Green tea catechins potentiate the effect of antibiotics and modulate adherence and gene expression in Porphyromonas gingivalis

ObjectivesA number of studies have brought evidence that green tea catechins may contribute to periodontal health. The objective of this study was to investigate the ability of a green tea extract and its principal constituent epigallocatechin-3-gallate (EGCG) to potentiate the antibacterial effects of antibiotics (metronidazole, tetracycline) against Porphyromonas gingivalis, and to modulate the adherence to oral epithelial cells and expression of genes coding for virulence factors and the high temperature requirement A (HtrA) stress protein in P. gingivalis.MethodsA broth microdilution assay was used to determine the antibacterial activity of the green tea extract and EGCG. The synergistic effects of either compounds in association with metronidazole or tetracycline were evaluated using the checkerboard technique. A fluorescent assay was used to determine bacterial adherence to oral epithelial cells. The modulation of gene expression in P. gingivalis was evaluated by quantitative RT-PCR. The Vibrio harveyi bioassay was used for monitoring quorum sensing inhibitory activity.ResultsThe MIC values of the green tea extract on P. gingivalis ranged from 250 to 1000 μg/ml, while those of EGCG ranged from 125 to 500 μg/ml. A marked synergistic effect on P. gingivalis growth was observed for the green tea extract or EGCG in combination with metronidazole. Both the green tea extract and EGCG caused a dose-dependent inhibition of P. gingivalis adherence to oral epithelial cells. On the one hand, green tea extract and EGCG dose-dependently inhibited the expression of several P. gingivalis genes involved in host colonization (fimA, hagA, hagB), tissue destruction (rgpA, kgp), and heme acquisition (hem). On the other hand, both compounds increased the expression of the stress protein htrA gene. The ability of the green tea extract and EGCG to inhibit quorum sensing may contribute to the modulation of gene expression.ConclusionsThis study explored the preventive and therapeutic potential of green tea catechins against periodontal disease. In addition to inhibit growth and adherence of P. gingivalis, a green tea extract and its main constituent EGCG was found to decrease the expression of genes coding for the major virulence factors.     

Thymus vulgaris L. extract has antimicrobial and anti-inflammatory effects in the absence of cytotoxicity and genotoxicity

ObjectivesThis study evaluated the biological effects of the T. vulgaris L. extract., such as antimicrobial activity on planktonic cultures and mono- and polymicrobial biofilms, cytotoxicity, anti-inflammatory activity and genotoxicity.MethodsMonomicrobial biofilms of Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans and Pseudomonas aeruginosa and polymicrobial biofilms composed by C. albicans with each bacterium were formed for 48 h and exposed for 5 min to the plant extract. Murine macrophages (RAW 264.7), human gingival fibroblasts (FMM-1), human breast carcinoma cells (MCF-7) and cervical carcinoma cells (HeLa) were also exposed to the plant extract for 5 min and the cell viability were analyzed by MTT, neutral red (NR) and crystal violet (CV) assays. Interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) produced by RAW 264.7 was quantified by ELISA, after 24 h exposure to the plant extract, both in the absence and presence of lipopolysaccharide (LPS) from Escherichia coli. Genotoxicity of the plant extract was evaluated by micronucleus formation (MN) in 1000 cells. The results were analyzed by T-Test or ANOVA and Tukey’s Test (P ≤ 0.05).ResultsAll biofilms showed significant reductions in CFU/mL (colony-forming units per milliliter). Cell viability was above 50% for all cell lines. Anti-inflammatory effect on the synthesis of IL-1β and TNF-α was observed. The MN was similar or lower than the control group in all cells.ConclusionsT. vulgaris L. extract was effective against all biofilms, promoted high cell viability, anti-inflammatory effect and presented no genotoxicity.     

