4D label-free quantitative proteomics analysis to screen potential drug targets of Jiangu Granules treatment for postmenopausal osteoporotic rats.

Background: Postmenopausal osteoporosis (PMOP) is a disease with a high prevalence in postmenopausal women and is characterized by an imbalance in bone metabolism, reduced bone mass, and increased risk of fracture due to estrogen deficiency. Jiangu granules (JG) is a compound prescription used in traditional Chinese medicine to treat PMOP. However, its definitive mechanism in PMOP is unclear. This study used a 4D label-free quantitative proteomics method to explore the potential therapeutic mechanism of JG in an ovariectomy (OVX) rats' model. Materials and methods: A rat model of PMOP was established by removing the ovaries bilaterally. Nine 3-month-old specific-pathogen-free female SD rats. The nine rats were randomly divided into 3 groups (n = 3 in each group): the sham-operated group (J), the ovariectomy group (NC), and the JG treatment (ZY) group. Proteins extracted from the bone tissue of the lumbar spine (L3, L4) of three groups of rats were analyzed by 4D label-free quantitative proteomics, and proteins differentially expressed after JG treatment and proteins differentially expressed after de-ovulation were intersected to identify proteins associated with the mechanism of PMOP by JG treatment. Result: There were 104 up-regulated and 153 down-regulated differentially expressed proteins (DEPs) in the J group vs. NC group, 107 up-regulated and 113 down-regulated DEPs in the J group vs. ZY group, and 15 up-regulated and 32 down-regulated DEPs in the NC group vs. ZY group. Six potential target proteins for JG regulation of osteoblast differentiation in OVX rats were identified by taking intersections of differential proteins in the J group vs. NC group and NC group vs. ZY group. Conclusion: JG may exert therapeutic effects by modulating the expression levels of target proteins associated with osteoblast differentiation to enhance osteoblast differentiation in OVX rats. These results further uncovered the target proteins and specific mechanisms of JG in treating PMOP, providing an experimental basis for the clinical application of JG in treating PMOP.

Screening of Differentially Expressed Genes and miRNAs in Hypothalamus and Pituitary Gland of Sheep under Different Photoperiods.

The reproduction of sheep is affected by many factors such as light, nutrition and genetics. The Hypothalamic-pituitary-gonadal (HPG) axis is an important pathway for sheep reproduction, and changes in HPG axis-related gene expression can affect sheep reproduction. In this study, a model of bilateral ovarian removal and estrogen supplementation (OVX + E2) was applied to screen differentially expressed genes and miRNAs under different photoperiods using whole transcriptome sequencing and reveal the regulatory effects of the photoperiod on the upstream tissues of the HPG axis in sheep. Whole transcriptome sequencing was performed in ewe hypothalamus (HYP) and distal pituitary (PD) tissues under short photoperiod 21st day (SP21) and long photoperiod 21st day (LP21). Compared to the short photoperiod, a total of 1813 differential genes (up-regulation 966 and down-regulation 847) and 145 differential miRNAs (up-regulation 73 and down-regulation 72) were identified in the hypothalamus of long photoperiod group. Similarly, 2492 differential genes (up-regulation 1829 and down-regulation 663) and 59 differential miRNAs (up-regulation 49 and down-regulation 10) were identified in the pituitary of long photoperiod group. Subsequently, GO and KEGG enrichment analysis revealed that the differential genes and target genes of differential miRNA were enriched in GnRH, Wnt, ErbB and circadian rhythm pathways associated with reproduction. Combined with sequence complementation and gene expression correlation analysis, several miRNA-mRNA target combinations (e.g., LHB regulated by novel-414) were obtained. Taken together, these results will help to understand the regulatory effect of the photoperiod on the upstream tissues of HPG in sheep.

Prophylactic effect of Trigonella foenum-graecum L. seed extract on inflammatory markers and histopathological changes in high-fat-fed ovariectomized rats.

