Overexpression of Bcl-2, SOCS 1, 3 and Cdh 1, 2 are associated with the early neoplasic changes in modified 4-nitroquinoline 1-oxide-induced murine oral cancer model.

BACKGROUND: The objective was to assess histopathological changes and the expression of proliferating cell nuclear antigen (PCNA), Bcl-2, suppressor of cytokine signaling (SOCS) 1 and 3, Vimentin, TWIST1, and Cdh 1 and 2 in early stages of experimental oral carcinogenesis process using a shorter period of exposure to 4-nitroquinoline oxide (4-NQO) model. METHODS: In this study, 20 rats were divided into control group (n = 10), sacrificed on the first day of the experiment, and experimental group (n = 10) treated with 50 ppm of 4-NQO solution dissolved in drinking water for 8 and 12 weeks. The histological sections were stained with H&E or subjected to immunohistochemistry for detecting PCNA, Bcl-2, SOCS 1 and 3, and STAT 3. Some specimens were used for verification of Vimentin expression, Cdh 1, Cdh 2, and TWIST1 by RT-qPCR. RESULTS: At both 8 and 12 weeks, morphological changes occurred mainly in the posterior portion of the tongue and were limited to the epithelial tissue, including moderate to severe dysplasia at 8 weeks, and severe dysplasia with exacerbation of atypical cells at 12 weeks. Expression of SOCS 1 and 3 increased from 8 to 12 weeks (P < 0.05), whereas STAT 3 expression was reduced mainly at 12 weeks (P < 0.05) in comparison with the control group. The expression of all epithelial-mesenchymal transition markers (EMT) was increased after 12 weeks, reaching statistical significance (P < 0.05) for Cdh 1 and 2. CONCLUSIONS: Together, the results suggested that overexpression of Bcl-2, SOCS 1 and 3, and Cdh 1 and 2 is associated with the early neoplasic changes in modified 4-nitroquinoline 1-oxide-induced murine oral cancer model.

Relevance of the myeloid differentiation factor 88 (MyD88) on RANKL, OPG, and nod expressions induced by TLR and IL-1R signaling in bone marrow stromal cells.

The myeloid differentiation factor 88 (MyD88) plays a pivotal role in Toll-like receptor (TLR)- and interleukin-1 receptor (IL-1R)-induced osteoclastogenesis. We examined the role of MyD88 on p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation and nucleotide-binding oligomerization domain (Nod) induction by lipopolysaccharide (LPS) and IL-1 beta, and their effect on receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) production in bone marrow stromal cell (BMSC). RANKL, Nod1, Nod2, NF-κB, and p38 protein levels were determined by Western blot. Nod2 was stimulated with muramyl dipeptide (MDP) prior to TLR4 stimulation with LPS. MyD88 deficiency markedly inhibited RANKL expression after LPS stimulation and increased OPG messenger RNA (mRNA) production. Also, MyD88 was necessary for NF-κB and p38 MAPK activation. MDP alone did not induce RANKL and OPG expressions; however, when combined with LPS, their expressions were significantly increased (p < 0.05). Our results support that MyD88 signaling has a pivotal role in osteoclastogenesis thought NF-κB and p38 activation. Nod2 and especially Nod1 levels were influenced by MyD88.

Chemopreventive activity of systemically administered curcumin on oral cancer in the 4-nitroquinoline 1-oxide model.

