Diferencia en la expresión del ligando de receptor activador para el factor nuclear K B, en tejido tumoral frente a la infección por diferentes patógenos periodontales / Difference in receptor activator of nuclear factor K B expression in tumoral tissue by different periodontal pathogens

La enfermedad periodontal es una de las principales causas de pérdida dentaria. Clínicamente, esta patología, mediada por la desregulación del sistema inmune producto de una disbiosis ocurrida en el surco gingival, inicia con la inflamación de la encía y evoluciona con el daño irreversible de los tejidos que rodean el diente. El hueso alveolar es uno de los tejidos afectados esta patología, esto debido a la activación de osteoclastos por la sobreexpresión de la proteína RANKL en el huésped. El propósito de este trabajo es determinar el nivel de sobreexpresión de RANKL, en un modelo de células tumorales U2OS, frente a la infección con Porphyromonas gingivalis y Prevotella intermedia. Para identificar el nivel de RANKL, se definieron cuatro grupos: Un grupo control, no tratado; Grupo PG, tratado con P. gingivalis; Grupo PI, tratado con P. Intermedia; y un grupo PG+PI, tratado con ambas bacterias. El nivel relativo de la proteína RANKL fue determinado en el sobrenadante y en los extractos celulares de manera independiente, mediante la técnica Western blot. En sobrenadantes, el grupo PG mostró mayores niveles de RANKL comparados con PI (p < 0,05). En extractos celulares los niveles fueron mayores en el grupo PG+PI (p < 0,05). El grupo PI mostró los niveles más bajos de RANKL. La infección polimicrobiana resulta en una mayor expresión de RANKL en células tumorales U2OS, mientras que frente a la infección P. gingivalis, se observó mayor cantidad de RANKL soluble.

SUMMARY: Periodontal disease is one of the main causes of tooth loss. Clinically, this pathology, mediated by the deregulation of the immune system due to a dysbiosis occurred in the gingival sulcus, begins with the inflammation of the gum and evolves with the irreversible damage of the tissues that surround the tooth. Alveolar bone is one of the most affected tissues by this disease, due to the activation of osteoclasts by the upregulation of RANKL in the host. The aim of this study is to determine the increase of RANKL, in a U2OS tumor cells model, inoculated with Porphyromonas gingivalis and Prevotella intermedia. To identify the level of RANKL, four groups were defined: A control group, not treated; PG group, treated with P.gingivalis; PI group, treated with P. intermedia; and a PG+PI group, treated with both bacteria. The relative level of RANKL was determined in the supernatant and cell extracts independently, using the Western blot technique. In supernatants, the PG group showed higher RANKL levels compared to PI (p < 0.05). In cell extracts the levels were higher in the PG+PI group (p < 0.05.). The PI group showed the lowest levels of RANKL.Polymicrobial infection results in a greater expression of of soluble RANKL was observed.

Beneficial effects of Cuscuta chinensis extract on glucocorticoid-induced osteoporosis through modulation of RANKL/OPG signals

Cuscuta chinensis Lam. (Convolvulaceae) is an important herbal medicine widely used to improve sexual function, treat osteoporosis, and prevent aging, and has been reported to exhibit anti-osteoporotic effects in vitro. However, the activity of Cuscuta chinensis Lam. on glucocorticoid-induced osteoporosis still remains unclear. The present study aimed to assess the protective effect and the underlying mechanism of action of Cuscuta chinensis extract (CCE) against glucocorticoid-induced osteoporosis in vivo. Sprague-Dawley rats were randomly divided into four groups as follows: control group, osteoporosis group, and 2 CCE-treated osteoporosis groups (100 mg·kg-1·day-1). Blood samples and femur bones were collected for immunohistochemistry, biochemical, mRNA expression, and western blot analysis. HPLC analysis revealed that chlorogenic acid, quercetin, and hyperin were the major constituents of CCE. The results indicated that CCE increased bone length, bone weight, and bone mineral density and suppressed dexamethasone (DEX)-induced reduction in body weight. In addition, TRAP staining indicated that CCE reduced osteoclasts in DEX-induced osteoporosis rats. Mechanistically, CCE treatment alleviated the increase of bone resorption markers and the decline of osteogenic markers, which might be partially mediated by regulation of RANKL/OPG and RunX2 pathways. These results suggest that CCE showed promising effects in the protection against glucocorticoid-induced osteoporosis through protecting osteoblasts and suppressing osteoclastogenesis.

