Porphyromonas endodontalis HmuY differentially participates in heme acquisition compared to the Porphyromonas gingivalis and Tannerella forsythia hemophore-like proteins.

Introduction: Porphyromonas gingivalis and Porphyromonas endodontalis belong to the Bacteroidota phylum. Both species inhabit the oral cavity and can be associated with periodontal diseases. To survive, they must uptake heme from the host as an iron and protoporphyrin IX source. Among the best-characterized heme acquisition systems identified in members of the Bacteroidota phylum is the P. gingivalis Hmu system, with a leading role played by the hemophore-like HmuY (HmuYPg) protein. Methods: Theoretical analysis of selected HmuY proteins and spectrophotometric methods were employed to determine the heme-binding mode of the P. endodontalis HmuY homolog (HmuYPe) and its ability to sequester heme. Growth phenotype and gene expression analysis of P. endodontalis were employed to reveal the importance of the HmuYPe and Hmu system for this bacterium. Results: Unlike in P. gingivalis, where HmuYPg uses two histidines for heme-iron coordination, other known HmuY homologs use two methionines in this process. P. endodontalis HmuYPe is the first characterized representative of the HmuY family that binds heme using a histidine-methionine pair. It allows HmuYPe to sequester heme directly from serum albumin and Tannerella forsythia HmuYTf, the HmuY homolog which uses two methionines for heme-iron coordination. In contrast to HmuYPg, which sequesters heme directly from methemoglobin, HmuYPe may bind heme only after the proteolytic digestion of hemoglobin. Conclusions: We hypothesize that differences in components of the Hmu system and structure-based properties of HmuY proteins may evolved allowing different adaptations of Porphyromonas species to the changing host environment. This may add to the superior virulence potential of P. gingivalis over other members of the Bacteroidota phylum.

A pulmonary abscess caused by Porphyromonas endodontalis infection:A case report and literature review.

Porphyromonas endodontalis is an oral anaerobic bacterium associated with periodontitis but seldomly been detected in other diseases. Only one case of respiratory disease caused by Porphyromonas endodontalis, pyopneumothorax, has been reported so far. A 53-year-old man with refractory periodontitis was admitted due to an indeterminate lung space-occupying lesion. Following mNGS analysis of the liquefaction necrotic area and solid component of the lesion through biopsy, Porphyromonas endodontalis and Parvimonas micra were detected. Therefore, the patient was diagnosed with an aspiration lung abscess and discharged after receiving effective antibacterial treatment. The Chest computed tomography (CT) scan revealed a remarkable improvement during outpatient follow-up. In this study, we applied mNGS to diagnose a case of lung abscess attributed to an uncommon bacterium successfully, suggesting that when patients complicated with periodontal diseases and clinical respiratory symptoms, the possibility of inhalation disease caused by oral pathogens should be considered.

Lipopolysaccharides from Porphyromonas endodontalis and Porphyromonas gingivalis promote angiogenesis via Toll-like-receptors 2 and 4 pathways in vitro.

AIM: Angiogenesis contributes to the development of apical periodontitis, periodontitis, and other oral pathologies; however, it remains unclear how this process is triggered. The aim was to evaluate whether lipopolysaccharide (LPS) from Porphyromonas endodontalis and Porphyromonas gingivalis induced angiogenesis-related effects in vitro via TLR2 and TLR4. METHODOLOGY: Porphyromonas endodontalis LPS (ATCC 35406 and clinical isolate) was purified with TRIzol, whereas P. gingivalis LPS was obtained commercially. The effects of the different LPS (24 h) in endothelial cell migration were analysed by Transwell assays, following quantification in an optical microscope (40×). The effects of LPS on FAK Y397 phosphorylation were assessed by Western blotting. Angiogenesis in vitro was determined in an endothelial tube formation assay (14 h) in Matrigel in the absence or presence of either LPS. IL-6 and VEGF-A levels were determined in cell supernatants, following 24 h treatment with LPS, and measured in multiplex bead immunoassay. The involvement of TLR2 and TLR4 was assessed with blocking antibodies. The statistical analysis was performed using STATA 12® (StataCorp LP). RESULTS: The results revealed that P. endodontalis LPS, but not P. gingivalis LPS, stimulated endothelial cell migration. Pre-treatment with anti-TLR2 and anti-TLR4 antibodies prevented P. endodontalis LPS-induced cell migration. P. endodontalis LPS promoted FAK phosphorylation on Y397, as observed by an increased p-FAK/FAK ratio. Both P. gingivalis and P. endodontalis LPS (ATCC 35406) induced endothelial tube formation in a TLR-2 and -4-dependent manner, as shown by using blocking antibodies, however, only TLR2 blocking decreased tube formation induced by P. endodontalis (clinical isolate). Moreover, all LPS induced IL-6 and VEGF-A synthesis in endothelial cells. TLR2 and TLR4 were required for IL-6 induction by P. endodontalis LPS (ATCC 35406), while only TLR4 was involved in IL-6 secretion by the other LPS. Finally, VEGF-A synthesis did not require TLR signalling. CONCLUSION: Porphyromonas endodontalis and P. gingivalis LPS induced angiogenesis via TLR2 and TLR4. Collectively, these data contribute to understanding the role of LPS from Porphyromonas spp. in angiogenesis and TLR involvement.

