Quantification of sphingosine-1-phosphate (S1P) in patients with periodontitis stage II grade B / Cuantificación de esfingosina 1 fosfato en pacientes con periodontitis estadío II grado B

Abstract The aim of this study was the quantification of Sphingosine-1-phosphate (S1P) in periodontal pockets of patients with periodontitis. This is an observational, descriptive, case-control study. Thirty subjects were selected: 15 controls and 15 cases. A periodontal study was conducted following the parameters of AAP 2017 for the diagnosis of periodontal diseases. A sample of saliva and gingival crevicular fluid was obtained from each subject and then analyzed with the Human S1P Elisa kit (MyBioSource #MBS2516132) accordingly to the manufacturer's instructions, in order to verify the presence of S1P and quantify it´s concentration when founded. Results showed a significant difference (p=0.05) between cases and controls. In the case of saliva samples, the concentration of S1P was higher than the ones found in the control group (72.94 ng/mL and 45.12 ng/mL). For GCF, a higher amount of S1P was found in patients with POD (20.09 ng/mL and 15.20 ng/mL). This work raises a possible route of bone metabolism, inflammatory process, and identification of periodontitis through oral quantification of S1P, however, future studies are needed.

Resumen El propósito de este estudio fue la cuantificación de Esfingosina-1-Fosfato (S1P) en las bolsas periodontales de pacientes con periodontitis. Estudio observacional, descriptivo de casos y controles. 30 sujetos fueron seleccionados de los cuales 15 controles y 15 casos. Se realizó un estudio periodontal completo siguiendo los parámetros establecidos por la AAP en 2017 para el diagnóstico de las enfermedades periodontales. Se tomaron muestras de saliva y de líquido crevicular gingival de cada sujeto estudiado y se analizaron con el ELISA kit humano para S1P (MyBiosource #MBS2516132) y de acuerdo con las instrucciones del fabricante, se realizó para cuantificar la presencia d S1P en las muestras estudiadas. Los resultados mostraron diferencia significativa (p=0.05) entre casos y controles. En el caso de las muestras de saliva, la concentración de S1P en controles fue mayor (72.94 ng/mL y 45.12 ng/mL). Para Líquido crevicular gingival, se encontró mayor cantidad de S1P en los pacientes con periodontitis (20.09 ng/mL y 15.20 ng/mL). Este estudio plantea una posible ruta de metabolismo óseo, proceso inflamatorio e identificación de la Periodontitis a través de la cuantificación oral de S1P, sin embargo se necesitan estudios futuros.

Kcnq1ot1 promotes osteogenic differentiation and suppresses osteoclast differentiation / 南方医科大学学报

OBJECTIVE@#To investigate the regulatory role of long non-coding RNA Kcnq1ot1 in osteoclast differentiation, osteogenic differentiation and osteoporosis.@*METHODS@#The expression of lnc-Kcnq1ot1, Bglap, Runx2, Alp, Bsp, Nfatc1, Mmp9, Ctsk and Oscar were detected by real-time quantitative PCR (qRT-PCR) in the femoral bones from mouse models of postmenopausal osteoporosis (ovariectomized mice, @*RESULTS@#The expression of lnc-Kcnq1ot1 was significantly upregulated during osteoblast differentiation but downregulated in the bone tissues of osteoporotic mice and during osteoclast differentiation (@*CONCLUSIONS@#Our data demonstrate that lnc-Kcnq1ot1 promotes osteogenic differentiation and alleviates osteoclast differentiation, suggesting the potential of lnc-Kcnq1ot1 as a therapeutic target against osteoporosis.

Effect of Kidney-tonifying Method on OPG/RANK/RANKL Signaling Pathway in Postmenopausal Osteoporosis Animal Model： A Meta-analysis / 中国实验方剂学杂志

