Increased Osteoprotegerin Expression Research Articles

N-acyl homoserine lactones lactonase est816 suppresses biofilm formation and periodontitis in rats mediated by Aggregatibacter actinomycetemcomitans

ABSTRACT Aims The current study aimed to explore the adjuvant therapeutic effect of N-acyl homoserine lactones (AHLs)-lactonase est816 on Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) biological behaviors and periodontitis progression. Methods The inhibitory properties of est816 were detected by live/dead bacterial staining, scanning electron microscope (SEM), crystal-violet staining and reverse-transcription quantitative PCR (RT-qPCR). Biocompatibility of est816 on human gingival fibroblasts (HGFs) and human gingival epithelial cells (HGEs) was evaluated by CCK8 and ELISA. The ligature-induced periodontitis model was established in rats. Micro computed tomography and immunohistochemical and histological staining served to evaluate the effect of est816 on the prevention of periodontitis in vivo. Results est816 significantly attenuated biofilm formation, reduced the mRNA expression of cytolethal distending toxin, leukotoxin and poly-N-acetyl glucosamine (PNAG) and downregulated expressions of interleukin-6 and tumor necrosis factor-α with low cell toxicity. In vivo investigations revealed est816 decreased alveolar bone resorption, suppressed matrix metalloproteinase-9 expression and increased osteoprotegerin expression. Conclusion est816 inhibited A. actinomycetemcomitans biofilm formation and virulence release, resulting in anti-inflammation and soothing of periodontitis in rats, indicating that est816 could be investigated in further research on periodontal diseases.

Stem cells derived from human exfoliated deciduous teeth-based media in a rat root resorption model

ObjectiveRoot resorption may occur during orthodontic treatment. Herein, we investigated the effect of a culture supernatant of stem cells derived from human exfoliated deciduous teeth on root resorption. DesignTwelve 8-week-old male Sprague–Dawley rats were used, and their maxillary first molars were pulled with excessive orthodontic force to induce root resorption. On days 1 and 7 after traction initiation, stem cells derived from human exfoliated deciduous teeth and alpha minimum essential medium (control group) were administered. After 14 days, the maxillary bone was evaluated for tooth movement. The expression of osteoprotegerin, receptor activator of nuclear factor κB ligand, tumor necrosis factor α, interleukin 1β, interleukin 6, and interleukin 17 was evaluated on the compression side and tension side. ResultsNo significant difference in tooth movement was observed between the two groups. Root resorption decreased in the group administered the culture supernatant compared with in the control. Immunohistochemical staining revealed increased osteoprotegerin expression and decreased receptor activators for nuclear factor κB ligand, tumor necrosis factor α, interleukin 1β, interleukin 6, and interleukin 17 on the compression side and tension side. ConclusionsAdministration of stem cells derived from human exfoliated deciduous teeth affected the expression of osteoprotegerin, receptor activator of nuclear factor κB ligand, tumor necrosis factor α, interleukin 1β, interleukin 6 and interleukin 17; hence, these stem cells may inhibit root resorption by regulating their expression.

Pangasius djambal gelatin increases osteoprotegerin, decreases RANK and RANKL after tooth extraction in vivo

Background: Osteoprotegerin (OPG) plays a role in preventing osteoclast formation and osteoclastic bone resorption by inhibiting the receptor activator nuclear factor-β and its ligand (RANK–RANKL) interaction that causes bone resorption. Pangasius djambal gelatin contains essential amino acids that may stimulate bone remodeling. Purpose: This study aimed to investigate the expressions of OPG, RANKL, and RANK following tooth extraction to ascertain whether OPG is associated with the RANKL and RANK ratios following gelatin induction. Methods: This study was a true experimental study with a simple, randomized post-test only control group design. Twenty-four albino rats (Rattus norvegicus) were divided into two groups (n = 4). The treatment group was treated with P. djambal gelatin following tooth extraction, while the control group was left untreated. Animal models undergoing tooth extraction were anesthetized intraperitoneally with ketamine (1,000 mg/10 ml up to 0.2 ml). The tooth was then extracted slowly so as not to break it. P. djambal gelatin was dripped (0.1 ml) into the socket after the extraction procedure. The association of OPG with RANKL and RANK immunohistochemistry expressions was evaluated after three, five, and seven days. Results: There was a significant increase in OPG expression on day 7 (P < 0.05) and a significant decrease in the expression of RANKL and RANK (P < 0.05) in the treatment group. Conclusion: The application of P. djambal gelatin could increase OPG expression, decrease RANKL and RANK expression in the socket following tooth extraction, and might be associated with the inhibition of bone resorption.