What Is the Microbial Profile in Persistent Endodontic Infections? A Scoping Review.

INTRODUCTION: This scoping review aimed to map the evidence about the microbiota found in persistent endodontic infections. METHODS: The study protocol was prospectively registered and is available at https://osf.io/3g2cp. The electronic search was performed in MEDLINE via PubMed, Lilacs, BBO, Scopus, Web of Science, Cochrane Library, and Embase. The eligibility criteria were based on the PCC acronym, where P (Population) represents patients with teeth presenting persistent endodontic infection, C (Concept) represents microbial profile, and C (Context) represents undergoing endodontic retreatment. Clinical studies that evaluated the microbial profile of samples collected from root canals of teeth undergoing retreatment, using classical or molecular methods, were included. Studies that did not show a minimum period of 1 year between primary endodontic treatment and retreatment or did not radiographically evaluate the quality of primary root canal filling were excluded. Two reviewers independently selected the articles and collected data. RESULTS: From a total of 957 articles, 161 were read in full, and 32 studies were included. The most prevalent species were Enterococcus faecalis, Parvimonas micra, Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Dialister invisus, Propionibacterium acnes, Tannerella forsythia, and Treponema denticola. Cases with symptomatology or inadequate root canal filling presented an increase in specific bacterial species compared to those with no symptomatology or adequate filling. A greater number of microorganisms was observed in teeth with inadequate coronal restoration compared to those with adequate restoration. CONCLUSIONS: Persistent endodontic infections have a polymicrobial profile identified by the commonly used methods for bacterial detection/identification and are subject to the limitations present in each of those methods.

Evaluation of PCR primers to identify Porphyromonas endodontalis in apical periodontitis clinical samples.

We aimed to evaluate whether two commonly used PCR primers are effective to identify P. endodontalis and discriminate from other prevalent black-pigmented bacteria in apical periodontitis (AP). Endodontic canal samples from patients with asymptomatic AP (n = 20) were collected and cultured in anaerobiosis. Two primer sets to detect P. endodontalis were selected from the literature and first analyzed for their specificity in silico; and then tested on clinical isolates in vitro and finally, in apical exudates ex vivo. The identity of P. endodontalis was verified by PCR and Sanger sequencing with universal primers for bacterial V3-V6 regions 16S rDNA. Only one primer set showed specificity only for P. endodontalis clones in silico and also was specific for P. endodontalis in vitro and ex vivo.

Lipopolysaccharide from Porphyromonas gingivalis, but Not from Porphyromonas endodontalis, Induces Macrophage M1 Profile.

