On Osteoprotegerin Research Articles

Exogenous iron caused osteocyte apoptosis, increased RANKL production, and stimulated bone resorption through oxidative stress in a murine model

Iron overload is a risk factor for osteoporosis due to its oxidative toxicity. Previous studies have demonstrated that an excessive amount of iron increases osteocyte apoptosis and receptor activator of nuclear factor κ-B ligand (RANKL) production, which stimulates osteoclast differentiation in vitro. However, the effects of exogenous iron supplementation-induced iron overload on osteocytes in vivo and its role in iron overload-induced bone loss are unknown. This work aimed to develop an iron overloaded murine model of C57BL/6 mice by intraperitoneal administration of iron dextran for two months. The iron levels in various organs, bone, and serum, as well as the microstructure and strength of bone, apoptosis of osteocytes, oxidative stress in bone tissue, and bone formation and resorption, were assessed. The results showed that 2 months of exogenous iron supplementation significantly increased iron levels in the liver, spleen, kidney, bone tissue, and serum. Iron overload negatively affected bone microstructure and strength. Osteocyte apoptosis and empty lacunae rate were elevated by exogenous iron. Iron overload upregulated RANKL expression but had no significant impact on osteoprotegerin (OPG) and sclerostin levels. Static and dynamic histologic analyses and serum biochemical assay showed that iron overload increased bone resorption without significantly affecting bone formation. Exogenous iron promoted oxidative stress in osteocytes in vivo and in vitro. Additional supplementation of iron chelator (deferoxamine) or N-acetyl-l-cysteine (NAC) partially alleviated bone loss, osteocyte apoptosis, osteoclast formation, and oxidative stress due to iron overload. These findings, in line with prior in vitro studies, suggest that exogenous iron supplementation induces osteoclastogenesis and osteoporosis by promoting osteocyte apoptosis and RANKL production via oxidative stress.

Interleukin-19 promotes bone resorption by suppressing osteoprotegerin expression in BMSCs in a lipopolysaccharide-induced bone loss mouse model

AimsOsteoporosis is characterized by decreased trabecular bone volume, and microarchitectural deterioration in the medullary cavity. Interleukin-19 (IL-19), a member of the IL-10 family, is an anti-inflammatory cytokine produced primarily by macrophages. The aim of our study was to investigate the effect of IL-19 on osteoporosis.MethodsBlood and femoral bone marrow suspension IL-19 levels were first measured in the lipopolysaccharide (LPS)-induced bone loss model. Small interfering RNA (siRNA) was applied to knock down IL-19 for further validation. Thereafter, osteoclast production was stimulated with IL-19 in combination with mouse macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). The effect of IL-19 was subsequently evaluated using tartrate-resistant acid phosphatase (TRAP) staining and quantitative real-time polymerase chain reaction (RT-qPCR). The effect of IL-19 on osteoprotegerin (OPG) was then assessed using in vitro recombinant IL-19 treatment of primary osteoblasts and MLO-Y4 osteoblast cell line. Finally, transient transfection experiments and chromatin immunoprecipitation (ChIP) experiments were used to examine the exact mechanism of action.ResultsIn the LPS-induced bone loss mouse model, the levels of IL-19 in peripheral blood serum and femoral bone marrow suspension were significantly increased. The in vivo results indicated that global IL-19 deletion had no significant effect on RANKL content in the serum and bone marrow, but could increase the content of OPG in serum and femoral bone marrow, suggesting that IL-19 inhibits OPG expression in bone marrow mesenchymal stem cells (BMSCs) and thus increases bone resorption.ConclusionIL-19 promotes bone resorption by suppressing OPG expression in BMSCs in a LPS-induced bone loss mouse model, which highlights the potential benefits and side effects of IL-19 for future clinical applications.Cite this article: Bone Joint Res 2023;12(11):691–701.

