RANK mRNA Expression Research Articles

Bone destruction in chronic otitis media is not mediated by the RANKL pathway or estrogen receptor-alpha.

Chronic otitis media with or without cholesteatoma progresses with various degrees of bone resorption and remodeling. Estrogen mediates osteoprotective effects through the receptor activator of NF-κB ligand (RANKL) pathway, which is mainly mediated by estrogen receptor-alpha (ER-α). The present study investigated the expression patterns of receptor activator of NF-κB (RANK), osteoprotegerin (OPG), RANKL, and ER-α in pathological tissue from patients with chronic otitis media to determine the roles of those factors in osteolytic mechanisms underlying the pathogenesis of chronic otitis media. Normal and pathological specimens from 18 patients with chronic otitis media were examined. There were no significant differences in RANK, OPG, RANKL, or ER-α mRNA expression between normal and pathological specimens of epithelial tissue. Our findings suggested that RANK, OPG, RANKL, and ER-α are not associated with the bone destruction in chronic otitis media; other cytokines may directly activate the osteoclasts in chronic otitis media.

Bone remodelling marker expression in grafted and ungrafted sheep tooth extraction sockets: A comparative randomised study

ObjectiveTo compare key markers of bone remodelling in a sheep tooth extraction model for sockets left to heal naturally or grafted with the bovine-derived xenograft Bio-Oss® covered with a collagen Bio-Gide® membrane. DesignRight side premolar teeth were removed from thirty Romney-cross ewes. Standardised sockets in each sheep were randomly allocated treatments, a grafted test and an empty control. At 4-, 8- and 16-weeks sheep were euthanized and tissue collected (N = 10/group). RANK, RANKL and OPG immunohistochemical analysis was performed (n = 3). RANK, RANKL, OPG, COL1A1, TIMP3, SP7 and MSX2 mRNA expression levels were determined using RT2-qPCR assays (n = 3). ResultsHistologically, more new woven bone was observed in the test group at all time points. Strong RANK and RANKL expression was found in both groups; at all time points with stronger RANK staining in the test group at 8 and 16 weeks. Strong OPG staining was localized to both osteoblasts and connective tissues. RANK receptor mRNA was expressed at a lower level in the test group (−4.26-fold; p = 0.02) at 4 weeks and SP7 at 16 weeks (−2.89-fold; p = 0.04). COL1A1 and TIMP3 mRNA expression increased significantly over time in the control group (p = 0.045, F = 5.4 and p = 0.003, F = 42.2 respectively). ConclusionSocket healing over time was comparable. The sheep tooth extraction model was found to be suitable for the evaluation of changes in the alveolar bone at the molecular level.

A novel Hoxd13 mutation causes synpolydactyly and promotes osteoclast differentiation by regulating pSmad5/p65/c-Fos/Rank axis

The mutations of HOXD13 gene have been involved in synpolydactyly (SPD), and the polyalanine extension mutation of Hoxd13 gene could lead to SPD in mice. In this study, a novel missense mutation of Hoxd13 (NM_000523: exon2: c.G917T: p.R306L) was identified in a Chinese family with SPD. The mice carrying the corresponding Hoxd13mutation were generated. The results showed that the homozygous mutation of Hoxd13 also caused SPD, but heterozygous mutation did not affect limbs development, which was different from that of SPD patients. With the increasing generation, the mice with homozygous Hoxd13 mutation presented more severe syndactyly. Western blotting showed that this mutation did not affect the protein expression of Hoxd13, suggesting that this mutation did not result in haploinsufficiency. Further analysis demonstrated that this homozygous Hoxd13mutation promoted osteoclast differentiation and bone loss, and enhanced the mRNA and protein expression of osteoclast-related genes Rank, c-Fos, and p65. Meanwhile, this homozygous Hoxd13 mutation elevated the level of phosphorylated Smad5 (pSmad5). Co-immunoprecipitation verified that this mutation attenuated the interaction between pSmad5 and HOXD13, suggesting that this mutation released more pSmad5. Inhibition of pSmad5 reduced the expression of Rank, c-Fos, and p65 despite in the mutation group. In addition, inhibition of pSmad5 repressed the osteoclast differentiation. ChIP assay confirmed that p65 and c-Fos could bind to the promoter of Rank. These results suggested that this novel Hoxd13 mutation promoted osteoclast differentiation by regulating Smad5/p65/c-Fos/Rank axis, which might provide a new insight into SPD development.

