Hyaluronan and chondroitin sulfate in chicken-vegetable bone broth delay osteoporosis progression.

Bone broth has recently gained worldwide recognition as a superfood that supplements several nutrients lacking in modern human diets; however, little is known of its efficacy on osteoporosis. Therefore, we aimed to identify the components of chicken-vegetable bone broth (CVBB) that are associated with osteoporosis prevention and verified the efficacy of these components using in vivo studies. In biochemical and cell biological experiments, CVBB was fractionated using ion exchange chromatography (IEC), and the effect of each IEC fraction on osteoclast differentiation was evaluated based on tartrate-resistant acid phosphatase (TRAP) activity, TRAP staining, and quantitative polymerase chain reaction analysis using mouse macrophage-like cells (RAW264 cell). In animal experiments, an ovariectomized (OVX) rat model was generated, followed by whole bone broth (OVX/CVBB) or IEC fraction (OVX/CVBB-Ext) administration and bone structural parameter characterization of OVX rat tibia based on micro-CT. Four CVBB fractions were obtained using IEC, and the fraction containing both hyaluronan and chondroitin sulfate (CVBB-Ext) led to the maximum inhibition of RAW264 cell differentiation. CVBB-Ext downregulated the expression of osteoclast differentiation marker genes. In animal experiments, the OVX group showed a clear decrease in bone density compared to that in the Sham operation group. The OVX/CVBB and OVX/CVBB-Ext groups showed increased bone mineral density and bone volume/tissue volume values compared to those in the OVX/control group. These results suggested that CVBB and CVBB-Ext slowed osteoporosis progression. Therefore, we conclude that hyaluronan and chondroitin sulfate in CVBB are key substances that impede osteoporosis progression. PRACTICAL APPLICATION: This study provides practical information on the effects of bone broth ingredients on osteoporosis to expand the current knowledge on the efficacy of bone broth, which is a widely consumed food. These results may help in the future development of bone broth as a dietary supplement for managing osteoporosis.

Non-targeted metabolomics strategy reveals the role of Geng-Nian-Shu in regulating ferroptosis in perimenopausal syndrome.

Ovariectomy (OVX) is usually accompanied by the occurrence of metabolic syndrome. Previous studies have shown that Geng-Nian-Shu (GNS) plays an important regulatory role in perimenopausal syndrome (PMS) rats. GNS is a traditional Chinese medicine (TCM) prescription which composed of Suanzaoren Decoction and Ganmai Dazao Decoction in "Jingui Yaolue" and Siwu Decoction in "Heji Jufang". Recently, metabolomics analysis has been used to identify slight changes in the metabolic profile and to help understand disease progression and therapeutic interventions in PMS. However, the mechanism of GNS in the treatment of PMS is still unknown. We purposed to study the metabolic characteristics of PMS by serum and fecal metabolomics, and revealed the internal mechanism of GNS regulating ferroptosis against PMS. The PMS model was established by surgical removal of 4/5 ovaries of rats. HPLC-Q-TOF/MS was used to analyze the metabolomics of rat plasma and feces to explore the potential mechanism of GNS in PMS. The expression of ferroptosis-related proteins in rat ovaries was detected by tissue Prussian blue staining, Elisa kit and Western blotting. Cluster analysis of differential metabolites in plasma and feces between the control group and the model group showed that organic acids and their derivatives, lipids and lipid molecules were mainly disturbed during PMS in rats. After GNS administration, 17 differential metabolites were adjusted, involving several major pathways, such as the tricarboxylic acid (TCA) cycle, biosynthesis of amino acids and biosynthesis of unsaturated fatty acids. Further, we found that GNS affected ferroptosis in ovarian cells by regulating endogenous substances in OVX rats. Our study provides new insights into the mechanism of OVX-induced metabolic syndrome based on non-targeted metabolomics. It provides new ideas for the development and application of GNS and the diagnosis and treatment of PMS.

MiR-210 promotes bone formation in ovariectomized rats by regulating osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells through downregulation of EPHA2.