Curcumin has therapeutic potential in preventing several types of cancer, including colon, liver, prostate, and breast. The goal of this study was to evaluate the chemopreventive activity of systemically administered curcumin on oral carcinogenesis induced by 4-nitroquinolone-1-oxide (4-NQO). A total of 50 male albino rats, Rattus norvegicus, (Holtzman), were divided into five groups (n = 10 per group). Four of these groups were exposed to 50 ppm 4-NQO in their drinking water ad libitum for 8 or 12 weeks, two groups were treated with curcumin by oral gavage at 30 or 100 mg/kg per day, and one group was treated with corn oil (vehicle) only. The negative control group was euthanized at baseline. Tongues of all animals were removed after euthanasia and used in the subsequent analysis because the tongue is the primary site of carcinogenesis in this model. Descriptive histological analysis and immunohistochemistry for PCNA, Bcl-2, SOCS1 e-3, and STAT3 were performed to assess the oncogenic process. The gene expression of Vimentin, E-cadherin, N-cadherin, or TWIST1 was assessed using RT-qPCR as a representative of epithelial-mesenchymal transition (EMT) events. The administration of curcumin at 100 mg/kg during the 12 weeks markedly decreased the expression of PCNA, Bcl-2, SOCS1 e -3, and STAT3. Curcumin also minimized the cellular atypia under microscopic analysis and diminished the expression of the genes associated with EMT. These findings demonstrate that the systemic administration of curcumin has chemopreventive activity during oral carcinogenesis induced by 4-NQO.

A novel chemically modified curcumin reduces severity of experimental periodontal disease in rats: initial observations.

Tetracycline-based matrix metalloproteinase- (MMP-) inhibitors are currently approved for two inflammatory diseases, periodontitis and rosacea. The current study addresses the therapeutic potential of a novel pleiotropic MMP-inhibitor not based on an antibiotic. To induce experimental periodontitis, endotoxin (LPS) was repeatedly injected into the gingiva of rats on one side of the maxilla; the contralateral (control) side received saline injections. Two groups of rats were treated by daily oral intubation with a chemically modified curcumin, CMC 2.24, for two weeks; the control groups received vehicle alone. After sacrifice, gingiva, blood, and maxilla were collected, the jaws were defleshed, and periodontal (alveolar) bone loss was quantified morphometrically and by µ-CT scan. The gingivae were pooled per experimental group, extracted, and analyzed for MMPs (gelatin zymography; western blot) and for cytokines (e.g., IL-1ß; ELISA); serum and plasma samples were analyzed for cytokines and MMP-8. The LPS-induced pathologically excessive bone loss was reduced to normal levels based on either morphometric (P = 0.003) or µ-CT (P = 0.008) analysis. A similar response was seen for MMPs and cytokines in the gingiva and blood. This initial study, on a novel triketonic zinc-binding CMC, indicates potential efficacy on inflammatory mediators and alveolar bone loss in experimental periodontitis and warrants future therapeutic and pharmacokinetic investigations.

Curcumin abrogates LPS-induced pro-inflammatory cytokines in RAW 264.7 macrophages. Evidence for novel mechanisms involving SOCS-1, -3 and p38 MAPK.

Curcumin is the active compound in the extract of Curcuma longa rhizomes with anti-inflammatory properties mediated by inhibition of intracellular signalling. SOCS and MAPKinases are involved in the signalling events controlling the expression of IL-6, TNF-α and PGE2, which have important roles on chronic inflammatory diseases. The aim was to assess if these pathways are involved in curcumin-mediated effects on LPS-induced expression of these cytokines in macrophages. RAW 264.7 murine macrophages were stimulated with Escherichia coli LPS in the presence and absence of non-cytotoxic concentrations of curcumin. Curcumin potently inhibited LPS-induced expression of IL-6, TNF-α and COX-2 mRNA and prevented LPS-induced inhibition of SOCS-1 and -3 expression and the inhibition of the activation of p38 MAPKinase by modulation of its nuclear translocation. In conclusion, curcumin potently inhibits expression of LPS-induced inflammatory cytokines in macrophages via mechanisms that involve modulation of expression and activity of SOCS-1 and SOCS-3 and of p38 MAPK.

Experimental development of bisphosphonate-related osteonecrosis of the jaws in rodents.

Osteonecrosis of the jaw (ONJ) following the use of bisphosphonates has become of increased interest in the scientific community, due in particular to its as-yet-unsolved pathogenesis. An experimental model of ONJ was induced in normal male rats [alendronate (ALN); 1 mg/Kg/day; n = 10] and matched controls (saline solution; n = 10). After 60 days of drug treatment, all animals were subjected to extractions of the left first lower molars and were euthanized at 3 and 28 days postsurgery. The following analyses were performed: (i) descriptive and quantitative (scores) histological evaluation, (ii) stereometry of distal sockets and (iii) biochemical measurement of C-telopeptide cross-linked collagen type I (CTX) and bone-specific alkaline phosphatase (BALP). The results showed that 28 days postsurgery the animals treated with ALN had areas of exposed and necrotic bone, associated with significant infection, especially in the interalveolar septum area and crestal regions, compared with controls. The levels of CTX, BALP and bone volume, as well as the degrees of inflammation and vascularization, were significantly reduced in these animals. Therefore, analysis of the data presented suggests that ALN therapy is associated with the development of osteonecrosis in the jaws of rodents after tooth extraction.