Apical Lesions of Endodontic Origin (ALEO) are initiated by polymicrobial endodontic canal infection. Porphyromonas gingivalis (Pg) and Porphyromonas endodontalis (Pe) lipopolysaccharides (LPS) can induce a pro-inflammatory macrophage response through their recognition by TLR2 and TLR4. However, polarization responses induced by Pg and/or Pe LPS in macrophages are not fully understood. We aimed to characterize the polarization profiles of macrophages differentiated from THP-1 cells following Pg and/or Pe LPS stimulation from reference strain and clinical isolates. A modified LPS purification protocol was implemented and the electrophoretic LPS profiles were characterized. THP-1 human monocytes differentiated to macrophages were stimulated with Pg and Pe LPS. Polarization profiles were characterized through cell surface markers and secreted cytokines levels after 24 h of stimulation. TLR2 and TLR4 cell surfaces and transcriptional levels were determined after 24 or 2 h of LPS stimulation, respectively. LPS from Pg induced a predominant M1 profile in macrophages evidenced by changes in the expression of the surface marker CD64 and pro-inflammatory cytokine profiles, TNF-α, IL-1ß, IL-6, and IL-12. Pe LPS was unable to induce a significant response. TLR2 and TLR4 expressions were neither modified by Pg or Pe LPS. Pg LPS, but not Pe LPS, induced a macrophage M1 Profile.

Machine Learning Based Metagenomic Prediction of Inflammatory Bowel Disease.

In this study, we investigate faecal microbiota composition, in an attempt to evaluate performance of classification algorithms in identifying Inflammatory Bowel Disease (IBD) and its two types: Crohn's disease (CD) and ulcerative colitis (UC). From many investigated algorithms, a random forest (RF) classifier was selected for detailed evaluation in three-class (CD versus UC versus nonIBD) classification task and two binary (nonIBD versus IBD and CD versus UC) classification tasks. We dealt with class imbalance, performed extensive parameter search, dimensionality reduction and two-level classification. In three-class classification, our best model reaches F1 score of 91% in average, which confirms the strong connection of IBD and gastrointestinal microbiome. Among most important features in three-class classification are species Staphylococcus hominis, Porphyromonas endodontalis, Slackia piriformis and genus Bacteroidetes.

[Effect of inhibition of Porphyromonas endodontalis on osteoblast differentiation].

PURPOSE: Porphyromonas endodontalis (P.e) is the dominant bacterium in the infected canal of pulpal and periapical disease.Lipopolysaccharides (LPS) in the outer membrane of the cell wall is an important toxicity factor of P.e. In this study, the effect of P.e-LPS on osteoblast differentiation was studied, and the pathogenic mechanism of P.e-LPS in periapical bone resorption disease was explored. METHODS: Porphyromonas endodontalis was cultured under anaerobic conditions. P.e-LPS was extracted by thermophenol water method, and then the extracted LPS was qualitatively analyzed by gel limulireagent method. Preosteoblast cell line MC3T3-E1 were induced to differentiate into osteoblasts by osteoblast differentiation medium (50 µg/mL ascorbic acid，6 mmol/L beta-glycerphosphate). Expressions of osteogenic differentiation genes including distal-less homeobox 5（DLX5）, runt-related transcription factor 2(Runx2), Osterix, bone sialoprotein (BSP), OCN(osteocalcin) and Collagen were detected by RT-PCR. The activity of alkaline phosphatase(ALP)， alizarin red staining and Von Kossa staining were used to determine the mineralization level of osteoblasts.The expression of TOLL-like receptor-4 (TLR-4), the receptor of P.e-LPS, was silenced by siRNA transfection. SPSS 11.0 software package was used for statistical analysis of the data. RESULTS: The mRNA expressions of osteogenic differentiation genes including DLX5, Runx2, Osterix, OCN, BSP, and Collagen were significantly decreased after treated with P.e-LPS (10 µg/mL) for 3 d, compared with the control group（P<0.05）.After treated with P.e-LPS (10 µg/mL) for 7 d or 14 d, respectively, ALP and alizarin red staining intensity was decreased. P.e-LPS was applied to the si-TLR-4 transfection group and the control group for 7,14 and 21 d, respectively. Compared with the control group, the expression level of osteogenic differentiation genes, ALP, alizarin red staining and Von Kossa staining intensity of si-TLR-4 group were significantly higher than those of the control group (P<0.05). CONCLUSIONS: P.e-LPS inhibits the differentiation of osteoblasts through TLR-4 receptor, thus participating in bone resorption process of periapical lesions.

[Resveratrol suppressed the expression of MIP-2 induced by lipopolysaccharide in osteoblasts via up-regulating SOCS-3].