PURPOSE: In osteoporosis, the balance between osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs) is disrupted. The osteogenic differentiation of bone marrow MSCs (BMSCs) is important for improving osteoporosis. The aim of this study was to explore the role and molecular mechanism of miR-210 in the balance of osteogenic/adipogenic differentiation of BMSCs in postmenopausal osteoporosis. METHODS: Postmenopausal osteoporosis rat models were constructed by ovariectomy (OVX). BMSCs were isolated from the femur in rats of Sham and OVX groups. MiR-210 was overexpressed and suppressed by miR-210 mimics and inhibitor, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative mRNA expression of miR-210, ephrin type-A receptor 2 (EPHA2), alkaline phosphatase (ALP), osterix (OSX), osteocalcin (Bglap), Runt-related transcription factor 2 (Runx2), peroxisome proliferator activated receptor gamma, and fatty acid binding protein 4 (FABP4) in each group of rat femoral tissues or BMSCs. Western blot was applied to detect the protein expression level of EPHA2 in rat femoral tissues and cells. Alizarin red S staining and oil red O staining were performed to assess the osteogenic and adipogenic differentiation of BMSCs, respectively. In addition, the targeting relationship between miR-210 and EPHA2 was verified by a dual luciferase gene reporter assay. RESULTS: The expression of miR-210 was significantly reduced in femoral tissues and BMSCs of OVX rats, and its low expression was associated with reduced bone formation. The osteogenic differentiation was enhanced in OVX rats treated with miR-210 mimic. Overexpression of miR-210 in transfected BMSCs was also found to significantly promote osteogenic differentiation and even inhibit adipogenic differentiation in BMSCs, while knockdown of miR-210 did the opposite. Further mechanistic studies showed that miR-210 could target and inhibit the expression of EPHA2 in BMSCs, thus promoting osteogenic differentiation and inhibiting adipogenic differentiation of BMSCs. CONCLUSION: MiR-210 promotes osteogenic differentiation and inhibits adipogenic differentiation of BMSCs by down-regulating EPHA2 expression. As it plays an important role in the osteogenic/adipogenic differentiation of osteoporosis, miR-210 can serve as a potential miRNA biomarker for osteoporosis.

[Adjuvant rice optimization of rice-steamed Rehmanniae Radix and its anti-osteoporosis effect].

The present study aimed to investigate the effect of various adjuvant rice on the quality of rice-steamed Rehmanniae Radix(RSRR) with Japonica rice, millet, yellow rice, black rice, and glutinous rice as raw materials, and analyze the anti-osteoporosis effect of RSRR by the optimal adjuvant rice. On the basis of the established UPLC-MS/MS method for the determination of the content of catalpol and rehmannioside D, comprehensive weighted scoring method was employed to evaluate the effect of various auxiliary rice on the quality of RSRR with the content of catalpol and rehmannioside D, character score, and taste score as indicators to optimize adjuvant rice. The osteoporosis model was induced by ovariectomy in rats. SD rats were randomly divided into a sham operation group, a model group, a positive control group, and low-dose and high-dose groups of Rehmanniae Radix, RSRR, steamed Rehmanniae Radix, and Epimedii Folium-RSRR. After treatment for 12 weeks, body weight, bone calcium content, and bone mineral density were mea-sured. The results showed that Japonica rice was selected as the optimal adjuvant due to the highest comprehensive score of RSRR steamed by Japonica rice. Rehmanniae Radix, RSRR, steamed Rehmanniae Radix, as well as Epimedii Folium-RSRR, could improve osteoporosis by increasing bone calcium content and bone mineral density. RSRR was superior to Rehmanniae Radix in improving osteo-porosis. However, there was no significant difference between RSRR and steamed Rehmanniae Radix. This study confirmed that Japo-nica rice was the optimal adjuvant rice of RSRR and verified the anti-osteoporosis effect of RSRR, which laid a foundation for further research on the pharmacological action and mechanism of RSRR.

Diosgenin reduces bone loss through the regulation of gut microbiota in ovariectomized rats.