Chronic Periodontitis and RANKL/OPG Ratio in Peri-Implant Mucosae Inflammation

Abstract tHistory of chronic periodontitis (CP) is a risk factor for oseointegration failure. The osteoclastogenesis system (RANK, RANKL and OPG) is critical for bone homeostatic control. We investigated the levels of OPG and RANKL in peri-implant tissues from volunteers with and without a history of CP and their association with mucosae inflammation. This is a single-blind case-contro study. Diagnosis of a history of CP and peri-implant examination was performed on 46 volunteers, divided into control (without history of CP, n=26) and CP group (with history of CP, n=20). Gingival biopsies were harvested during implant exposure. Quantitative PCR evaluated OPG/RANKL mRNA expressions. OPG and RANKL proteins were analyzed by western blot and immunohistochemistry assay. The chi-square test analyzed the significance of nominal variables between groups while continuous variables were analyzed by T-test or Mann-Whitney test, after Shapiro-Wilk test evaluation. The 2-ΔΔCT Livak method calculation evaluated the gene expression. Values of p<0.05 were considered statistically significant. Volunteers with CP history had 23 times higher chance of developing mucosae inflammation. High mucosae levels of RANKL (p=0.04) and RANKL/OPG (p=0.001) mRNA expressions were observed in CP group. CP volunteers showed increased RANKL protein levels in opposition to decreased OPG expression. Even without active periodontitis, volunteers with a history of CP had elevated gingival levels of RANKL/OPG and higher correlation with peri-implant mucosae inflammation and implant loss.

Resumo A história de periodontite crônica (CP) é um fator de risco para falhas na osseointegração. O sistema de osteoclastogênese (RANK, RANKL e OPG) é crucial para o controle da homeostase óssea. O objetivo deste estudo foi investigar os níveis de OPG e RANKL no tecido peri-implantar de voluntários com e sem histórico de CP e sua associação com inflamação da mucosa. Este é um estudo tipo caso-controle. O exame para diagnóstico de CP e na região peri-implantar foi realizado em 46 voluntários, divididos em controle (sem história CP, n=26) e grupo CP (com histórico de CP, n=20). Descartes gengivais foram obtidos durante a exposição do implante. PCR quantitativo avaliou a expressão do RNAm de OPG/RANKL. As proteínas OPG e RANKL foram analisadas por western blot e imunohistoquímica. O teste do qui-quadrado analisou a significância entre as variáveis nominais enquanto as variáveis contínuas foram analisadas pelo teste-t e Mann-Whitney, após o teste de Shapiro-wilk. O método do Livak 2--ΔΔCT avaliou a expressão gênica. Os voluntários com CP apresentaram 23 vezes mais chances de desenvolver inflamação da mucosa. Expressão elevada no RNAm de RANKL (p=0.04) e RANKL/OPG (p=0.001) foram observadas no grupo CP. Voluntários com CP mostraram aumento dos níveis da proteína RANKL em contraste com diminuída expressão de OPG. Mesmo sem periodontite ativa, voluntários com histórico de CP apresentaram elevado nível gengival de RANKL/OPG e alta correlação com inflamação peri-implantar e perda do implante.