Probiotic intervention influences the salivary levels of Matrix Metalloproteinase (MMP)-9 and Tissue Inhibitor of metalloproteinases (TIMP)-1 in healthy adults

ObjectiveTo study the effect of orally administered Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus rhamnosus GG on the salivary levels of Matrix Metalloproteinases (MMP)-8, MMP-9 and of Tissue Inhibitor of Metalloproteinases (TIMP)-1 in healthy adults. Furthermore, the correlations between MMP-8, MMP-9 and TIMP-1 and plaque and gingival indices, salivary mutans streptococci and lactobacilli counts, and stimulated saliva secretion rate were analysed.DesignThe salivary samples originated from a randomized controlled trial where healthy student volunteers consumed probiotic or placebo lozenges twice a day for four weeks. The saliva samples were collected and clinical parameters measured at the baseline and at the end of the original study. For this study, the salivary levels of MMP-8, MMP-9 and TIMP-1 were analysed with immunofluorometric assay (IFMA) and enzyme-linked immunosorbent assay (ELISA).ResultsIn the probiotic group (n = 29), salivary MMP-9 levels increased (p < 0.01) and TIMP-1 levels decreased (p < 0.01) significantly during the intervention. Furthermore, MMP-9/TIMP-1 ratio differed significantly from the baseline level (p < 0.01). These changes were not observed in the control group (n = 31). In the whole data, salivary MMP-9 and gingival index correlated (r = 0.260, p < 0.05 at baseline and r = 0.354, p < 0.01 at the end of the study). Intergroup differences or correlations with other clinical parameters were not found. Probiotic consumption did not affect the saliva flow rate.ConclusionsIncreased MMP-9 and decreased TIMP-1 levels in saliva may indicate that probiotics have immunomodulatory effects in the oral cavity. Furthermore, increased salivary MMP-9 levels may be an indication of the defensive potential of matrix metalloproteinases.     

Does green tea consumption improve the salivary antioxidant status of smokers?

Objective Considering the higher rate of oral cancer, and reduction in salivary antioxidants in smokers as indicated in previous studies, antioxidant- containing nutrients such as green tea, seem to be beneficial in counteracting against oxidative stress in this group. This study assessed the salivary total antioxidant alteration in smokers compared to nonsmokers, after short-tem (7 days) and long-term (3 weeks), green tea drinking.DesignIn this experimental study, 20 volunteer moderate-to-heavy male smokers, and 20 matched healthy non-smokers were selected to participate, according to the inclusion criteria. Participants were instructed to drink two cups of green tea per day, by dissolving 2 g of green tea in 150 ml of hot water for each cup. After saliva collection, antioxidant capacity of saliva was measured at baseline, after 7 days, and after 21 days. Statistical evaluation was done by SPSS 21, using paired samplet tests, one-way ANOVA and Bonferroni tests.Results At day zero nonsmokers had a higher antioxidant capacity than smokers (686.6 ± 62.22 vs. 338.8 ± 59.9) mM/50 μl, P < 0.001. There was also a significant difference between two groups in salivary total antioxidant capacity after one week and three weeks of green tea consumption (P < 0.001). However, there was an upward trend in both smokers and non-smokers over the study period (after tea drinking). In addition, a significant difference was found in total antioxidant capacity alteration in smokers compared to non-smokers from baseline to day 21.ConclusionsResults support the effectiveness of green tea consumption in salivary antioxidants enhancement in smokers, in both the short- and long term.     