PURPOSE: To explore whether resveratrol dependents on the production of suppressor of cytokine signaling suppressor 3 (SOCS-3) in inhibiting mRNA production of macrophage inflammatory protein-2 (MIP-2) in osteoblasts induced by lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e). METHODS: MC3T3-E1 cells were treated with different concentrations of resveratrol (0, 5, 10 and 20 µmol/L) and 20 µmol/L resveratrol for different time( 0, 10, 30, 60, 120 and 180 min). The expression of SOCS-3 protein was detected by Western blot. MC3T3-E1 cells were transfected with mouse SOCS3 siRNA (si-SOCS-3) and control siRNA(si-control). Reverse transcription real-time PCR(real-time RT-PCR) and Western blot was used to detect the silencing efficiency of SOCS-3. Cells were stimulated by 20 µg/mL P.e-LPS for 24 h after transfection, in the absence or presence of 20 µmol/L resveratrol for 1 h , and the changes of MIP-2 mRNA were determined by real-time RT-PCR. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. RESULTS: Treatment of MC3T3-El cells with different concentrations of resveratrol caused a significant increase in SOCS-3 protein expression in a dose-dependent manner. During the observation time of 180 min, SOCS-3 protein expression was the highest at 20 µmol/L resveratrol-treated osteoblasts for 60 min. The silencing efficiency of SOCS-3 mRNA was 63.7%. Transfection with SOCS-3 siRNA increased MIP-2 mRNA expression in LPS-stimulated MC3T3-E1 cells and negated the inhibitory effects of resveratrol on LPS-induced MIP-2 mRNA expression(P<0.05). CONCLUSIONS: Resveratrol inhibits the expression of MIP-2 mRNA in osteoblasts induced by P.e-LPS by up-regulating the expression of SOCS-3 protein.

Systemic and Extraradicular Bacterial Translocation in Apical Periodontitis.

Apical periodontitis is an inflammatory disease of microbial etiology. It has been suggested that endodontic bacterial DNA might translocate to distant organs via blood vessels, but no studies have been conducted. We aimed first to explore overall extraradicular infection, as well as specifically by Porphyromonas spp; and their potential to translocate from infected root canals to blood through peripheral blood mononuclear cells. In this cross-sectional study, healthy individuals with and without a diagnosis of apical periodontitis with an associated apical lesion of endodontic origin (both, symptomatic and asymptomatic) were included. Apical lesions (N=64) were collected from volunteers with an indication of tooth extraction. Intracanal samples (N=39) and respective peripheral blood mononuclear cells from apical periodontitis (n=14) individuals with an indication of endodontic treatment, as well as from healthy individuals (n=14) were collected. The detection frequencies and loads (DNA copies/mg or DNA copies/µL) of total bacteria, Porphyromonas endodontalis and Porphyromonas gingivalis were measured by qPCR. In apical lesions, the detection frequencies (%) and median bacterial loads (DNA copies/mg) respectively were 70.8% and 4521.6 for total bacteria; 21.5% and 1789.7 for Porphyromonas endodontalis; and 18.4% and 1493.9 for Porphyromonas gingivalis. In intracanal exudates, the detection frequencies and median bacterial loads respectively were 100% and 21089.2 (DNA copies/µL) for total bacteria, 41% and 8263.9 for Porphyromonas endodontalis; and 20.5%, median 12538.9 for Porphyromonas gingivalis. Finally, bacteria were detected in all samples of peripheral blood mononuclear cells including apical periodontitis and healthy groups, though total bacterial loads (median DNA copies/µL) were significantly higher in apical periodontitis (953.6) compared to controls (300.7), p<0.05. Porphyromonas endodontalis was equally detected in both groups (50%), but its bacterial load tended to be higher in apical periodontitis (262.3) than controls (158.8), p>0.05; Porphyromonas gingivalis was not detected. Bacteria and specifically Porphyromonas spp. were frequently detected in endodontic canals and apical lesions. Also, total bacteria and Porphyromonas endodontalis DNA were detected in peripheral blood mononuclear cells, supporting their plausible role in bacterial systemic translocation.

