitro and observed the influence of FTO on ovariectomized (OVX) mice model to mimic postmenopausal osteoporosis in vivo. Results found that FTO was up-regulated in BMMs from OVX mice. Double immunofluorescence assay showed co-localization of FTO with tartrate-resistant acid phosphatase (TRAP) in femurs of OVX mice. FTO overexpression enhanced TRAP-positive osteoclasts and F-actin ring formation in RAW264.7 cells upon RANKL stimulation. The expression of osteoclast differentiation-related genes, including nuclear factor of activated T cells c1 (NFATc1) and c-FOS, was upregulated in BMMs and RAW264.7 cells after FTO overexpression. FTO overexpression induced the phosphorylation and nuclear translocation of factor-kappa B (NF-κB) p65 in BMMs and RAW264.7 cells exposed to RANKL. ChIP and dual-luciferase assays revealed that FTO overexpression contributed to RANKL-induced binding of NF-κB to NFATc1 promoter. Rescue experiments suggested that FTO overexpression-mediated osteoclast differentiation was suppressed after intervention with a NF-κB inhibitor pyrrolidine dithiocarbamate. Further in vivo evidence revealed that FTO knockdown increased bone trabecula and bone mineral density, inhibited bone resorption and osteoclastogenesis in osteoporotic mice. Collectively, our research demonstrates that downregulated FTO inhibits bone resorption and osteoclastogenesis through NF-κB inactivation, which provides a novel reference for osteoporosis treatment.     

Osteoprotective effect of combination therapy of low-dose oestradiol with G15, a specific antagonist of GPR30/GPER in ovariectomy-induced osteoporotic rats

Identified and cloned in 1996 for the first time, G protein-coupled oestrogen receptor (ER) 30 (GPR30/GPER) has been a hot spot in the field of sex hormone research till now. In the present study, we examined the effects of low-dose oestradiol (E2) combined with G15, a specific antagonist of GPR30 on ovariectomy (OVX)-induced osteoporosis in rats. Female Sprague–Dawley (SD) rats undergoing OVX were used to evaluate the osteoprotective effect of the drugs. Administration of E2 [35 μg/kg, intraperitoneally (ip), three times/week) combining G15 (160 μg/kg, ip, three times/week) for 6 weeks was found to have prevented OVX-induced effects, including increase in bone turnover rate, decrease in bone mineral content (BMC) and bone mineral density (BMD), damage of bone structure and the aggravation in biomechanical properties of bone. The therapeutic effect of these two drugs in combination was better than that of E2 alone. Meanwhile, the administration of G15 prevented body weight increase or endometrium proliferation in the rats. In conclusion, administration of low-dose E2 combining G15 had a satisfactory bone protective effect for OVX rats, without significant influence on body weight or the uterus. This combination therapy may be an effective supplement of drugs in prevention and treatment for postmenopausal osteoporosis.