Effect of the Combination of Demineralization Freeze Dried Dentin Matrix (DFDDM) and Moringa oleifera Lam on Osteoprotegerin (OPG) and Receptor Activator of Nuclear Factor Kapa B Ligand (RANKL) as a Marker of Bone Remodeling

Background: The socket preservation has important role in alveolar bone resorption after tooth extraction. Various graft materials can be used in socket preservation must have osteoconductive, osteoinductive and osteoproliferation properties. Autogenous bone graft from dentin can be used because it has good osteoinductive and osteoconductive properties. One of the active substances of Moringa oleifera (MO) is flavonoids that have several beneficial characteristics as an anti‑inflammatory. The combination of anti-inflammatory with MO extract and Demineralization Freeze Dried Dentin Matrix (DFDDM) is expected to provide a good response to bone formation. Aim: To determine the potential of combination Moringa oleifera and DFDDM in formation of OPG and RANKL expression post extraction cavia cobaya’s tooth. Method: The mandibular incisors of 45 Cavia cobaya were extracted and divided into five groups subjected to different socket preservation treatments sequentially with MO, DFDDM, combination of MO and DFDDM, Gamacha® and polyethylene glycol (PEG). The cavia cobaya were examined on days 7, 14 and 21 after which the specimens were sacrificed and examined using an immunohistochemical technique. The resulting data were then analyzed using one‑way ANOVA and Tukey’s honestly significant difference tests. Result: The One Way Anova test results showed a significant difference in OPG and RANKL between the groups (p

Effect of enhanced masticatory force on OPG, RANKL and MGF in alveolar bone of ovariectomized rats.

Menopause induces oral bone loss, leading to various oral diseases. Mastication importantly affects bone metabolism in the jawbone.Objective:To analyze the effect of enhanced masticatory force on osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), and mechano–growth factor (MGF) in alveolar bone of ovariectomized rats and to study the mechanics mechanism of the alveolar bone of ovariectomized rats response to enhanced masticatory force.Methodology:Thirty Sprague Dawley rats were randomly divided into three groups: sham–operation group (fat around the removed ovary + normal hard diet), model group (ovariectomy + normal hard diet), and experimental group (ovariectomy + high hard diet). It was a 2–month experiment. Enzyme–linked immunosorbent assay (ELISA) detected serum estradiol (E2), osteocalcin (BGP) and alkaline phosphatase (ALP) in rats. Bone histomorphometric indices in the third molar region of maxilla were detected by micro-CT; protein expressions of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Western blot; and gene expression of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Quantitative Real–Time PCR.Results:Comparing with model group, serum E2 in experimental group increased but not significantly, serum BGP and serum ALP in experimental group decreased but not significantly, OPG in experimental group in alveolar bone increased significantly, RANKL in experimental group in alveolar bone decreased significantly, RANKL/OPG ratio in experimental group decreased significantly, MGF in experimental group in alveolar bone increased significantly, bone volume to total volume fraction increased significantly in experimental group, trabecular thickness increased significantly in experimental group, and trabecular separation decreased significantly in experimental group.Conclusion:Enhanced masticatory force affected the expression of OPG, RANKL, and MGF in alveolar bone of ovariectomized rats, improved the quality of jaw bone of ovariectomized rats, and delayed oral bone loss by ovariectomy.

Effects of hyperbaric oxygen on the expression of osteoprotegerin and integrin β1 in rabbit＇s mandibular distraction gap

Objective To investigate the effect of hyperbaric-oxygen therapy on osteoprotegerin (OPG) and integrin β1 (Itgβ1) expression during mandibular distraction osteogenesis. Methods Forty New-Zealand rabbits were used. The animals were randomly divided into two groups (experimental group and control group) with 20 animals each; after accomplished osteotomy and implant distraction devices on mandible bilaterally for 3 days of latency period, the device was activated at the rate of 0.8 mm per day for 10 days. All animals in the experimental group were subjected to hyperbaric oxygen for 90 minutes once a day since the beginning of distraction, and lasted for four weeks. In control group, all the distractors were activated following the same distraction protocol as the experimental group, but without hyperbaric oxygen therapy. Five animals of each group were sacrificed at 10th day after distraction, 7th, 14th and 28th day of consolidation, respectively. The lengthened mandibles were harvested and processed for immunohistochemical examinations to detect OPG and Itgβ1 expression in the distraction gap. Semi-quantitative analysis was carried out by image analysis software. Results OPG staining was mainly located in the membrane and cytoplasm of osteoprogenitor cells and osteoblasts in the distraction zones. The expression of OPG increased after distraction accomplished and reached to the peak at 7th day of consolidation, and then decreased gradually. At every time point, the level of expression of OPG in the experimental group was remarkably higher than those in control group. There were significant differences between the experimental group and control group (P 0.05). Conclusions Hyperbaric oxygen therapy can up-regulate the expression of OPG and Itgβ1 in the distraction gap, which may promote osteoblast differentiation, proliferation, enhance osteoblast function, and new bone formation in distraction gap. Key words: Rabbits; Traction; Mandible; Integrin beta1; Osteoprotegerin; Hyperbaric-oxygen