Characterizing the Role of Calcium Sensing Receptor in the Progression of Obesity-Mediated Aggressive Prostate Cancer Phenotype

Obesity increases the risk of advanced prostate cancer (PCa). The calcium sensing receptor (CaSR) has been shown to be responsive to obesity-mediated cytokines and is upregulated in metastatic PCa. This study used a novel in vitro approach, involving the exposure of PCa cells to sera, from obese or normal weight males, and to CaSR inhibitor NPS-2143. Cell viability was determined using MTT assay. MMP-9 activity and invasion were assessed using zymography and invasion chambers, respectively. Microscopy was used to visualize EMT proteins. qRT-PCR and immunoblot analysis were used to quantify changes in genes and proteins important for tumorigenesis. Exposure to obese sera increased the proliferation, and the invasive capacity of PCa cells and de-localized epithelial-mesenchymal transition markers, which were attenuated with CaSR inhibition. Exposure to obese sera upregulated mRNA expression of PTHrP and protein expression of COX-2, IL-6, and CaSR. Inhibition of CaSR downregulated the mRNA expression of PTHrP and RANK, and protein expression of pERK and TNF-α. Obesity was shown to increase invasion and upregulate the expression of genes and proteins involved in PCa tumorigenesis. CaSR inhibition downregulated the expression of several of these factors. Thus, CaSR is a potentially important protein to target in obesity-mediated PCa progression.

Effects of Co-exposure to Fluoride and Arsenic on TRAF-6 Signaling and NF-κB Pathway ofBoneMetabolism.

Little is known about the combined effect of fluoride (F)and arsenic (As) on bone metabolism. This study aims to explore the effect of co-exposure to F and As on the expressions of TNF receptor-associated factor 6 (TRAF-6), nuclear factor-kappa B (NF-κB), and the related factors in cell and animal experiments. With the rats exposed to different doses of F, As, and combined F-As, we found that F exposure doses were positively correlated with the protein expression of receptor activator of nuclear factor-kappa B ligand (RANKL), receptor activator of nuclear factor-kappa B (RANK), TRAF-6, NF-κB, and nuclear factor of activated T cells (NFAT-c1) (P < 0.001). As exposure doses were negatively correlated with RANK, TRAF-6, NF-κB, and NFAT-c1 (P < 0.001). The effect of F and As interaction on the protein expression of RANKL, TRAF-6, NF-κB, and NFAT-c1 was significant in bone tissue (P < 0.05). In the cellular experiment, F could promote the mRNA expression of RANK, TRAF-6, and NFAT-c1. A higher concentration of As could inhibit the mRNA expression of Tartrate-resistant acid phosphatase (TRAP), RANK, TRAF-6, and NFAT-c1. The effect of F and As interaction on the mRNA expression of TRAP, RANK, TRAF-6, and NFATc1 in osteoclasts was significant (P < 0.001). In conclusion, the expression of TRAF-6 and NF-κB pathway was affected by F and As co-exposure in osteogenic differentiation, and As could antagonize the promoting effect of F on the expression of TRAF-6, TRAP, RANKL, RANK, NF-κB, and NFAT-c1 in these exposure levels. These results could provide a scientific basis for understanding the interaction of F and As in bone formation.

Histological Grade and Tumor Stage Are Correlated with Expression of Receptor Activator of Nuclear Factor Kappa b (Rank) in Epithelial Ovarian Cancers.

The receptor activator of nuclear factor kappa B (RANK) is becoming recognized as a master regulator of tumorigenesis, yet its role in gynecological cancers remains mostly unexplored. We investigated whether there is a gradation of RANK protein and mRNA expression in epithelial ovarian cancer (EOC) according to malignancy and tumor staging. Immunohistochemical expression of RANK was examined in a cohort of 135 (benign n = 29, borderline n= 23 and malignant n = 83) EOCs. Wild type and truncated RANK mRNA isoform quantification was performed in a cohort of 168 (benign n = 26, borderline n = 13 and malignant n = 129) EOCs. RANK protein and mRNA values were increased in malignant vs. benign or borderline conditions across serous, mucinous and endometrioid cancer subtypes. Additionally, a trend of increased RANK values with staging was observed for the mucinous and serous histotype. Thus, increased expression of RANK appears associated with the evolution of disease to the onset of malignancy in EOC. Moreover, in some EOC histotypes, RANK expression is additionally associated with clinicopathological markers of tumor aggressiveness, suggesting a role in further progression of tumor activity.

