Litsea japonica Leaf Extract Suppresses Proinflammatory Cytokine Production in Periodontal Ligament Fibroblasts Stimulated with Oral Pathogenic Bacteria or Interleukin-1ß.

Periodontal disease, a chronic disease caused by bacterial infection, eventually progresses to severe inflammation and bone loss. Regulating excessive inflammation of inflamed periodontal tissues is critical in treating periodontal diseases. The periodontal ligament (PDL) is primarily a connective tissue attachment between the root and alveolar bone. PDL fibroblasts (PDLFs) produce pro-inflammatory cytokines in response to bacterial infection, which could further adversely affect the tissue and cause bone loss. In this study, we determined the ability of Litsea japonica leaf extract (LJLE) to inhibit pro-inflammatory cytokine production in PDLFs in response to various stimulants. First, we found that LJLE treatment reduced lipopolysaccharide (LPS)-induced pro-inflammatory cytokine (interleukin-6 and interleukin-8) mRNA and protein expression in PDLFs without cytotoxicity. Next, we observed the anti-inflammatory effect of LJLE in PDLFs after infection with various oral bacteria, including Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. These anti-inflammatory effects of LJLE were dose-dependent, and the extract was effective following both pretreatment and posttreatment. Moreover, we found that LJLE suppressed the effect of interleukin-1 beta-induced pro-inflammatory cytokine production in PDLFs. Taken together, these results indicate that LJLE has anti-inflammatory activity that could be exploited to prevent and treat human periodontitis by controlling inflammation.

Detection and function of lipopolysaccharide and its purified lipid A after treatment with auxiliary chemical substances and calcium hydroxide dressings used in root canal treatment.

AIM: To investigate the influence of auxiliary chemical substances (ACSs) and calcium hydroxide [Ca(OH)2 ] dressings on lipopolysaccharides (LPS)/lipid A detection and its functional ability in activating Toll-like receptor 4 (TLR4). METHODOLOGY: Fusobacterium nucleatum pellets were exposed to antimicrobial agents as following: (i) ACS: 5.25%, 2.5% and 1% sodium hypochlorite solutions (NaOCl), 2% chlorhexidine (CHX) (gel and solution) and 17% ethylenediaminetetraacetic acid (EDTA); (ii) intracanal medicament: Ca(OH)2 paste for various periods (1 h, 24 h, 7 days, 14 days and 30 days); (iii) combination of substances: (a) 2.5% NaOCl (1 h), followed by 17% EDTA (3 min) and Ca(OH)2 (7 days); (b) 2% CHX (1 h), afterwards, 17% EDTA (3 min) followed by Ca(OH)2 (7 days). Saline solution was the control. Samples were submitted to LPS isolation and lipid A purification. Lipid A peaks were assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrom (MALDI-TOF MS) whilst LPS bands by SDS-PAGE separation and silver staining. TLR4 activation determined LPS function activities. Statistical comparisons were carried out using one-way anova with Tukey-Kramer post-hoc tests at the 5% significance level. RESULTS: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of control lipid A demonstrated the ion cluster at mass/charge (m/z) 1882 and an intense band in SDS-PAGE followed by silver staining of control LPS. In parallel, LPS control induced a robust TLR4 activation when compared to ACS (P ≤ .001). 5.25% NaOCl treatment led to the absence of lipid A peaks and LPS bands, whilst no changes occurred to lipid A/LPS after treatment with others ACS. Concomitantly, 5.25% NaOCl-treated LPS did not activate TLR4 (P < .0001). As for Ca(OH)2 , lipid A was not detected by MALDI-TOF nor by gel electrophoresis within 24 h. LPS treated with Ca(OH)2 was a weak TLR4 activator (P < .0001). From 24 h onwards, no significant differences were found amongst the time periods tested (P > 0.05). The addition of Ca(OH)2 for 7 days to cells treated either with 2.5% NaOCl or 2% CHX led to the absence of lipid A peaks and LPS bands, leading to a lower activation of TLR4. CONCLUSION: 5.25% NaOCl and Ca(OH)2 dressings from 24 h onwards were able to induce both, loss of lipid A peaks and no detection of LPS bands, rendering a diminished immunostimulatory activity through TLR4.

Protective effects of grape seed proanthocyanidins against oxidative stress induced by lipopolysaccharides of periodontopathogens.

BACKGROUND: During phagocytosis or stimulation with bacterial components, macrophages activate various cell processes, including the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are critical for successful defense against invading organisms. Increased levels of ROS/RNS create oxidative stress that results in tissue and bone destruction. Grape seed proanthocyanidins have been reported to possess a wide range of biologic properties against oxidative stress. In the present study, we investigated the effects of a grape seed proanthocyanidin extract (GSE) and commercial polyphenols on the production of ROS and RNS and on the protein expression of inducible nitric oxide synthase (iNOS) by murine macrophages stimulated with lipopolysaccharides (LPS) of periodontopathogens. METHODS: Macrophages (RAW 264.7) were treated with non-toxic concentrations of either GSE or commercial polyphenols (gallic acid [GA] and [-]-epigallocatechin-3-gallate [EGCG]) and stimulated with LPS of Actinobacillus actinomycetemcomitans or Fusobacterium nucleatum, and iNOS expression was evaluated by immunoblotting. Nitric oxide (NO) production was quantified using the colorimetric Griess assay, whereas ROS production was measured with the fluorescent 123-dihydrorhodamine dye. RESULTS: GSE strongly decreased NO and ROS production and iNOS expression by LPS-stimulated macrophages. GA also revealed a strong inhibitory effect on NO production without affecting iNOS expression but slightly increasing ROS production. EGCG showed an inhibitory effect on NO and ROS production and on iNOS expression by macrophages. CONCLUSION: Our findings demonstrate that proanthocyanidins have potent antioxidant properties and should be considered a potential agent in the prevention of periodontal diseases.

[Effect and mechanism of garlic juice and hydrogen peroxide on the degradation of lipopolysaccharide].

OBJECTIVE: To elucidate the effect and mechanism of garlic juice and hydrogen peroxide on the degradation of lipopolysaccharide (LPS). METHODS: Hot phenol-water method, phenol-chloroform-petroleum ether procedure, limulus lysate test, lowry's ash spetrographical examination and gas-liquid chromatography etc. were used in this study. RESULTS: The sequence of degradation effect was 30% hydrogen peroxide (H), the most powerful, followed by garlic juice (G), 1:1 diluted G and 3% H, their effects were dose dependent and G group was time dependent. The mechanism of H on LPS degradation was fractionization of phosphoryl in position 1 from lipid A, while that of G was complex, it could bound LPS molecule and influenced its effect besides LPS hydrolysis. CONCLUSION: The study may imply that the degradation position and mechanism on LPS are different and remain to be elucidated.