Background and aim: Trigonella foenum-graecum L. seeds (TFG) are used as spices in Indian cuisine. In Indian traditional medicine, TFG is used to treat diabetes, dyslipidemia, obesity, arthritis, cancer, digestive disorders, and postmenopausal conditions. Pathophysiology of postmenopausal diseases involves low-grade systemic inflammation. The purpose of this study is to investigate the prophylactic effect of petroleum ether fraction of TFG-extract (PE-TFG) on inflammatory markers, and histopathological changes in ovariectomized rats (OVX-rats) fed with a high-fat diet (HFD). Experimental procedure: OVX female Sprague Dawley rats were used for the study. Three weeks after ovariectomy, rats were randomized in different groups and administered PE-TFG, atorvastatin, diosgenin, 17ß-estradiol for 12 weeks along with HFD. The sham-operated rats (S.OVX) were fed with a standard pellet diet. At the end of 12-weeks, rats were sacrificed, and blood samples were used to estimate lipid profile, glucose, hepatic markers, TNF-α, and leptin. Liver, kidney, and common carotid artery were isolated for testing oxidative stress markers, mRNA expression of adiponectin, PPAR-γ, and histopathological changes. Results: Administration of PE-TFG significantly decreased (P < 0.05) total cholesterol, LDL, hepatic markers, leptin, TNF-α and improved mRNA expression of adiponectin and PPAR-γ in HFD-fed OVX-rats. Further, micro and macro hepatic steatosis, inflammation, glomerular hypertrophy, degenerated tubules in kidney, increased tunica intima, and media thickness of common carotid artery and the pathological changes were not significant upon PE-TFG administration compared to S.OVX-rats. Conclusion: PE-TFG protects cellular inflammation and metabolic alternations in HFD-fed OVX-rats and thus can be explored further in postmenopausal diseases as a prophylactic agent.

Apigenin, a Single Active Component of Herbal Extract, Alleviates Xerostomia via ERα-Mediated Upregulation of AQP5 Activation.

Xerostomia is a common symptom in menopausal women, suggesting the role of sex steroids in disease development. Shreds of literature had reported the potential use of herbal extracts to relieve xerostomia. However, a cocktail of multiple components in herbal extract makes it difficult to understand the exact mechanism of action. Aquaporin5 (AQP5), the specific aquaporin expressed in salivary glands, plays an important role in salivary secretion as a downstream of estrogen signaling. In this study, we aimed to unravel a single active herbal component as a therapeutic for xerostomia and investigate its mechanism of action. The effects of apigenin (flavonoid), dauricine (alkaloids), protopine (alkaloids), and lentinan (polysaccharides) on AQP5 transcription were screened in vitro. Only apigenin robustly induced AQP5 transcription and expression, and this effect was even robust compared to the effect of estradiol (E2, a positive control). Overexpression of estrogen receptor α (ERα) in the human salivary gland cell line (HSG) upregulated the AQP5 transcription and expression and the knockdown ERα reversed this effect, suggesting the role of ERα signaling on AQP5 activation in HSG cells. Docking results showed apigenin-specific binding sites in ERα. We further analyzed the therapeutic effect of apigenin on ovariectomized mice as a xerostomia model. The saliva secretion in the xerostomia group was reduced to one-third of the sham group, whereas the apigenin or E2 treatment for 12 weeks reversed this effect. Meanwhile, the water consumption in the xerostomia group was augmented obviously compared to the sham group, whereas the water consumption in the apigenin and E2 group was declined to the level of the sham group. Immunohistochemistry of submandibular glands revealed the downregulation of AQP5 expression in xerostomia mice compared to control. Apigenin, or E2 treatment, upregulated AQP5 expression in xerostomia mice. In conclusion, apigenin, a single active component of herbal extract, upregulated AQP5 expression in HSG cells via activation of ERα signaling and restored saliva flow rates in OVX mice. These results revealed apigenin as a single active component of herbal extract with the potential to treat xerostomia.

Yukmijihwang-Tang Suppresses Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL)-Induced Osteoclast Differentiation and Prevents Ovariectomy (OVX)-Mediated Bone Loss.