Diosgenin (DIO) is an aglycone of steroid saponins acquired from plants, including Dioscorea alata, Smilax China, and Trigonella foenum graecum, acting as an anti-osteoporosis, anti-diabetic, anti-hyperlipidemic, anti-inflammatory. Recent studies have demonstrated that DIO reduces bone loss. This study aimed to investigate the effects of DIO on the gut microbiota (GM) of ovariectomized (OVX) osteoporotic rats. Female Sprague-Dawley rats were randomly divided into sham operation (sham + vehicle group) or ovariectomy. For 12 weeks, OVX rats were treated using a vehicle (OVX + vehicle group) and DIO (OVX + DIO group). Subsequently, ELISA was conducted to determine serum estradiol levels, micro-CT scanning was performed to evaluate bone quality, and feces were collected for metagenomics sequencing to examine the structure and function of GM. Raw reads were filtered to remove chimera sequences. Operational taxonomic units (OTUs) were clustered in the filtered reads. A Venn diagram analysis was conducted to study the common and unique OTUs in the sham + vehicle, OVX + vehicle, and OVX + DIO groups. LEfSe analysis was conducted to evaluate the specific GM of the three groups. The GM functions were analyzed using the KEGG and CAZy databases. After a 12-week treatment, DIO administration prevented OVX-induced weight gain and increased the estradiol levels. DIO treatment improved the bone microstructure and structural parameters of rat tibias. Metagenomics sequencing results identified 1139, 1207, and 1235 operational taxonomic units (OTUs) in the sham + vehicle, OVX + vehicle, and OVX + DIO groups, respectively. The percentage of common OTUs was 41.2%. Treatment with DIO restored the composition of GM in OVX rats by increasing the abundance of Coriobacteriia Adlercreutzia, Romboutsia, and Romboutsia_idealis and reducing the abundance of Betaproteobacteria, Gammaproteobacteria, Methanobacteria, Bacteroides, Phocaeicola, Alistipes, Bacteroids_uniformis, Bacteroids_xylanisolvens. The anti-osteoporosis effect of DIO can be regulated through environmental information processing, organismal Systems, Cellular Processes, human diseases, metabolism, and genetic information processing. Meanwhile, treatment with DIO improved GM homeostasis by increasing the metabolism of carbohydrates, other amino acids, and glycans and reducing translation, energy metabolism, and nucleotide metabolism. DIO can reduce bone loss by regulating the structural composition and function of GM, a novel strategy for preventing osteoporosis.

Oral Administration of Artemisia argyi Polysaccharide Increases Estrogen Level and Maintains Blood Lipid Homeostasis in Ovariectomized Rats.

INTRODUCTION: Artemisia argyi polysaccharide (AAP) has a beneficial effect on menstruation-related symptoms and the potential regulation of lipid metabolism. It is expected to be a safe and effective ingredient for estrogen deficiency and lipid metabolic disorders. Here, we investigate the effect of AAP on body weight gain, estrogen level, and blood lipid changes in ovariectomized (OVX) rats. METHODS: Thirty-six female Wistar rats were randomly divided into six treatment groups, including a sham-operated (Sham) group, OVX group, estrogen replacement (OVX + E2) group, and AAP treatment (OVX + 125, 250, 500 mg/kg AAP) group. The body weight and feed intake were recorded every week. The level of estrogen and blood lipid was determined. The gene expressions and protein expressions of estrogen receptors (ERs), fatty acid synthetase (FAS), acetyl CoA carboxylase 2 (ACC2), and 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) were determined. RESULTS: AAP treatment significantly decreased the body weight gain and average daily food intake of rats in the OVX group. Treatment with AAP significantly increased the relative weight of the uterus, plasma estrogen level, and the gene expression and protein expression of ER-α in the uterus. For blood lipids, plasma levels of triglyceride, total cholesterol, and low-density lipoprotein cholesterol were significantly reduced by AAP treatment in OVX rats. AAP treatment decreased the expression of FAS and HMGR in the liver of OVX rats. Furthermore, AAP treatment significantly increased the gene expression of ACC2, the protein expression of P-ACC2, and the ratio of P-ACC2/ACC2. CONCLUSION: In summary, AAP treatment exerts beneficial effects on body weight gain and lipid metabolism disorder induced by ovariectomy through increasing estrogen levels, inhibiting FAS, and promoting fatty acid oxidation.

Analysis of Intervention of Erxiantang in Ovariectomized Rats by LC-MS Serum Metabolomics / 中国实验方剂学杂志