Effect of estrogen receptor-α gene polymorphism on serum osteoprotegerin and calcaneus bone density in postmenopausal women in Guangxi Zhuang nationality

Objective To investigate the effect of estrogen receptor-α gene polymorphism on osteoprotegerin(OPG)and calcaneus bone density in early and late postmenopausal women in Guangxi Zhuang nationality, in order to provide the theoretical basis for the early prevention and treatment of postmenopausal osteoporosis caused by estrogen receptor gene-induced osteoprotegerin reduction. Methods The broadband ultrasound attenuation in the right heel bone was measured by quantitative ultrasound bone densitometry in 621 postmenopausal women of Guangxi Zhuang nationality.Peripheral blood mononuclear cells and their DNA were extracted.Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)was used to detect the estrogen receptor-α gene polymorphism.Serum osteoprotegerin level was determined by enzyme-linked immunosorbent assay.The differences of data distribution of OPG and calcaneus bone density were compared between five items of 3 genotypes and 2 alleles at the same age group of 40-, 45-, 50-, 55-, 60-, 65-, 70-, 75-80 years. Results Women aged 60 years and over versus those aged 40-59 years showed that serum osteoprotegerin(OPG)and bone mineral density(BMD)were decreased(P 0.05). Women aged 60 years and over showed that ER-α Puv Ⅱ polymorphism of allele big P versus p, Pp, pp, PP in the same age groups had significantly decreased serum osteoprotegerin and bone mineral density.Women aged 65 years and over showed that ER-α XbaⅠpolymorphism of heterozygote Xx versus xx, XX, X, x in the same age groups had a significantly increased serum osteoprotegerin and bone mineral density(P<0.05). Conclusions Women with big P allele of ER-α PvuⅡpolymorphism have low serum osteoprotegerin level and a decreased bone mineral density, who are prone to postmenopausal osteoporosis.Thus, P allele of ER-α PvuⅡpolymorphism is a causative agent.More attention should be paid to early prevention and treatment.But, women with the Xx heterozygote of ER-α XbaⅠpolymorphism have high serum osteoprotegerin level and an increased bone mineral density, who are not easy to suffer from postmenopausal osteoporosis.Therefore, Xx heterozygote of ER-α XbaⅠpolymorphism is a protective agent.The prevention and treatment of postmenopausal osteoporosis should be individualized based on estrogen receptor-α gene polymorphism. Key words: Zhuang nationality; Receptors, estrogen; Polymorphism, single nucleotide; Osteoprotegerin

The effects of P-gp and CYP450 modulated by rifampicin on the steroid-induced osteonecrosis of the femoral head.

This study investigated the effects of rifampicin-modulated P-glycoprotein (P-gp) and cytochrome P450 (CYP450) activity on the development of steroid-induced osteonecrosis of the femoral head. Thirty-two rabbits were equally divided into four groups: control group, oral administration group, intramuscular injection group, and local release group, in which rifampicin-loaded artificial bone graft was implanted in the left femur cavity and blank bone graft was implanted in the right femur cavity. Dexamethasone was given 1week after rifampicin administration. Peripheral P-gp activity and hepatic CYP450 content were investigated 4weeks later. Hematoxylin and eosin, Massson, and tetracycline-fluorescence staining of the femoral head were compared. In vitro, the effects of intracellular dexamethasone concentration modulated by P-gp on osteoprotegerin (OPG)/receptor activator of nuclear factor κB ligand (RANKL) expression and differentiation of mesenchymal stem cells were further investigated. Peripheral P-gp activity and hepatic CYP450 content in the oral administration group and the intramuscular injection group were significantly higher than those in the local release group. P-gp activity of mesenchymal stem cells in rifampicin-implanted femoral head was significantly higher than that in the blank control. Histological study showed that rifampicin could prevent steroid-induced bone loss and lipid formation, and promote new bone formation and maturation. In vitro study confirmed that intracellular dexamethasone concentration modulated by P-gp could influence the OPG/RANKL ratio and the differentiation of mesenchymal stem cells. Enhanced levels of peripheral P-gp and hepatic CYP450 can reduce the incidence of steroid-induced osteonecrosis of the femoral head. P-gp activity locally enhanced by rifampicin decreases the intracellular steroid concentration, but rifampicin does not have significant effects on peripheral P-gp and hepatic CYP450.

Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

ObjectiveTo investigate the influence of Notch signaling on osteoprotegerin (OPG) expression in a human oral squamous cell carcinoma cell line. MethodsActivation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a γ-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG mRNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software. ResultsJagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 mRNA expression, confirming the intracellular activation of Notch signaling. OPG mRNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a γ-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 mRNA levels, and a marked increase in OPG protein expression was observed. These results implied that Notch signaling regulated OPG expression in HSC-4 cells. However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line (HSC-5) or a human head and neck squamous cell carcinoma cell line (HN22). ConclusionsNotch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

Jagged1 inhibits osteoprotegerin expression by human periodontal ligament cells

Notch signaling regulates bone homeostasis. The present study investigated the effect of Jagged1 on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) expression in human periodontal ligament stromal (hPDL) cells. hPDL cells were seeded on to indirect immobilized Jagged1 surfaces. OPG expression was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Lentiviral small hairpin RNA particles against NOTCH2 were employed to inhibit NOTCH2 expression. Osteoclast formation was evaluated using RAW264.7 cells. An influence of exogenous OPG on osteogenic differentiation was determined by real-time polymerase chain reaction and Alizarin Red S staining. Jagged1 significantly enhanced HES1 and HEY1mRNA expression in a dose-dependent manner. Furthermore, OPG mRNA and protein levels dramatically decreased upon exposing hPDL cells to Jagged1. However, RANKL mRNA levels were not significantly different. There was also no difference in M-CSF and MCP-1mRNA expression. A γ-secretase inhibitor and cycloheximide treatment rescued Jagged1-attenuated OPG expression. Furthermore, shNOTCH2 overexpressing hPDL cells did not exhibit a decrease in OPG expression upon exposure to Jagged1, implying the involvement of NOTCH2 in the regulatory mechanism. Culturing RAW264.7 cells with conditioned medium from Jagged1-treated hPDL cells enhanced osteoclast formation compared with those cultured with conditioned medium of the control group. Lastly, OPG treatment did not influence osteogenic differentiation by hPDL cells. These results suggest that Jagged1 activates Notch signaling in hPDL cells, leading to decreased OPG expression. This may imply an indirect role of Jagged1 on the regulation of osteoclast differentiation via hPDL cells.

Roles and Clinical Applications of OPG and TRAIL as Biomarkers in Cardiovascular Disease.

Cardiovascular diseases (CVD) remain the major cause of death and premature disability in Western societies. Assessing the risk of CVD is an important aspect in clinical decision-making. Among the growing number of molecules that are studied for their potential utility as CVD biomarkers, a lot of attention has been focused on osteoprotegerin (OPG) and its ligands, which are receptor activator of nuclear factor κB ligand (RANKL) and TNF-related apoptosis-inducing ligand. Based on the existing literature and on our experience in this field, here we review what the possible roles of OPG and TRAIL in CVD are and their potential utility as CVD biomarkers.

Resveratrol reduces prostaglandin E1-stimulated osteoprotegerin synthesis in osteoblasts: Suppression of stress-activated protein kinase/c-Jun N-terminal kinase

Resveratrol, a natural polyphenol mainly existing in red grapes and berries, possesses beneficial effects on human being. We have previously reported that prostaglandin E1 (PGE1) stimulates vascular endothelial growth factor synthesis via activation of p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) but not p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the PGE1-effect on osteoprotegerin (OPG) synthesis and the effect of resveratrol on the synthesis in MC3T3-E1 cells. PGE1 induced the expression levels of OPG mRNA and stimulated the OPG release. Resveratrol significantly reduced the PGE1-induced OPG release and the mRNA expression. SRT1720, an activator of SIRT1, suppressed the release of OPG. The protein levels of SIRT1 were not up-regulated by resveratrol with or without PGE1. Both SB203580 and SP600125, a specific p38 MAP kinase inhibitor and a specific SAPK/JNK inhibitor, respectively, but not PD98059, a specific MEK inhibitor, reduced the PGE1-stimulated OPG release. Resveratrol or SRT1720 failed to affect the phosphorylation of p38 MAP kinase. On the contrary, PGE1-induced phosphorylation of SAPK/JNK was significantly attenuated by both resveratrol and SRT1720. Our results strongly suggest that resveratrol inhibits PGE1-stimulated OPG synthesis via suppressing SAPK/JNK but not p38 MAP kinase in osteoblasts.

