DNMT3B (rs2424913) polymorphism is associated with systemic lupus erythematosus alone and with co-existing periodontitis in a Brazilian population.

The association between Periodontitis and Systemic Lupus Erythematosus (SLE) has been primarily based on their similar pathophysiology and both are associated with genetic polymorphisms. OBJECTIVES: To investigate an association between the methylation-related gene polymorphisms DNMT3B (rs2424913) and MTHFR (rs1801133) to Systemic Lupus Erythematosus (SLE) and Periodontitis. METHODOLOGY: In total, 196 individuals of all genders aged 24 to 60 years old were allocated into four groups based on their systemic and periodontal status, namely: Healthy control (n=60), periodontitis (n=51), SLE (n=47), and SLE + periodontitis (n=38). Individuals with SLE were stratified according to disease activity (SLEDAI) in inactive or active. We performed polymorphism analysis using PCR-RFLP with genomic DNA from mouthwash. We analyzed data using Fisher's Exact, Chi-square test, and regression models. RESULTS: Periodontal status were similar in subjects with periodontitis alone and combined with SLE. SLE patients with periodontitis had a longer SLE diagnosis than SLE only (p=0.001). For DNMT3 B polymorphism, the periodontitis, SLE, and Inactive SLE + periodontitis groups showed a higher frequency of T allele and TT genotypes compared to healthy controls (p<0.05). Regression analyses showed that the TT genotype is a strong risk factor for periodontitis (OR=4.53; CI95%=1.13-18.05) and also for SLE without periodontitis (OR=11.57; CI95%=3.12-42.84) and SLE with periodontitis (OR=5.27; CI95%=1.25-22.11) when compared to control. CONCLUSION: SLE patients with periodontitis had a longer length of SLE diagnosis. The DNMT3B (rs2424913) polymorphism was associated with periodontitis and SLE alone or combined with periodontitis. Our study contributes to understanding the genetic mechanisms involved in periodontitis and SLE susceptibility.

In vitro antibacterial and anti-inflammatory effects of Anacardium occidentale L. extracts and their toxicity on PBMCs and zebrafish embryos.

This study evaluated the in vitro antimicrobial and immunomodulatory action of crude extracts from Anacardium occidentale L. (cashew tree) leaves and bark, and to determine their toxicity to peripheral-blood mononuclear cells (PBMCs) and to zebrafish embryos and larvae. Chemical analysis of extracts was performed by proton nuclear magnetic resonance (1H-NMR). The antibacterial activity was evaluated against selected bacteria strains by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Cytotoxicity of the extracts was assessed using resazurin method, while the effect on production of ROS by PMN leukocytes was measured by luminol. Embryotoxicity to zebrafish was assessed using the fish embryo acute toxicity test (FET) and quantification of toxicity marker enzymes (AChE, LDH, and GST). 1H-NMR results showed anacardic acid as the main component of the extracts. All bacterial species tested were sensitive to the extracts, with MICs ranging from 312.5 to 10,000 µg/mL. Streptococcus mutans and Escherichia coli were the most susceptible species. The extracts promoted cell viability above 75% at concentrations from 1.25 to 80 µg/mL. Both extracts reduced zymosan-induced ROS (p < 0.05) at concentrations of 1, 8, and 80 µg/mL compared to the control. In vivo, there were embryotoxic effects in zebrafish embryos exposed to both extracts through the presence of lethal and sublethal endpoints. The samples also acted by inhibiting the activities of biomarker enzymes. The A. occidentale L. bark and leaf extracts showed antimicrobial potential and modulated ROS production in vitro, but these also showed embryotoxic effects to zebrafish.

DNMT3B (rs2424913) polymorphism is associated with systemic lupus erythematosus alone and with co-existing periodontitis in a Brazilian population