Up-regulated expression of CD147 gene in malignant bone tumor and the possible induction mechanism during osteoclast formation

It is increasingly evident that the microenvironment of bone can influence cancer phenotype in many ways that favor growth in bone. CD147, a transmembrane protein of the immunoglobulin (Ig) superfamily, was identified independently in different species and has many designations across different species. However, expression levels of CD147 mRNA in bone cancer have not been described. In this study, we have used real-time fluorescence quantification (RT-PCR) to demonstrate CD147 expression in malignant bone cancer and benign bone tumor tissues. The results suggested that the expression of CD147 gene was significantly up-regulated in malignant bone cancer. Moreover, we found that over-expressed RANKL progressively enhanced osteoclast formation up to 48 h, which suggested that RANKL could promote the formation of osteoclast, indicating that both CD147 and RANKL play important roles in the formation of osteoclasts. Furthermore, the expressions of four osteoclast specific expression genes, including TRACP, MMP-2, MMP-9 and c-Src, were analyzed using RT-PCR. The results indicated that four osteoclast-specific expression genes were detectable in all osteoclast with different treatments. However, the highest expression level of these four osteoclast-specific expression genes appears in the CD147+ RANKL group and the lowest expression level of these four osteoclast-specific expression genes appears with si-RANKL treatment. Characterization of the role of CD147 in the development of tumors should lead to a better understanding of the changes occurring at the molecular level during the development and progression of primary human bone cancer.

Rankl expression predicts poor prognosis in gastric cancer patients: results from a retrospective and single-center analysis

The receptor activator of nuclear factor κB ligand (RANKL)/RANK pathway plays an important role in the prognosis of several solid tumor types, but its role in gastric cancer prognosis has been poorly characterized. A total of 116 gastric cancer patients who underwent surgical resection were enrolled in this study. Expressions of RANKL and RANK in gastric cancer tissues were detected using immunohistochemical staining. Thirty-eight patients (33%) showed a high level of RANKL expression and 61 patients (53%) showed a high level of RANK expression. There was a positive correlation between expressions of RANKL and RANK (P=0.014, r=0.221). A high level of RANKL expression indicated shorter overall survival (OS) (P=0.008), and was associated with a higher pathological tumor/lymph node/metastasis (pTNM) stage (P=0.035), while no significant correlation was detected between RANK expression and clinicopathological parameters. RANKL also predicted poor prognosis in patients with high RANK expression (P=0.008) and Bormann's type III/IV (P=0.002). Furthermore, RANKL expression correlated with pTNM stage according to high RANK expression (P=0.009), while no significance was found in patients with low RANK expression (P=1.000). Together, our results revealed that high expression of RANKL could predict worse outcomes in gastric cancer especially combined with RANK detection, and thereby this pathway could be a useful prognostic indicator of gastric cancer.

Evolution of Periodontal Disease: Immune Response and RANK/RANKL/OPG System

Abstract The aim of this study was to evaluate markers of bone loss and immune response present in evolution of periodontal disease. One hundred and two Wistar rats were divided into three animals groups: PD0, without ligation and PD15 days and PD60 days, submitted to ligation placement with a sterile 3-0 silk cord in the cervical region of the upper first molar on both sides. Samples were obtained from the gingival tissue for histomorphometric analysis, immunohistochemical analysis of RANK, RANKL, OPG, characterization of the inflammatory infiltrate, quantification of nitric oxide, MCP-1, RANTES, IP10 chemokines, and expression of the TGF-b1, VEG, and bFGF. The number of inflammatory cells in gingival tissue was higher in PD60 samples. The collagen content and the area occupied by birefringent collagen fibers were lower for PD60. Differential leukocyte counting showed that there was a significantly higher polymorphonuclear influx in group PD15, while PD60 showed a greater number of lymphocytes. PD60 showed higher RANTES, IP-10, MCP-1 gene transcripts, as well as a higher nitric oxide concentration. Clinical evaluation revealed that the PD60 group presented an increase in furcal area. In conclusion, in this animal model the increase of RANK/RANKL and HGF markers is related to a specific immune response, and probably contributed to the evolution of periodontal disease. Investigating the effect of these biomarkers can help in targeted therapy for bone resorption, since blocking these can inhibit bone loss.