ObjectiveTo investigate the changes of endogenous metabolites in serum of ovariectomized rats and the effect of Erxiantang on them based on liquid chromatography-mass spectrometry（LC-MS）. MethodTwenty-four healthy female SD rats were randomly divided into sham-operated group， model group and Erxiantang group（7.5 g·kg-1）， with 8 rats in each group. Bilateral ovarian tissues were excised in the model and Erxiantang groups， and small pieces of adipose tissues were excised in the abdominal cavity of the sham-operated group bilaterally， and gastric administration was started 2 weeks after surgery， and equal volumes of distilled water were gavaged in the sham-operated and model groups. After 12 weeks of administration， blood was collected from abdominal aorta， and non-targeted metabonomics was performed on rat serum by LC-MS， and orthogonal partial least squares-discriminant analysis（OPLS-DA） was used to screen differential metabolites. Metabolic pathway analysis was performed based on Kyoto Encyclopedia of Genes and Genomes（KEGG）， and the levels of key enzymes of metabolic pathways were verified by enzyme-linked immunosorbent assay（ELISA）. ResultThe results of metabonomics showed that 82 differential metabolites between the model group and the sham-operated group were glycerophospholipids， fatty acyls， steroids and steroid derivatives， of which the most significant difference was glycerophospholipids. At the same time， Erxiantang could call back 65 out of 82 differential metabolites， of which 11 were statistically significant， mainly phosphatidylcholine（PC） and lysophosphatidylcholine（LysoPC） in glycerophospholipids， followed by corticosterone and 11-deoxycortisol in steroids and steroid derivatives. Metabolic pathway analysis showed that the pathways of glycerophospholipid metabolism and steroid hormone biosynthesis in model group were changed， and were recovered after the administration of Erxiantang. ELISA results showed that compared with the sham-operated group， serum levels of cholinephosphate cytidylytransferase（CCT）， secretory phospholipase A2（sPLA2） and lysophosphatidylcholine acyltransferase（LPCAT）， which were the key metabolic enzymes of glycerophospholipid metabolite PC and LysoPC， were significantly decreased in the model group（P<0.05， P<0.01）， and choline phosphotransferase 1（CPT1） levels decreased but the difference was not statistically significant， compared with the model group， the levels of CCT， sPLA2 and CPT1 were significantly increased in Erxiantang group（P<0.01）. In addition， compared with the sham-operated group， the levels of cholesterol（TC）， triglyceride（TG） and low density lipoprotein cholesterol（LDL-C） were significantly increased in the model group（P<0.01）， the high density lipoprotein cholesterol（HDL-C） level was decreased（P<0.05）， compared with the model group， the levels of TC， TG and LDL-C were significantly decreased and the level of HDL-C was significantly increased in Erxiantang group（P<0.01）. ConclusionEndogenous metabolites and related metabolic pathways in ovariectomized rats were altered， and Erxiantang can reverse some of the different metabolites and related pathways， such as regulating glycerophospholipid metabolism by regulating metabolic enzymes CCT， sPLA2 and CPT1 to increase the levels of PC and LysoPC， and then improve the pathological changes such as lipid metabolism disorder in ovariectomized rats.

Multiscale Analysis on Changes in Bone Microstructure of Osteoporotic Rats / 医用生物力学

Objective To study changes in bone microstructure of osteoporotic rats by multiscale analysis. Methods A total of 20 5-month-old female SD rats were randomly divided into two groups, i.e., ovariectomy (OVX) group (n=12) and the SHAM group (n=8), respectively. The rats in OVX group were subjected to bilateral ovariectomy and became osteoporosis models after 8 weeks, while sham operation was performed for the SHAM group. Changes in microstructure of cortical bone and cancellous bone at tissue scale, and osteocyte lacunar-canalicular network (LCN) and extracellular matrix (ECM) at cell scale were quantitatively analyzed using Micro-CT and SR-Nano-CT. Results At tissue scale, the cross-sectional area of cortical bone in OVX group was significantly higher than that in SHAM group (P<0.05), and the bone mineral density (BMD) and thickness of cortical bone were not significantly different from those in SHAM group. The trabecular BMD, bone volume fraction, trabecular thickness and trabecular number in OVX group were significantly decreased in comparison with SHAM group (P<0.01), while the trabecular separation was significantly increased (P<0.01). At cell scale, there was no significant difference in the semiaxes of lacunae between OVX group and SHAM group, but the thickness of lacunae and the diameter of canaliculi in OVX group were significantly increased in comparison with SHAM group (P<0.05). At the same time, the porosity of cortical bone in OVX group was significantly higher than that in SHAM group at cell scale (P<0.05). Conclusions The bone microstructure in OVX group varied to different extents at tissue and cell scales. At tissue scale, the cancellous bone loss was severe, while the cortical bone had fewer changes. At cell scale, porosity of the lacunar-canalicular network significantly increased, which directly affected the BMD and strength of cortical bone. Multiscale analysis on changes in bone microstructure of OP rats has potential application value for clinical diagnosis and pathological analysis of osteoporosis.

Adjuvant rice optimization of rice-steamed Rehmanniae Radix and its anti-osteoporosis effect / 中国中药杂志