Quantitative Analysis of Candidate Endodontic Pathogens and Their Association with Cause and Symptoms of Apical Periodontitis in a Sudanese Population.

OBJECTIVE: To investigate the prevalence of key endodontic pathogens and their association with the clinical features and the cause of apical periodontitis. METHODS: The study population included patients referred to Khartoum Dental teaching Hospital, Sudan for endodontic treatment. Samples were collected from single-rooted teeth carious or traumatised teeth with clinical and radiographic evidence of apical periodontitis. The endodontic pathogens Porphyromonas endodontalis, Fusobacterium nucleatum and Treponema denticola were quantified by real time polymerase chain reaction (qPCR). The prevalence of each species was identified at both a low detection threshold (>50 bacteria) and a high detection threshold (>1000 bacteria). RESULTS: 75 patients (mean age 30.1 yrs SD 10.1) were included in the analysis. The most prevalent bacterium at both the low and high threshold was F. nucleatum followed by T. denticola at the low threshold and P. endodontalis at the high threshold. There was no association with symptoms at the low detection threshold, but at high threshold P. endodontalis was associated with swelling, adjusted odds ratio (OR), 9.32 95%CI 1.11- 78.66, P=0.04. All species were more prevalent in apical periodontitis due to caries only at the low detection threshold, OR=5.01 (P=0.006) for T. denticola; 4.84 (P=0.01) for F. nucleatum; and 3.62 (P=0.03) for P. endodontalis. CONCLUSION: There was a high prevalence of the F. nucleatum, T. denticola and P. endodontalis in apical periodontitis associated with caries. None of these bacterial were associated with pain but the presence of P. endodontalis at high levels was associated with swelling.

Surveying bovine digital dermatitis and non-healing bovine foot lesions for the presence of Fusobacterium necrophorum, Porphyromonas endodontalis and Treponema pallidum.

BACKGROUND: Non-healing bovine foot lesions, including non-healing white line disease, non-healing sole ulcer and toe necrosis, are an increasingly important cause of chronic lameness that are poorly responsive to treatment. Recent studies have demonstrated a high-level association between these non-healing lesions and the Treponema phylogroups implicated in bovine digital dermatitis (BDD). However, a polymicrobial aetiology involving other gram-stain-negative anaerobes is suspected. METHODS: A PCR-based bacteriological survey of uncomplicated BDD lesions (n=10) and non-healing bovine foot lesions (n=10) targeting Fusobacterium necrophorum, Porphyromonas endodontalis, Dichelobacter nodosus and Treponema pallidum/T. paraluiscuniculi was performed. RESULTS: P. endodontalis DNA was detected in 80.0% of the non-healing lesion biopsies (p=<0.001) but was entirely absent from uncomplicated BDD lesion biopsies. When compared to the BDD lesions, F. necrophorum was detected at a higher frequency in the non-healing lesions (33.3% vs 70.0%, respectively), whereas D. nodosus was detected at a lower frequency (55.5% vs 20.0%, respectively). Conversely, T. pallidum/T. paraluiscuniculi DNA was not detected in either lesion type. CONCLUSION: The data from this pilot study suggest that P. endodontalis and F. necrophorum should be further investigated as potential aetiological agents of non-healing bovine foot lesions. A failure to detect syphilis treponemes in either lesion type is reassuring given the potential public health implications such an infection would present.

[Effect of resveratrol on the expression of MIP-2 in P.e-LPS-induced mouse osteoblasts].