Alleviative Effects of Exercise on Bone Remodeling in Fluorosis Mice.

Fluorine is widely present in nature in the form of fluoride. Prolonged high-dose fluoride exposure can cause skeletal fluorosis, resulting in osteosclerosis, osteoporosis or osteomalacia. It has been proved that exercise is one of the important factors affecting the health of the bone and promoting bone formation. To investigate the effects of exercise on bone remodeling in fluorosis mice, 120 male 3-week-old ICR mice were randomly divided into four groups: control group (C), exercise group (E), fluoride group (F), fluoride plus exercise group (F + E). After 8-week physical exercise and/or fluoride exposure, we evaluated the content of fluorine, the histopathological structure and microstructure of femur, bone metabolism biochemical indexes and oxidative stressrelated parameters, and the mRNA and protein levels of genes in BMP-2/Smads and OPG/RANKL/RANK signaling pathways. Our results showed that 100mg/L NaF exposure increased the accumulation of fluoride in bone, altered histology of bone, and enhanced the activities of ALP and TRACP. Meanwhile, excessive fluoride induced oxidative stress in bone tissue by increasing the content of ROS and MDA, and decreasing the activities of antioxidant enzymes. In addition, the results of qRT-PCR suggested that NaF significantly increased the mRNA expression of BMP-2, Smad-5, Col IA1, Col IA2, OPG, RANKL and RANK, as well as the elevated proteins of OPG, RANKL and RANK. However, these fluoride-induced changes were alleviated after moderate exercise. Taken together, these findings indicated that moderate exercise decreased the toxicity of fluoride by reducing the accumulation of fluorine in the body to relieve the bone damage caused by fluorosis.

Inhibition Effect of Zoledronate on the Osteoclast Differentiation of RAW264.7 Induced by Titanium Particles.

Objective This study is aimed at studying the effect of zoledronate (ZOL) on the differentiation of osteoclast precursor RAW264.7 cells induced by titanium (Ti) particles and explores the possibility of preventing and treating periprosthetic osteoporosis using ZOL. Methods RAW264.7 cells were cultured in vitro. Ti particles were prepared. The cell proliferation curve of RAW264.7 cells was plotted using the MTT assay to find the best concentration of ZOL for intervention. The cells were divided into three groups: control, Ti particles, and Ti particles+ZOL. The cell morphology was observed using tartaric acid–resistant acid phosphatase (TRAP) staining, and the activity of TRAP in cell supernatant was determined using the biochemical method. The number of bone resorption lacunae was detected using toluidine blue staining. The mRNA expression of RANK, NFATcl, CAII, and MMP-9 was detected using real-time polymerase chain reaction. The protein expression of RANK, NFATcl, and MMP-9 was detected using Western blot analysis. Results Ti particles stimulated the differentiation of RAW264.7 cells into osteoclasts. They also increased the activity of TRAP, number of bone resorption lacunae, and mRNA and protein expression of RANK, NFATcl, and MMP-9. However, ZOL could suppress the effect of TI particles on the osteoclast differentiation of RAW264.7 cells. Conclusions ZOL could effectively inhibit the differentiation of RAW264.7 cells into osteoclasts induced by Ti particles, decrease the activity of TRAP, reduce the number of bone resorption lacunae, and decrease the mRNA and protein expression of RANK, NFATcl, and MMP-9. Hence, it may be a promising candidate for preventing and treating periprosthetic osteoporosis after the artificial joint operation.

