Protective effects of oridonin against osteoporosis by regulating immunity and activating the Wnt3a/ß-catenin/VEGF pathway in ovariectomized mice.

This study was performed with the aim of investigating the effect of oridonin (ORI) on estrogen deprivation-induced osteoporosis in mice and its mechanism. Animal experiments were used in this work to validate the anti-osteoporotic efficacy of ORI. Morphometric analysis was performed by micro-CT. A special protein meter was used to detect the content of immunoglobulin lgM, immunoglobulin lgG, complement C3 and C4 in the serum of mice. The expression of CD4+CD25+Foxp3+ Treg cell and CD4+/CD8+ lymphocyte subsets in mice was detected by flow cytometry. ELISA was used to detect the content of insulin-like growth factor (IGF-1), tumor necrosis factor (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6). In addition, key signaling molecules in the Wnt3a/ß-catenin signaling pathway were detected by Western blotting. The results showed that compared with the model group, the contents of calcium and phosphorus in the femurs of mice in the ORI groups were increased, and the spleen coefficient was decreased. The ALP activity in the serum of mice in the high and medium dose ORI groups was decreased, and the uterine coefficient was increased. ORI significantly increased the maximum bending load and the maximum bending stress of the femurs of mice, increased the number of trabeculae, and repaired the bone microstructure. At the same time, ORI could significantly increase the levels of immunoglobulin (lgG and lgM) and complement (C3 and C4), increase the activity of peritoneal macrophages in mice, increase the expression of CD4+CD25+Foxp3+ Tregs and CD4+/CD8+ in the spleen, increase the content of IGF-1, reduce the content of TNF-α, IL-1 and IL-6 and increase the expression levels of VEGF, Wnt3a, p-GSK3ß/GSK3ß and ß-catenin/Lamin in the femoral tissue. These results indicated that ORI might regulate the expression of VEGF through the Wnt3a/ß-catenin signaling pathway, improve the immunity of mice, maintain the balance of the immune system, and promote angiogenesis, thereby improving the bone mineral density and bone tissue morphology of mice and playing an anti-osteoporotic role.

[Effects of ERα gene overexpression on bone mineral density and calcium and phosphorus metabolism inovariectomized osteoporosis mice].

Objective: To investigate the effects of estrogen receptor α (ERα) gene overexpression on bone metabolism and calcium and phosphorus metabolism in ovariectomized osteoporosis mice, and to provide experimental basis for targeted gene therapy of osteoporosis. Methods: Thirty SPF female mice were randomly divided into sham operation group, model group and ERα overexpression group with 10 mice in each group. After the model was established, the ERα overexpression group was transfected with recombinant adenovirus vector carrying mouse ERα gene by intraspinal injection. The model group was transfected with empty virus, and the sham operation group was not treated. The expression of ERα gene in bone tissue of mice was detected by quantitative Real-time PCR (qRT-PCR). Bone mineral density (BMD) of mouse femur was measured after modeling. Trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular segregation (Tb.Sp), bone volume fraction (BV/TV) and biomechanical strength of femur were measured by micro-CT scanning. Serum levels of calcium (Ca), phosphorus (P), osteocalcin (BGP) and alkaline phosphatase (ALP) were measured by automatic biochemical analyzer. The expressions of tissue inhibitor of metalloproteinases 1 (TIMP-1) and monocyte chemotactic protein 1 (MCP-1) in bone homogenate were detected by Immunohistochemistry. Results: Compared with sham operation group, the expression level of ERα gene in bone tissue of model group was decreased significantly, the levels of BMD, BV/TV, Tb. Th, maximum load, rigidity coefficient, Ca and P were decreased, while the levels of Tb. Sp, BGP and ALP were increased significantly (P＜0.05). Compared with the sham operation group, the expression level of TIMP-1 protein in the bone tissue of the model group was significantly decreased, while that of MCP-1 protein was increased, while that of the ERα overexpression group was increased while that of MCP-1 was decreased (P＜0.05).The levels of ERα gene expression, BMD, BV/TV, TB. Th, maximum load, rigidity coefficient, Ca and P in the ERα overexpression group were significantly higher than those in the model group, while Tb. Sp, BGP and ALP were significantly lower (P＜0.05). Compared with the sham operation group, mean optical density of TIMP-1 in the bone tissue of the model group was significantly decreased, while that of MCP-1 was significantly increased, and that of the ERα overexpression group was significantly increased while that of MCP-1 was significantly decreased (P＜0.05). Conclusion: ERα gene overexpression can improve osteoporosis by regulating bone mineral density, bone parameters, bone metabolism, calcium and phosphorus metabolic indicators and the expression levels of TIMP-1 and MCP-1 in tissues.

[Low-magnitude vibration promotes osteogenesis of osteoblasts in ovariectomized osteoporotic rats via the estrogen receptor α].