The present study aimed to investigate the effect of various adjuvant rice on the quality of rice-steamed Rehmanniae Radix(RSRR) with Japonica rice, millet, yellow rice, black rice, and glutinous rice as raw materials, and analyze the anti-osteoporosis effect of RSRR by the optimal adjuvant rice. On the basis of the established UPLC-MS/MS method for the determination of the content of catalpol and rehmannioside D, comprehensive weighted scoring method was employed to evaluate the effect of various auxiliary rice on the quality of RSRR with the content of catalpol and rehmannioside D, character score, and taste score as indicators to optimize adjuvant rice. The osteoporosis model was induced by ovariectomy in rats. SD rats were randomly divided into a sham operation group, a model group, a positive control group, and low-dose and high-dose groups of Rehmanniae Radix, RSRR, steamed Rehmanniae Radix, and Epimedii Folium-RSRR. After treatment for 12 weeks, body weight, bone calcium content, and bone mineral density were mea-sured. The results showed that Japonica rice was selected as the optimal adjuvant due to the highest comprehensive score of RSRR steamed by Japonica rice. Rehmanniae Radix, RSRR, steamed Rehmanniae Radix, as well as Epimedii Folium-RSRR, could improve osteoporosis by increasing bone calcium content and bone mineral density. RSRR was superior to Rehmanniae Radix in improving osteo-porosis. However, there was no significant difference between RSRR and steamed Rehmanniae Radix. This study confirmed that Japo-nica rice was the optimal adjuvant rice of RSRR and verified the anti-osteoporosis effect of RSRR, which laid a foundation for further research on the pharmacological action and mechanism of RSRR.

Prebiotics improve osteoporosis indicators in a preclinical model: systematic review with meta-analysis.

CONTEXT: Studies using experimental models have demonstrated that prebiotics are involved in antiosteoporotic mechanisms. OBJECTIVE: This study was conducted to determine the impact of supplementation with prebiotics in the basal diet of ovariectomized rats with induced osteoporosis as a preclinical model. METHODS: A comprehensive systematic search was carried out in the electronic databases PubMed, Science Direct, Web of Science, Scielo, and Google through March 2022 for studies that investigated the impact of prebiotics on bone mineral density (BMD), bone mineral content (BMC), and bone biomechanics. RESULTS: The search returned 844 complete articles, abstracts, or book chapters. After detailed screening, 8 studies met the inclusion criteria. Rats (n = 206), were randomly divided between control and treatment groups. Weighted mean differences (WMDs) with the 95%CIs were used to estimate the combined effect size. Compared with the control group, dietary intake of prebiotics significantly increased bone density in the BMD subgroups, with WMDs as follows: 0.03 g/cm3, 95%CI, 0.01-0.05, P < 0.00001, n = 46; and 0.00 g/cm2, 95%CI, 0.00-0.02, P < 0.00001, n = 81; total BMD: WMD, 0.01, 95%CI, 0.01-0.02, P < 0.00001, n = 127; bone content in BMC: WMD, 0.02 g, 95%CI, 0.00-0.04, P = 0.05, n = 107; and the 3-point-bend test: WMD, 15.20 N, 95%CI, 5.92-24.47, P = 0.00001, n = 120. CONCLUSION: Prebiotics improve indicators of osteoporosis, BMD, BMC, and bone biomechanics in ovariectomized rats. More studies are needed to increase the level of evidence. SYSTEMIC REVIEW REGISTRATION: Systematic Review Protocol for Animal Intervention Studies.

Protective effect of isoflavone enriched soy ß-conglycinin on osteoporosis in ovariectomized rats.

Research shows that the consumption of soybean foods can reduce the incidence rate of bone fractures in women after menopause. The aim of this study was to investigate the effects of different complex of soy ß-conglycinin (7S) and isoflavones (7S-ISO) on osteoporosis in ovariectomized rats. All treatments were administrated intragastrically to the groups every afternoon for 3 months. The treatments were administrated at 1 mL·(100 g)-1 , the animals were given 50 mg·kg-1 ·d-1 ISO, and the concentration of protein was about 2 wt. %. The bone mineral density (BMD) and the bone biomechanics results of left tibia' maximum load in the 7S-ISO group is significantly higher than in the ovariectomized group and the 7S group (p < .05). Otherwise, the serum tartrate-resistant acid phosphatase (s-TRACP), serum osteocalcin (s-BGP), and serum estradiol (s-E2 ) levels in 7S-ISO were all significantly different from the OVX, OVX + casein, and the OVX + 7S group (p < .05). The serum calcium (s-Ca) level was not significantly different among all the groups. 7S-ISO may exhibit moderate estrogenic activities and as compared to 7S and ISO in osteoporosis (OP) of ovariectomized rats. PRACTICAL APPLICATIONS: The effects of soy proteins on the health of females have always been a concern. It has been extensively reported soy 7S globulin (7S) as a type of trimer glycoprotein can depress blood fats. The aim of this study was to investigate the effects of different complex of soy ß-conglycinin and isoflavones (ISO), the main storage proteins and polyphenols in soy, on osteoporosis in ovariectomized rats.

Systemic administration with melatonin in the daytime has a better effect on promoting osseointegration of titanium rods in ovariectomized rats.