LncRNA NORAD promotes bone marrow stem cell differentiation and proliferation by targeting miR-26a-5p in steroid-induced osteonecrosis of the femoral head

BackgroundSteroid-induced osteonecrosis of the femoral head (SONFH) is a devastating orthopedic disease, which seriously affects the quality of life of patients. The study aims to investigate the effects of LncRNA NORAD on SONFH.MethodsHuman bone marrow-derived mesenchymal stem cells (hBMSCs) were isolated from the proximal femur of patients during routine orthopedic surgery and then cultured with dexamethasone (Dex) and transfected with NORAD overexpression vector, siRNA-NORAD and miR-26a-5p mimics. The mRNA expression of NORAD, miR-26a-5p, OPG, RANK, and RANKL was detected by RT-qPCR. Cell proliferation and apoptosis was measured by CCK-8 assay and flow cytometry, respectively. The protein expression of RUNX2, OPG, RANK, and RANKL was detected by western blot. The dual-luciferase reporter gene assay was performed to confirm the binding between NORAD and miR-26a-5p.ResultsNORAD expression was downregulated in SONFH tissues, while miR-26a-5p expression was upregulated. Overexpression of NORAD improved DEX-induced inhibition of proliferation and differentiation, and promotion of apoptosis in hBMSCs, while knockdown of NORAD led to the opposite results. Moreover, NORAD improved DEX-induced inhibition of proliferation and differentiation, and promotion of apoptosis by regulation of miR-26a-5p in hBMSCs.ConclusionsNORAD expression was downregulated in SONFH tissues, while miR-26a-5p expression was upregulated. NORAD improved DEX-induced inhibition of proliferation and differentiation, and promotion of apoptosis by regulation of miR-26a-5p in hBMSCs.

Deep learning enables discovery of highly potent anti-osteoporosis natural products.

A pre-trained self-attentive message passing neural network (P-SAMPNN) model was developed based on our anti-osteoclastogenesis dataset for virtual screening purpose. Validation processes proved that P-SAMPNN model was significantly superior to the other base line models. A commercially available natural product library was virtually screened by the P-SAMPNN model and resulted in confirmed 5 hits from 10 selected virtual hits. Among the confirmed hits, compounds AP-123/40765213 and AE-562/43462182 are the nanomolar inhibitors against osteoclastogenesis with a new scaffold. Further studies indicate that AP-123/40765213 and AE-562/43462182 significantly suppress the mRNA expression of RANK and downregulate the expressions of osteoclasts-related genes Ctsk, Nfatc1, and Tracp. Our work demonstrated that P-SAMPNN method can guide phenotype-based drug discovery.

Lactobacillus Fermentum ZS40 prevents secondary osteoporosis in Wistar Rat.

Using retinoic acid to inducer, we successfully established a rat model of secondary osteoporosis and verified the preventive effect of Lactobacillus fermentum ZS40 (ZS40) on secondary osteoporosis. Serum biochemical indicators showed that ZS40 can effectively slow down bone resorption caused by retinoic acid, increase blood content of calcium, phosphorus, bone alkaline phosphatase, bone gla protein, and insulin‐like growth factor 1, and decrease blood content of tartrate‐resistant acid phosphatase (TRAP) 5b. qRT‐PCR results showed that ZS40 could upregulate mRNA expressions of β‐catenin, Wnt10b, Lrp5, Lrp6, Runx2, ALP, RANKL, and OPG, and downregulate mRNA expression of DKK1, RANK, TRACP, and CTSK in the rats’ spinal cord. Results following TRAP staining showed that ZS40 could slow down retinoic acid‐induced formation of osteoclasts. Micro‐CT results showed that ZS40 could reduce Tb.Sp, increase BV/TV, Tb.N, Tb.Th, and ultimately increase bone mineral density of rats in vivo. These findings indicate that ZS40 might have a potential role in preventing retinoic acid‐induced secondary osteoporosis in vivo.

RANK-RANKL signaling upregulates Il-10 mRNA expression in mucosal Candida infection in vivo

Osteoprotegerin (OPG) prevents binding of receptor activator of nuclear factor-kappa B ligand (RANKL) to RANK. Recent studies have reported that immune cell RANK-RANKL interactions are critical to the infection process. Candida albicans is an opportunistic pathogenic fungus and a common cause of candidiasis. This study utilized an orally inoculated mouse model of C. albicans infection to determine whether superficial or systemic candidiasis was associated with alterations in RANK/RANKL/OPG expression. Invasive systemic C. albicans infection increased serum OPG levels in mice. In addition, tongue Opg, Rankl, and Rank mRNA expression were upregulated in mice with superficial oral cavity C. albicans infection. Moreover, administration of exogenous soluble RANKL upregulated Rank and interleukin-10 (Il-10) mRNA in superficially infected tissue, suggesting suppression of localized inflammation. Taken together, these findings suggested that RANK/RANKL/OPG signaling contributes to the pathogenesis of candidiasis. This is the first in vivo study to identify a relationship between this opportunistic infection and the RANK/RANKL/OPG axis.