Objective:As the problem of global aging intensifies，postmenopausal osteoporosis （PMOP） has become a global health problem among females. At present，the commonly used biological agents have been proved not suitable for long-term use due to multiple adverse reactions. Several Meta-analyses have confirmed the good safety and effectiveness of kidney-tonifying method against PMOP，but its therapeutic mechanism remains unclear. The purpose of this Meta-analysis was to evaluate the effect of kidney-tonifying method on osteoclastogenesis inhibitory factor（OPG）/receptor activator of nuclear transcription factor （NF）-<italic>κ</italic>B （RANK）/receptor activator of NF-<italic>κ</italic>B ligand （RANKL） signaling pathway in PMOP animal model，so as to provide an experimental basis for the treatment of PMOP with kidney-tonifying method. Method:The related articles were retrieved from PubMed，Ovid Medline，Embase，China National Knowledge Infrastructure （CNKI），Chongqing Weipu Database for Chinese Technical Periodicals （VIP），and Wanfang Data Knowledge Service Platform with the retrieval time set from their inception to January 2020. The quality of each included article was evaluated using the SYRCLE's risk of bias tool. Then RevMan 5.3 was utilized for Meta-analysis according to the Cochrane systematic review methodology. Result:Thirty-two studies involving 619 rats were included. The quality score of these studies ranged from 3 to 5 points. The results of the Meta-analysis indicated obvious advantages of kidney-tonifying method in increasing bone mineral density （BMD）［standardized mean difference （SMD）=2.01，95% confidence interval（CI）=1.50-2.52，<italic>P</italic><0.000 01］），serum OPG level （SMD=3.33，95% CI=2.59-4.07，<italic>P</italic><0.000 01），and OPG mRNA expression （SMD=11.81，95% CI=7.49-16.13，<italic>P</italic><0.000 01），promoting OPG protein production （SMD=4.95，95% CI=3.09-6.81，<italic>P</italic><0.000 01），reducing serum RANKL（SMD=-4.88，95% CI=-6.01--3.75，<italic>P</italic><0.000 01） and RANK levels （SMD=-7.30，95% CI=-9.53--5.07，<italic>P</italic><0.000 01），and down-regulating RANKL （SMD=-6.22，95%CI=-8.95--3.49，<italic>P</italic><0.000 01） and RANK mRNA （SMD=-3.18，95% CI=-6.19--0.18，<italic>P</italic><0.05） expression and RANKL protein expression in bone tissue （SMD=-3.99，95% CI=-5.47--2.50，<italic>P</italic><0.000 01）. Conclusion:The kidney-tonifying method has been proved to possess potential advantages in regulating the balance of OPG/RANK/RANKL signaling pathway in PMOP animal model. Nevertheless，more large-sample sized，properly designed，and high-quality animal experiments are still needed for further verification.

Polyherbal formula SC-E3 inhibits rheumatoid arthritis activity in a mouse model of type-II collagen-induced arthritis / 中西医结合学报

OBJECTIVE@#SC-E3 is a polyherbal formula that contains five medicinal herbs used frequently in traditional herbal medicine. In our previous study, we demonstrated the antioxidant and anti-inflammatory effects of SC-E3. The present study examined the effects of SC-E3 in a mouse model of type-II collagen-induced arthritis (CIA).@*METHODS@#In vivo, male DBA/1J mice were immunized by intradermal injection of bovine type-II collagen and complete or incomplete Freund's adjuvant, to induce arthritis. SC-E3 was orally administered daily for 23 days. In vitro, bone marrow-derived macrophages (BMMs) were treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) in the absence or presence of SC-E3.@*RESULTS@#Administrations of SC-E3 were found to have anti-arthritic effects in the joints of CIA mice, as evidenced by reduced paw swelling, bone erosion and deformation, inflammatory cell infiltration, and inflammation in synovial membrane. SC-E3 also reduced serum levels of tumor necrosis factor-α, interleukin-1β, aspartate aminotransferase and alanine aminotransferase. Furthermore, tartrate-resistant acid phosphatase-positive osteoclast numbers in the joints were significantly lower in SC-E3-treated CIA mice than in CIA mice. In addition, the differentiations of BMMs to multinucleated osteoclasts induced by M-CSF and RANKL stimulation were dose-dependently reduced by SC-E3.@*CONCLUSION@#These results suggest that SC-E3 possesses substantial anti-arthritic activity because it inhibits pro-inflammatory cytokines and osteoclastogenesis, and that SC-E3 has potential therapeutic use for the treatment of rheumatoid arthritis.

Efeito do consumo de álcool no desenvolvimento de lesão periapical induzida em ratos: uma análise microtomográfica / The Effect of Alcohol Consumption on the Development of Periapical Lesion Induced in Rats: a Micro-CT Analysis