PURPOSE: To investigate the effect of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) on the expression of macrophage inflammatory protein-2 (MIP-2) mRNA and protein levels in MC3T3-E1 cells and the influence of resveratrol on the expression of MIP-2 protein in P.e-LPS induced cells. METHODS: MC3T3-E1 cells were treated with different concentrations of P.e-LPS(0-50 mg/L) and 20 mg/L P.e-LPS for different time (0-48 h). The expression of MIP-2 mRNA and protein was detected by real-time RT-PCR and enzyme linked immunosorbent assay (ELISA). MC3T3-E1 cells were pretreated with resveratrol for 1 h in the presence of 20 mg/L P.e-LPS for 24 h，which was detected by ELISA. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. RESULTS: Treatment of MC3T3-El cells with different concentrations of P.e-LPS(0-50 mg/L) caused a significantly increase in MIP-2 mRNA and protein expression in dose-dependent manners.The expression of MIP-2 protein increased from (41.86±2.49) ng/L to (3126.74±158.30) ng/L, and the difference was significant(P<0.05). In the observation time (0-48 h), the impact of 20 mg/L P.e-LPS on induction of MIP-2 in MC3T3-El cells exhibited a time-dependent manner. At 48 h, the maximal induction of MIP-2 protein expression was (2102.55±123.27) ng/L(P<0.01). Incubation of cells with 10 µmol/L resveratrol for 1h significantly decreased the expression of MIP-2 protein from (1805.33±67.54) ng/L to（813.82±47.21) ng/L, and the difference was significant(P<0.05). CONCLUSIONS: The results suggest that P.e-LPS may mediate MIP-2 expression in MC3T3-E1 cells, and resveratrol has a significant inhibitory effect on this process.

microRNA-146a and Hey2 form a mutual negative feedback loop to regulate the inflammatory response in chronic apical periodontitis.

Chronic apical periodontitis (CAP) is defined as chronic inflammation of the dental pulp and root canal system. Porphyromonas endodontalis lipopolysaccharide ( P. endodontalis LPS) plays an important role in inducing an inflammatory response in CAP. microRNA-146a (miR-146a) is a key regulator of inflammation and is induced by LPS. Hairy and enhancer-of-split related with YRPW motif 2 (Hey2) has been confirmed to be induced by the Notch signaling pathway, which is involved in tooth development, pulp regeneration, and repair after injury. Our study aimed to investigate the functional role of miR-146a via the targeting of Hey2 in CAP as well as the underlying mechanism. Compared with 13 healthy controls, miR-146a and Hey2 expressions were significantly higher in 20 patients with CAP. In addition, miR-146a, Hey2, interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α expressions were significantly increased in MC3T3-E1 cells stimulated with different concentrations (0-20 µg/mL) of P. endodontalis LPS for different amounts of time (0-48 hours). Moreover, miR-146a, which acts as an anti-inflammatory mediator, negatively regulated the expression of IL-6, IL-1ß, and TNF-α, and Hey2 was confirmed as a target gene of miR-146a by a luciferase reporter assay. Hey2 also negatively regulated miR-146a, IL-6, IL-1ß, and TNF-α expressions, and P. endodontalis LPS strongly induced Hey2 recruitment to the IL-6 promoter (-400 ~ -200 bp). These findings suggest that miR-146a and Hey2 form a mutual negative feedback regulatory loop, demonstrating a novel mechanism that regulates inflammatory responses in CAP.

Resveratrol Suppresses Matrix Metalloproteinase-2 Activation Induced by Lipopolysaccharide in Mouse Osteoblasts via Interactions with AMP-Activated Protein Kinase and Suppressor of Cytokine Signaling 1.

Porphyromonas endodontalis (P. endodontalis) lipopolysaccharide (LPS) is associated with the progression of bone resorption in periodontal and periapical diseases. Matrix metalloproteinase-2 (MMP-2) expression and activity are elevated in apical periodontitis and have been suggested to participate in bone resorption. Therefore, inhibiting MMP-2 activation may be considered a therapeutic strategy for treating apical periodontitis. Resveratrol is a natural non-flavonoid polyphenol that has been reported to have antioxidant, anti-cancer, and anti-inflammatory properties. However, the capacity of resveratrol to protect osteoblast cells from P. endodontalis LPS insults and the mechanism of its inhibitory effects on MMP-2 activation is poorly understood. Here, we demonstrate that cell viability is unchanged when 10 mg L-1P. endodontalis LPS is used, and MMP-2 expression is drastically induced by P. endodontalis LPS in a concentration- and time-dependent manner. Twenty micromolar resveratrol did not reduce MC3T3-E1 cell viability. Resveratrol increased AMP-activated protein kinase (AMPK) phosphorylation, and Compound C, a specific AMPK inhibitor, partially abolished the resveratrol-mediated phosphorylation of AMPK. In addition, AMPK inhibition blocked the effects of resveratrol on MMP-2 expression and activity in LPS-induced MC3T3-E1 cells. Treatment with resveratrol also induced suppressor of cytokine signaling 1 (SOCS1) expression in MC3T3-E1 cells. SOCS1 siRNA negated the inhibitory effects of resveratrol on LPS-induced MMP-2 production. Additionally, resveratrol-induced SOCS1 upregulation was reduced by treatment with compound C. These results demonstrate that AMPK and SOCS1 activation are important signaling events during resveratrol-mediated inhibition of MMP-2 production in response to LPS in MC3T3-E1 cells, and there is crosstalk between AMPK and SOCS1 signaling.