Resumo Este estudo avaliou marcadores de perda óssea e da resposta imune presentes na evolução da doença periodontal. Cento e dois ratos Wistar foram divididos em três grupos de animais: PD0, sem ligadura e PD15 dias e PD60 dias, submetidos a colocação de ligadura com um fio de seda estéril 3-0 na região cervical do primeiro molar superior em ambos os lados. Foram obtidas amostras de tecido gengival para análise histomorfométrica, análises imunohistoquímicas de RANK, RANKL, OPG, caracterização do infiltrado inflamatório, quantificação de óxido nítrico, expressão de quimiocinas MCP-1, RANTES, IP10 e do TGF-b1, VEGF e bFGF . O número de células inflamatórias no tecido gengival foi maior nas amostras PD60. O teor de colágeno na área ocupada pelas fibras de colágeno birrefringentes foram menores para PD60. A contagem diferencial de leucócitos mostrou que houve um influxo polimorfonuclear significativamente maior no grupo PD15, enquanto que PD60 mostrou número maior de linfócitos. PD60 apresentou transcritos de genes RANTES, IP-10, MCP-1 mais elevados, bem como uma maior concentração de óxido nítrico. A avaliação clínica revelou que o grupo PD60 apresentou aumento da área óssea exposta na região da furca. Em conclusão, neste modelo animal o aumento dos marcadores RANK/RANKL e HGF está relacionado a uma resposta imunológica específica e provavelmente contribuiu para a evolução da doença periodontal. Investigar o efeito destes biomarcadores pode ajudar na terapia dirigida para a reabsorção óssea, uma vez que bloquear estes pode inibir a perda óssea.

Effects of triethylene glycol dimethacrylate (TEGDMA) on the odontoclastic differentiation ability of human dental pulp cells

Abstract Objectives: The primary purpose of this study was to examine the effects of triethylene glycol dimethacrylate (TEGDMA) on odontoclastic differentiation in the dental pulp tissue. Material and Methods: The effects of different TEGDMA dosages on the odontoclastic differentiation capability of dental pulp cells were analyzed in vitro using the following methodologies: i) flow cytometry and tartrate-resistant acid phosphatase (TRAP) staining; ii) apoptotic effects using Annexin V staining; iii) mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor (NF)-kB ligand (RANKL) genes by quantitative Real-time PCR (qRT-PCR); and iv) OPG and RANKL protein expression by enzyme-linked immunosorbent assay (ELISA). Results: TEGDMA caused relatively less odontoclastic differentiation in comparison with the control group; however, odontoclastic differentiation augmented with increasing doses of TEGDMA (p<0.05). The mRNA and protein expression of OPG was lower in TEGDMA treated pulp cells than in the control group (p<0.05). While the mRNA expression of RANKL remained unchanged compared to the control group (p>0.05), its protein expression was higher than the control group (p<0.05). In addition, TEGDMA increased the apoptosis of dental pulp cells dose dependently. Conclusions: TEGDMA reduced the odontoclastic differentiation ability of human dental pulp cells. However, odontoclastic differentiation ratios increased proportionally with the increasing dose of TEGDMA.

The effects of chitosan oligosaccharides on OPG and RANKL expression in a rat osteoarthritis model

Abstract Purpose: To investigate the effect of chitosan oligosaccharides (COS) against osteoarthritis (OA) and preliminarily discuss the osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL) and RANK expression in a rat OA model. Methods: Thirty-six 6-week-old Male SD rats were randomly divided into three groups: sham-operated group(CON), OA-induction group(OA), COS intervention group(n=12/group). At 4 weeks after the operation, COS (50 ul) intervention weekily for consecutive 5 weeks. The OA and CON groups received an injection of 50 ul physiological saline. At death, 11 weeks following surgery, cartilage was harvested and total RNA and protein were extracted. Both the morphological changes of the cartilage were observed and harvested the total RNA and protein. Meanwhile, the expression of OPG, RANKL and RANK in cartilage were determined. Results: The expression of OPG and RANKL were both enhanced in the cartilage of the OA model. Compared with the OA group, COS treatment improved the cartilage damage (both extent and grade). Furthermore, the COS group showed highly OPG and lower RANKL. Simultaneously, COS treatment upregulated the ratio of OPG/RANKL and downregulated the RANKL/RANK. Conclusion: Chitosan oligosaccharides may be used as a unique biological agent to prevent and treat osteoarthritis, and this effect is associated with modulation of the expression of osteoprotegerin and receptor activator of NF-κB ligand.