Objetivo: O objetivo deste estudo foi avaliar a influência do consumo crônico de álcool no aumento da destruição óssea periapical em ratos. Material e métodos: Foram selecionados 12 ratos machos da linhagem Wistar, aleatoriamente divididos nos grupos controle e álcool (n=6). Os ratos do grupo álcool foram submetidos à auto-administração de solução alcoólica contendo 25% de álcool puro. O grupo controle recebeu apenas água filtrada durante o estudo. Após as 5 semanas de adaptação do grupo álcool, todos os ratos foram anestesiados e a polpa dos seus primeiros molares inferiores esquerdos foi exposta à cavidade oral para indução da lesão periapical. Após 28 dias de exposição pulpar, os ratos foram sacrificados por sobredose de anestesia, e suas mandíbulas foram removidas e seccionadas para avaliação microtomográfica. As hemimandíbulas esquerdas foram fixadas e escaneadas no microtomógrafo SkyScan 1173 (Bruker, Kontich, Belgium). O tamanho da lesão periradicular foi medido a partir das imagens de microtomografia computadorizada (micro-CT), onde foram calculados a área de superfície e o volume da lesão. Também foram avaliadas a taxa percentual de ganho de peso e a ingestão de sólidos/líquidos dos grupos. Os dados foram estatisticamente analisados utilizando o teste t de Student (p<0,05). Resultados: Os animais do grupo controle tiveram uma maior taxa percentual de ganho de peso corporal e de ingestão tanto de sólidos como de líquidos (p<0,05). As lesões periapicais apresentaram maior volume e maior área nos animais do grupo álcool, em comparação ao grupo controle (p<0,05). Conclusão: O consumo crônico de álcool contribuiu para o aumento da destruição óssea periapical em casos de periodontite apical.

Objective: the aim of this study was to evaluate the influence of alcohol consumption on the increase of periapical bone destruction in rats. Material and Methods: the sample included 12 Wistar male rats, randomly assigned into a control group and an alcohol group (n=6). Rats in the alcohol group were submitted to self-administration of a 25% pure alcoholic solution. The control group received only filtered water throughout the study. After 5 weeks of adaptation to the alcohol dose, all animals were anesthetized and the pulps of their mandibular left first molar were exposed to the oral cavity to induce periapical lesion. Twenty-eight days after the pulp exposure, those rats were euthanized due to overdose of anesthesia and their mandibles were removed and sectioned to obtain a micro-computed tomographic (micro-CT) scan. The rats' left hemimandibles were fixed and scanned on the SkyScan 1173 (Bruker, Konitch, Belgium) microtomograph. The size of the periradicular lesions was measured from the images obtained on the micro-CT and the surface area and volume were calculated. It was also evaluated the weight gain rate and the ingestion of solid/liquid of both groups. Data were analyzed by the Student's t-test (p<0.05). Results: the control group showed higher rates of weight gain and ingested more solid and liquid than the alcohol group (p<0.05). Periapical lesions found in the alcohol group had higher volume and surface area than the ones of the control group (p<0.05). Conclusion: the chronic consumption of alcohol contributed to the increase of periapical bone destruction in cases of apical periodontitis

Micro-ARNs y células óseas / Micro RNAs and bone cells

Los micro-ARNs (miARNs) son pequeñas moléculas de ARN no codificante (de aproximadamente 15-25 nucleótidos), que regulan la expresión de genes involucrados en numerosas funciones biológicas, a través de la inhibición o degradación de un ARN mensajero diana. La homeostasis ósea se mantiene por el balance entre la formación osteoblástica y la resorción osteoclástica. La sobreexpresión o inhibición de miARNs específicos afecta la proliferación, diferenciación y actividad de osteoblastos, osteocitos y osteoclastos. Estas acciones son llevadas a cabo modulando la expresión de distintos factores transcripcionales y moléculas de señalización de las vías esenciales para la osteoblastogénesis u osteoclastogénesis. Estos efectos modifican el balance entre la formación y la resorción, determinando cambios en la homeostasis ósea. Esta revisión enumera una serie de miARNs que participan en la homeostasis ósea. Profundizando en el conocimiento de los mecanismos por medio de los cuales los miARNs actúan sobre el hueso, podrían revelarse nuevos usos potenciales futuros, entre los que se encuentran su utilidad como nuevos biomarcadores óseos o como agentes terapéuticos para el tratamiento de trastornos metabólicos óseos, pérdida de masa ósea o enfermedades óseas. (AU)

MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules (of approximately 15­25 nucleotides), which regulate the expression of genes controlling numerous biological functions, through the inhibition or degradation of the target messenger RNA. Bone homeostasis is maintained by a balance between osteoblastic bone formation and osteoclastic bone resorption. The overexpression or inhibition of specific miRNAs affects cell proliferation, differentiation and activity of osteoblast, osteocytes and osteoclast. This action is done by modulating the expression of different transcription factors and signaling molecules of the most relevant pathways of osteoblastogenesis or osteoclastogenesis. This effect is able to modify the balance between bone formation and resorption, determining changes in bone homeostasis. The present review is an overview of a series of miRNAs involved in bone homeostasis. An in depth knowledge of the mechanisms by which miRNAs act on bone may reveal potential uses in the future as new bone biomarkers or therapeutic agents for treating metabolic bone disorders, bone loss and bone diseases. (AU)