Curcumin modulates the immune response associated with LPS-induced periodontal disease in rats.

Curcumin is a plant-derived dietary spice ascribed various biological activities. Curcumin therapeutic applications have been studied in a variety of conditions, but not on periodontal disease. Periodontal disease is a chronic inflammatory condition initiated by an immune response to micro-organisms of the dental biofilm. Experimental periodontal disease was induced in rats by injecting LPS in the gingival tissues on the palatal aspect of upper first molars (30 µg LPS, 3 times/week for 2 weeks). Curcumin was administered to rats daily via oral gavage at 30 and 100 mg/kg body weight. Reverse transcriptase-qPCR and ELISA were used to determine the expression of IL-6, TNF-α and prostaglandin E(2) synthase on the gingival tissues. The inflammatory status was evaluated by stereometric and descriptive analysis on hematoxylin/eosin-stained sections, whereas modulation of p38 MAPK and NK-κB signaling was assessed by Western blot. Curcumin effectively inhibited cytokine gene expression at mRNA and protein levels, but NF-κB was inhibited only with the lower dose of curcumin, whereas p38 MAPK activation was not affected. Curcumin produced a significant reduction on the inflammatory infiltrate and increased collagen content and fibroblastic cell numbers. Curcumin potently inhibits innate immune responses associated with periodontal disease, suggesting a therapeutic potential in this chronic inflammatory condition.

Influence of antiplatelet drugs in the pathogenesis of experimental periodontitis and periodontal repair in rats.

BACKGROUND: Platelets contain an array of biologic mediators that can modulate inflammation and repair processes including proinflammatory mediators and growth factors. Previous studies have shown that periodontitis and periodontal repair are associated with platelet activation. We hypothesized that drug-induced platelet inactivation may interfere in the processes of inflammation and repair in experimental periodontitis in rats by suppressing the release of biologic mediators from platelets to the site of injury. METHODS: To measure the effects on periodontitis, ligatures were placed around first molars, and aspirin (Asp, 30 mg/kg) or clopidogrel (Clo, 75 mg/kg) was given intragastrically once daily for 15 days. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and thromboxane A(2) levels were measured by enzyme-linked immunosorbent assay. To evaluate the effects of antiplatelet drugs on periodontal repair, ligatures were removed after 15 days of periodontitis induction, and Asp or Clo were administered beginning the following day for 15 days. Periodontal repair was assessed by microcomputed tomography. RESULTS: On periodontitis phase, Asp and Clo significantly reduced levels of TNF-α and Il-6 (P <0.05), but only Asp decreased thromboxane A(2) (P <0.05). Asp and Clo decreased inflammatory infiltration; however, this reduction was more pronounced with Clo treatment (P <0.05). Histometric analysis showed that Asp and Clo impaired alveolar bone resorption. During the repair phase and after removal of the ligatures, microcomputed tomography analysis demonstrated that treatment with Asp and Clo did not impair alveolar bone repair. CONCLUSION: Systemic administration of Asp and Clo attenuates the inflammation associated with periodontitis without affecting the repair process when stimulus is removed.

Differential regulation of MMP-13 expression in two models of experimentally induced periodontal disease in rats.