Yukmijihwang-tang (YJ) has been used to treat diabetes mellitus, renal disorders, and cognitive impairment in traditional medicine. This study aimed to evaluate the anti-osteoporotic effect of YJ on ovariectomy (OVX)-induced bone loss in a rat and receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated osteoclast differentiation in bone marrow macrophages (BMMs). YJ reduced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) in an osteoclast/osteoblast co-culture system by regulating the ratio of RANKL/osteoprotegerin (OPG) by osteoblasts. Overall, YJ reduced TRAP-positive cell formation and TRAP activity and F-actin ring formation. Analysis of the underlying mechanisms indicated that YJ inhibited the activation of the nuclear factor of activated T cell cytoplasmic 1 (NFATc1) and c-Fos, resulting in the suppression of osteoclast differentiation-related genes such as TRAP, ATPase, H+ transporting, lysosomal 38 kDa, V0 subunit d2, osteoclast-associated receptor, osteoclast-stimulatory transmembrane protein, dendritic cell-specific transmembrane protein, matrix metalloproteinase-9, cathepsin K, and calcitonin receptor. YJ also inhibited the nuclear translocation of NFATc1. Additionally, YJ markedly inhibited RANKL-induced phosphorylation of signaling pathways activated in the early stages of osteoclast differentiation including the p38, JNK, ERK, and NF-κB. Consistent with these in vitro results, the YJ-administered group showed considerably attenuated bone loss in the OVX-mediated rat model. These results provide promising evidence for the potential novel therapeutic application of YJ for bone diseases such as osteoporosis.

Genistein and exercise treatment reduced NASH related HDAC3, IL-13 and MMP-12 expressions in ovariectomized rats fed with high fat high fructose diet.

BACKGROUND AND AIM: Genistein (GEN) and exercise (Ex) may be regarded as an alternative treatment for non-alcoholic steatohepatitis (NASH). However, the mechanisms behind their therapeutic effects in NASH are not well-understood. EXPERIMENTAL PROCEDURE: This study investigated the roles of histone deacetylase (HDAC)3 and interleukin-(IL-)13 in the NASH model of ovariectomized (OVX) rats fed with high fat high fructose (HFHF) diet. RESULTS AND CONCLUSION: Nine weeks after being fed with HFHF diet, severe NASH pathology with mild fibrosis were seen along with an increase in HDAC3, IL-13 and matrix metalloelastase (MMP-12) expressions in OVX rats. Five weeks of either GEN or Ex treatments abrogated the increase in both HDAC3 and IL-13 expressions in OVX rats fed with HFHF diet and ameliorated NASH features, liver fibrosis and MMP-12 expression. The combination of Gen and Ex, however, did not provide additional benefits on NASH features in OVX rats fed with HFHF diet. These results suggested that GEN and Ex treatments improved HFHF diet induced NASH in OVX rats through the suppression of HDAC3, IL-13 and MMP-12 expression.

Pharmacological properties of durva swaras (Cynodon dactylon L. Pers.) in an ovariectomised rat model mimicking chronic menopausal syndrome.

Hormonal replacement therapy (HRT), as the first-line management of chronic menopausal syndrome (CMS) in women, has limited application due to adverse effects. We aimed to evaluate the therapeutic potential of a herbal alternative (HALT), durva swaras (DS) of Cynodon dactylon L. Pers., in a CMS rat model. Female Sprague-Dawley rats were subjected to Sham and ovariectomy (OVX) surgery. OVX rats received either 0.11 mg/kg oestrogen as a positive treatment control or 1 (DS1), 2 (DS2), and 4 (DS3) g/kg DS for 160 days. Vaginal smear tests indicated the menopausal status. Routine clinical examinations, weekly body weights (BW), serum calcium, proinflammatory cytokines, and reproductive hormones levels were monitored. Clinical chemistry, body composition, bone mineral density (BMD), uterotrophic response, bone morphometry, and histopathology of major organs were evaluated. BW of OVX rats increased by 18-25% compared to Sham. Total fat and fat percentage were significantly elevated in the oestrogen group compared to DS2, DS3, and OVX group. DS treatment groups showed the levels of TNF- α was slightly reduced, while IL-1ß and IL-6 levels were significantly reduced (P < 0.05) compared to the oestrogen treated group. DS treatment restored serum calcium levels, while BMD, bone quality, osteoblast/osteoclast ratio, and collagen levels improved in both DS and oestrogen treatment groups. The uterotrophic assay demonstrated non-oestrogenic activity of DS. Endometrial hyperplastic change was observed in oestrogen-treated rats. The preclinical non-oestrogenic activity of DS has therapeutic potential in CMS through anti-inflammatory and osteo-protective effects. Further clinical research into DS, as a viable HALT to HRT, is required.