Eurycoma longifolia, a promising suppressor of RANKL-induced differentiation and activation of osteoclasts: An in vitro mechanistic evaluation

Background: Eurycoma longifolia (E. longifolia) has gained remarkable recognition due to its promisingefficacy of stimulating bone formation in androgen-deficient osteoporosis. Numerous in vivo studies haveexplored the effects of E. longifolia on osteoporosis; however, the in vitro cellular mechanism was notdiscovered yet.Objectives: The present study was aimed to investigate the effect of E. longifolia on the proliferation,differentiation and maturation of osteoclasts and the translational mechanism of inhibition of osteoclastogenesis using RAW 264.7 cells as an in vitro osteoclastic model.Materials and methods: Having assessed cytotoxicity, the cell viability, cell proliferation rate and osteoclastic differentiation capacity of E. longifolia was investigated by evaluating the tartrate-resistant acidphosphatase (TRAP) activity in receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL)-inducedosteoclasts. Taken together, the time-mannered expression of osteoclast-related protein biomarkers suchas matrix metallopeptidase-9 (MMP-9), cathepsin-K, TRAP, nuclear factor of activated T-cells cytoplasmic1 (NFATc1), superoxide (free radicals) generation and superoxide dismutase activity were also measuredto comprehend the mechanism of osteoclastogenesis.Results: E. longifolia did not show significant effects on cytotoxicity and cell proliferation of RAW 264.7cells; however, a significant inhibition of cells differentiation and maturation of osteoclasts was observed.Moreover, a significant down-regulation of RANKL-induced TRAP activity and expression of MMP-9,cathepsin-K, TRAP, NFATc1 and generation of superoxide and enhanced superoxide dismutase activitywas observed in E. longifolia treated cell cultures.Conclusion: We anticipated that E. longifolia that enhances bone regeneration on the one hand andsuppresses osteoclast’s maturation on the other hand may have great therapeutic value in treatingosteoporosis and other bone-erosive diseases such as rheumatoid arthritis and metastasis associatedwith bone loss.© 2017 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Publishing Services byElsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Periapical bone response to bacterial lipopolysaccharide is shifted upon cyclooxygenase blockage

Abstract Objectives: Infection, inflammation and bone resorption are closely related events in apical periodontitis development. Therefore, we sought to investigate the role of cyclooxygenase (COX) in osteoclastogenesis and bone metabolism signaling in periapical bone tissue after bacterial lipopolysaccharide (LPS) inoculation into root canals. Methodology: Seventy two C57BL/6 mice had the root canals of the first molars inoculated with a solution containing LPS from E. coli (1.0 mg/mL) and received selective (celecoxib) or non-selective (indomethacin) COX-2 inhibitor. After 7, 14, 21 and 28 days the animals were euthanized and the tissues removed for total RNA extraction. Evaluation of gene expression was performed by qRT-PCR. Statistical analysis was performed using analysis of variance (ANOVA) followed by post-tests (α=0.05). Results: LPS induced expression of mRNA for COX-2 (Ptgs2) and PGE2 receptors (Ptger1, Ptger3 and Ptger4), indicating that cyclooxygenase is involved in periapical response to LPS. A signaling that favours bone resorption was observed because Tnfsf11 (RANKL), Vegfa, Ctsk, Mmp9, Cd36, Icam, Vcam1, Nfkb1 and Sox9 were upregulated in response to LPS. Indomethacin and celecoxib differentially modulated expression of osteoclastogenic and other bone metabolism genes: celecoxib downregulated Igf1r, Ctsk, Mmp9, Cd36, Icam1, Nfkb1, Smad3, Sox9, Csf3, Vcam1 and Itga3 whereas indomethacin inhibited Tgfbr1, Igf1r, Ctsk, Mmp9, Sox9, Cd36 and Icam1. Conclusions: We demonstrated that gene expression for COX-2 and PGE2 receptors was upregulated after LPS inoculation into the root canals. Additionally, early administration of indomethacin and celecoxib (NSAIDs) inhibited osteoclastogenic signaling. The relevance of the cyclooxygenase pathway in apical periodontitis was shown by a wide modulation in the expression of genes involved in both bone catabolism and anabolism.