Expression and ratio of receptor activator of nuclear factor kappa-B ligand and osteoprotegerin following application of Nigella sativa/bovine bone graft combination in post tooth extraction sockets.

The aim of this study was to analyze the induction effect of a combination of N. sativa and bovine bone graft on the expression and ratio of receptor activator of nuclear factor kappa-B ligand expression (RANKL) and osteoprotegerin (OPG) on alveolar bone socket preservation on days 7 and 14. The research incorporated a posttest-only control group design. A total of 56 Cavia cobaya were divided into four groups: a control group, an N. sativa group, a bovine bone graft group, and a combined N. sativa and bovine bone graft group. The lower incisors of the C. cobaya were extracted with material subsequently being applied to the resulting socket. After the 7th and 14th days, the experimental animals were terminated to enable observation of the socket. Following processing, the tissue was subjected to immunohistochemistry staining consisting of RANKL and OPG antibodies before being observed under a light microscope at × 400. Statistical analysis was carried out using the one-way ANOVA and Tukey's honestly significant difference tests. A combination of N. sativa and bovine bone graft reduced both RANKL expression and the RANKL/OPG ratio while increasing OPG expression in comparison to the other groups. In all the results obtained, the N. sativa and bovine bone graft combination was significant (P < 0.05) when compared to the control group on both the 7th and 14th days. A combination of N. sativa and bovine bone graft reduced both RANKL expression and the RANKL/OPG ratio while increasing OPG expression.

Beneficial regulation of vitamin D3-rich extract from the processing by-products of Penaeus sinensis on preosteoblastic MC3T3-E1 cells and improvement of bone health in VD-deficient mice.

Vitamin D (VD) plays an important role in preventing osteoporosis. However, knowledge of the osteogenic effect of VD3 from shrimp processing by-products is limited. In this study, a VD3-rich extract from Penaeus sinensis processing by-products was prepared by saponification and liquid-liquid extraction combined with solid phase extraction for purification. The activity of purified VD3-rich extract (PPs-VD3) on MC3T3-E1, a preosteoblastic cell line, was determined. Furthermore, the improvement effect of PPs-VD3 on bone health of VD-deficient mice was investigated. PPs-VD3 stimulated the proliferation and differentiation of MC3T3-E1 cells. Compared to the same concentration of the VD3 standard, mineralization of MC3T3-E1 cells increased after 14 d or 21 d of PPs-VD3 treatment. Western blotting showed that PPs-VD3 significantly upregulated the protein levels of bone morphogenetic protein 2 and runt-related transcription factor 2 compared to the VD3 standard. Furthermore, PPs-VD3 treatment activated the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) signaling pathway in MC3T3-E1 cells, especially increased OPG expression was detected at day 3 to day 14 compared to the VD3 standard treatment. More than ten medium and long chain fatty acids were detected in PPs-VD3, of which n-3 polyunsaturated fatty acids (PUFA) constituted 38.83 ± 8.61%, and the n-3/n-6 PUFA ratio in PPs-VD3 was 2.84 ± 0.23. The femoral trabeculae number and thickness of VD-deficient mice increased after 3 weeks of PPs-VD3 treatment. The changes of parameters associated with bone resorption including parathyroid hormone, bone mineral density and tartrate resistant acid phosphatase revealed the contribution of PPs-VD3 treatment in improving bone remodeling in VD-deficient mice. Our results suggest that PPs-VD3 could have potential prospects in alleviating osteoporosis or promotion of bone health.