The purpose of this study was to investigate the effect of low-magnitude vibration on osteogenesis of osteoblasts in ovariectomized rats with osteoporosis via estrogen receptor α(ERα). The mRNA expression of osteogenic markers were examined with qRT-PCR, based on which the optimal vibration parameter for promoting osteogenesis was determined (45 Hz × 0.9 g, g = 9.8 m/s2). Then we loaded the optimal vibration parameter on the osteoblasts of ovariectomized rats with osteoporosis. The protein expression of osteogenic markers and ERα were detected with Western blot; the distribution of ERα was examined with immunofluorescence technique. Finally, through inhibiting the expression of ERα with estrogen receptor inhibitor ICI182780, the protein and mRNA expression of osteogenic markers were examined. First, the results showed that low-magnitude vibration could promote the expression of osteogenic markers and ERα in osteoblasts of ovariectomized rats with osteoporosis (P < 0.05), and make ERα transfer to the nucleus. On the other hand, the results also showed that after inhibiting the expression of ERα in osteoblasts of ovariectomized rats with osteoporosis, the protein and mRNA expression of osteogenic marker were decreased (P < 0.05). In our study, low-magnitude vibration played an important role in the osteogenesis of osteoblasts in ovariectomized rats with osteoporosis through increasing the expression and causing translocation of ERα. Furthermore, it provides a theoretical basis for the application of low-magnitude vibration in the prevention and treatment of postmenopausal osteoporosis.

Effects of resveratrol on bone metabolism and bone turnover related indexes in ovariectomized osteoporosis rats.

This study aimed to investigate the effects of resveratrol (RES) on bone metabolism and bone turnover related indexes in ovariectomized osteoporosis rats. 48 clean grade adult healthy unmated female SD rats were randomly divided into 6 groups, including normal control group (NCG), osteoporosis model group (OP MG), estrogen treatment group (17ß-E2 group), RES low dose group (RES-L), RES medium-dose group (RES-M) and RES high dose group (RES-H). The rats in NCG and OP MG were given distilled water once a day and the rats in the other two groups were given 17ß-E2 and resveratrol respectively. The levels of serum calcium (S-Ca), serum phosphorus (S-P), urinary calcium (U-Ca/Cr) and urinary phosphorus (U-P/Cr) were measured with an automatic biochemistry analyzer. The levels of serum osteocalcin (BGP), alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), type I amino front-end peptide (PINP), type I collagen strong carboxyl peptide (CTX-I), urine deoxypyridinoline (DPD) and serum estrogen were detected by enzyme-linked immunosorbent assay (ELISA). In the OP group, the serum estrogen levels, S-Ca and S-P decreased significantly and the expression of U-Ca/Cr and U-P/Cr increased significantly (P< 0.05). Compared with the OP group, the expression of S-Ca and S-P increased significantly and the expression of U-Ca/Cr and U-P/Cr decreased significantly (p< 0.05) after treatment. The levels of TRAP, BGP, DPD and CTX-I in the OP group increased significantly (P< 0.05). Compared with the OP group, the levels of TRAP decreased significantly (P< 0.05). The levels of PINP and ALP in OP MG increased significantly (P< 0.05). IP and ALP increased in the middle and lower levels (P< 0.05). The bone mineral density of the OP group decreased significantly (P< 0.05). Resveratrol can affect the changes in bone turnover in ovariectomized rats, promote bone formation in low estrogen state and inhibit bone resorption. Resveratrol may have a protective effect on the bone of ovariectomized rats.

Low-magnitude vibration promotes osteogenesis of osteoblasts in ovariectomized osteoporotic rats via the estrogen receptor / 生物医学工程学杂志

The purpose of this study was to investigate the effect of low-magnitude vibration on osteogenesis of osteoblasts in ovariectomized rats with osteoporosis via estrogen receptor α(ERα). The mRNA expression of osteogenic markers were examined with qRT-PCR, based on which the optimal vibration parameter for promoting osteogenesis was determined (45 Hz × 0.9 g, g = 9.8 m/s

High-Throughput Metabolomics Evaluate the Efficacy of Total Lignans From Acanthophanax Senticosus Stem Against Ovariectomized Osteoporosis Rat.

Postmenopausal osteoporosis (PMOP) is a common clinical illness in postmenopausal women, but there is no effective drug at present. Metabolomics approach was used to explore the potential biomarkers of PMOP and evaluate the efficacy and therapeutic targets of total lignans in the stem of Acanthophanax senticosus (ASSL) on the ovariectomized osteoporosis model rats. UPLC/MS and pattern recognition methods were used for serum metabolites discovery to illustrate the pathological mechanism of PMOP model rats, and then revealing the intervention effect of ASSL. The pattern recognition result showed that serum metabolic profiles of the sham operation group and the model group were clustered clearly, and 16 potential biomarkers were finally identified (7 in positive ion mode and 9 in negative ion mode), and they are involved in 15 related metabolic pathways. After oral administration of ASSL, 10 biomarkers were found to be significantly up-regulated and mainly regulated metabolic pathways include unsaturated fatty acid biosynthesis, linoleic acid metabolism, and arachidonic acid metabolism, primary bile acid synthesis, tyrosine metabolism, etc. Our study demonstrated that the ASSL could affect the endogenous metabolites related metabolic mechanism, provides a pharmacological basis of the ASSL for PMOP treatment.