Effects of IL-10 and glucose on expression of OPG and RANKL in human periodontal ligament fibroblasts

The effects of interleukin-10 (IL-10) and glucose on mRNA and protein expression of osteoprotegerin (OPG), and its ligand, receptor activator of nuclear factor-κB ligand (RANKL), were investigated in human periodontal ligament fibroblasts (HPDLFs). Primary HPDLFs were treated with different concentrations of IL-10 (0, 1, 10, 25, 50, and 100 ng/mL) or glucose (0, 5.5, 10, 20, 30, and 40 mmol/L). Changes in mRNA and protein expression were examined using the reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. After IL-10 treatment, mRNA and protein levels of OPG were increased, while mRNA and protein levels of RANKL were decreased (P<0.05), both in a concentration-dependent manner. Glucose stimulation had the opposite concentration-dependent effect to that of IL-10 on OPG and RANKL expression. IL-10 upregulated OPG expression and downregulated RANKL expression, whereas high glucose upregulated RANKL and downregulated OPG in HDPLFs. Abnormal levels of IL-10 and glucose may contribute to the pathogenesis of periodontal disease.

Static compression regulates OPG expression in periodontal ligament cells via the CAMK II pathway

ABSTRACT Objective This study aimed to investigate the potential role of CAMK II pathway in the compression-regulated OPG expression in periodontal ligament cells (PDLCs). Material and Methods The PDL tissue model was developed by 3-D culturing human PDLCs in a thin sheet of poly lactic-co-glycolic acid (PLGA) scaffolds, which was subjected to static compression of 25 g/cm2 for 3, 6 and 12 h, with or without treatment of KN-93. After that, the expression of OPG, RANKL and NFATC2 was investigated through real-time PCR and western blot analysis. Results After static compression, the NFATC2 and RANKL expression was significantly up-regulated, while partially suppressed by KN-93 for 6 and 12 h respectively. The OPG expression was significantly down-regulated by compression in 3 h, started to elevate in 6 h, and significantly up-regulated in 12 h. The up-regulation after 12 h was significantly suppressed by KN-93. Conclusions Long-term static compression increases OPG expression in PDLCs, at least partially, via the CAMK II pathway.

Elevated serum osteoprotegerin levels in women: friend or foe? / Níveis séricos elevados de osteoprotegerina na mulher: aliado ou inimigo?

SUMMARY Introduction: osteoprotegerin has emerged as a new candidate for the treatment of osteoporosis. However, high levels of osteoprotegerin have been linked to vascular calcification, an independent and well-defined risk factor for cardiovascular disease (CVD) and mortality. Thus, the action of osteoprotegerin in these situations has been questioned. Objective: to evaluate the effect of osteoprotegerin (OPG) on the human body, especially in bone tissue and in vascular diseases. Methods: the scientific databases consulted were PubMed-Medline and Cochrane, using keywords (MeSH terms) grouped into the following syntaxes: (Osteoprotegerin OR Osteoclastogenesis Inhibitory Factor OR Receptors, Tumor Necrosis Factor, Member 11b OR Tumor Necrosis Factor Receptor Superfamily, Member 11b OR FDCR-1 Protein OR FDCR 1 Protein OR OCIF Protein OR Follicular Dendritic Cell-Derived Receptor-1) AND (Bones AND Bone OR Bones AND Bone Tissue OR Bones OR Bone Tissue OR Cardiovascular Diseases). Results: Osteoprotegerin is present in various organs and binds to two ligands: nuclear factor kB (RANKL) related to the differentiation of osteoclasts, and tumor necrosis factor related to the apoptosis-inducing ligand (TRAIL). OPG inhibits the regulation effects of nuclear factor kB on inflammation and on the skeletal and vascular systems, preventing the apoptosis induced by TRAIL, being related to the preservation of bone tissue. Conclusion: a deeper knowledge of the mechanisms involved in the association between OPG serum levels, bone integrity and cardiovascular disease can provide important data for future therapeutic interventions.