Effect of Olive oil on Osteoprotegerin Expression on the Tension side of Gingival Crevicular Fluid in the Orthodontic Tooth Movement of Old and Young Guinea Pigs

Background: Olive oil is a natural source of phytoestrogens and antioxidants similar to mammalian estrogen. The substance increases osteoblast differentiation and osteoprotegerin (OPG) expression. Objective of the study: To examine the effect of giving olive oil on OPG expression of gingival crevicular fluid in the orthodontic tooth movement among old and young guinea pigs. Methodology: The four groups of guinea pigs (n = 3) consisted of old guinea pigs given olive oil (OO), old controls (OC), the young given olive oil (YO), and young controls (YC). The teeth were movedreciprocally to distal by a 0.35 N open coil spring which was attached to the bracket of the lower incisors. The YO group was given olive oil at 0.7ml/day dosage and the OO group had 1.86ml olive oil administered per day until day 14. OPG expression was analyzed in gingival crevicular fluid (GCF) sample using the enzyme-linked immunosorbent assay (ELISA) on days 0, 7, and 14. Retrieved data was analyzed using Three-way ANOVA Statistic Test followed by Post Hoc LSD Test. Results: The statistical test showed that olive oil increased OPG expression in old and young guinea pigs (p&lt;0.05), and OPG OO expression was comparable to that in YC day 7. OPG YO expression was higher than OO (p &lt;0.05). Increasing duration of olive oil application led to higher OPG expression (p &lt;0.05). There was a correlation between olive oil and age and duration of administration (p &lt;0.05). Conclusion: Olive oil increased the OPG expression on GCF of old and young guinea pigs during orthodontic tooth movement, with the highest OPG expression in YO day 14.

Voluntary physical activity mitigates alveolar bone loss in mice with ligature-induced experimental periodontitis

ObjectiveTo investigate the effect of voluntary physical activity (VPA) on inflammatory profile and the progression of experimental periodontal disease (PD) in mice. MethodsMale C57BL/6 mice were randomly distributed into Control; VPA; PD and PD/VPA groups. We registered VPA (total volume of revolutions) and average speed (revolutions/minute) in a free running wheel for 30 days. On the 15th day, animals from the PD and PD/VPA groups received ligatures on the upper second molars bilaterally. On the 30th day animals were euthanized, and PD progression was assessed by measuring alveolar bone loss (ABL - the linear distance between the cemento-enamel junction and the alveolar bone crest on the teeth buccal surface). Gene expression of RANKL (kappa nuclear factor B receptor) OPG (osteoprotegerin), IL-1β (interleukin 1 beta), IL-6 (interleukin 6) and TNF-α (tumor necrosis factor alpha) were evaluated by real-time PCR (quantitative Polymerase Chain Reaction - relative gene expression). ResultsThe total volume of physical activity and the activity speed decreased along the seven days after ligature-placement (p < 0.05), returning to a similar pattern in relation to VPA group. Ligature placement produced significant bone resorption, and increased RANKL, IL-1β, IL-6 and TNF-α expression. VPA reduced ABL (p < 0,05) and the expression of TNF-α and IL-1β, whereas increased OPG expression. ConclusionAnimals induced to PD with access to the VPA wheel presented both lower gingival inflammation and less alveolar bone resorption in comparison to animals without access to the wheel.

Therapeutic Effect of Matrine on Collagen-Induced Arthritis Rats and Its Regulatory Effect on RANKL and OPG Expression.