Abstract The association between Periodontitis and Systemic Lupus Erythematosus (SLE) has been primarily based on their similar pathophysiology and both are associated with genetic polymorphisms. Objectives: To investigate an association between the methylation-related gene polymorphisms DNMT3B (rs2424913) and MTHFR (rs1801133) to Systemic Lupus Erythematosus (SLE) and Periodontitis. Methodology: In total, 196 individuals of all genders aged 24 to 60 years old were allocated into four groups based on their systemic and periodontal status, namely: Healthy control (n=60), periodontitis (n=51), SLE (n=47), and SLE + periodontitis (n=38). Individuals with SLE were stratified according to disease activity (SLEDAI) in inactive or active. We performed polymorphism analysis using PCR-RFLP with genomic DNA from mouthwash. We analyzed data using Fisher's Exact, Chi-square test, and regression models. Results: Periodontal status were similar in subjects with periodontitis alone and combined with SLE. SLE patients with periodontitis had a longer SLE diagnosis than SLE only (p=0.001). For DNMT3 B polymorphism, the periodontitis, SLE, and Inactive SLE + periodontitis groups showed a higher frequency of T allele and TT genotypes compared to healthy controls (p<0.05). Regression analyses showed that the TT genotype is a strong risk factor for periodontitis (OR=4.53; CI95%=1.13-18.05) and also for SLE without periodontitis (OR=11.57; CI95%=3.12-42.84) and SLE with periodontitis (OR=5.27; CI95%=1.25-22.11) when compared to control. Conclusion: SLE patients with periodontitis had a longer length of SLE diagnosis. The DNMT3B (rs2424913) polymorphism was associated with periodontitis and SLE alone or combined with periodontitis. Our study contributes to understanding the genetic mechanisms involved in periodontitis and SLE susceptibility.

Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages.

Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 µM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 µM (p<0.05) and 250 µM (p<0.01). The POH increased ROS production at both 10 µM and 100 µM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 µM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 µM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.

miR-9-1 gene methylation and DNMT3B (rs2424913) polymorphism may contribute to periodontitis.

BACKGROUND: Genetic and epigenetic changes have been associated with periodontitis in various genes; however, little is known about genes involved in epigenetic mechanisms and in oxidative stress. OBJECTIVE: This study aims to investigate the association of polymorphisms C677T in MTHFR (rs1801133) and -149C→T in DNMT3B (rs2424913), as well as the methylation profiles of MTHFR, miR-9-1, miR-9-3, SOD1, and CAT with periodontitis. The association between polymorphisms and DNA methylation profiles was also analyzed. METHODOLOGY: The population studied was composed of 100 nonsmokers of both sexes, divided into healthy and periodontitis groups. Genomic DNA was extracted from the epithelial buccal cells, which were collected through a mouthwash. Polymorphism analysis was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while methylation-specific PCR (MSP) or combined bisulfite restriction analysis techniques were applied for methylation analysis. RESULTS: For DNMT3B, the T allele and the TT genotype were detected more frequently in the periodontitis group, as well as the methylated profile on the miR-9-1 promoter region. There was also a tendency towards promoter region methylation on the CAT sequence of individuals with periodontal disease. CONCLUSION: The polymorphism -149C→T in DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis.

miR-9-1 gene methylation and DNMT3B (rs2424913) polymorphism may contribute to periodontitis

Abstract Genetic and epigenetic changes have been associated with periodontitis in various genes; however, little is known about genes involved in epigenetic mechanisms and in oxidative stress. Objective: This study aims to investigate the association of polymorphisms C677T in MTHFR (rs1801133) and −149C→T in DNMT3B (rs2424913), as well as the methylation profiles of MTHFR, miR-9-1, miR-9-3, SOD1, and CAT with periodontitis. The association between polymorphisms and DNA methylation profiles was also analyzed. Methodology: The population studied was composed of 100 nonsmokers of both sexes, divided into healthy and periodontitis groups. Genomic DNA was extracted from the epithelial buccal cells, which were collected through a mouthwash. Polymorphism analysis was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while methylation-specific PCR (MSP) or combined bisulfite restriction analysis techniques were applied for methylation analysis. Results: For DNMT3B, the T allele and the TT genotype were detected more frequently in the periodontitis group, as well as the methylated profile on the miR-9-1 promoter region. There was also a tendency towards promoter region methylation on the CAT sequence of individuals with periodontal disease. Conclusion: The polymorphism −149C→T in DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis.

Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages

Abstract Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 μM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 μM (p<0.05) and 250 μM (p<0.01). The POH increased ROS production at both 10 μM and 100 μM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 μM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 μM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.

Effect of avocado/soybean unsaponifiables on periodontal repair in rats with arthritis and induced periodontitis.

