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.

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.

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.

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.

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.