Extracellular ATP is a key modulator of alveolar bone loss in periodontitis

Periodontal diseases are initiated by pathogenic bacterial biofilm activity that induces a host inflammatory cells immune response, degradation of dento gingival fibrous tissue and its detachment from root cementum. It is well accepted, that osteoclastic alveolar bone loss is governed exclusively through secretion of proinflammatory cytokines. Nevertheless, our findings suggest that once degradation of collagen fibers by MMPs occurs, a drop of cellular strains cause immediate release of ATP from marginal gingival fibroblasts, cell deformation and influx of Ca + 2. Increased extracellular ATP (eATP) by interacting with P2 × 7 purinoreceptors, present on fibroblasts and osteoblasts, induces generation of receptor activator of nuclear factor kB ligand (RANKL) that further activates osteoclastic alveolar bone resorption and bone loss. In addition, increased eATP levels may amplify inflammation by promoting leukocyte recruitment and NALP3-inflammasome activation via P2 × 7. Then, the inflammatory cells secrete cytokines, interleukin IL-1, TNF and RANKL that further trigger alveolar bone resorption. Moreover, eATP can be secreted from periodontal bacteria that may further contribute to inflammation and bone loss in periodontitis. It seems therefore, that eATP is a key modulator that initiates the pathway of alveolar bone resorption and bone loss in patients with periodontal disease. In conclusion, we propose that strain release in gingival fibroblasts aligned on collagen fibers, due to activity of MMP, activates release of ATP that triggers the pathway of alveolar bone resorption in periodontitis. We predict that by controlling the eATP interaction with its cellular purinoreceptors will reduce significantly bone loss in periodontitis.     

Prophylactic effect of green tea and Nigella sativa extracts against fenitrothion-induced toxicity in rat parotid gland

ObjectiveThe aim of the present study was to compare between two antioxidant treatments in prevention of fenitrothion induced toxicity on rat parotid salivary gland.DesignForty adult male Wistar rats with an average weight of 120–150 g were randomised into 4 groups, control (group I), fenitrothion administration (group II), fenitrothion administration 1 h after green tea extract or Nigella sativa oil extract administration (groups III and IV respectively). The rats were then sacrificed after 28 days. The submandibular salivary glands were examined histologically, immunohistochemically and ultrastructurally.ResultsHistopathologically the fenitrothion group showed sign of acini degeneration represented by loss of normal architecture (amalgamation). The nuclei of the acinar cells revealed different sizes and shape (polymorphism). The acini relatively preserved their shape in both prophylactic groups (III and IV). Histomorphometric analysis showed significant increase in the optical density of caspase-3 cleaved activity in all experimental groups (p = 0.0001). A significant difference was observed between both prophylactic experimental groups III and IV (p = 0.0039). Ultrastructurally, the nuclei of serous acini in group II appeared pyknotic with segregation of chromatin. Condensation of the chromatin at the periphery of the nucleus was observed in the nuclei of group III, Clumping of the chromatin with darkly stained pyknotic nucleus was detected in group IV.ConclusionsIn a rat model the administration of natural antioxidants could be of beneficial effect on prevention of cytotoxicity induced by organophosphorous compounds. However, green tea showed more promising results than that of Nigella sativa.     

Effects of alendronate on human osteoblast-like MG63 cells and matrix metalloproteinases

ObjectiveThe objective of this study was to examine the effects of alendronate on the expression and activity of matrix metalloproteinases (MMPs) and the expression of the tissue inhibitors of MMPs (TIMPs) from human osteoblast-like MG63 cells.Materials and methodsMG63 cells were exposed to various concentrations of alendronate. Cell proliferation and cytotoxicity were evaluated by water-soluble tetrazolium-1 and lactate dehydrogenase, respectively. MG63-mediated collagen degradation was assessed utilising Type I collagen assays. Conditioned media and membrane extracts were collected for Western blot analyses of select MMPs and TIMPs. Gelatin zymography gels were incubated with alendronate to assess its effects on MMP-2 activity.ResultsAlendronate affected MG63 proliferation and cytotoxicity at concentrations equal to/or greater than 10^?5 M (all p < 0.05). There were no significant differences in the collagen degrading ability of treated cells at non-toxic levels vs. untreated cells. Alendronate had no effects on the expression of MMP-2 or MT1-MMP (membrane type-1 MMP) in the conditioned media or membrane extracts, and of MMP-1 or TIMP-2 in the conditioned media. TIMP-2 in the membrane extracts was not detectable. MMP-2 activity in the zymograms was inhibited by 10^?3 and 10^?2 M alendronate.ConclusionAlendronate at 10^?5 M or higher was toxic to the cells. Alendronate at 10^?8 to 10^?6 M did not alter the expression of MMP-1, MMP-2, MT1-MMP or TIMP-2, as well as did not alter collagen degradation. Alendronate inhibited MMP-2 activity at 10^?3 and 10^?2 M in the zymograms. In conclusion, non-toxic levels of alendronate (10^?8 to 10^?6 M) did not alter MMP expression in MG63 cells or inhibit MMP-2 activity.     