OBJECTIVE: This study aimed to evaluate the effect of avocado/soybean unsaponifiables (ASU) on periodontal repair in rats with induced periodontitis and arthritis. METHODOLOGY: Forty-five rats were submitted to periodontitis induction by insertion of ligatures into the upper second molars, maintained for 15 days. These animals were randomly allocated to 3 groups according to the presence of induced arthritis (ART) and the application of the ASU: Control (CTR) group-healthy animals, where saline solution was administered; ART-animals with induced arthritis, where saline solution was administered; ART/ASU-animals with induced arthritis, where ASU (0.6 mg/ kg) was administered. The drugs were administered daily by gavage and the animals were euthanized after 7, 15 and 30 days of the ligature removal. Bone resorption, inflammatory infiltrate composition and marker proteins expression of the differentiation and formation of osteoclasts (RANKL and TRAP) were assessed. RESULTS: The ART/ASU group presented higher bone volume than the ART group at 7 and 30 days after the ligature removal. Furthermore, the ART group presented higher quantity of inflammatory cells and expression of TRAP and RANKL than the other groups. CONCLUSION: ASU administration improves the repair of periodontal tissues in an experimental periodontitis model in rats with induced arthritis.

Systemic administration of curcumin or piperine enhances the periodontal repair: a preliminary study in rats.

OBJECTIVES: Studies have documented the anti-inflammatory effects of spices, which may be related to treatment of chronic diseases. The purpose of this study was to evaluate the influence of curcumin and piperine and their association on experimental periodontal repair in rats. MATERIALS AND METHODS: Periodontitis was induced via the installation of a ligature around the first molar. After 15 days, the ligatures were removed, and the rats were separated into groups (12 animals per group): (i) curcumin, (ii) piperine, (iii) curcumin+piperine, (iv) corn oil vehicle, and (v) control group (animals had ligature-induced periodontitis but were not treated). The compounds were administered daily, for 15 days by oral gavage. Animals were euthanized at 5 and 15 days, and hemimaxillae and gingival tissues were harvested. Bone repair was assessed by µCT (microcomputer tomography). Histological sections were stained with hematoxylin/eosin (H/E) for the assessment of cellular infiltrate or picrosirius red for quantification of collagen content, and subjected to immunohistochemistry for detecting NF-ĸB. Gingival tissues were used to evaluate levels of TGF-ß and IL-10 (ELISA). RESULTS: Curcumin and piperine increased the TGF-ß level, significantly improved the collagen repair, and decreased the cellularity and activation of NF-ĸB in the periodontal tissues, but only curcumin caused a significant increase in early bone repair. CONCLUSION: Curcumin and piperine promoted a substantive effect on tissue repair; however, there was not synergistic effect of compounds administered in combination. CLINICAL RELEVANCE: Curcumin and piperine stimulates the tissue repair and may be potential candidates for the treatment of periodontal disease.

Effect of avocado/soybean unsaponifiables on periodontal repair in rats with arthritis and induced periodontitis

Abstract Objective: This study aimed to evaluate the effect of avocado/soybean unsaponifiables (ASU) on periodontal repair in rats with induced periodontitis and arthritis. Methodology: Forty-five rats were submitted to periodontitis induction by insertion of ligatures into the upper second molars, maintained for 15 days. These animals were randomly allocated to 3 groups according to the presence of induced arthritis (ART) and the application of the ASU: Control (CTR) group-healthy animals, where saline solution was administered; ART-animals with induced arthritis, where saline solution was administered; ART/ASU-animals with induced arthritis, where ASU (0.6 mg/ kg) was administered. The drugs were administered daily by gavage and the animals were euthanized after 7, 15 and 30 days of the ligature removal. Bone resorption, inflammatory infiltrate composition and marker proteins expression of the differentiation and formation of osteoclasts (RANKL and TRAP) were assessed. Results: The ART/ASU group presented higher bone volume than the ART group at 7 and 30 days after the ligature removal. Furthermore, the ART group presented higher quantity of inflammatory cells and expression of TRAP and RANKL than the other groups. Conclusion: ASU administration improves the repair of periodontal tissues in an experimental periodontitis model in rats with induced arthritis.

In Vitro Effect of Cinnamomum zeylanicum Blume Essential Oil on Candida spp. Involved in Oral Infections.