Objective To investigate the effect of matrine on rats with collagen-induced arthritis (CIA) and its regulatory effect on receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) expression. Methods Wistar rats (n = 6) and CIA rats (n = 30) were randomly divided into six groups: healthy, CIA control, low/medium/high matrine (25, 50, or 100 mg/kg, once per day for six weeks), and methotrexate (MTX) (2 mg/kg, once per week for six weeks). The degree of joint damage was evaluated by X-ray and HE staining. Bone marrow suppression was assessed by routine blood analysis. In addition, the levels of serum RANKL and OPG in the rats were measured by ELISA. Results The level of joint swelling and degree of joint damage assessed by ankle swelling measurements, AI score, X-ray, and HE staining were alleviated in the CIA rats treated with MTX or different doses of matrine. Furthermore, no obvious inhibitory effect was observed on the bone marrow of the CIA rats, regardless of the dose of matrine or treatment with 2 mg/kg MTX (P > 0.05). The levels of OPG in serum and the ratio of OPG/RANKL were higher, and RANKL expression was lower in the low/medium/high matrine group compared with that of the CIA control group. The serum levels of OPG and OPG/RANKL ratio increased with the matrine dose, while the opposite was observed for RANKL expression. Conclusion Matrine treatment was associated with a lower degree of bone destruction, increased OPG expression and OPG/RANKL ratio, and decreased RANKL expression in CIA rats. Thus, matrine may represent a novel drug candidate for the treatment of RA.

Effects of photobiomodulation on bone remodeling in an osteoblast-osteoclast co-culture system.

The general bone anabolic effect of photobiomodulation (PBM) is largely accepted. As a result, PBM therapy is expected to be beneficial in the medical fields of dentistry and bone healing. However, most of the previous in vitro studies on PBM and bone metabolism were performed with single-cell cultures of osteoclast-lineage cells or osteoblast-lineage cells. In the present study, the bone-modulating effects of PBM were evaluated in an in vitro osteoblast/osteoclast co-culture system. Mouse bone marrow-derived macrophages (BMMs) and mouse calvarial pre-osteoblasts cells were purified and used as precursor cells for osteoclasts and osteoblasts, respectively. The PBM effects on single-cell culture of osteoclasts or osteoblasts as well as co-culture were examined by 1.2J/cm2 low-level Ga-Al-As laser (λ = 808 ± 3nm, 80 mW, and 80mA; spot size, 1cm2; NDLux, Seoul, Korea) irradiation for 30s at daily intervals throughout culture period. At the end of culture, the osteoclast differentiation and osteoblast differentiation were assessed by TRAP staining and ALP staining, respectively. The expressions of osteoclastogenic cytokines were evaluated by RT-PCR and Western blot analyses. Under the single-cell culture condition, PBM enhanced osteoblast differentiation but had minor effects on osteoclast differentiation. However, in the co-culture condition, its osteoblastogenic effect was maintained, and osteoclast differentiation was substantially reduced. Subsequent RT-PCR analyses and western blot results revealed marked reduction in receptor activator of NF-κB ligand (RANKL) expression and elevation in osteoprotegerin (OPG) expression by PBM in co-cultured cells. More importantly, these alterations in RANKL/OPG levels were not observed under the single-cell culture conditions. Our results highlight the different effects of PBM on bone cells based on culture conditions. Further, our findings suggest the indirect anti-osteoclastogenic effect of PBM, which is accompanied by a decrease in RANKL expression and an increase in OPG expression.

Metformin: Is It the Well Wisher of Bone Beyond Glycemic Control in Diabetes Mellitus?

Both diabetes mellitus and osteoporosis constitute a notable burden in terms of quality of life and healthcare costs. Diabetes mellitus affecting the skeletal system has been gaining attention in recent years and is now getting recognized as yet another complication of the disease, known as diabetic bone disease. As this condition with weaker bone strength increases fracture risk and reduces the quality of life, so much attention is being paid to investigate the molecular pathways through which both diabetes and its therapy are affecting bone metabolism. Out of many therapeutic agents currently available for managing diabetes mellitus, metformin is one of the most widely accepted first choices worldwide. The purpose of this review is to describe the effects of biguanide-metformin on bone metabolism in type 2 diabetes mellitus including its plausible mechanisms of action on the skeleton. In vitro studies suggest that metformin directly stimulates osteoblasts differentiation and may inhibit osteoclastogenesis by increasing osteoprotegerin expression, both through activation of the AMPK signaling pathway. Several studies in both preclinical and clinical settings report the favorable effects of metformin on bone microarchitecture, bone mineral density, bone turnover markers, and fracture risk. However, animal studies were not specific in terms of the diabetic models used and clinical studies were associated with several confounders. The review highlights some of these limitations and provide future recommendations for research in this area which is necessary to better understand the role of metformin on skeletal outcomes in diabetes.