Down-regulation by resveratrol of basic fibroblast growth factor-stimulated osteoprotegerin synthesis through suppression of Akt in osteoblasts.

It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2) on osteoprotegerin (OPG) synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP) kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation.

Influence of Calcium Hydroxide–loaded Microcapsules on Osteoprotegerin and Receptor Activator of Nuclear Factor Kappa B Ligand Activity

IntroductionCalcium hydroxide (Ca[OH]2) microcapsules were synthesized to allow controlled release of Ca(OH)2. The aim of this study was to evaluate the influence of Ca(OH)2 microcapsules on osteoprotegerin (OPG) activity, receptor activator of nuclear factor kappa B ligand (RANKL) activity, and the OPG/RANKL ratio compared with pure Ca(OH)2 powder and Vitapex (Neo Dental Chemical Products Co Ltd, Tokyo, Japan). MethodsOne formula of Ca(OH)2 microcapsules was evaluated, and pure Ca(OH)2 powder was used as a control. A commonly used Ca(OH)2 medication containing an oily vehicle (Vitapex) was also evaluated, and the in vitro release profile of Vitapex was studied. The human osteosarcoma cell line MG63 was used to evaluate the influence of Ca(OH)2 microcapsules, pure Ca(OH)2 powder, and Vitapex on OPG and RANKL activity. The relative messenger RNA (mRNA) expression of OPG and RANKL was determined by real-time polymerase chain reaction. The protein expression of OPG and RANKL in supernatants was measured using enzyme-linked immunosorbent assay. ResultsVitapex prolonged the release of Ca(OH)2 compared with pure Ca(OH)2 powder, and the release rate of Vitapex was faster than that of the microcapsules. The OPG/RANKL ratio in the microcapsules group was up-regulated at both the mRNA and protein levels compared with the negative control group and the pure Ca(OH)2 powder group. The ratio in the Vitapex group was lower than the microcapsule group both at the mRNA and protein levels. ConclusionsCa(OH)2 microcapsules increased the expression of OPG although they did not increase the expression of RANKL compared with pure Ca(OH)2 powder and Vitapex. This increase in expression led to an increase in the OPG/RANKL ratio and eventual inhibition of osteoclast activity.

Estradiol induces osteoprotegerin expression by human dental pulp cells.

Estrogen deficiency is associated with increased inflammation related periapical bone resorption. The present study aimed to evaluate the effect and intracellular mechanism(s) of estrogen on osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) expression in human dental pulp cells (HDPs). HDPs were treated with estradiol at a concentration of 0.1-10 μM. The results showed that estradiol induced OPG expression at both the mRNA and protein levels in a dose-dependent manner. However, no influence on RANKL expression was observed. An estrogen receptor (ER) inhibitor failed to attenuate the estradiol-induced OPG expression. Furthermore, ER-α and ER-β agonists did not simulate estradiol's effects on OPG expression by HDPs. However, a significant OPG upregulation was observed in HDPs treated with an estradiol-BSA conjugate or a GPR30 agonist. An ERK inhibitor significantly enhanced estradiol-induced OPG expression, whereas a p38 inhibitor markedly attenuated this expression. In conclusion, OPG expression by HDPs may be regulated by estradiol binding a membrane receptor and the balance between the ERK and p38 signaling pathways.

Regulation by resveratrol of prostaglandin E2-stimulated osteoprotegerin synthesis in osteoblasts.

Resveratrol is a natural polyphenol found in red grape skins, berries and red wine. Accumulating evidence suggests that resveratrol has various beneficial effects on the human body. In the present study, we investigated the effects of prostaglandin E(2) (PGE(2)) on osteoprotegerin (OPG) synthesis and the effects of resveratrol on OPG synthesis in osteoblast-like MC3T3-E1 cells. PGE(2) significantly stimulated both the release of OPG and the mRNA expression levels of OPG, as shown by OPG assay and real-time RT-PCR, respectively. Resveratrol markedly suppressed the release and the mRNA levels of OPG induced by PGE(2). On the contrary, SRT1720, an activator of sirtuin 1 (SIRT1), hardly affected the PGE(2)-induced release of OPG. PD98059 [a specific inhibitor of the upstream kinase that activates p44/p42 mitogen-activated protein (MAP) kinase], SB203580 (a specific inhibitor of p38 MAP kinase) and SP600125 [a specific inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK)], reduced the PGE(2)-induced release of OPG. Resveratrol attenuated the PGE(2)-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK. However, SRT1720 failed to affect the phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK induced by PGE(2). These results strongly suggest that resveratrol reduces PGE(2)-stimulated OPG synthesis through the inhibition of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK in osteoblasts, and that these suppressive effects are independent of the activation of SIRT1.