The present study demonstrates the antifungal potential of chemically characterized essential oil (EO) of Cinnamomum zeylanicum Blume on Candida spp. biofilm and establishes its mode of action, effect on fungal growth kinetics, and cytotoxicity to human cells. The minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) values varied from 62.5 to 1,000 µg/mL, and the effect seems to be due to interference with cell wall biosynthesis. The kinetics assay showed that EO at MICx2 (500 µg/mL) induced a significant (p < 0.05) reduction of the fungal growth after exposure for 8 h. At this concentration, the EO was also able to hinder biofilm formation and reduce Candida spp. monospecies and multispecies in mature biofilm at 24 h and 48 h (p < 0.05). A protective effect on human red blood cells was detected with the EO at concentrations up to 750 µg/mL, as well as an absence of a significant reduction (p > 0.05) in the viability of human red blood cells at concentrations up to 1,000 µg/mL. Phytochemical analysis identified eugenol as the main component (68.96%) of the EO. C. zeylanicum Blume EO shows antifungal activity, action on the yeast cell wall, and a deleterious effect on Candida spp. biofilms. This natural product did not show evidence of cytotoxicity toward human cells.

Antifungal Activity, Mode of Action, Docking Prediction and Anti-biofilm Effects of (+)-ß-pinene Enantiomers against Candida spp.

AIMS: The objective of this study was to investigate the effectiveness of (+)-ß-pinene inhibition on Candida spp. growth, aiming at elucidation of the mechanism of action; to determine fungal cell enzyme binding activity (through molecular docking simulations) and its effects on biofilm reduction. METHODS: Candida strains (n=25) from referenced and clinical origins, either susceptible or resistant to standard clinical antifungals, were tested for determination of Minimum Inhibitory Concentration (MIC); Minimum Fungicidal Concentration (MFC); and microbial death curves upon treatment with (+)-ß-pinene; the effects of (+)-ß-pinene on the cell wall (sorbitol assay), membrane ergosterol binding, and effects on biofilm were evaluated by microdilution techniques. We also evaluated the interactions between (+)-ß-pinene and cell wall and membrane enzymes of interest. RESULTS: The MIC values of (+)-ß-pinene ranged from <56.25 to 1800 µmol/L. The MIC of (+)-ß-pinene did not increase when ergosterol was added to the medium, however it did increase in the presence of sorbitol, leading to a doubled MIC for C. tropicalis and C. krusei. The results of the molecular docking simulations indicated better interaction with delta-14-sterol reductase (-51 kcal/mol). (+)-ß-pinene presents anti-biofilm activity against multiples species of Candida. CONCLUSION: (+)-ß-pinene has antifungal activity and most likely acts through interference with the cell wall; through molecular interaction with Delta-14-sterol reductase and, to a lesser extent, with the 1,3-ß- glucan synthase. This molecule was also found to effectively reduce Candida biofilm adhesion.

Effect of Avocado/Soybean Unsaponifiables (ASU) on Osseointegration in Rats with Experimental Arthritis.

PURPOSE: To evaluate the influence of the use of avocado/soybean unsaponifiables (ASU) on osseointegration of implants in animals with experimental arthritis. MATERIALS AND METHODS: One hundred twenty rats were randomly divided into four groups: CTR, healthy animals and saline solution administration; ASU, healthy animals and ASU administration; ART, arthritic animals and saline solution administration; and ART/ASU, arthritic animals and ASU administration. The solutions were administered daily by gavage, beginning 7 days before the surgical procedures until the completion of the experimental period (15, 30, and 60 days after the placement of the implants in the tibia). The osseointegration of the implants was evaluated by histometric analysis (bone-to-implant contact [% BIC], bone area between the threads [% BBT]) and biomechanical analysis. Microcomputed tomography (micro-CT) analysis was used to assess bone volume in the vicinity of the implant. Immunohistochemistry analysis was performed to assess the expression of osteocalcin and transforming growth factor beta 1 (TGF-ß1). RESULTS: The ART/ASU group showed a decreased percentage of bone in the area around the implant compared with the ASU and ART groups (15 and 30 days). The ART/ASU group showed increased removal torque values (30 days) and % BIC and % BBT (30 to 60 days) compared with the ART group. The ASU group had increased % BIC values compared with the ART and CTR groups (60 days). The CTR group had the highest expression of osteocalcin, while the ASU group presented the highest expression of TGF-ß1 at 60 days. CONCLUSION: The ASU administration improved the osseointegration, particularly in animals with induced arthritis.