Gamabufotalin Inhibits Osteoclastgenesis and Counteracts Estrogen-Deficient Bone Loss in Mice by Suppressing RANKL-Induced NF-κB and ERK/MAPK Pathways.

Osteolytic bone disease is a condition of imbalanced bone homeostasis, characterized mainly by excessive bone-resorptive activity, which could predispose these populations, such as the old and postmenopausal women, to developing high risk of skeletal fragility and fracture. The nature of bone homeostasis is the coordination between the osteoblasts (OBs) and osteoclasts (OCs). Abnormal activation of osteoclasts (OCs) could compromise the bone homeostasis, constantly followed by a clutch of osteolytic diseases, including postmenopausal osteoporosis, osteoarthritis, and rheumatoid arthritis. Thus, it is imperatively urgent to explore effective medical interventions for patients. The traditional Chinese medicine (TCM) gamabufotalin (CS-6) is a newly identified natural product from Chansu and has been utilized for oncologic therapies owing to its good clinical efficacy with less adverse events. Previous study suggested that CS-6 could be a novel anti-osteoporotic agent. Nevertheless, whether CS-6 suppresses RANK-(receptor activator of nuclear factor-κ B ligand)/TRAF6 (TNF receptor-associated factor 6)-mediated downstream signaling activation in OCs, as well as the effects of CS-6 on OC differentiation in vivo, remains elusive. Therefore, in this present study, we aimed to explore the biological effects of CS-6 on osteoclastogenesis and RANKL-induced activation of related signaling pathways, and further to examine the potential therapeutic application in estrogen-deficient bone loss in the mice model. The results of in vitro experiment showed that CS-6 can inhibit RANKL-induced OC formation and the ability of bone resorption in a dose-dependent manner at both the early and late stages of osteoclastogenesis. The gene expression of OC-related key genes such as tartrate-resistant acid phosphatase (TRAP), CTSK, DC-STAMP, MMP9, and ß3 integrin was evidently reduced. In addition, CS-6 could mitigate the systemic estrogen-dependent bone loss and pro-inframammary cytokines in mice in vivo. The molecular mechanism analysis suggested that CS-6 can suppress RANKL/TRAF6-induced early activation of NF-κB and ERK/MAPK signaling pathways, which consequently suppressed the transcription activity of c-Fos and NFATc1. Taken together, this present study provided ample evidence that CS-6 has the promise to become a therapeutic candidate in treating osteolytic conditions mediated by elevated OC formation and bone resorption.

Effects of Xian-Ling-Gu-Bao capsule on the gut microbiota in ovariectomized rats: Metabolism and modulation.

Xian-Ling-Gu-Bao capsule (XLGB) has been proven to prevent and treat osteoporosis. However, as a long-term oral formula, XLGB's effects on the metabolic capacity, structure and function of gut microbiota have yet to be elucidated in ovariectomized (OVX) rats. Our objectives were to evaluate the capacity of gut microbiota for metabolizing XLGB ingredients and to assess the effect of this prescription on gut microbiota. Herein, an integrated analysis that combined ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and ultrahigh-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-TQD-MS) was conducted to determine the metabolic capacity of gut microbiota. The effects of XLGB on gut microbiota were explored by metagenomic sequencing in OVX rats. Fecal samples from each group were collected after intragastric administration for three months. In total, 64 biotransformation products were fully characterized with rat gut microbiota from the OVX group and the XLGB group. The deglycosylation reaction was the main biotransformation pathway in core structures in the group that was incubated with XLGB. Compared with the OVX group, different biotransformation products and pathways of the XLGB group after incubation for 2 h and 8 h were described. After three months of feeding with XLGB, the domesticated gut microbiota was conducive to the production of active absorbed components via deglycosylation, such as icaritin, psoralen and isopsoralen. Comparisons of the gut microbiota of the OVX and XLGB groups showed differences in the relative abundances of the two dominant bacterial divisions, namely, Firmicutes and Bacteroidetes. The proportion of Firmicutes was significantly lower and that of Bacteroidetes was significantly higher in the XLGB group. This result demonstrated that XLGB could provide a basis for the treatment of osteoporosis by regulating lipid and bile acid metabolism. In addition, the increase in Lactobacillus, Bacteroides and Prevotella could be an important factor that led to easier production of active absorbed aglycones in the XLGB group. Our observation provided further evidence of the importance of gut microbiota in the metabolism and potential activity of XLGB.