OBJECTIVE: Evaluate expression of MMP-13 during the course of two models experimentally induced periodontal disease in rats. DESIGN: Expression of MMP-13 at mRNA and protein levels was studied, respectively, by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Two experimental models were used: LPS injections and ligature placement. 30mug of LPS from Eschericia coli was injected twice a week into the palatal aspect of upper molars. Ligatures were placed at the gingival margin around lower first molars. Controls received injections of PBS vehicle and no ligatures on lower molars. Samples were collected 5, 15 and 30 days after initiation of periodontal disease and processed for extraction of total RNA, total protein, and routinely processed for histology. RESULTS: Both experimental models produced a significant increase on the inflammatory infiltrate that paralleled elevated levels of MMP-13 mRNA and protein at 5 and 15 days. The LPS model was associated with a sustained level of inflammation and increased MMP-13 mRNA throughout the 30 days, whereas the ligature model showed a decrease on the severity of inflammation and MMP-13 mRNA at the 30-day period. Interestingly, MMP-13 protein levels were diametrically contrary to the mRNA levels. CONCLUSION: MMP-13 expression during LPS- and ligature-induced experimental periodontal disease follows the increase on severity of inflammation at the earliest periods. At 30 days, there is a decrease on the severity of inflammation on the ligature model associated with decreased MMP-13 mRNA. There is a lack of transcription-translation coupling of MMP-13 gene in both experimental models.

Biochemical evaluation of glycemic levels of long-term tacrolimus therapy in rats.

One of the more serious complications following transplantation is the development of post-transplantation diabetes mellitus (PTDM), which has a major impact on the quality of life, with effects ranging from the control of glycemia times to increased susceptibility to infections and cardiovascular complications. It has been suggested that immunosuppressive therapy, mainly tacrolimus therapy, may be an important factor in the development of PTDM. There is a lack of studies that explore the effects of long-term tacrolimus on PTDM in animal protocols. The objective of this study was therefore to evaluate the effects of long-term therapy with tacrolimus in rats. One group was treated with tacrolimus, injected subcutaneously, in a daily dose of 1 mg/kg of body weight. The chosen dose was sufficient to achieve therapeutic tacrolimus serum levels. The experimental periods were 60, 120, 180 and 240 days. One group was used as control and received daily subcutaneous injections of saline solution during all periods. A tendency towards increased glycemia levels during the initial periods (60 and 120 days) was observed. However, at 180 and 240 days, the glycemia levels were not statistically different from that of the control group of the same period. It may thus be concluded that the deleterious effects of tacrolimus therapy on glycemia may be a time-related side effect.

Biochemical evaluation of glycemic levels of long-term tacrolimus therapy in rats

One of the more serious complications following transplantation is the development of post-transplantation diabetes mellitus (PTDM), which has a major impact on the quality of life, with effects ranging from the control of glycemia times to increased susceptibility to infections and cardiovascular complications. It has been suggested that immunosuppressive therapy, mainly tacrolimus therapy, may be an important factor in the development of PTDM. There is a lack of studies that explore the effects of long-term tacrolimus on PTDM in animal protocols. The objective of this study was therefore to evaluate the effects of long-term therapy with tacrolimus in rats. One group was treated with tacrolimus, injected subcutaneously, in a daily dose of 1 mg/kg of body weight. The chosen dose was sufficient to achieve therapeutic tacrolimus serum levels. The experimental periods were 60, 120, 180 and 240 days. One group was used as control and received daily subcutaneous injections of saline solution during all periods. A tendency towards increased glycemia levels during the initial periods (60 and 120 days) was observed. However, at 180 and 240 days, the glycemia levels were not statistically different from that of the control group of the same period. It may thus be concluded that the deleterious effects of tacrolimus therapy on glycemia may be a time-related side effect.