Suppression by resveratrol of prostaglandin D2-stimulated osteoprotegerin synthesis in osteoblasts

Resveratrol, a natural polyphenol with health-related properties mainly existing in grape skins and red wine, possesses beneficial effects on human being. We have previously reported that prostaglandin D2 (PGD2) stimulates heat shock protein 27 (HSP27) induction via activation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism behind the effect of PGD2 on osteoprotegerin (OPG) synthesis and the effect of resveratrol on the OPG synthesis in MC3T3-E1 cells. PGD2 significantly stimulated both the OPG release and the expression levels of OPG mRNA. Resveratrol and SRT1720, an activator of SIRT1, markedly suppressed the PGD2-induced OPG release and the mRNA levels of OPG. PD98059, a specific MEK inhibitor, SB203580, a specific p38 MAP kinase inhibitor, and SP600125, a specific SAPK/JNK inhibitor suppressed the PGD2-stimulated OPG release. PGD2-induced phosphorylation of p38 MAP kinase and SAPK/JNK was attenuated by resveratrol or SRT1720. However, resveratrol or SRT1720 failed to affect the phosphorylation of myosin phosphatase-targeting subunit-1 (MYPT-1), a downstream substrate of Rho-kinase and p44/p42 MAP kinase. These results strongly suggest that resveratrol suppresses PGD2-stimulated OPG synthesis through inhibiting p38 MAP kinase and SAPK/JNK in osteoblasts, and that the suppressive effect is exerted at the point downstream of Rho-kinase but upstream of p38 MAP kinase or SAPK/JNK.

Effect of heparin on osteoprotegerin content and cells apoptosis in cervical loop zone of rat lower incisors

Objective To investigate the effect of heparin on osteoprotegerin (OPG) content and cells apoptosis in the rat cervical loop zone of rat lower incisors.Methods Twenty-four Sprague-Dawley (SD) rats aged 4 weeks were divided into two groups randomly.The experimental group was subcutaneously injected with heparin at the abdomen per day by the dose of 1 U/g,and the control group was injected with normal sodium (NS) by the same volume.All of the rats were sacrificed after 4 weeks,their right femurs were obtained for measurement of the bone mineral density (BMD),and the mandibles were harvested,decalcified and made into paraffin sections,which would be dealt with hematoxylin and eosin (HE)staining,OPG immunohistochemical staining,and TdT-mediated dUTP nick end labeling (TUNEL) staining.The professional software Image Pro Plus was used to semi-quantitatively analyze the content of OPG.The lower incisor cerv;ical loops were separated and prepared to be observed by transmission electron microscopy (TEM).Results The right femur BMD [(0.185 ±0.006) g/cm2] in experimental group was lower than that in control group [(0.196 ±0.011) g/cm2,P ＜0.05].The Absorbance (A) value of OPG was 0.235 3 ± 0.035 0 in the experimental group and 0.267 3 ± 0.044 4 in control group (P ＞ 0.05).A few odontoblasts and stratum intermedium cells in control group were apoptosis-positive by TUNEL staining;in contrast,there was no apoptosis in the experimental group.The results of TEM examination were similar with TUNEL staining.Conclusion Heparin could lead to osteoporosis and inhibit the apoptosis of odontogenic cells in the rat lower incisor.However,heparin has no significant effect on the OPG content. Key words: Heparin ; Osteoprotegerin ; Apoptosis ; Cervical loop tissue

Resveratrol suppresses prostaglandin F2α-induced osteoprotegerin synthesis in osteoblasts: Inhibition of the MAP kinase signaling

Resveratrol, a natural polyphenol abundantly found in grape skins and red wine, possesses various beneficial properties for human health. In the present study, we investigated the mechanism underlying the effects of prostaglandin F2α (PGF2α) on osteoprotegerin (OPG) synthesis and of resveratrol on the OPG synthesis in osteoblast-like MC3T3-E1 cells. PGF2α stimulated both the release of the OPG protein and the expression of OPG mRNA. Treatment with PD98059, SB203580 and SP600125, specific inhibitors of MEK1/2, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) all suppressed the OPG release induced by PGF2α. Resveratrol also significantly reduced the PGF2α-stimulated OPG release and the mRNA levels of OPG. Similarly, treatment with SRT1720, an activator of SIRT1, also suppressed the PGF2α-stimulated OPG release. Resveratrol and SRT1720 both attenuated the phosphorylation of p44/p42 MAP kinase, MEK1/2, Raf-1, p38 MAP kinase and SAPK/JNK induced by PGF2α. These findings strongly suggest that resveratrol suppresses PGF2α-stimulated OPG synthesis by inhibiting the MAP kinase pathways in osteoblasts, and that the effect is mediated via SIRT1 activation.