Scopolin Attenuates Osteoporotic Bone Loss in Ovariectomized Mice.

Bone remodeling is a renewal process regulated by bone synthesis (osteoblasts) and bone destruction (osteoclasts). A previous study demonstrated that Lycii radicis cortex (LRC) extract inhibited ovariectomized (OVX)-induced bone loss in mice. This study investigated the anti-osteoporotic effects of bioactive constituent(s) from the LRC extract. The effective compound(s) were screened, and a single compound, scopolin, which acts as a phytoalexin, was chosen as a candidate component. Scopolin treatment enhanced alkaline phosphatase activity and increased mineralized nodule formation in MC3T3-E1 pre-osteoblastic cells. However, osteoclast differentiation in primary-cultured monocytes was reduced by treatment with scopolin. Consistently, scopolin treatment increased osteoblast differentiation in the co-culture of monocytes (osteoclasts) and MC3T3-E1 (osteoblast) cells. Scopolin treatment prevented bone mineral density loss in OVX-induced osteoporotic mice. These results suggest that scopolin could be a therapeutic bioactive constituent for the treatment and prevention of osteoporosis.

Anti-Osteoporotic Effects of the Herbal Mixture of Cornus officinalis and Achyranthes japonica In Vitro and In Vivo.

Osteoporosis is a porous bone disease caused by bone density loss, which increases the risk of fractures. Cornus officinalis (CO) and Achyranthes japonica (AJ) have been used as traditional herbal medicine for various disorders in East Asia. Although the anti-osteoporotic effects of single extract of CO and AJ have already been reported, the synergistic effect of a combined mixture has not been studied. In this study, we investigated the effects of a CO and AJ herbal mixture on osteoporosis in in vitro and in vivo models. The results demonstrate that treatment with the CO and AJ mixture significantly promoted osteoblast differentiation of MC3T3-E1 mouse preosteoblasts through the upregulation of osteoblastic differentiation-associated genes such as alkaline phosphatase (Alpl), runt-related transcription factor 2 (Runx2), and bone gamma-carboxyglutamic acid-containing protein (Bglap), while the mixture significantly inhibited differentiation of osteoclasts isolated from primary-cultured mouse monocytes. In addition, oral administration of CO and AJ mixture significantly prevented bone mineral density loss and trabecular bone structures in an ovariectomy-induced osteoporotic mouse model. These results suggest that the combination treatment of CO and AJ mixture might be a beneficial therapy for osteoporosis.

Pyrroloquinoline Quinone Prevents Estrogen Deficiency-Induced Osteoporosis by Inhibiting Oxidative Stress and Osteocyte Senescence.

Accumulating studies have shown that oxidative stress increases with aging, which is related to the pathophysiology of postmenopausal osteoporosis. Pyrroloquinoline quinone (PQQ) is a natural anti-oxidant with anti-oxidative and anti-aging effects. However, it is unclear whether PQQ has a protective role against estrogen deficiency-induced osteoporosis. Here, we evaluated the efficacy of PQQ on bone mineral density, bone microarchitecture, bone turnover and biomechanical strength in ovariectomy (OVX)-induced osteoporosis mouse model. Although dietary PQQ supplement did not affect serum E2 levels and uterine weight in OVX mice, it could prevent OVX-induced bone loss and improve bone strength by inhibiting oxidative stress, osteocyte senescence and senescence-associated secretory phenotype (SASP), subsequently promoting osteoblastic bone formation and inhibiting osteoclastic bone resorption, which was comparable to treatment with exogenous estrogen. The results from our study provide experimental evidence for the clinical use of PQQ to prevent estrogen deficiency-induced osteoporosis.