TNF-α stimulates the expression of RANK during orthodontic tooth movement

ObjectiveThis study aimed to evaluate the effects of tumor necrosis factor (TNF)-α on receptor activator of nuclear factor-κB (RANK) expression in osteoclast precursors in vitro and during orthodontic tooth movement (OTM) in vivo. DesignWe assessed whether TNF-α influenced RANK expression levels in osteoclast precursors in vitro by real-time PCR and western blotting. For in vivo experiments, TNF-α was subcutaneously injected into mouse calvariae daily for 5 days. Mice were sacrificed and RANK expression was evaluated by real-time PCR and immunohistochemistry. For OTM, a nickel-titanium closed-coil spring was fixed between the upper incisors and upper-left first molar to move the first molar in the mesial direction in wild-type (WT) and TNFR1/TNFR2-deficient (TNFRsKO) mice. After OTM, the number of RANK-positive cells on the compression side was evaluated by immunohistochemistry. ResultsRANK levels were enhanced in TNF-α-treated osteoclast precursors in vitro. RANK mRNA expression levels and the number of RANK-positive cells were higher in TNF-α-injected mice than in phosphate-buffered saline-injected mice. RANK-positive cells increased on the compression side of the alveolar bone in WT mice because of the mechanical loading. In addition, the number of RANK-positive cells on the compression side was significantly higher in WT mice than in TNFRsKO mice after OTM. ConclusionThese results suggest that TNF-α induces RANK expression in vitro and at baseline in vivo, as well as on the compression side during OTM.

Multitarget-based cotreatment with cilostazol and celecoxib synergistically suppresses collagen-induced arthritis in mice by enhancing interleukin-10 expression

Cilostazol exerts potent anti-inflammatory effects and celecoxib, a COX-2 specific inhibitor, improves the unsatisfactory profile of NSAIDs. It was aimed to assess the anti-arthritic potential of celecoxib add-on for cilostazol therapy in collagen induced arthritis (CIA), and to elucidate the implication of interleukin (IL)-10 in the action of cilostazol and celecoxib cotreatment. Cotreatment of RAW 264.7 cells with 10 μM cilostazol and 0.3 μM celecoxib synergistically suppressed RANKL-induced increases in RANK mRNA and protein levels. When cultured in the presence of RANKL for 5 days, RANKL-stimulated expressions of osteoclastogenic genes (OSCAR, DC-STAMP, and cathepsin K mRNA) and the expression of RANK mRNA were markedly elevated. Furthermore, these gene expressions, including that of RANK, were significantly suppressed by cotreatment with cilostazol (10 μM) and celecoxib (0.3 μM). In addition, this co-treatment strongly down-regulated RANKL-induced NFATc1 protein and TRAP activity (key osteoclastogenic factors), and these down-regulations were significantly prevented by pretreating cells with IL-10 neutralizing antibody. Furthermore, increased osteoclast formation and extensive resorption pit formation by bone marrow-derived monocytes obtained from C57BL/6 mice cultured in the presence of M-CSF/RANKL were markedly suppressed by cilostazol and celecoxib cotreatment. Consequently, hindlimb paw thicknesses in DBA/1J CIA mice were significantly reduced by cilostazol (10 mg/kg/d) and celecoxib (5 mg/kg/d) cotreatment. These results were accompanied by synergistic suppression of cartilage depletion and bone erosion and reductions in arthritis scores in the CIA mice. In conclusion, serum IL-10 levels in these mice were markedly increased by cilostazol and celecoxib cotreatment, whereas elevated serum IL-1β levels were markedly reduced. Cotreatment with low-dose cilostazol and celecoxib may ensure the synergistic anti-arthritic potential.