RESUMO Introdução: a osteoprotegerina (OPG) tem surgido como uma nova candidata para o tratamento da osteoporose; no entanto, níveis elevados de OPG têm sido relacionados à calcificação vascular, um fator de risco independente e bem definido para doença cardiovascular (DCV) e mortalidade. Assim, a ação da OPG nessas situações tem sido questionada. Objetivo: avaliar a ação da OPG no corpo humano, em especial no tecido ósseo e nas doenças vasculares. Métodos: as bases de informação científica consultadas foram PubMed-Medline e Cochrane, utilizando-se palavras-chave (MeSH terms) agrupadas nas seguintes sintaxes: (Osteoprotegerin OR Osteoclastogenesis Inhibitory Factor OR Receptors, Tumor Necrosis Factor, Member 11b OR Tumor Necrosis Factor Receptor Superfamily, Member 11b OR FDCR-1 Protein OR FDCR 1 Protein OR OCIF Protein OR Follicular Dendritic Cell-Derived Receptor-1) AND (Bones AND Bone OR Bones AND Bone Tissue OR Bones OR Bone Tissue OR Cardiovascular Diseases). Resultados: a OPG está presente em vários órgãos e une-se a dois ligantes: o fator nuclear kB (RANKL), relacionado com a diferenciação dos osteoclastos, e o fator de necrose tumoral, relacionado ao ligante indutor de apoptose (TRAIL). Assim, a OPG inibe os efeitos da regulação do fator nuclear kB na inflamação e nos sistemas esquelético e vascular, prevenindo a apoptose induzida pelo TRAIL, estando relacionada com a preservação do tecido ósseo. Conclusão: um conhecimento mais aprofundado sobre os mecanismos envolvidos na associação entre os níveis séricos da OPG, integridade óssea e doenças cardiovasculares podem proporcionar dados importantes para futuras intervenções terapêuticas.

Serum from patients with ankylosing spondylitis can increase PPARD, fra-1, MMP7, OPG and RANKL expression in MG63 cells

OBJECTIVES: To explore the effects of serum from patients with ankylosing spondylitis on the canonical Wnt/β-catenin pathway and to assess whether the serum has an osteogenic effect in MG63 cells. METHODS: MG63 cells were cultured with serum from 45 ankylosing spondylitis patients, 30 healthy controls, or 45 rheumatoid arthritis patients. The relative PPARD, fra-1, MMP7, OPG and RANKL mRNA levels were measured using quantitative real-time polymerase chain reaction. Associations between gene expression and patient demographics and clinical assessments were then analyzed. RESULTS: MG63 cells treated with serum from ankylosing spondylitis patients had higher PPARD, fra-1, MMP7 and OPG gene expression than did cells treated with serum from controls or rheumatoid arthritis patients (all p<0.05). RANKL expression was higher in MG63 cells treated with serum from patients with ankylosing spondylitis or rheumatoid arthritis than in those treated with serum from controls (both p<0.05). The OPG/RANKL ratio was also higher in MG63 cells treated with serum from ankylosing spondylitis patients than in those treated with serum from controls (p<0.05). No associations were found between the expression of the five genes and the patient demographics and clinical assessments (all p>0.05). CONCLUSIONS : Serum from ankylosing spondylitis patients increases PPARD, fra-1, MMP7, OPG and RANKL expression and the OPG/RANKL ratio in MG63 cells; these effects may be due to the stimulatory effect of the serum on the Wnt pathway.