AIMS: This study examined whether systemic administration of melatonin would have different effects on osseointegration in ovariectomized (OVX) rats, depending on whether this was administered during the day or night. METHODS: In this study, a titanium rod was implanted in the medullary cavity of one femoral metaphysis in OVX rats, and then the rats were randomly divided into four groups: Sham group (Sham, n = 10), OVX rat group (OVX, n = 10), melatonin day treatment group (OVX + MD, n = 10), and melatonin night treatment group (OVX + MN, n = 10). The OVX + MD and OVX + MN rats were treated with 30 mg/kg/day melatonin at 9 am and 9 pm, respectively, for 12 weeks. At the end of the research, the rats were killed to obtain bilateral femora and blood samples for evaluation. RESULTS: Micro-CT and histological evaluation showed that the bone microscopic parameters of femoral metaphysis trabecular bone and bone tissue around the titanium rod in the OVX + MD group demonstrated higher bone mineral density, bone volume fraction, trabecular number, connective density, trabecular thickness, and lower trabecular speculation (p = 0.004) than the OVX + MN group. Moreover, the biomechanical parameters of the OVX + MD group showed higher pull-out test and three-point bending test values, including fixation strength, interface stiffness, energy to failure, energy at break, ultimate load, and elastic modulus (p = 0.012) than the OVX + MN group. In addition, the bone metabolism index and oxidative stress indicators of the OVX + MD group show lower values of Type I collagen cross-linked C-telopeptide, procollagen type 1 N propeptide, and malondialdehyde (p = 0.013), and higher values of TAC and SOD (p = 0.002) compared with the OVX + MN group. CONCLUSION: The results of our study suggest that systemic administration with melatonin at 9 am may improve the initial osseointegration of titanium rods under osteoporotic conditions more effectively than administration at 9 pm.Cite this article: Bone Joint Res 2022;11(11):751-762.

Experimental study on the effects of simvastatin in reversing the femoral metaphyseal defects induced by sodium valproate in normal and ovariectomized rats.

Introduction: Long-term treatment with antiepileptic drugs may cause secondary osteoporosis. The present study investigated the influence of simvastatin (SIM) in reversing the effects of valproate on bone defect healing in normal and ovariectomized (OVX) rats. Methods: Bone defects in femora were established in seven experimental groups of rats: control (vehicle), sodium valproate (SVP; 300 mg/kg/d), SVP plus SIM (25 mg/kg/d), sham control (sham), OVX, OVX SVP and OVX SVP plus SIM. All rats were euthanized at 8 weeks after bone defect creation. Results: Micro-CT, biomechanical and histological evaluations demonstrated lower bone strength and delayed bone healing in the SVP therapy group compared with the SVP plus SIM therapy group. Biochemical and immunohistochemical results showed that osteocalcin (OCN), collagen I (Col I) and procollagen type I N-terminal propeptide (P1NP) levels decreased, tartrate-resistant acid phosphatase type 5 precursor (TRACP-5b) expression increased, and Dickkopf-1 (DKK-1) and receptor activator of nuclear factor-κ B ligand (RANKL) expression were upregulated in the SVP therapy rats compared with the SVP plus SIM therapy group. Bone loss was exacerbated by OVX, but the effect of SIM in ameliorating bone loss was also more marked in the OVX rats. Conclusions: This study indicated lower bone strength and delayed healing of bone defects in rats given SVP therapy, especially the OVX SVP treatment group. In contrast, treatment with SIM was effective in enhancing bone strength and promoting bone defect repair and showed significant influence on promoting osteogenesis and inhibiting osteoclastogenesis.

Comprehensive metabolite profiling of Phoenix rupicola pulp and seeds using UPLC-ESI-MS/MS and evaluation of their estrogenic activity in ovariectomized rat model.