Effect of ProRoot MTA® and Biodentine® on osteoclastic differentiation and activity of mouse bone marrow macrophages

Abstract Objectives This investigation aimed to assess the differentiation inhibitory effects of ProRoot MTA® (PMTA) and Biodentine® (BIOD) on osteoclasts originated from murine bone marrow macrophages (BMMs) and compare these effects with those of alendronate (ALD). Materials and Methods Mouse BMMs were cultured to differentiate into osteoclasts with macrophage colony-stimulating factor and receptor activator of NF-κB (RANKL), treated with lipopolysaccharide. After application with PMTA, BIOD, or ALD, cell toxicities were examined using WST-1 assay kit, and RANKL-induced osteoclast differentiation and activities were determined by resorption pit formation assay and tartrate-resistant acid phosphate (TRAP) staining. The mRNA levels of osteoclast activity-related genes were detected with quantitative real time polymerase chain reaction. Expressions of molecular signaling pathways were assessed by western blot. All data were statistically analyzed with one-way ANOVA and Tukey's post-hoc test (p<0.05). Results Mouse BMMs applied with PMTA, BIOD, or ALD showed highly reduced levels of TRAP-positive osteoclasts. The BIOD treated specimens suppressed mRNA expressions of cathepsin K, TRAP, and c-Fos. Nonetheless, it showed a lower effect than PMTA or ALD applications. Compared with ALD, PMTA and BIOD decreased RANKL-mediated phosphorylation of ERK1/2 and IκBα. Conclusions PMTA and BIOD showed the inhibitory effect on osteoclast differentiation and activities similar to that of ALD through IκB phosphorylation and suppression of ERK signaling pathways.

Pentamidine Inhibits Titanium Particle-Induced Osteolysis In Vivo and Receptor Activator of Nuclear Factor-κB Ligand-Mediated Osteoclast Differentiation In Vitro

BACKGROUND: Wear debris-induced osteolysis leads to periprosthetic loosening and subsequent prosthetic failure. Since excessive osteoclast formation is closely implicated in periprosthetic osteolysis, identification of agents to suppress osteoclast formation and/or function is crucial for the treatment and prevention of wear particle-induced bone destruction. In this study, we examined the potential effect of pentamidine treatment on titanium (Ti) particle-induced osteolysis, and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. METHODS: The effect of pentamidine treatment on bone destruction was examined in Ti particle-induced osteolysis mouse model. Ti particles were implanted onto mouse calvaria, and vehicle or pentamidine was administered for 10 days. Then, calvarial bone tissue was analyzed using micro-computed tomography and histology. We performed in vitro osteoclastogenesis assay using bone marrow-derived macrophages (BMMs) to determine the effect of pentamidine on osteoclast formation. BMMs were treated with 20 ng/mL RANKL and 10 ng/mL macrophage colony-stimulating factor in the presence or absence of pentamidine. Osteoclast differentiation was determined by tartrate-resistant acid phosphatase staining, real-time polymerase chain reaction, and immunofluorescence staining. RESULTS: Pentamidine administration decreased Ti particle-induced osteoclast formation significantly and prevented bone destruction compared to the Ti particle group in vivo. Pentamidine also suppressed RANKL-induced osteoclast differentiation and actin ring formation markedly, and inhibited the expression of nuclear factor of activated T cell c1 and osteoclast-specific genes in vitro. Additionally, pentamidine also attenuated RANKL-mediated phosphorylation of IκBα in BMMs. CONCLUSION: These results indicate that pentamidine is effective in inhibiting osteoclast formation and significantly attenuates wear debris-induced bone loss in mice.

Tetrabromobisphenol A Promotes the Osteoclastogenesis of RAW264.7 Cells Induced by Receptor Activator of NF-kappa B Ligand In Vitro

BACKGROUND: Tetrabromobisphenol A (TBBPA), one of the most widely used brominated flame-retardants, is a representative persistent organic pollutants group. Studies on TBBPA toxicity have been conducted using various target cells; however, few studies have investigated TBBPA toxicity in bone cells. Therefore, this study investigated the in vitro effects of TBBPA on osteoclasts, a cell type involved in bone metabolism. METHODS: RAW264.7 cells were cultured in medium containing 50 ng/mL receptor activator of nuclear factor kappa B ligand (RANKL) and varying concentrations of TBBPA. To evaluate the effects of TBBPA on the differentiation and function of osteoclasts, osteoclast-specific gene expression, tartrate-resistant acid phosphatase (TRAP) activity, bone resorbing activity, mitochondrial membrane potential (MMP) and mitochondrial superoxide were measured. RESULTS: The presence of 20 μM TBBPA significantly increased TRAP activity in RANKL-stimulated RAW264.7 cells, the bone resorbing activity of osteoclasts, and the gene expression of Akt2, nuclear factor of activated T-cells cytoplasmic 1, and chloride channel voltage-sensitive 7. However, TBBPA treatment caused no change in the expression of carbonic anhydrase II, cathepsin K, osteopetrosis-associated transmembrane protein 1, Src, extracellular signal-related kinase, GAB2, c-Fos, or matrix metalloproteinase 9. Furthermore, 20 μM TBBPA caused a significant decrease in MMP and a significant increase in mitochondrial superoxide production. CONCLUSION: This study suggests that TBBPA promotes osteoclast differentiation and activity. The mechanism of TBBPA-stimulated osteoclastogenesis might include increased expression of several genes involved in osteoclast differentiation and reactive oxygen species production.