Activation of protein kinase C accelerates murine osteoclastogenesis partly via transactivation of RANK gene through functional AP-1 responsive element in RANK gene promoter

Receptor activator of NF-κB (RANK) expressed on osteoclasts and their precursors is a receptor for RANK ligand (RANKL). Signals transduced by RANKL-RANK interaction induce genes essential for the differentiation and function of osteoclasts. We have cloned a basic promoter region of the mouse RANK gene and have analyzed the transcription machinery by transcription factors such as PU.1 (−480), and MITF (−100). Here, we examined the regulatory mechanisms of RANK gene transcription through AP-1 binding site, agagctca (−240). RANK mRNA expression in pre-osteoclastic RAW264.7 cells was induced by Phorbol12-myristate13-acetate (PMA) and suppressed by protein kinase C (PKC) inhibitor calphostin C. In RAW264.7 cells, Fos knockdown by siRNA blocked the inducible effect of PMA on RANK expression. By EMSA, an oligonucleotide (−246/-238) showed DNA protein binding, the specificity of which was confirmed by block-shift assay with an anti-Fos antibody and by the addition of the excess of a cold consensus probe. Co-transfection with a Fos expression vector showed that Fos increased RANK promoter activity 6-fold in RAW264.7 cells, and the addition of PU.1 and MITF superinduced the activity more than twenty-fold by the addition of PU.1 and MITF. Mutagenesis of the putative AP-1 site (−240) blocked the inducible effect of Fos on promoter activity. Taken together, these results indicate that during the differentiation of bone marrow mono-nucleated cells into osteoclast precursors, RANK transcription is positively regulated by Fos/AP-1 through the binding element of its gene promoter, supporting the concept that Fos activation by continuous CSF-1 stimulation on macrophages triggers initial expression of RANK and, later, a positive feedback loop by RANKL-RANK interaction.

Expression of the RANK/RANKL/OPG system in the human intervertebral disc: implication for the pathogenesis of intervertebral disc degeneration

BackgroundThe expression of the receptor activator of nuclear factor kappa B (RANK) /RANK ligand (RANKL) /osteoprotegerin (OPG) system and its association with the progression of intervertebral disc (IVD) degeneration has recently been reported in a human IVD. However, the effect of the RANK/RANKL/OPG system on the matrix metabolism of human IVD cells, especially on the expression of catabolic factors relevant to IVD degeneration, remains unknown. The purpose of this study was to examine the expression of the RANK/RANKL/OPG system, and then to evaluate the effect of this system on the expression of catabolic factors by human IVD cells.MethodsAnnulus fibrosus (AF) and nucleus pulposus (NP) cells isolated by sequential enzyme digestion from human IVD tissues obtained during spine surgeries were monolayer cultured. The expression of the RANK/RANKL/OPG system was determined using immunohistochemical methods and real-time polymerase chain reaction (PCR). To evaluate the influence of interleukin-1 beta (IL-1β) stimulation on the mRNA expression of RANK, RANKL, and OPG, recombinant human IL-1β (rhIL-1β) was administered in the culture media of IVD cells. To examine the influence of RANKL signaling on the expression of matrix metalloprotease-3 (MMP-3), MMP-13, and IL-1β, the cells were cultured with exogenous recombinant human RANKL (rhRANKL), recombinant human OPG (rhOPG) or anti-human RANKL mouse monoclonal antibody (ahRANKL-mAB) with or without rhIL-1β.ResultsImmunoreactivity to RANK/RANKL/OPG and the mRNA expression of the three genes were obviously identified in both AF and NP cells. rhIL-1β stimulation significantly upregulated the mRNA expression level of RANK/RANKL/OPG. The mRNA expression of catabolic factors was significantly upregulated by stimulation of rhRANKL in the presence of rhIL-1β. On the other hand, the administration of either rhOPG or ahRANKL-mAB significantly suppressed the mRNA expression of catabolic factors that had been upregulated by rhIL-1β stimulation. The suppressive effect of ahRANKL-mAB against rhIL-1β stimulation was also confirmed by the protein expression of MMP-3.ConclusionsThe present study showed that the RANK/RANKL/OPG system may be involved in the progression of IVD degeneration. This study also suggested the potential use of anti-RANKL monoclonal antibody and OPG as therapeutic agents to suppress the progression of IVD degeneration.