Rehmanniae Radix Preparata suppresses bone loss and increases bone strength through interfering with canonical Wnt/ß-catenin signaling pathway in OVX rats.

Rehmanniae Radix Preparata (RRP) improves bone quality in OVX rats through the regulation of bone homeostasis via increasing osteoblastogenesis and decreasing osteoclastogenesis, suggesting it has a potential for the development of new anti-osteoporotic drugs. INTRODUCTION: Determine the anti-osteoporotic effect of RRP in ovariectomized (OVX) rats and identify the signaling pathway involved in this process. METHODS: OVX rats were treated with RRP aqueous extract for 14 weeks. The serum levels of tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor kappa-Β ligand (RANKL), alkaline phosphatase (ALP), and osteoprotegerin (OPG) were determined by ELISA. Bone histopathological alterations were evaluated by H&E, Alizarin red S, and Safranin O staining. Bone mineral density (BMD) and bone microstructure in rat femurs and lumbar bones were determined by dual-energy X-ray absorptiometry and micro-computed tomography. Femoral bone strength was detected by a three-point bending assay. The expression of Phospho-glycogen synthase kinase 3 beta (p-GSK-3ß), GSK-3ß, Dickkopf-related protein 1 (DKK1), cathepsin K, OPG, RANKL, IGF-1, Runx2, ß-catenin, and p-ß-catenin was determined by western blot and/or immunohistochemical staining. RESULTS: Treatment of OVX rats with RRP aqueous extract rebuilt bone homeostasis demonstrated by increasing the levels of OPG as well as decreasing the levels of TRAP, RANKL, and ALP in serum. Furthermore, RRP treatment preserved BMD and mechanical strength by increasing cortical bone thickness and epiphyseal thickness as well as improving trabecular distribution in the femurs of OVX rats. In addition, RRP downregulated the expression of DKK1, sclerostin, RANKL, cathepsin K, and the ratio of p-ß-catenin to ß-catenin, along with upregulating the expression of IGF-1, ß-catenin, and Runx2 and the ratio of p-GSK-3ß to GSK-3ß in the tibias and femurs of OVX rats. Echinacoside, jionoside A1/A2, acetoside, isoacetoside, jionoside B1, and jionoside B2 were identified in the RRP aqueous extract. CONCLUSION: RRP attenuates bone loss and improves bone quality in OVX rats partly through its regulation of the canonical Wnt/ß-catenin signaling pathway, suggesting that RRP has the potential to provide a new source of anti-osteoporotic drugs.

Madecassoside inhibits estrogen deficiency-induced osteoporosis by suppressing RANKL-induced osteoclastogenesis.

Osteoporosis is the most common osteolytic disease characterized by excessive osteoclast formation and resultant bone loss, which afflicts millions of patients around the world. Madecassoside (MA), isolated from Centella asiatica, was reported to have anti-inflammatory and antioxidant activities, but its role in osteoporosis treatment has not yet been confirmed. In our study, MA was found to have an inhibitory effect on the RANKL-induced formation and function of OCs in a dose-dependent manner without cytotoxicity. These effects were attributed to its ability to suppress the activity of two transcription factors (NFATc1 and c-Fos) indispensable for osteoclast formation, followed by inhibition of the expression of bone resorption-related genes and proteins (Acp5/TRAcP, CTSK, ATP6V0D2/V-ATPase-d2, and integrin ß3). Furthermore, we examined the underlying mechanisms and found that MA represses osteoclastogenesis by blocking Ca2+ oscillations and the NF-κB and MAPK pathways. In addition, the therapeutic effect of MA on preventing bone loss in vivo was further confirmed in an ovariectomized mouse model. Therefore, considering its ability to inhibit RANKL-mediated osteoclastogenesis and the underlying mechanisms, MA might be a potential candidate for treating osteolytic bone diseases.

A bone-targeting delivery system carrying osteogenic phytomolecule icaritin prevents osteoporosis in mice.