Dates have been consumed since ancient times as functional foods which beside their high nutritional value possess various biological activities. Phoenix rupicola T. Anderson (Cliff date palm) produces non-conventional edible dates, however, due to low natural abundance, these dates aren't commercially important as the dates of Phoenix dactylifera L. The present study was designed to evaluate the phytochemical constituents as well as the estrogenic activity of P. rupicola dates. UPLC-ESI-MS/MS approach was used to study the metabolite profile of the 70% aqueous methanol extracts of P. rupicola dates (pulps and seeds) for the first time. A total of fifty-five metabolites were tentatively identified in both extracts, belonging to different classes, chiefly phenolic compounds viz. procyanidins, flavonoid glycosides, hydroxycinnamic acid derivatives, as well as, fatty acids, organic acids and sphingolipids. Acute toxicity studies revealed that the studied extracts were safe at oral doses up to 2 g/kg. Besides, they possessed significant (P < 0.05) estrogenic activity in ovariectomized rat model, as compared to ovariectomized (OVX) and reference standard (17ß-estradiol; OVX-E) groups. Moreover, the extracts showed significant improvement on bone metabolism, lipid profile, liver and kidney functions. In silico docking study revealed that various metabolites possessed high binding affinities to both ERs, where 2-palmitoyl glycerol (-10.28 Kcal/mol) and aminotetradecanetriol (-9.61 Kcal/mol) showed the strongest affinities to Erα and Erß, respectively. Thus, it can be concluded that P. rupicola pulp and seeds possess bioactive phytoconstituents comparable to those in P. dactylifera and can be used as a safe and efficient natural estrogen substitute in postmenopausal women.

Early effects of ovariectomy on bone microstructure, bone turnover markers and mechanical properties in rats.

BACKGROUND: Fragility fracture is one of the most serious consequences of female aging, which can increase the risk of death. Therefore, paying attention to the pathogenesis of postmenopausal osteoporosis (PMOP) is very important for elderly women. METHODS AND MATERIALS: Forty 12-week-old female rats were divided into two groups including the ovariectomy (OVX) group and the control group. Four rats in each group were selected at 1, 4, 8, 12 and 16 weeks after operation. Vertebral bones and femurs were dissected completely for micro-Computed Tomography (micro-CT) scanning, biological modulus detection and histomorphological observation. RESULTS: In OVX group, bone volume/total volume (BV/TV), bone trabecular connection density (Conn.D) and trabecular bone number (Tb.N) decreased significantly with time (P < 0.05). The elastic modulus of femur in OVX group was lower than that in control group, but there was no significant difference between them (P > 0.05). Over time, the tartrate resistant acid phosphatase (TRAP), osteocalcin (BGP), type I procollagen amino terminal propeptide (PINP) and type I collagen carboxy terminal peptide (CTX-I) in OVX group increased significantly (P < 0.05). The micrographs of the OVX group showed sparse loss of the trabecular interconnectivity and widening intertrabecular spaces with time. CONCLUSION: The bone loss patterns of vertebral body and femur were different in the early stage of estrogen deficiency. The bone turnover rate of OVX rats increased, however the changes of biomechanical properties weren't obvious.

Salidroside protects against osteoporosis in ovariectomized rats by inhibiting oxidative stress and promoting osteogenesis via Nrf2 activation.

BACKGROUND: Osteoporosis (OP) is characterized as low bone mass, bone microarchitecture breakdown and bone fragility. The increase of oxidative stress could lead to breakdown in the balance of bone formation and resorption which gives rise to OP. Nrf2 is a transcription factor which takes part in oxidative stress and recently was reported that it can regulate the occurrence of OP. Salidroside (SAL) with the efficacies of anti-oxidation, anti-aging and bone-protection is one of the active ingredients in Ligustri Lucidi Fructus, a traditional Chinese medicinal herb. Nevertheless, few studies have explored the potential mechanism of SAL preventing OP development from the perspective of oxidative stress intervention. PURPOSE: This study aimed to investigate the pharmacological effect and molecular mechanisms of SAL on OP. STUDY DESIGNS AND METHODS: A tert-butyl hydroperoxide (t-BHP)-induced oxidative stress model was applied for investigating the effects of SAL in vitro, and an ovariectomized (OVX) model was used for in vivo study on the effect of SAL for OP. Related pharmacodynamic actions and molecular mechanisms of SAL were explored in both rat osteoblasts (ROBs) and OVX rats. Network biology and cell metabolomics were performed for further investigating the correlation and association among potential biomarkers, targets and pathways. RESULTS: SAL reduced levels of ROS and lipid peroxidation (LPO), increased activities of antioxidant enzymes like GPx and SOD, and enhanced osteogenic differentiation in t-BHP-induced ROBs and OVX rats. Mechanistic studies showed SAL prevented OP development and reduced oxidative damage in ROBs and OVX rats through up-regulating Nrf2 expression and facilitating its nuclear translocation. The joint analysis of network biology and cell metabolomics revealed that galactose metabolism and fatty acid metabolism could be the major influenced pathways following treatment with SAL. CONCLUSION: SAL could protect against OP by inhibiting oxidative stress, promoting osteogenesis through the up-regulation of Nrf2 and intervening galactose metabolism and fatty acid metabolism. Our study implied that SAL may be a potential drug to treat OP.