Effect of salmon calcitonin on RANK/RANKL/OPG osteolysis pathway in human macrophages after particle induction

Objective To explore the effect of salmon calcitonin on the receptor activator of NF-κB/receptor activator of NF-κB ligand/osteoprotegerin (RANK/RANKL/OPG) osteolysis pathway in human macrophages after particles induction. Methods The polyethylene wear particles were extracted from the periacetabular boundary membrane tissue of a patient with hip prosthesis loosening. The optimal reaction cell concentration of human macrophages to polyethylene wear particles (with a concentration of 0.1 mg/ml) was measured by methyl thiazolyl tetrazolium (MTT) assay. Particles were used to stimulate human macrophages, while salmon calcitonin with different drug concentrations was used for intervention. They were randomly divided into five groups, with six parts in each group: Group A, control group; Group B, particle group; Group C, particle+ salmon calcitonin (10-8 mol/L) group; Group D, particle+ salmon calcitonin (10-10 mol/L) group; Group E, particle+ salmon calcitonin (10-12 mol/L) group. After cocultured for 48 hours, quantitative polymerase chain reaction (qPCR) assay was used to detect the mRNA expression of RANK, RANKL and OPG in macrophages. Results After induction with particles, Group B had a higher expression in RANK and RANKL, and lower OPG expression than Group A. After salmon calcitonin intervention, the expression of RANK and RANKL were significantly decreased, and OPG expression was significantly increased. Group B had the highest RANKL/OPG rate. After drug intervention, the RANKL/OPG rate in C, D, E group were reduced. Conclusions The particles can induce the transformation of macrophages into osteoclasts. In addition to directly inhibiting osteoclast activity, salmon calcitonin prevents macrophage from differentiating into osteoclasts through modulating RANKL/RANK/OPG signaling pathway. Key words: Calcitonin; Macrophages; Receptor activator of nuclear factor-kappa B; RANK ligand; Osteoprotegerin; Osteolysis; In vitro

Immunohistochemical and mRNA expression of RANK, RANKL, OPG, TLR2 and MyD88 during apical periodontitis progression in mice.

Objective To evaluate and correlate, in the same research, the mRNA expression and the staining of RANK, RANKL, OPG, TLR2 and MyD88 by immunohistochemistry in the apical periodontitis (AP) progression in mice.Material and Methods AP was induced in the lower first molars of thirty-five C57BL/6 mice. They were assigned to four groups according to their euthanasia periods (G0, G7, G21 and G42). The jaws were removed and subjected to histotechnical processing, immunohistochemistry and real-time reverse transcription-PCR (qRT-PCR). Data were analyzed with parametric and nonparametric tests (α=0.05).Results An increase of positive immunoreactivity for RANK, RANKL, OPG, TLR2 and MyD88 was observed over time (p<0.05). The RANKL expression was different between the groups G0 and G42, G21 and G42 (p=0.006), with G42 presenting the higher expression in both comparations. The OPG expression was statistically different between the groups G0 and G7, G7 and G21 and G7 and G42 (p<0.001), with G7 presenting higher expression in all the time points. The TLR2 expression was different between the groups G0 and G42 (p=0.03), with G42 showing the higher expression. The MyD88 expression presented a statistical significant difference between groups G7, G21 and G42 compared with G0 (p=0.01), with G0 presenting the smallest expression in all the comparisons. The Tnfrsf11/Tnfrsf11b (RANKL/OPG) ratio increased with the AP progression (p=0.002). A moderate positive correlation between MyD88 and RANKL (r=0.42; p=0.03) and between MyD88 and TLR2 (r=0.48; p<0.0001) was observed.Conclusion The expression of the RANK, RANKL, OPG, MyD88 and TLR2 proteins as well as the ratio Tnfrsf11/Tnfrsf11b (RANKL/OPG) increased with AP progression. There was also a moderate positive correlation between the expression Myd88-Tnfrsf11 and Tlr2-Myd88, suggesting the relevance of Tlr2-Myd88 in bone loss due to bacterial infection.

Cytokine expression in response to root repair agents.