Targeting delivery system has been widely used in packaging drugs for medical therapies attributed to its high efficiency and efficacy. A Traditional Chinese Medicine (TCM) formula consisting of Herba Epimedii has previously been shown to effectively treat postmenopausal osteoporosis. We have subsequently found that icaritin, which was a flavonoid isolated from both Herba Epimedii and its serum metabolites after oral administration, inhibited the adipogenic capacity of bone mesenchymal stem cells (BMSCs) while promoted their osteogenesis. However, previous pharmacokinetic analyses have shown that icaritin had a short half-life in blood and only trace amounts of the molecule reach the bone tissue. To overcome this limitation, we developed a bone-targeting liposome containing an oligopeptide of eight aspartate residues (Asp8), which had previously been shown to specifically target the bone, encapsulating icaritin. In vivo, we found that the Asp8-icaritin-liposome enhanced bone formation in ovariectomized mice compared to an icaritin-liposome control lacking the Asp8 moiety. Through in vitro mechanistic studies we further found that icaritin inhibited adipogenesis through an Akt/GSK-3ß/ß-catenin signaling pathway. Taken together, our study shows that Asp8-liposome as a bone-targeting delivery system is effective to carry an osteogenic phytomolecule for facilitating and enhancing its therapeutic effects on the prevention of estrogen depletion-induced osteoporosis.

Beneficial Function of Taurine on Bone Metabolism in Alcohol-Fed OVX Rat Model.

The present study investigates the effects of taurine on bone markers and bone mineral density (BMD) in alcohol-fed ovariectomized (OVX) rat model. We divided twenty four rats into Sham and OVX groups. These two groups were thereafter subdivided into two groups: control and experimental diet containing 2 g/kg of taurine. BMD and bone mineral content (BMC) were estimated by PIXImus. As bone markers, we measured serum calcium, phosphorus, ALP activity, osteocalcin and urine calcium, phosphorus and DPD crosslinks value. The results were as follows: weight gain showed no significant difference and serum calcium concentration was in normal range. Urine DPD crosslink value was significantly decreased in taurine-fed group (p < 0.05). Serum ALP activity and osteocalcin levels, and urine phosphorus concentration did not show any differences among groups. Also the mineral density and content of spinal and femural bone did not show any differences among groups. However, the femur BMD was significantly increased in taurine-fed group (p < 0.05). In conclusion, taurine supplemented diets may have positive results on bone metabolism in alcohol-fed OVX rat model.

Liver Antioxidant Enzyme Activities Increase After Taurine in Ovariectomized Rats.

The present study was performed to know the effects of taurine on the lipid level of plasma and liver, lipid peroxidation and antioxidative enzyme activities of liver tissue in ovariectomized (OVX) rats fed cholesterol. Twenty-four female SD rats (200 ± 5 g) were grouped; sham and ovariectomy groups, which were each randomly subgrouped; fed control and control supplemented with taurine (20 g/kg diet). The serum total cholesterol, TG (triglyceride), LDL-cholesterol, athrogenic index, and HDL-cholesterol of taurine diet group were not statistically different. Also the levels of liver total cholesterol, triglyceride were not considerably different in different diets. The lipid peroxidation of malondialdehyde concentration was considerably lower in taurine-feeding group than control-feeding group in ovariectomy group. The superoxide dismutase activity in liver tissue was significantly higher in rats fed taurine than in rats fed control diet in OVX rats. GSH-Px (glutathione peroxidase) activity was statistically greater at the rats fed taurine diets compared to rats fed control diet in ovariectomy group. Activity of catalase was higher in taurine group than in control group in ovariectomy group, but it was not significantly different. In conclusion, taurine supplementation was beneficial on antioxidative enzyme activities of liver tissue in ovariectomized rats fed cholesterol.

Taurine May Modulate Bone in Cholesterol Fed Estrogen Deficiency-Induced Rats.