Topical application of ointment containing 0.5% green tea catechins suppresses tongue oxidative stress in 5-fluorouracil administered rats

ObjectiveThe purpose of this study was to investigate the preventive effects of topical application of green tea catechins on tongue oxidative stress induced by 5-fluorouracil (5-FU) administration in rats.DesignMale Wistar rats (n = 28, 8 weeks old) were divided into four groups of seven rats each: a negative control group (saline administration and application of ointment without green tea catechins), a positive control group (5-FU administration and application of ointment without green tea catechins), and two experimental groups (5-FU administration and application of ointment containing 0.1% or 0.5% green tea catechins). Topical application of each ointment to the ventral surface of the tongue was performed once a day for 5 days. The level of 8-hydroxydeoxyguanosine (8-OHdG) was determined to evaluate oxidative stress. Fluorescence staining was also performed to confirm nuclear factor erythroid 2-related factor 2 (Nrf2) translocation to the nucleus.ResultsAfter the experimental period, the ratios of 8-OHdG-positive cells in the ventral tongue tissue were higher in the positive control group than in the negative control group (P < 0.05). On the other hand, those in the 0.5% green tea catechin group, but not in the 0.1% green tea catechin group, were lower than the positive control group (P < 0.05). In addition, Nrf2 translocation to the nucleus was greater in the 0.5% green tea catechin group than in the positive control group (P < 0.05).ConclusionsTopical application of ointment containing 0.5% green tea catechins could prevent tongue oxidative stress in 5-FU administered rats, via up-regulation of the Nrf2 signaling pathway.     

Association of thalassemia major and gingival inflammation: A pilot study

ObjectivesThis cross-sectional study aimed to investigate the relationship between thalassemia major (TM) and gingival inflammation through the salivary, serum, and gingival crevicular fluid (GCF) levels of matrix metalloproteinase (MMP)-8, MMP-9 and tissue inhibitor of MMP (TIMP)-1.MethodsBiofluid samples and full-mouth clinical periodontal recordings were obtained from 29 otherwise healthy patients with TM and 25 systemically healthy (SH) individuals. Biofluid samples were evaluated by immunofluorometric assay (IFMA) and enzyme-linked immunoassays (ELISAs). Data were tested statistically by Kolmogorov Simirnov, Mann–Whitney U tests, Spearman correlation analysis.ResultsAge, smoking status, bleeding on probing, plaque index were similar in the study groups, but probing depth, gender data exhibited significant differences (p = 0.037 for both). Salivary MMP-8, MMP-9, TIMP-1 concentrations were significantly higher in the TM than SH group (p = 0.014; p < 0.001; p = 0.042, respectively). Serum TIMP-1 concentrations were significantly higher; MMP-8/TIMP-1, MMP-9/TIMP-1 molar ratios were significantly lower in the TM than SH group (p < 0.001; p = 0.005; p = 0.022, respectively). Very few GCF samples revealed biochemical data above the detection limits. Numerous correlations were found between clinical periodontal parameters and biochemical data.ConclusionsIt may be suggested that TM may exacerbate the local inflammatory response as manifested in salivary MMP-8, MMP-9, TIMP-1 levels.     

Profiling cytokine levels in chlorhexidine and EGCG-treated odontoblast-like cells

Objective

To screen the effect of two compounds, chlorhexidine diacetate (CHX) and epigallocatechin-gallate (EGCG), on the levels of cytokines produced by odontoblast-like cells (MDPC-23).