Uma das mais sérias complicações pós-transplante é o desenvolvimento de Diabetes Mellitus Pós-Transplante (DMPT), que irá produzir um grande impacto na qualidade de vida, com variações do controle glicêmico, aumentando a susceptibilidade a infecções e complicações cardiovasculares. Tem sido sugerido que a terapia imunossupressora, principalmente com tacrolimo, pode ser um importante fator no desenvolvimento de DMPT. Existem atualmente poucos estudos explorando os efeitos de um longo período de terapia com tacrolimo sobre DMPT em protocolos animais. Portanto, o objetivo deste estudo foi avaliar o efeito glicêmico de uma terapia por longo período com tacrolimo em ratos. Um grupo foi tratado com tacrolimo com doses diárias subcutâneas de 1 mg/kg de peso corporal. A escolha dessa dose foi suficiente para a obtenção dos níveis séricos terapêuticos desejados com tacrolimo. Os períodos experimentais foram 60, 120, 180 e 240 dias. Outro grupo foi usado como controle e recebeu injeções salinas subcutâneas diariamente durante todos os períodos. Houve uma tendência ao aumento do nível glicêmico nos períodos iniciais (60 e 120 dias). Entretanto, após 180 e 240 dias, os níveis de glicemia não foram estatisticamente diferentes dos obtidos nos grupos controles de mesmo período. Assim, pode-se concluir que os efeitos glicêmicos adversos provocados pela terapia com tacrolimo podem ser relacionados ao tempo.

Avaliação longitudinal de pacientes com disfunções têmporomandibulares tratados com placas oclusais, aconselhamento e farmacoterapia / Longitudinal evaluation of temporomandibular disorders patients managed with occlusal splints, counsellinfg and medications

Tendo em vista o amplo uso das placas oclusais em pacientes com DTM e a mínima utilização de aconselhamento/farmacoterapia como modalidades terapêuticas, este trabalho teve como objetivoavaliar os pacientes tratados na Clínica de Oclusão e Dores Orofaciais da Faculdade de Odontologia deAraraquara - UNESP, para verificar o percentual de pacientes tratados com placas oclusais e farmacoterapia/aconselhamento, bem como o índice de sucesso das modalidades terapêuticas empregadas. A amostra inicial foi de 258 pacientes (63 do gênero masculino e do 195 gênerofeminino), sendo que 62 (24%) tiveram indicação para o uso de placa oclusal e aconselhamento e para 196 (76%) foi recomendado apenas o aconselhamento e/ou farmacoterapia, sem uso de nenhum tipode placa oclusal. Quanto ao sucesso das terapias empregadas, houve melhora da sintomatologia dolorosa em 79,2% dos pacientes que usaram a placa oclusal comparado a 75% dos pacientes aos quais foi indicado apenas aconselhamento e/ou farmacoterapia. Após 2 a 3 anos da finalização dotratamento, 41,6% dos pacientes não utilizavam mais as placas oclusais e 58,4% só a utilizavam durante a noite. A conclusão foi que a maior parte dos pacientes da Clínica de Dores Orofaciais foi tratada com aconselhamento e/ou farmacoterapia (76%) sem a utilização de placas oclusais. Ambas as modalidades se mostraram eficazes na redução dos sinais e sintomas de DTM. Sendo assim, pelo índice de sucesso obtido e ainda pelos pacientes não utilizarem mais as placas com o decorrer do tempo, o uso do aconselhamento associado à farmacoterapia deveria ser uma modalidade terapêutica mais explorada pelos profissionais

Taking into consideration that occlusal splints have been largely used for the management of TMD signsand symptoms and that self-care/medications are rarely utilized, the aim of this study was to evaluate patients treated at the Occlusion,TMDand Orofacial Pain Clinic, Araraquara School of Dentistry, in orderto obtain the number of patients treated with occlusal splints, self-care and medications, as well as the index of effectiveness of the modalities that had been indicated. Study sample was comprised of 258 patients (63 male and 195 female). 62 (24%) had indication for the use of an occlusal splint and self-care, and for the others 196 patients (76%) only self-care and medications were utilized, with no use of anykind of occlusal splint. Regarding the effectiveness of the treat modalities, occlusal splint was effective in the reduction of TMD pain in 79, 2% of the patients as opposed to 75% of the patients who were treated with self-care and medications only. After 2 to 3 years of occlusal splint therapy, 41,6% of the patients were not using the splint anymore and the other 58,4% were using it just at bedtime, not in an everyday basis. Authors concluded that for the most part of the patients treated at our Clinic, self-care and medications were effective (76%) without the use of an occlusal splint. All the groups had the same success rate. Therefore, since both therapies were effective and considering the fact that patients do not use an occlusal splint for the rest of their lives, the use of self-care associated with medications should be more utilized in the clinical management of TMD symptoms