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.

HMGB1 localization during experimental periodontitis.

AIM: This study sought to investigate the in vitro expression profile of high mobility group box 1 (HMGB1) in murine periodontal ligament fibroblasts (mPDL) stimulated with LPS or IL-1ß and in vivo during ligature- or LPS-induced periodontitis in rats. MATERIAL AND METHODS: For the in vivo study, 36 rats were divided into experimental and control groups, and biopsies were harvested at 7-30 d following disease induction. Bone loss and inflammation were evaluated. HMGB1 expression was assessed by immunohistochemistry, qPCR, and Western blot. RESULTS: Significant increases in mPDL HMGB1 mRNA occurred at 4, 8, and 12 h with protein expression elevated by 24 h. HMGB1 mRNA expression in gingival tissues was significantly increased at 15 d in the LPS-PD model and at 7 and 15 d in the ligature model. Immunohistochemical staining revealed a significant increase in the number of HMGB1-positive cells during the experimental periods. CONCLUSION: The results show that PDL cells produce HMGB1, which is increased and secreted extracellularly after inflammatory stimuli. In conclusion, this study demonstrates that HMGB1 may be associated with the onset and progression of periodontitis, suggesting that further studies should investigate the potential role of HMGB1 on periodontal tissue destruction.

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.

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.

Expressão de MMP-13 e ativação de vias de sinalização intracelular em dois modelos de doença periodontal induzida experimentalmente em ratos / MMP-13 expression and signaling pathways activated in two models of experimentally-induced periodontal disease in rats

A progressão da doença periodontal é marcada pela excessiva produção de citocinas que, por sua vez, promove o aumento de outros mediadores inflamatórios, entre os quais, de metaloproteinases de matriz (MMPs). MMP-13 é uma colagenase de regulação complexa que tem sido relacionada à degradação da matriz extracelular (ECM) e à reabsorção óssea em diversas condições inflamatórias, incluindo doença periodontal e artrite reumatóide. A regulação da expressão gênica requer a ativação de várias vias de sinalização através da interação de receptores celulares específicos a estímulos externos, como antígenos bacterianos e citocinas derivadas do hospedeiro. A complexidade da rede de citocinas estabelecida durante a progressão da doença periodontal depende das vias de sinalização ativadas, as quais são influenciadas pela natureza do estímulo extracelular. Considerando o papel fundamental das vias de sinalização no controle da expressão gênica de citocinas e a relevante atividade de MMP-13 na doença periodontal, este estudo avaliou a expressão de MMP-13 e as vias de sinalização ativadas durante o curso de dois modelos de doença periodontal induzida experimentalmente. A expressão de MMP-13 nos níveis de RNA mensageiro (mRNA) e proteína foram avaliados por RT-PCR e Western Blot, respectivamente. A cinética de ativação das vias de sinalização intracelular relacionadas à expressão de mediadores inflamatórios também foi verificada por Western Blot. Estes achados foram relacionados à severidade da reação inflamatória determinada por estereometria. Dois modelos experimentais foram usados: injeção de LPS e colocação de ligadura. Injeções de LPS de Eschericia coli foram realizadas na região palatina de molares superiores 2 vezes por semana (30 µg por aplicação). Ligaduras foram colocadas na região cervical dos primeiros molares inferiores. O grupo controle recebeu injeções de PBS (veículo) na gengiva palatina dos primeiros molares superiores, enquanto ligaduras não foram colocadas nos molares inferiores. Amostras foram coletadas 5, 15 e 30 dias após a indução da doença periodontal e processadas para extração de RNA total e proteína, assim como rotineiramente processadas para análise histológica/estereométrica. Um aumento significante da severidade da inflamação foi observado em ambos os modelos já no período de 5 dias. Entretanto, o modelo de ligadura mostrou uma diminuição da intensidade da inflamação aos 30 dias, que não foi observada no modelo de injeção de LPS. O perfil de expressão dos níveis de mRNA de MMP-13, assim como a cinética das vias de sinalização ativadas durante o curso da doença periodontal foram distintos segundo o modelo experimental mas, em geral, acompanharam a severidade do processo inflamatório. De forma interessante, não houve correlação entre os níveis protéicos e de mRNA de MMP-13 em ambos os modelos, sugerindo uma regulação pós­transcricional da expressão de MMP-13 in vivo. Além disso, p38 e ERK MAPkinases, assim como NF- B foram ativadas nos dois modelos de doença periodontal, embora com cinética distinta; enquanto ativação de STAT3 e STAT5 foi verificada apenas no modelo de ligadura. Estes achados sugerem uma ativação diferencial de receptores celulares em cada forma de indução da doença periodontal, o que determina diferenças temporais e qualitativas nas redes de sinalização intracelular ativadas e influencia a regulação da expressão gênica de MMP-13 em cada modelo experimental