Taurine is thought to affect bone in rats favorably. However, studies on the actions of this estrogen deficiency and high cholesterol diet factors on the bone metabolism are limited. In this study, the protective effect of taurine on bone was determined. Thirty-two 42 days old female SD rats were placed in individual stainless cages. Given to rats was fed to chow (Samyang Corporation, South Korea) and deionized water for a 4 days adaptation period. After the period of adaptation, Half of the rats were induced estrogen deficiency model by ovariectomy (OVX), and the left rats with sham-operated were used control (SHAM). For six weeks, the OVX and SHAM rats had separately a 2% taurine supplemented diet with ad libitum in both the water and the food. DEXA for small animals (PIXImus, GE Lunar co, Wisconsin) was used to determine spinal and femoral bone. The concentrations of serum calcium and phosphorus were also measured. The monitoring of bone formation was done by determining the serum ALP and osteocalcin. Urinary DPD the values were determined as index of bone resorption. Statistical measure was done with SAS (version 9.3). A lower overall intake of the daily food was observed in non-ovariectomized rats than in the OVX rats. At sacrifice, a much greater body weight was observed in ovariectomized group compare to non-operated group. That difference was absent in both fed taurine SHAM and OVX rats. Serum calcium and phosphorus were not statistically different by taurine supplementation. Urinary excretion of calcium was not effected by taurine supplementation. Serum ALP and was significantly decreased by taurine in OVX rats (p < 0.05). For the spine BMD and BMC, there was no difference among SHAM and OVX rats by taurine. Spine BMC per body weight of taurine groups were higher than control groups (p < 0.1). No significant difference was observed after taurine supplementation in femur BMD and BMC. The analysis of the results suggest that taurine supplementation modulates the bone mineral contents in postmenopausal model rats fed with high cholesterol diet.

Formononetin, a methoxy isoflavone, enhances bone regeneration in a mouse model of cortical bone defect.

The bone regeneration and healing effect of formononetin was evaluated in a cortical bone defect model that predominantly heals by intramembranous ossification. For this study, female Balb/c mice were ovariectomised (OVx) and a drill-hole injury was generated in the midfemoral bones of all animals. Treatment with formononetin commenced the day after and continued for 21 d. Parathyroid hormone (PTH1-34) was used as a reference standard. Animals were killed at days 10 and 21. Femur bones were collected at the injury site for histomorphometry studies using microcomputed tomography (µCT) and confocal microscopy. RNA and protein were harvested from the region surrounding the drill-hole injury. For immunohistochemistry, 5 µm sections of decalcified femur bone adjoining the drill-hole site were cut. µCT analysis showed that formononetin promoted bone healing at days 10 and 21 and the healing effect observed was significantly better than in Ovx mice and equal to PTH treatment in many aspects. Formononetin also significantly enhanced bone regeneration as assessed by calcein-labelling studies. In addition, formononetin enhanced the expression of osteogenic markers at the injury site in a manner similar to PTH. Formononetin treatment also led to predominant runt-related transcription factor 2 and osteocalcin localisation at the injury site. These results support the potential of formononetin to be a bone-healing agent and are suggestive of its promising role in the fracture-repair process.

Synergistic attenuation of ovariectomy-induced bone loss by combined use of fish oil and 17ß-oestradiol.

Oestrogen and n-3 PUFA, especially EPA and DHA, have been reported to have beneficial effects on bone loss. Thus, the purpose of the present study was to investigate the synergistic bone-protective mechanism of combined treatments of EPA+DHA supplementation and oestrogen injection in ovariectomised rats. Rats were fed a modified American Institute of Nutrition-93G diet with 0 %, 1 % or 2 % n-3 PUFA (EPA+DHA) relative to the total energy intake for 12 weeks. Rats were surgically ovariectomised at week 8, and after a 1-week recovery period rats were injected with either 17ß-oestradiol-3-benzoate (E2) or maize oil for the last 3 weeks. Combined use of n-3 PUFA and E2 synergistically increased femoral cortical bone volume, bone mineral content and the bone expression of runt-related transcription factor 2 (RUNX2), but decreased the bone expression of IL-1ß. Both n-3 PUFA and E2 decreased the bone expressions of IL-7, TNF-α and PPAR-γ, and increased the bone expression of oestrogen receptor-α. n-3 PUFA in the presence of E2 and E2 alone significantly decreased the bone expressions of IL-1ß and IL-6 and increased the bone expression of RUNX2. E2 significantly decreased the serum levels of bone turnover markers and the bone expression of receptor activator of NF-κB ligand, but decreased the bone expression of osteoprotegerin. The combined use of n-3 PUFA and E2 exerted synergistic bone-protective efficacy through up-regulation of RUNX2, an essential transcription factor for bone formation, as well as the suppression of bone-resorbing cytokine IL-1ß.