Expressão imuno-histoquímica de proteínas relacionadas com a absorção óssea em carcinoma epidermoide de palato duro e língua / Immunohistochemical expression of proteins related to bone resorption in squamous cell carcinoma of the hard palate and tongue

O carcinoma epidermóide oral (CEO) apresenta uma tendência marcante de invadir o osso quando localizado em palato duro e rebordo. O mecanismo preciso desta invasão permanece incompletamente descrito, apesar de sugerirem na literatura que a destruição óssea, invasão e metástase seja mediada por osteoclastos e não diretamente por células do carcinoma. As moléculas RANK/RANKL /OPG são fundamentais na oesteoclastogênese, assim como a IL-6 que regula suas expressões. O objetivo desta pesquisa foi avaliar a expressão imuno-histoquímica de fatores de reabsorção óssea (RANKL e OPG) e da citocina (IL-6) no carcinoma epidermóide de palato duro (com invasão óssea) e língua (sem invasão óssea), correlacionandoas com os parâmetros clinicopatológicos e prognósticos. A amostra foi constituída por 30 carcinomas epidermóides com invasão óssea (localizados no palato) e 31 sem invasão óssea (localizados na língua). Foram avaliados a intensidade e a média das células tumorais, estromais e inflamatórias imunomarcadas para os anticorpos anti-RANKL, anti-OPG e anti-IL-6, no front de invasão e no centro tumoral. O escore (s) da imunorreatividade das células foi estabelecido através da multiplicação do percentual de células positivas (P) pelo valor da intensidade da marcação (I) (S = P x I), em cinco campos (400×). A análise da expressão da proteína RANKL foi significativamente mais expressa (p=0,002) nas células inflamatórias, com tendência há uma maior expressão nas células dos carcinomas epidermóides do palato duro. Entre os parâmetros clinicopatológicos foi observado associação do RANKL com o pior prognóstico, com significância estatística apenas para o estágio avançado do tumor (p= 0,033). A OPG demonstrou fraca expressão tanto nos casos de língua (0,77 ±1,85) quanto de palato duro (1,32 ± 2,48), com ausência de significância estatística (p>0,05). Em relação aos parâmetros clinicopatológicos a OPG apresentou tendência de associação com o pior prognóstico, com associação estatística significante para o óbito (p=0,048) e invasão perivascular (p=0,047). A IL-6 foi significantemente mais expressa (p<0,001) em células tumorais e inflamatórias nos carcinomas epidermóides de palato duro. E dentre os parâmetros clinicopatológicos, a IL-6 apresentou tendência de associação com o bom prognóstico, com diferença estatística para a ausência de metástase e as células tumorais (p = 0,020), estromais (p = 0,027) e inflamatórias (p = 0,017). Com base nos resultados pode-se concluir que a IL-6 pode ser utilizada como um marcador do carcinoma epidermóide oral com invasão óssea, e que a relação RANKL/OPG está alterada no carcinoma epidermóide oral. (AU)

Oral squamous cell carcinoma (CEO ) has a marked tendency to invade the bone when located in the palate and lip . The precise mechanism of this invasion is still not completely described , although the literature suggests that the bone destruction ABSTRAT , invasion and metastasis is mediated by osteoclasts and not directly by carcinoma cells . The molecules RANK / RANKL / OPG are fundamental in oesteoclastogênese, and IL-6 that regulates its expression. The objective of this research was to evaluate the immunohistochemical expression of factors of bone resorption ( RANKL and OPG ) and cytokine ( IL - 6 ) in squamous cell carcinoma of the hard palate (with bone invasion ) and tongue (without bone invasion ) , correlating with the clinicopathological parameters and prognosis. The sample consisted of 30 squamous cell carcinoma with bone invasion (located on the palate) and 31 without bone invasion (located on the tongue). Were evaluated and the average intensity of the tumor cells , stromal and inflammatory immunostained for anti-RANKL antibodies , anti- OPG and anti -IL- 6 at the invasive front and center of the tumor. The score (s) of the immunoreactivity of cells was established by multiplying the percentage of positive cells (P) for marking intensity value (I) (S = P x I) in five fields (× 400). The analysis of the expression of RANKL protein was expressed significantly higher (p = 0.002) in inflammatory cells, there is a trend to higher expression in squamous cell carcinomas of the hard palate. Among the clinicopathologic parameters was observed association of RANKL with the worst prognosis, with statistical significance only for the advanced stage of the tumor (p = 0.033). The OPG showed weak expression both in cases of tongue (0.77 ± 1.85) and hard palate (1.32 ± 2.48), with no statistical significance (p > .05). In relation to clinicopathological parameters OPG tended association with worse prognosis, with a statistically significant association for death (p = 0.048) and perivascular invasion (p = 0.047). IL- 6 was expressed significantly more (p < 0.001) in tumor and inflammatory cells in squamous cell carcinoma of the hard palate. In addition, among the clinicopathologic parameters, IL- 6 showed a tendency of association with good prognosis, with a statistical difference for the absence of metastasis and tumor cells (p = 0.020), stromal (p = 0.027) and inflammatory (p = 0.017). Based on the results it can be concluded that IL -6 may be used as a marker of oral squamous cell carcinoma with bone invasion, and the ratio RANKL / OPG is changed in oral squamous cell carcinoma. (AU)