Naringin increases osteoprotegerin expression in fibroblasts from periprosthetic membrane by the Wnt/\u03b2-catenin signaling pathway

BackgroundThe osteoclast bone resorption is critical in aseptic loosening after joint replacement. The balance between activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) is considered to play a central role in osteoclast maturation. Fibroblasts from the periprosthetic membrane express RANKL and promote osteoclast formation. Studies have demonstrated that naringin inhibited osteoclastogenesis and wear particle-induced osteolysis. In this study, the naringin-induced OPG/RANKL effects and its underlying mechanism were studied in fibroblasts from periprosthetic membrane.MethodsFibroblasts were isolated from the periprosthetic membrane during hip arthroplasty for revision due to aseptic loosening. Fibroblasts were cultured and treated with or without naringin and DKK-1 (the classical inhibitor of Wnt/β-catenin signaling pathway). OPG and RANKL mRNA and protein levels, gene expression of β-catenin, and cyclin D1, which participate in the Wnt signaling pathway, were examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay.ResultsThe mRNA and protein levels of OPG were enhanced by naringin in a dose-dependent manner compared to that of the non-treated control. In contrast, naringin did not affect the expression of RANKL. Importantly, DKK-1 attenuated OPG expression in fibroblasts under naringin treatment. Moreover, naringin stimulated the gene expression of β-catenin and cyclin D1 in fibroblasts, and the effect could be inhibited by DKK-1.ConclusionThe results indicated that naringin enhanced OPG expression through Wnt/β-catenin signaling pathway in fibroblasts from periprosthetic membrane, which may be useful to inhibit periprosthetic osteolysis during aseptic loosening after total joint arthroplasty.

The Impact of Hyperbaric Oxygen Therapy on Serum C-Reactive Protein Levels, Osteoprotegerin Expression, and Osteoclast Numbers in Induced-Periodontitis Diabetic Rats.

Objectives This study aimed to examine the impact of hyperbaric oxygen therapy (HBOT) on serum C-reactive protein (CRP) levels, osteoclast numbers, and osteoprotegerin (OPG) expression in periodontitis-induced diabetic rats Materials and Methods This study constituted an in vivo laboratory-based experiment incorporating a posttest only control group design. Thirty male Wistar rats were divided into three groups of research subjects: a healthy group (K0), periodontitis-induced diabetic group (K1), and periodontitis-induced diabetic group treated with HBOT for 7 days (K2). After treatment, the subjects were sacrificed to determine the level of serum CRP by the ELISA method. Immunohistochemical analysis was conducted to check the level of OPG expression, while a histological analysis was undertaken to quantify the number of osteoclasts. Statistical Analysis The data was analyzed using a one-way ANOVA and Least Significant Difference (LSD) test on which a result of p < 0.05 was considered statistically significant. Results HBOT appreciably decreased serum CRP levels, significantly enhancing OPG expression in periodontitis-induced diabetic ( p < 0.05) and decreasing the number of osteoclasts in -periodontitis-induced diabetic ( p > 0.05). Conclusion HBOT reduced the serum CRP level, increased OPG expression, and decreased osteoclast numbers in periodontitis-induced diabetic rats.