Piperlongumine suppressed osteoclastogenesis in RAW264.7 macrophages

Piperlongumine (PL) is a natural product found in long pepper (Piper longum). The pharmacological effects of PL are well known, and it has been used for pain, hepatoprotection, and asthma in Oriental medicine. No studies have examined the effects of PL on bone tissue or bone-related diseases, including osteoporosis. The current study investigated for the first time the inhibitory effects of PL on osteoclast differentiation, bone resorption, and osteoclastogenesis-related factors in RAW264.7 macrophages stimulated by the receptor activator for nuclear factor-κB ligand (RANKL). Cytotoxicity was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and osteoclast differentiation and bone resorption were confirmed by tartrate-resistant acid phosphatase (TRAP) staining and pit formation analysis. Osteoclast differentiation factors were confirmed by western blotting. PL exhibited toxicity in RAW264.7 macrophages, inhibiting osteoclast formation and bone resorption, in addition to inhibiting the expression of osteoclastogenesis-related factors, such as tumor necrosis factor receptor-associated factor 6 (TRAF6), c-Fos, and NFATc1, in RANKL-stimulated RAW264.7 macrophages. These findings suggest that PL is suitable for the treatment of osteoporosis, and it serves as a potential therapeutic agent for various bone diseases.

Intravenous Immunoglobulin Controls Th17 Cell-Mediated Osteoclastogenesis

The purpose of this study was to determine the regulatory role of intravenous Ig (IVIg) in Th17 cytokine–induced RANK ligand (RANKL) expression and osteoclast (OC) differentiation from OC precursors (pre-OC). Human CD14⁺ monocytes were isolated and stimulated by Th17 cytokines (IL-17, IL-21, and IL-22) and RANKL expression was investigated using a real-time PCR. CD14⁺ monocytes were incubated with RANKL, Th17 cytokines, and M-CSF, with/without IVIg, and OC differentiation was determined by counting tartrate-resistant acid phosphatase-positive multinucleated cells. OC differentiation was investigated after monocytes were cocultured with Th17 cells in the presence of IVIg. Th17 cell differentiation was determined using enzyme-linked immunosorbent assay and flow cytometry after CD4⁺ T cells were cultured with IVIg under Th17 condition. Th17 cytokines stimulated monocytes to express RANKL and IVIg suppressed the Th17 cytokine-induced RANKL expression. OCs were differentiated when pre-OC were cocultured with RANKL or Th17 cytokines and IVIg reduced the osteoclastogenesis. IVIg also decreased osteoclastogenesis when pre-OC were cocultured with Th17 cells. IVIg decreased both Th17 and Th1 cell differentiation while it did not affect Treg cell differentiation. In summary, IVIg inhibited Th17 cytokine-induced RANKL expression and OC differentiation. IVIg reduced osteoclastogenesis when monocytes were cocultured with Th17 cells. IVIg also reduced Th17 polarization. IVIg could be a new therapeutic option for Th17 cell–mediated osteoclastogenesis.

Effects of the cathepsin K inhibitor with mineral trioxide aggregate cements on osteoclastic activity

OBJECTIVES: Root resorption is an unexpected complication after replantation procedures. Combining anti-osteoclastic medicaments with retrograde root filling materials may avert this resorptive activity. The purpose of this study was to assess effects of a cathepsin K inhibitor with calcium silicate-based cements on osteoclastic activity. METHODS: MC3T3-E1 cells were cultured for biocompatibility analyses. RAW 264.7 cells were cultured in the presence of the receptor activator of nuclear factor-kappa B and lipopolysaccharide, followed by treatment with Biodentine (BIOD) or ProRoot MTA with or without medicaments (Odanacatib [ODN], a cathepsin inhibitor and alendronate, a bisphosphonate). After drug treatment, the cell counting kit-8 assay and Alizarin red staining were performed to evaluate biocompatibility in MC3T3-E1 cells. Reverse-transcription polymerase chain reaction, tartrate-resistant acid phosphatase (TRAP) staining and enzyme-linked immunosorbent assays were performed in RAW 264.7 cells to determine the expression levels of inflammatory cytokines, interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2). Data were analyzed by one-way analysis of variance and Tukey's post hoc test (p < 0.05). RESULTS: Biocompatibility results showed that there were no significant differences among any of the groups. RAW 264.7 cells treated with BIOD and ODN showed the lowest levels of TNF-α and PGE2. Treatments with BIOD + ODN were more potent suppressors of inflammatory cytokine expression (p < 0.05). CONCLUSION: The cathepsin K inhibitor with calcium silicate-based cement inhibits osteoclastic activity. This may have clinical application in preventing inflammatory root resorption in replanted teeth.