Effects of a Mikania laevigata extract on bone resorption and RANKL expression during experimental periodontitis in rats

OBJECTIVES: The Mikania laevigata extract (MLE) (popularly known in Brazil as "guaco") possesses anti-inflammatory properties. In the present study we tested the effects of MLE in a periodontitis experimental model in rats. We also investigated possible mechanisms underlying such effects. MATERIAL AND METHODS: Periodontal disease was induced by a ligature placed around the mandibular first molars of each animal. Male Wistar rats were divided into 4 groups: non-ligated animals treated with vehicle; non-ligated animals treated with MLE (10 mg/kg, daily); ligature-induced animals treated with vehicle and ligature-induced animals treated with MLE (10 mg/kg, daily). Thirty days after the induction of periodontal disease, the animals were euthanized and mandibles and gingival tissues removed for further analysis. RESULTS: Morphometric analysis of alveolar bone loss demonstrated that MLE-treated animals presented a decreased alveolar bone loss and a lower expression of the activator of nuclear factor-κB ligand (RANKL) measured by immunohistochemistry. Moreover, gingival tissues from the MLE-treated group showed decreased neutrophil migration myeloperoxidase (MPO) assay. CONCLUSIONS: These results indicate that MLE may be useful to control bone resorption during progression of experimental periodontitis in rats.

Triiodothyronine (T3) does not induce Rankl expression in rat Ros 17/2.8 cells

Osteoclastogenesis may be regulated via activation of the RANK/RANKL (receptor activator of nuclear factor-kappa B/ receptor activator of nuclear factor-kappa B ligand) system, which is mediated by osteoblasts. However, the bone loss mechanism induced by T3 (triiodothyronine) is still controversial. In this study, osteoblastic lineage rat cells (ROS 17/2.8) were treated with T3 (10-8 M, 10-9 M, and 10-10 M), and RANKL mRNA (messenger RNA) expression was measured by semiquantitative RT-PCR. Our results show that T3 concentrations used did not significantly enhance RANKL expression compared to controls without hormone treatment. This data suggests that other mechanisms, unrelated to the RANK/RANKL system, might be to activate osteoclast differentiation in these cells.

A osteoclastogênese pode ser regulada via ativação do sistema RANK/RANKL (receptor ativador do fator nuclear kapa B/ ligante do receptor do fator nuclear kapa B), que é mediado pelos osteoblastos. Entretanto, o mecanismo de perda óssea induzido pelo T3 (triiodotironina) ainda é controverso. Neste estudo, a linhagem osteoblástica de células de rato ROS 17/2.8 foi tratada com T3 (10-8 M, 10-9 M e 10-10 M), e a expressão do mRNA do RANKL foi medida por RT-PCR semiquantitativo. Nossos resultados mostraram que as concentrações de T3 utilizadas não induziram significativamente a expressão do RANKL, comparado ao controle (sem tratamento hormonal). Estes dados sugerem que outros mecanismos, não relacionados ao sistema RANK/RANKL, são usados para ativar a diferenciação osteoclástica nestas células.