[Effect of Er-xian decoction on femur proteomics in ovariectomized osteoporosis rats].

This experiment was mainly aimed to investigate the effect of Er-xian decoction on osteoporosis and the femur proteomics in ovariectomized rats with osteoporosis. The female SD rats were randomly divided into sham operation group, model group, Alendronate group (1 mg•kg⁻¹), Er-xian decoction group (in dose of 8 g•kg⁻¹) according to their weight. The rats in sham operation group and model group were gavaged with normal saline; the rats in Alendronate group were gavaged with the Alendronate at the dose of 1 mg•kg⁻¹ and the rats in Er-xian decoction group were gavaged with Er-xian decoction at the dose of 8 g•kg⁻¹, once a day for continuous 90 days. Then the femoral bone mineral density (BMD) was detected. The femoral bone proteins were detected by NanoLC-LTQ-Orbitrap system, identified by Protein Discovery software, and the intensity of differentially expressed proteins were quantitated by SIEVE software. The results showed that Er-xian decoction could significantly improve femoral BMD in ovariectomized rats. As compared with model group, 41 differentially expressed proteins whose variation trend was consistent with the sham operation group, were found in Er-xian decoction group, mainly including biological oxidation related protein, signal transduction pathway related protein, proteins involved in aliphatic acid metabolism, cytoskeleton related protein, proteins involved in energy metabolism, and proteins involved in glucose metabolism etc. The osteoporosis could be prevented and cured by Er-xian decoction. The differentially expressed proteins such as carbonic anhydrase 2 and integrin ß1 may be the action targets for Er-xian decoction.

Effect of Er-xian decoction on femur proteomics in ovariectomized osteoporosis rats / 中国中药杂志

This experiment was mainly aimed to investigate the effect of Er-xian decoction on osteoporosis and the femur proteomics in ovariectomized rats with osteoporosis. The female SD rats were randomly divided into sham operation group, model group, Alendronate group (1 mg•kg⁻¹), Er-xian decoction group (in dose of 8 g•kg⁻¹) according to their weight. The rats in sham operation group and model group were gavaged with normal saline; the rats in Alendronate group were gavaged with the Alendronate at the dose of 1 mg•kg⁻¹ and the rats in Er-xian decoction group were gavaged with Er-xian decoction at the dose of 8 g•kg⁻¹, once a day for continuous 90 days. Then the femoral bone mineral density (BMD) was detected. The femoral bone proteins were detected by NanoLC-LTQ-Orbitrap system, identified by Protein Discovery software, and the intensity of differentially expressed proteins were quantitated by SIEVE software. The results showed that Er-xian decoction could significantly improve femoral BMD in ovariectomized rats. As compared with model group, 41 differentially expressed proteins whose variation trend was consistent with the sham operation group, were found in Er-xian decoction group, mainly including biological oxidation related protein, signal transduction pathway related protein, proteins involved in aliphatic acid metabolism, cytoskeleton related protein, proteins involved in energy metabolism, and proteins involved in glucose metabolism etc. The osteoporosis could be prevented and cured by Er-xian decoction. The differentially expressed proteins such as carbonic anhydrase 2 and integrin β1 may be the action targets for Er-xian decoction.

Therapeutic effects of modified Bushen Huoxue granules on ovariecto-mized osteoporosis rats / 中国病理生理杂志

AIM: To observe the therapeutic effect of modified Bushen Huoxue granules (MBHG) on ovariec-tomized osteoporosis rats.METHODS: Female SD rats were randomly given sham operation (n =10) and ovariectomy, and then the model rats were further randomly divided into model group, MBHG treatment groups at doses of 200 mg/kg, 100 mg/kg and 50 mg/kg respectively, and positive control (estradiol valerate) group, with 10 rats in each group by intra-gastric administration for 12 weeks.The morphology, area, thickness, spacing and area percentage of trabecular bone in the rats were observed.The serum levels of calcium (Ca), phosphorus (P) and alkaline phosphatase (ALP) were mea-sured by automatic analyzer.Bone mineral density (BMD) was analyzed.Serum estradiol (E2 ), osteocalcin (BGP), os-teoprotegerin (OPG), receptor activator of nuclear factor-κB (RANK) and receptor activator of nuclear factor κB ligand (RANKL) levels were detected by ELISA.RESULTS: Compared with model group, trabecular bone significantly widened in all treatment groups with large number, and net-like structure restored partially.The thickness, area and area percenta-ges of trabecular bone in treatments groups were higher than those in model group,and trabecular spacing was less than that in model group (P <0.05).The serum Ca, P, E2 and OPG, and femoral BMD were significantly higher in treatment groups than those in model group, and the levels of ALP, BGP, RANK and RANKL were significantly lower than those in model group (P <0.01).CONCLUSION: MBHG has a significant therapeutic effect on ovariectomized osteoporosis rats. The mechanism may be related to the regulation of OPG and inhibition of RANKL secretion.