Baicalin inhibits root resorption during tooth movement in a rodent model

ObjectiveBaicalin mediates bone metabolism and has shown protective activity against periodontal tissue damage in a rat model of periodontitis. Therefore, we hypothesized that baicalin may inhibit the root resorption that occurs during orthodontic tooth movement and examined its effect on the histological changes in periodontal tissue that occur during tooth movement. MethodsFirst molars of rats were subjected to traction using excessive orthodontic force to produce a root resorption model. Rats in the baicalin group received baicalin for 3 weeks during tooth movement, and the amount of first molar movement on day 21 after the initiation of traction was measured by three-dimensional micro-computed tomography analysis. After tooth movement, tissue samples from the mesial and tension sides were collected, and successive horizontal sections were prepared and examined using hematoxylin–eosin and tartrate-resistant acid phosphatase (TRAP) staining and immunohistochemical staining for the receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG). The severity of root resorption was also determined by histological analysis. ResultsThere was no significant intergroup difference in tooth movement during the experimental exaggerated tooth movement. In comparison with the control group, the baicalin-treated group showed increased OPG expression, suppressed RANKL expression, and significantly fewer TRAP-positive cells in the first molars. The root resorption area was significantly smaller in the baicalin group. ConclusionsTreatment with baicalin prevented root resorption without preventing tooth movement. Baicalin may be useful for the management of root resorption during orthodontic treatment.

Role of osteoprotegerin in the regulation of dental epithelial‑mesenchymal signaling during tooth development

Dental epithelial-mesenchymal signaling is crucial for tooth development, but the detailed mechanism is not fully understood. Using microarray analysis, it was revealed that the expression of osteoprotegerin, an important factor regulating bone remodeling, significantly increased after removal of the dental epithelium. Immunohistochemical staining revealed that osteoprotegerin expression within the dental mesenchyme was quite low during the prenatal period, but significantly increased after birth. To investigate the influence of osteoprotegerin upon tooth development, first-molar tooth germs from embryonic day 14.5 (E14.5) Chinese Kunming mice were treated with different concentrations of osteoprotegerin. It was revealed that osteoprotegerin could inhibit the expression of odontogenic markers while promoting the expression of osteogenic markers, thereby disrupting tooth morphogenesis. These findings were further supported by in vitro and in vivo cultures. Finally, quantitative reverse transcription-polymerase chain reaction and immunofluorescence studies revealed that, after osteoprotegerin treatment, the activity of the wingless/integrated (Wnt)/β-catenin pathway increased, indicating that increased osteoprotegerin expression in prenatal tooth development could lead to uncontrolled upregulation of the Wnt/β-catenin pathway.

Hypoxia, hypoxia-inducible transcription factors and oxygen-sensing prolyl hydroxylases in bone development and homeostasis.

To summarize the role of hypoxia signaling in skeletal cells. Hypoxia occurs at several stages during bone development. Skeletal cells, like chondrocytes and osteoblasts, respond to this challenge by stabilizing the hypoxia inducible transcription factor HIF, which induces the expression of angiogenic factors and promotes glycolysis. The increased delivery of oxygen and nutrients, together with metabolic adaptations, prevent chondrocyte cell death in the growth plate and promote bone formation by osteoblasts. However, excessive HIF levels have to be avoided during bone development as the resulting metabolic maladaptations cause skeletal dysplasia. Recent studies show that HIF also targets other genes to increase bone mass: it decreases osteoclastogenesis by increasing osteoprotegerin expression and represses sclerostin expression by epigenetic mechanisms, resulting in increased bone formation and decreased resorption. Moreover, increased HIF signaling in osteolineage cells promotes primary and metastatic breast tumor growth, and induces erythropoietin (EPO) production, resulting in polycythemia. Finally, HIF can directly or indirectly through increasing EPO levels, induce the expression and processing of FGF23 and may thereby affect mineral homeostasis and vitamin D metabolism. HIF signaling in skeletal cells not only affects their behavior but also influences erythropoiesis and possibly mineral homeostasis.

I-BET151 suppresses osteoclast formation and inflammatory cytokines secretion by targetting BRD4 in multiple myeloma.