Exposure to Porphyromonas gingivalis LPS during macrophage polarisation leads to diminished inflammatory cytokine production

ObjectiveThe objective of the present study was to determine the effects of concurrent LPS and cytokine priming, reflective of the in vivo milieu, on macrophage production of key periodontitis associated cytokines TNF, IL-1β and IL-6.DesignTHP-1 cells were pre-treated with combinations of Porphyromonas gingivalis and Escherichia coli lipopolysaccharide (LPS), concurrently with polarising cytokines IFNγ and IL-4, or PMA as a non-polarised control. Production of key periodontitis associated cytokines in response to subsequent LPS challenge were measured by enzyme − linked immunosorbent assay.ResultsCompared with cells incubated with IFNγ or IL-4 alone in the “polarisation” phase, macrophages that were incubated with LPS during the first 24 h displayed a down-regulation of TNF and IL-1β production upon secondary LPS treatment in the “activation” phase. In all three macrophage populations (M0, M1 and M2), pre-treatment with P. gingivalis LPS during the polarisation process led to a significant decrease in TNF production in response to subsequent activation by LPS (p = 0.007, p = 0.002 and p = 0.004, respectively). Pre-treatment with E. coli LPS also led to a significant down-regulation in TNF production in all three macrophage populations (p < 0.001). Furthermore, the presence of E. coli LPS during polarisation also led to the down-regulation of IL-1β in the M1 population (p < 0.001), whereas there was no measurable effect on IL-1β production in M0 or M2 macrophages. There was no significant effect on IL-6 production.ConclusionsMacrophages become refractory to further LPS challenge, whereby production of key periodontitis associated cytokines TNF and IL-1β is reduced after exposure to LPS during the polarisation phase, even in the presence of inflammatory polarising cytokines. This diminished cytokine response may lead to the reduced ability to clear infection and transition to chronic inflammation seen in periodontitis.     

The cytotoxic effects of three different bisphosphonates in-vitro on human gingival fibroblasts,osteoblasts and osteogenic sarcoma cells

IntroductionOsteonecrosis of the jaw (ONJ) is an emerging condition in patients undergoing long-term administration of bisphosphonates (BP) for the treatment of osteoporosis and hypercalcaemia associated with malignancy, multiple myeloma, and metastatic breast and prostate cancers. This is a follow-up study, its purpose was to examine the effects in-vitro of intravenous zoledronic acid (ZOL) and pamidronate (PAM) and oral alendronate (FOS) on the human oral cavity using gingival fibroblasts and osteoblasts cells and, in addition, osteogenic sarcoma cells (SaOS-2-cells).Materials and methodsHuman gingival fibroblasts, osteoblasts and SaOS-2-cells were seeded on multiple 6-well plates at a density of 5 × 10⁵ cells in a 4-week cell culture. Four different concentrations (1, 5, 10, 20 μM) of each BP (ZOL, PAM, FOS) and pyrophosphate were used in this study.ResultsAll BP decreased collagen production and lowered cell proliferation in-vitro. ZOL was the component with most inhibitory effect.ConclusionThe findings in this study suggest that ZOL, PAM and FOS generally diminish cell proliferation and collagen production of human gingival fibroblasts, osteoblasts and SaOS-2-cells. The present follow-up study shows that not only ZOL and PAM but also FOS have a strong inhibitory effect on collagen production and cell survival in-vitro.     