Impact of Pueraria candollei var. mirifica and its potent phytoestrogen miroestrol on expression of bone-specific genes in ovariectomized mice

Miroestrol (MR) is a highly active phytoestrogen isolated from tuberous root of Pueraria candollei var. mirifica (PM). Modulatory effects of PM and MR on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) mRNAs which are bone-specific genes were investigated in ovariectomized female ICR mice. After ovariectomy, expression of OPG mRNA was suppressed but that of RANKL was induced. Estradiol benzoate (E2) recovered OPG expression to the level comparable to the sham while that of RANKL was suppressed in ovariectomized mice. PM crude extract (PME) significantly down-regulated the expression of RANKL mRNA with no change in the OPG level whereas MR elevated the expression of OPG mRNA with lowering level of RANKL mRNA, resulting in the increased OPG/RANKL ratio, and consequently lead to lowering progression of osteoporosis at molecular level. These findings revealed potential of PME and MR on bone loss prevention via increasing the ratio of OPG to RANKL (osteoformation/osteoresorption) in liver of ovariectomized mice. Therefore, using PME and MR as alternative hormone replacement therapy of E2 might be beneficial recommended due to advantageous on regulation of osteoporosis related genes.

Denervation affects the influence of CGRP on OPG/RANKL system in the fracture healing process: an experimental study in rats

Objective To study the influence of sensory/motor nerve injury on fracture healing and the influence of calcitonin gene related peptide (CGRP) on osteoprotegerin (OPG)/receptor activator nuclear factor kappa B ligand (RANKL) system during the fracture healing process through the establishment of selective transection of sensory/motor nerve in rats with tibia fracture as an animal model and to elucidate the peripheral nerve injury regulation mechanism during fracture healing. Methods Sixty Wistar rats were divided into 3 groups randomly:motor nerve (ventral root) transection + tibia fracture group (anterior rhizotomy group,ART group) ; sensory nerve (dorsal root) transection + tibia fracture group (poeterior rhizotomy group,PRT group) ;simple tibia fracture group (sham operated group,SO group).The transverse tibia fracture was treated by intramedullary nailing.The rats were sacrificed at postoperative day 7,14,21 and 28 respectively to harvest the bony calluses 5 mm proximal and 5 mm distal to the fracture site.The expression of CGRP,OPG and RANKL was detected by immunohistochemistry and analyzed by Image-proplus 6.0 software.The comparison among the groups was analyzed using SPSS 16.0. Results Strong positive expression of CGRP was seen at each time point in SO group.At postoperative day 14 and 21,the differences of CGRP expression between SO group,ART group and PRT group were significant ( P ＜ 0.05).OPG expression was strongest at postoperative day 7 in SO group,while remaining high even with gradual decrease.The differences in OPG expression were significant ( P ＜ 0.05 ) at postoperative day 14 between PRT group and SO group and between PRT group and ART group,while that difference was seen between SO group and PRT group and between SO group and ART group at postoperative day 21.RANKL expression in the SO group peaked at postoperative day 21,and later gradually decreased.At postoperative day 14 the differences between PRT group and ART group and between PRT group and SO group were significant ( P ＜0.05),while at postoperative day 21 the differences between SO group and ART group and between SO group and PRT group were significant ( P ＜ 0.05). Conclusion During the fracture healing process,the influence of sensory nerve fibers was significantly higher than that of the motor nerve fibers.CGRP can regulate the OPG/RANKL expression ratio,thus affecting the healing process.Denervation (especially the sensory nerves) causes reduced CGRP regulation on OPG/RANKL expression,which is not favorable for fracture healing.Complete innervation is a necessary condition for the normal healing of fractures. Key words: Sensory nerve; Motor nerve; Calcitonin gene related peptide; Osteoprotegerin; Receptor activator nuclear factor kappa B ligand