Background: Multiple myeloma (MM) is an incurable hematologic cancer, accompanied by excessive osteoclast formation and inflammatory cytokine secretion. The mechanisms by which bromodomain and extra-terminal domain (BET) protein inhibitor I-BET151 regulates osteoclast differentiation and inflammatory cytokine secretion in MM are largely unknown. Methods: The isolated peripheral blood mononuclear cells from normal or patients with MM were treated with receptor activator of NF-κB ligand (RANKL) and M-CSF to induce osteoclast differentiation. RAW 264.7 cells were treated with RANKL. I-BET151 was applied to investigate the effects of BRD4 inhibition on osteoclast formation and inflammatory cytokine secretion. Osteoclast formation was determined by tartrate-resistant acid phosphatase (TRACP) staining. The expression of osteoclast-specific genes TRACP, matrix metalloproteinase-9 (MMP-9), cathepsin K (Ctsk), and c-Src was tested using quantitative real-time PCR. And the level of inflammatory cytokines TNF-α, IL-1β, and IL-6 was assessed by ELISA. Tumor necrosis factor receptor-associated factor 6 (TRAF6), BRD4, nuclear and cytoplasm p65, IκB-α, nuclear factor of activated T cells cytoplasmic (NFATc1), and osteoprotegerin (OPG) expression were measured by Western blotting. RNAi technology was applied to knock down BET family member BRD4. Results: I-BET151 dose-dependently suppressed osteoclast formation, inhibited the levels of osteoclast-specific genes TRACP, MMP-9, Ctsk, and c-Src and inflammatory cytokines TNF-α, IL-1β, and IL-6 secretion in peripheral blood mononuclear cells and RAW 264.7. I-BET151 inhibited the protein levels of BRD4 and NFATc1, increased OPG expression, and suppressed IκB-α degradation and p65 nuclear translocation. Further, the effects of I-BET151 on osteoclast formation, osteoclast-specific genes expression, inflammatory cytokine secretion, and NF-κB inhibition were promoted by BRD4 knockdown. Conclusion: I-BET151 inhibits osteoclast formation and inflammatory cytokine secretion by targetting BRD4-mediated RANKL-NF-κB signal pathway and BRD4 inhibition might be beneficial for MM treatment.

A Novel Small Molecule Which Increases Osteoprotegerin Expression and Protects Against Ovariectomy-Related Bone Loss in Rats.

The ratio of osteoprotegerin (OPG) to the receptor activator of NF-κB ligand (RANKL) is a key determinant in the regulation of bone metabolism. The study was performed to screen novel anti-osteoporotic drugs regulating OPG/RANKL ratio and evaluate their effect on bone metabolism. According to the screening results and in vitro results, we found a small molecule, E09241, significantly increased the ratio of OPG/RANKL by mainly increasing OPG expression. Our in vitro studies showed that E09241 increased the alkaline phosphatase (ALP) activity of mouse osteoblasts, promoted mineralization, and increased the expression of osteogenic differentiation-related genes. In addition, we observed that E09241 inhibited RANKL-induced osteoclast differentiation and reduced the expression of osteoclast differentiation-related proteins nuclear factor of activated T cells c1 (NFATc1) and matrix metalloproteinase 9 (MMP-9). More importantly, E09241 exerted therapeutic protection against bone loss in ovariectomized rats in vivo. This protective effect was confirmed to be achieved by inhibiting bone resorption and promoting bone formation in vivo. Mechanistically, E09241 regulates OPG expression through canonical Wnt/β-catenin signaling. Our findings suggest that E09241 is a promising small-molecule compound for treating osteoporosis with a dual effect on osteoblasts and osteoclasts.

Connexin43 regulates osteoprotegerin expression via ERK1/2 -dependent recruitment of Sp1