Inhibition of Streptococcus mutans biofilm formation using extracts from Assam tea compared to green tea

ObjectiveStreptococcus mutans, a gram-positive oral bacterium, has been identified as one of the principal etiological agents of human dental caries. To clarify the nature of the difference anti-biofilm effect against S. mutans between Assam tea from Camellia sinensis var. assamica, partially fermented, and green tea from Camellia sinensis, non-fermented, active agents from the teas were purified.MethodsEffects of Assam tea and green tea samples on biofilm were assessed by using the conventional titer plate method and the human saliva-coated hydroxyapatite discs. The purification and identification of inhibitors were performed by using ultrafiltration with centrifugal filter devices and high performance liquid chromatography.ResultsAssam tea has stronger biofilm inhibition activity against S. mutans than green tea. A substance of <10 kDa in mass in Assam tea had a high concentration of galloylated catechins and a stronger biofilm inhibiting activity than green tea. In contrast, substances >10 kDa in mass from green tea included higher concentrations of polysaccharides composed of galacturonic acid, such as pectin, that enhance biofilm formation.ConclusionsThe higher concentrations of galloylated catechins in Assam tea may assist in prevention of dental caries, whereas in green tea, this mode of inhibition was likely offset by the presence of pectin. Purification of catechins in partially fermented Assam tea with lower-molecular-weight polysaccharide than pectin may be useful for developing oral care products such as toothpaste and oral care gel pastes.     

Porphyromonas gingivalis antigens differently participate in the proliferation and cell death of human PBMC

ObjectiveModulation of cell-mediated immunity by microorganisms in periodontal diseases has been widely studied; however, the proliferative activity and/or programmed death of mononuclear cells under periodontopathogenic stimuli are not yet well understood. The aim of this study was to investigate in vitro proliferation and death of peripheral blood mononuclear cells (PBMC) upon stimulation with Porphyromonas gingivalis (Pg) antigens.DesignIn 19 patients with chronic periodontitis (CP) and 16 controls without periodontitis (NP) the following clinical parameters were evaluated: bleeding on probing, probing depth, and clinical attachment level. PBMC were cultured under Pg stimuli and apoptosis/necrosis and proliferation assays were carried out for 18 and 48 h, respectively. Fluorescence of labelled cells was determined using flow cytometry.ResultsPBMC of CP and NP subjects exhibited a lower proliferative response to Pg LPS (p < 0.05) and HmuY protein (p < 0.001) compared with non-stimulated cells. Early apoptosis was induced by Pg LPS (p < 0.01) and Pg extract (p < 0.05), whilst all antigens induced late apoptosis (Pg LPS: p < 0.001; Pg extract: p < 0.001; HmuY: p < 0.01) and necrosis (Pg LPS: p < 0.01; Pg extract: p < 0.001; HmuY: p < 0.001). Pg LPS induced higher late apoptosis than HmuY (p < 0.05). Only Pg LPS-induced necrosis tended to be higher in CP compared with NP.ConclusionsThe inhibitory effect of cell proliferation caused by Pg LPS and HmuY protein is not observed when these antigens comprise Pg extract. Despite induced apoptosis, some still unknown mechanism determines the inflammatory outcome in cell death stimulated by HmuY.     

Epigallocatechin-3-gallate (EGCG) enhances the therapeutic activity of a dental adhesive

ObjectivesThe purpose of this study was to evaluate the antibacterial potential and physicochemical properties of a dental adhesive incorporated with epigallocatechin-3-gallate (EGCG) in different concentration over time.MethodsEGCG was incorporated at a ratio of 100, 200, and 300 μg/ml into a dental adhesive. The effects of the cured adhesives on the growth of Streptococcus mutans were determined by direct contact test immediately or one month later and by scanning electron microscopy (SEM), respectively. Microtensile bond strength (μTBS) test was used to test the mechanical property of the adhesives immediately or six months later. The degree of conversion (DC) of the adhesives was evaluated by Fourier Transform Infrared Spectroscopy (FTIR).ResultsCompared with negative control, the 200 μg/ml and 300 μg/ml EGCG-incorporated dental adhesive were found to exhibit inhibitory effect on the growth of S. mutans. The μTBS of the EGCG-incorporated dental adhesive was higher than the control. The DC of the adhesive system was not affected by the addition of EGCG.Conclusions200 μg/ml EGCG incorporated dental adhesives could accomplish therapeutic goals that play in antimicrobial function whilst keeping the durability of resin–dentine bond.