Humanin suppresses receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation via AMP-activated protein kinase activation

Humanin (HN) is a mitochondrial peptide that exhibits cytoprotective actions against various stresses and diseases. HN has been shown to induce the phosphorylation of AMP-activated protein kinase (AMPK), which is a negative regulator of receptor activator of nuclear factor-κB ligand (RANKL). However, the role of HN in osteoclastogenesis or other skeletal disorders remains unknown. Here, we examined whether HN regulates osteoclastogenesis via AMPK activation using bone marrow-derived macrophage (BMM) cultures. Our results show that HN inhibited RANKL-induced osteoclast formation and reduced the expression of genes involved in osteoclastogenesis, including nuclear factor of activated T-cells cytoplasmic 1, osteoclast-associated receptor, cathepsin K, and tartrate-resistant acid phosphatase. Moreover, HN increased the levels of phosphorylated AMPK protein; compound C, an AMPK inhibitor, recovered HN-induced osteoclast differentiation. In addition, we found that HN significantly decreased the levels of RANKL-induced reactive oxygen species in BMMs. Therefore, these results indicate that HN plays an important role in osteoclastogenesis and may function as an inhibitor of bone disorders via AMPK activation.

SPA0355 prevents ovariectomy-induced bone loss in mice

Estrogen withdrawal in post-menopausal women leads to overactivation of osteoclasts, which contributes to the development of osteoporosis. Inflammatory cytokines are known as one of mechanisms of osteoclast activation after estrogen deficiency. SPA0355 is a thiourea derivative that has been investigated for its antioxidant and anti-inflammatory activities. However, its efficacy in bone resorption has not been previously investigated. The aim of this study was to investigate the impact of SPA0355 on the development of osteoporosis and to explore its mode of action. In vitro experiments showed that SPA0355 inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in primary bone marrow-derived macrophages. This effect appears to be independent of estrogen receptor activation as ICI 180,782 failed to abrogate its effects on osteoclasts. Further signaling studies revealed that SPA0355 suppressed activation of the MAPKs, Akt, and NF-κB pathways. SPA0355 also increased osteoblastic differentiation, as evidenced by its effects on alkaline phosphatase activity and mineralization nodule formation. Intraperitoneal administration of SPA0355 to ovariectomized mice prevented bone loss, as verified by three-dimensional images and bone morphometric parameters derived from µCT analysis. Noticeably, SPA0355 did not show hepatotoxicity and nephrotoxicity and also had little effect on hematological parameters. Taken together, the results indicate that SPA0355 may protect against bone loss in ovariectomized mice by stimulation of osteoblast differentiation and by inhibition of osteoclast resorption. Therefore, SPA0355 is a safe and potential candidate for management of postmenopausal osteoporosis.

Regulation of Osteoclast Differentiation by Cytokine Networks

Cytokines play a pivotal role in maintaining bone homeostasis. Osteoclasts (OCs), the sole bone resorbing cells, are regulated by numerous cytokines. Macrophage colony-stimulating factor and receptor activator of NF-κB ligand play a central role in OC differentiation, which is also termed osteoclastogenesis. Osteoclastogenic cytokines, including tumor necrosis factor-α, IL-1, IL-6, IL-7, IL-8, IL-11, IL-15, IL-17, IL-23, and IL-34, promote OC differentiation, whereas anti-osteoclastogenic cytokines, including interferon (IFN)-α, IFN-β, IFN-γ, IL-3, IL-4, IL-10, IL-12, IL-27, and IL-33, downregulate OC differentiation. Therefore, dynamic regulation of osteoclastogenic and anti-osteoclastogenic cytokines is important in maintaining the balance between bone-resorbing OCs and bone-forming osteoblasts (OBs), which eventually affects bone integrity. This review outlines the osteoclastogenic and anti-osteoclastogenic properties of cytokines with regard to osteoimmunology, and summarizes our current understanding of the roles these cytokines play in osteoclastogenesis.