In bone, connexin43 expression in cells of the osteoblast lineage plays an important role in restraining osteoclastogenesis and bone resorption. While there is a consensus around the notion that the anti-osteoclastogenic factor, osteoprotegerin, is a driver of this effect, how connexin43 regulates osteoprotegerin gene expression is unclear. Here, we show that loss of connexin43 decreased osteoprotegerin gene expression and reduced ERK1/2 activation. Conversely, overexpression of connexin43 increased osteoprotegerin expression and enhanced ERK1/2 activation. This increase in phospho-ERK1/2 is required for connexin43 to induce transcription from the osteoprotegerin proximal promoter. Connexin43 increased promoter activity via a specific 200 base pair region of the osteoprotegerin promoter located at −1486 to −1286 with respect to the transcriptional start site, a region which includes four Sp1 binding elements. Further, activation of this promoter region required an intact functional connexin43, as hypomorphic or dominant negative connexin43 mutant constructs, including one with increased hemichannel activity, were unable to stimulate osteoprotegerin expression as strongly as wild type connexin43. Using chromatin immunoprecipitations, we show that connexin43 expression enhanced the recruitment of Sp1, but not Runx2, to the osteoprotegerin proximal promoter. In total, these data show that connexin43-dependent gap junctional communication among osteoblast cells permits efficient ERK1/2 activation. ERK1/2 signaling promotes the recruitment of the potent transcriptional activator, Sp1, to the osteoprotegerin proximal promoter, resulting in robust transcription of anti-osteoclastogenic factor, osteoprotegerin.

Expression of Osteoprotegrin and Osteoclast Level in Chronic Apical Periodontitis Induced with East Java Propolis Extract.

Introduction:The objective of this animal study was to promote East Java propolis as a potential natural intracanal medicament for periapical chronic apical periodontitis bone resorption through evaluating the expression of osteoprotegrin (OPG) and osteoclast level.Methods and Materials:Propolis extract was produced using a maceration procedure. Thirty Wistar rats were divided into three groups. In group I, the control group, the first upper right molar constituted a healthy tooth. In group II, containing rodents with experimentally chronicapical periodontitis, infection with Enterococcus faecalis ATCC29212 106 CFU was performed. In group III, the treatment group, after being injected with E faecalis, 10 μL propolis was applied. It required 21 days to induce post-pulp chronic apical periodontitis infection. The rats were euthanised for immunohistochemical examination in order to measure the expression of OPG and to count histologically the number of osteoclast. Result:The expression of OPG and osteoclast constituted 17.5±1.58 and 6.4±0.96 in group I, 10±2 and 16.2±1.31 in group II and 17±1.69 and 7.5±1.08 in group III. Group I presented the highest level of OPG expression but the lowest level of osteoclast expression. There were significant differences between groups II and III and group I regarding OPG and osteoclast expression (P<0.05). Conclusion:East Java Propolis was a potential intracanal medicament promoting an increase in osteoprotegerin expression and a decrease in the number of osteoclasts thereby inhibiting osteoclastogenesis.

RANK-L is a Potential Therapeutic Target in Homogeneous Atherosclerotic Plaques

Objective and design: The late stages of carotid atherosclerosis are responsible for increased local stiffness, suggesting the need for a therapeutic target that affect either plaque composition and arterial stiffness. There is a lack of data on the role of the local arterial stiffness, assessed by radio-frequency based system and its relationship with the molecular profile of plaques. Subjects: In this study we enrolled 18 consecutive patients undergoing carotid endoarterectomy, with homogeneus or heterogeneous plaques, as established by Doppler-ultrasound and local pulse-wave velocity was assessed before surgery. Methods: In carotid plaque specimens, we evaluated inflammasome (NLRP3), Receptor Activator of Nuclear Factor κB (RANK) and its natural ligand (RANK-L), Osteoprotegerin (OPG), and other inflammatory and apoptotic molecules by Western Blotting and qPCR analysis. In addition, lipid peroxidation of arterial specimens was assessed by TBARS assay. Results: In heterogeneus plaques we observed increased OPG expression (p=0.04), positively correlated with lipid peroxidation values (r=0.511, p=0.03); increased levels of RANK (p=0.02), and other inflammatory and apoptotic molecules. RANK-L protein was augmented in homogeneus plaques (p=0.01) and correlated to s-index (r=0.514, p=0.03) and PWV (r=0.525, p=0.03) values. Conclusions: Our data provide evidence that increased local PWV and s-index might identify plaque evolution towards calcification.