The hallmark of destructive periodontal disease progression is the overproduction of cytokines which promotes the increased expression of other inflammatory mediators such as, MMPs. MMP-13 is a collagenase of complex gene regulation that has been implicated on ECM degradation and bone resorption in several inflammatory conditions, including periodontal disease and rheumatoid arthritis. Regulation of gene expression requires the activation of several signaling pathways through receptor-ligand binding of external stimuli represented by bacterial antigens and/or host-derived cytokines. The complexity of the cytokine network established during periodontal disease progression results from the signaling pathways activated, which are determined by the nature of external stimuli. Thus, considering the fundamental role of signaling pathways on regulation of cytokine gene expression and the relevant role of MMP-13 in periodontal disease, this study evaluated the expression of MMP-13 and the signaling pathways activated during the course of two experimentallyinduced periodontal disease models. Expression of MMP-13 at mRNA and protein levels was evaluated by reverse transcription polymerase chain reaction (RT-PCR) and Western Blot, respectively. The activation kinetics of some signaling pathways that are related to the expression of inflammatory mediators was also verified by Western Blot. The two experimental models used were: LPS injections and placement of ligatures. Bi-weekly injections of Eschericia coli LPS were done into the palatal aspect of upper molars (30 µg per injection). Ligatures were placed at the cervical portion of both lower first molars. The control animals received injections of PBS vehicle on the palatal gingiva of upper molars, whereas no ligatures were placed on the lower molars. Samples were collected 5, 15 and 30 days after beginning of periodontal disease induction and processed for extraction of total RNA and protein, as well as routinely processed for histology/estereometry. A significant increase on the severity of inflammation was observed in both experimental models already at the 5-day period. However, the ligature model presented a significant decrease on the intensity of inflammation at the 30-day period, which was not observed with the LPS injection model. MMP-13 expression profile, as well as the kinetic of signaling pathways activated during periodontal disease were somewhat different according to each experimental model, however paralleled the severity of inflammation. Interestingly, there was no transcription-translation coupling for MMP-13 in both models, suggesting a post­transcriptional regulation of MMP13 expression in vivo. Moreover, p38 and ERK MAPkinases, as well as NF-kB were activated in both periodontal disease models, however with different kinetics, whereas activation of STAT3 and STAT5 was verified only on the ligature model. These findings suggest a differential activation of cellular receptors in each model of experimentally-induced periodontal disease, which results in temporal and qualitative differences on the signaling network and influences MMP-13 gene regulation in each experimental model

Cytotoxic effects of different concentrations of chlorhexidine.

PURPOSE: To evaluate the cytotoxic effects of different concentrations of Chlorhexidine (Chx) to the odontoblast cell line MDPC-23. METHODS: The odontoblast-like cells were seeded (30,000 cells/cm2) in 60 wells of 24-well dishes and then incubated in contact with the following experimental and control solutions: Group 1: 0.0024% Chx; Group 2: 0.004% Chx; Group 3: 0.02% Chx; Group 4: Phosphate buffer saline solution (PBS, negative control); and Group 5: 0.06% H2O2 (positive control). Cell metabolic activity was measured by MTT assay and the cell morphology was analyzed by SEM. RESULTS: The cytotoxic effects of Chx are dose-dependent. The reduction in the cell metabolism for Groups 1, 2, and 3 was 24.8%, 29.9% and 70.8%, respectively. No statistical difference was observed between the Groups 1 and 2 in which no significant cell morphology changes occurred. Consequently, it was concluded that 0.02% Chx solution presents high cytotoxicity to the odontoblast-like cells MDPC-23. On the other hand, 0.0024% and 0.004% Chx causes slight cytopathic effects to the cultured cells.