Lysophosphatidic acid enhances breast cancer cells-mediated osteoclastogenesis

Lysophosphatidic acid (LPA) is known to play a critical role in breast cancer metastasis to bone. In this study, we tried to investigate any role of LPA in the regulation of osteoclastogenic cytokines from breast cancer cells and the possibility of these secretory factors in affecting osteoclastogenesis. Effect of secreted cytokines on osteoclastogenesis was analyzed by treating conditioned media from LPA-stimulated breast cancer cells to differentiating osteoclasts. Result demonstrated that IL-8 and IL-11 expression were upregulated in LPA-treated MDA-MB-231 cells. IL-8 was induced in both MDA-MB-231 and MDA-MB-468, however, IL-11 was induced only in MDA-MB-231, suggesting differential LPARs participation in the expression of these cytokines. Expression of IL-8 but not IL-11 was suppressed by inhibitors of PI3K, NFkB, ROCK and PKC pathways. In the case of PKC activation, it was observed that PKCδ and PKCμ might regulate LPA-induced expression of IL-11 and IL-8, respectively, by using specific PKC subtype inhibitors. Finally, conditioned Medium from LPA-stimulated breast cancer cells induced osteoclastogenesis. In conclusion, LPA induced the expression of osteolytic cytokines (IL-8 and IL-11) in breast cancer cells by involving different LPA receptors. Enhanced expression of IL-8 by LPA may be via ROCK, PKCu, PI3K, and NFkB signaling pathways, while enhanced expression of IL-11 might involve PKCδ signaling pathway. LPA has the ability to enhance breast cancer cells-mediated osteoclastogenesis by inducing the secretion of cytokines such as IL-8 and IL-11.

Amentoflavone inhibits debris-induced osteolysis and osteoclastogenesis through blocking MAPK pathway / 实用医学杂志

Objective To investigate the effect of amentoflavone(AMF)on inhibiting wear debris-in-duced osteolysis in vivo and RANKL-induced osteoclastogenesis in vitro. Methods Twenty-four male C57BL/J6 mice were randomly divided into four groups:the control group,titanium group,the low and high concentration group.Micro-CT and histological analysis were performed.CCK-8 assay was used to determin the effect of AMF on the proliferation of BMMs.TRAP staining and bone resorption assays were used to investigate the effect of AMF on osteoclastogenesis and function. The effect of AMF on prevention MAPK signaling was detected by Western blot assay. Results AMF could prevent osteolysis in vivo and suppress osteoclastogenesis,bone resorption without cytotoxicity in vitro.AMF suppressed MAPK signaling pathways.Conclusion AMF could inhibit osteoclastogenesis and titanium debris-induced osteolysis through suppressing MAPK signaling.

Effects of water extracts of Davallia formosana on ovariectomized mice / 中草药·英文版

Objective: The Taiwanese native fern, Davallia formosana, is used to treat bone diseases in traditional Chinese medicine, but, very few animal tests to verify. To study how this fern and its active components alter bone metabolism. Methods: The effects of low and high doses of D. formosana water extracts (DFW, 50 and 200 mg/(kg·d)) and (-)-epicatechin-3-O-D-allopyranoside isolates of DFW (ECAP, 0.06 and 2.5 mg/(kg·d)) on protein levels of important indicators of osteoclast and osteoblast activities were examined. Ovariectomized mice were used as a model of osteoporosis. Bone morphogenetic protein 2 (BMP-2), collagen 1 (COL-1), alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) were analyzed using western blotting of femur tissue. Interleukin (IL)-1β and tumor necrosis factor (TNF)-α were measured in the serum, and hematoxylin and eosin staining were used to assess bone morphology. Results: Treatment with DFW or ECAP significantly induced BMP-2, COL-1, ALP, RUNX2, OCN, and OPN protein expression, indicating stimulation of osteogenesis. The treatments also reduced IL-1β and TNF-α expression, indicating inhibition of osteoclastogenesis. In histological examinations, mice treated with DFW or ECAP had more bone trabeculae. The results demonstrate that DFW and ECAP inhibit osteoclast differentiation and promote osteoblast differentiation, and effectively ameliorate ovariectomy (OVX)-induced osteoporosis. Conclusion: ECAP is a major component of DFW, which may have therapeutic potential for the treatment of diseases associated with excessive osteoclastic and insufficient osteogenic activity. ECAP may not be the specific active molecule because it is unstable in the stomach. However, the major active molecule needs to be confirmed using a relevant pharmacological model.