MSTN is an important myokine for weight-bearing training to attenuate bone loss in ovariectomized rats

Weight-bearing training, as one of resistance exercises, is beneficial to bone health. Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. Animals lacking MSTN show increased bone mineral density (BMD). The aim of this study was to investigate the preventive effect of weight-bearing training on bone loss in ovariectomized rats and whether it was related to MSTN. In this study, the rats were randomly assigned to three group: Sham-ovariectomized (Sham), ovariectomized (OVX), ovariectomized and weight-bearing training (OWT). The rats in the OWT group ran at 20-m/min bearing with 35% of their body weight for 6 days/week. After 10 weeks, compared with the OVX group, weight-bearing training increased the BMD of total femur and trabecular bone by 8.13% and 57.44%, respectively. The OVX-induced destruction of bone microarchitecture including the thickness and number of trabeculae and bone volume fraction was all significantly improved (9.26%, 47.68%, 63.03%) in the OWT group. The OVX-induced degradation of bone mechanical properties was significantly enhanced in the OWT group (maximum load increased by 35.46%, stiffness increased by 89.19%, energy absorption increased by 53.4%; elastic modulus increased by 26.3%). Ten-week weight-bearing training also significantly upregulated the mRNA and protein expression of Wnt1 and β-catenin, which is crucial in bone development. Compared with the Sham group, MSTN in serum and muscle increased in the OVX group, but it decreased in the OWT group compared with the OVX group. Its receptor ActRIIB and downstream molecules Smad2/3 in the OVX group were downregulated in bone by weight-bearing training. The results indicated that MSTN is an important myokine for weight-bearing training to attenuate bone loss in ovariectomized rats. (AU)

Species-level gut microbiota analysis in ovariectomized osteoporotic rats by Shallow shotgun sequencing.

Gut microbiota was verified to regulate bone metabolism and was closely associated with osteoporosis. Using 16S rRNA sequencing, gut microbiota at genus level such as Helicobacter, Bacteroides, and Prevotella were found to increase in the osteoporotic animals and people. However, the changes of species-level gut microbiota and related functional alterations were still unknown. Female SD rats were divided into the ovariectomized (OVX) group and the control group, and the fecal samples were collected at 4, 8, and 12 weeks to analyze the information of gut microbiota. Using Shallow shotgun sequencing, we compared the species-level gut microbiota structure, composition, and functional pathways of the OVX group with the control group. Alpha diversity of the OVX rats were significantly decreased than those in the control group. Beta diversity showed that samples in the two groups could be distinguished in each coordinate at different time points. Furthermore, the relative abundance of gut microbiota at species-level and differential analysis found that bacteria species such as Helicobacter rodentium, Lachnospiraceae bacterium 10 1, and Lachnospiraceae bacterium A4 were markedly increased in the OVX rats. Furthermore, differential analysis of KEGG functional pathway revealed that lysine metabolism was enriched in the OVX group.In conclusion, gut microbiota were significantly altered in structure and composition estrogen-deficiency osteoporotic rats at the species level. Functional metabolism of gut microbiota was also changed in osteoporotic group. These changes in gut microbiota at the species level might be closely associated with osteoporosis caused by estrogen deficiency.

Histomorphological and Biochemical Analysis of Rat Model of Menopausal Skin Aging.

The study examined the skin histomorphology and biochemistry in mature ovariectomized rats treated and not treated with estrogen. Biochemical parameters (superoxide dismutase, malondialdehyde, and hydroxyproline content) were measured in dorsal skin samples collected in 50 days after surgery. The morphology of dorsal skin was analyzed under a microscope. In ovariectomized rats, the skin levels of superoxide dismutase and hydroxyproline were significantly lower, while the superoxide dismutase content was significantly higher than in shamoperated animals (p<0.05). Estrogen therapy significantly increased the levels of superoxide dismutase and hydroxyproline and reduced superoxide dismutase level in ovariectomized rats in comparison with the corresponding parameters in untreated ovariectomized animals (p<0.05). Histomorphological analysis of the skin from non-treated ovariectomized rats revealed reduced vascularization and lower density of papillary capillaries in comparison with these parameters in sham-operated controls; estrogen treatment prevented these changes. We concluded that ovariectomized rats can be employed as a model of aging skin in menopause.

Behavioral effects of Aronia melanocarpa fruit juice in a rat model of ovariectomy-induced estrogen deficit.

INTRODUCTION: The ovariectomized rat is a model used to mimic the changes in female organism during menopause. Aroniamelanocarpa fruit juice (AMFJ) is extremely rich in phenolic substances (procyanidins, flavonoids and phenolic acids).