To evaluate the expression of TNF-α, IL-6, IFN-γ, TGF-β, IL-4, IL-10, RANKL, RANK and OPG on mouse calvarial bone treated with MTA, Geristore® and Emdogain® . Bone wounds were made on the heads of C57BL/6 mice, breaking the periosteum and the cortical surface of the calvaria. Each repair agent was inserted into sectioned Eppendorf microtubes and placed on the bone wound, and soft tissues were sutured. At 14 and 21days, animals were sacrificed and the treated region was dissected. The calvaria bone was removed, and RNA was extracted. mRNA expression of the aforementioned cytokines was assessed using real-time PCR. Data were analysed by nonparametric methods, including the Mann-Whitney and Kruskal-Wallis tests (P<0.05). Following treatment with Emdogain® and MTA, mRNA expression of RANKL, RANK and OPG increased significantly (P<0.05) between days 14 to 21. Geristore® did not alter the basal expression of these mediators during the same period of evaluation. Whilst treatment with Emdogain® did cause a significant increase in TNF-α mRNA expression between days 14 and 21 (P<0.05), treatment with MTA did not alter the basal expression of this cytokine at either experimental time point. However, TNF-α mRNA expression was down-regulated significantly at day 21 (P<0.05) when Geristore® was applied. A significant increase in the mRNA expression of IL-6, TGF-β, IL-10, IL-4 and IFN-γ was observed with Emdogain® and MTA treatment between days 14 to 21, whereas Geristore® reduced significantly the expression of IL-6, TGF-β and IL-4 (P<0.05). The clinical indication of these repair agents depends on the root resorption diagnosis. Whilst MTA and Emdogain® induce a pro- and anti-inflammatory response early and late, respectively, Geristore® was not associated with an inflammatory reaction when compared with both repair agents.

Carbon Monoxide Releasing Molecule 3 Inhibits Osteoclastogenic Differentiation of RAW264.7 Cells by Heme Oxygenase-1

Background/Aims: Increased osteoclastogenic differentiation may disrupt the balance of bone resorption and formation, giving rise to bone defective disease. The study aimed to investigate the influence of carbon monoxide releasing molecule 3 on osteoclastogenic differentiation of RAW264.7 cells, and explore the possible mechanism underlying the regulatory effect. Methods: Influence of CORM-3 on the proliferation of RAW264.7 cells was determined by CCK-8 assay. RAW264.7 cells were divided into four groups: Control group; Osteoclastogenic differentiation group, in which cells were induced osteoclastogenic differentiation in medium supplemented with 100μg/L RANKL and 50μg/L M-CSF; Degassed CORM-3-osteoclastogenic differentiation group, in which cells were pretreated with 200μmol/L degassed CORM-3 for 6hrs, and then induced osteoclastogenic differentiation; CORM-3-osteoclastogenic differentiation group, in which cells were pretreated with 200μmol/L CORM-3, and then induced osteoclastogenic differentiation. The mRNA and protein expression of RANK, TRAP, MMP-9, Cts-K and HO-1 of the cells during the osteoclastogenic differentiation was checked by RT-qPCR and Western blot. The induced osteoclasts were identified by TRAP staining. The HO-1 expression of the RAW264.7 cells was silenced by lentivirus transfection, and the expression of RANK, TRAP, MMP-9 and Cts-K was examined by RT-qPCR and Western blot. Results: CORM-3 promoted the proliferation of RAW264.7 cells at the concentration of 200μmol/L. Pretreatment with CORM-3, but not degassed CORM-3, significantly decreased the mRNA and protein expression of osteoclast-specific marker TRAP, RANK, MMP-9 and Cts-K induced by RANKL and M-CSF on day 5, 7 and 9 during the osteoclastogenic differentiation (P< 0.05). After HO-1 was silenced by lentivirus transfection, the mRNA and protein expression of TRAP, RANK, MMP-9 and Cts-K in group with CORM-3 pretreatment maintained the same level as in osteoclastogenic differentiation group. Conclusion: CORM-3 inhibits osteoclastogenic differentiation of RAW264.7 cells via releasing CO. The inhibitory effect is mediated partially by HO-1 pathway. The results suggest the potential application of CORM-3 on some bone defective diseases.