[Effects of Porphyromonas endodontalis lipopolysaccharides on the expression of monocyte chemotactic protein-1 in mouse osteoblasts].

PURPOSE: To investigate the effects of lipopolysaccharide (LPS) extracted from Porphyromonas endodontalis (P.endodontalis) on expression of monocyte chemotactic protein-1 (MCP-1) mRNA and protein in MC3T3-E1 cells and the role of p38 mitogen-activated　protein　kinase (MAPK) and nuclear factor-kB(NF-kB）in the process. METHODS: MC3T3-E1 cells were treated with different concentrations of P.endodontalis LPS(0-50mg/L) and 20 mg/L P.endodontalis LPS for different hours (0-48 h). The expression of MCP-1 mRNA was detected by real-time reverse transcription PCR(RT-PCR) and protein was detected by enzyme-1inked immunosorbent assay (ELISA). MC3T3-E1 cells were pretreated with SB203580 (inhibitor of p38MAPK) and BAY11-7082 (inhibitor of NF-kB) for 1h, and then were treated with 20 mg/L P.endodontalis LPS for 24 h, the expression of MCP-1 mRNA was also detected by RT-PCR. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. RESULTS: The level of MCP-1 mRNA and protein increased significantly after treatment with different concentrations of P.endodontalis LPS (0-50 mg/L), which indicated that P.endodontalis LPS induced osteoblasts to express MCP-1 in a dose dependent manners. During the observation time (0-48 h), the impact of 20 mg/L P.endodontalis LPS on induction of MCP-1 in MC3T3-E1 cells exhibited a time-dependent manner. The expression of MCP-1 mRNA decreased significantly after pretreated with 10 mol/L SB203580 and BAY11-7082 for 1 h,and the inhibitory effect of SB203580 was stronger than BAY11-7082. CONCLUSIONS: P.endodontalis LPS may induce the expression of MCP-1 mRNA and protein in MC3T3-E1 cells through the signaling pathway of p38MAPK and NF-kB.

Porphyromonas endodontalis reactivates latent Epstein-Barr virus.

AIM: To determine whether Porphyromonas endodontalis can reactivate latent Epstein-Barr virus (EBV). METHODOLOGY: The concentrations of short-chain fatty acids (SCFAs) in P. endodontalis culture supernatants were determined using high-performance liquid chromatography. A promoter region of BamHI fragment Z leftward open reading frame 1 (BZLF-1), which is a transcription factor that controls the EBV lytic cycle, was cloned into luciferase expression vectors. Then, the luciferase assay was performed using P. endodontalis culture supernatants. Histone acetylation using Daudi cells treated with P. endodontalis culture supernatants was examined using Western blotting. BZLF-1 mRNA and BamHI fragment Z EB replication activator (ZEBRA) protein were also detected quantitatively using real-time polymerase chain reaction (PCR) and Western blotting. Surgically removed periapical granulomas were examined to detect P. endodontalis, EBV DNA, and BZLF-1 mRNA expression using quantitative real-time PCR. Statistical analysis using Steel tests was performed. RESULTS: The concentrations of n-butyric acid in P. endodontalis culture supernatants were significantly higher than those of other SCFAs (P = 0.0173). Using B-95-8-221 Luc cells treated with P. endodontalis culture supernatants, the luciferase assay demonstrated that P. endodontalis induced BZLF-1 expression. Hyperacetylation of histones was also observed with the culture supernatants. BZLF-1 mRNA and ZEBRA protein were expressed by Daudi cells in a dose-dependent manner after the treatment with P. endodontalis culture supernatants. P. endodontalis and BZLF-1 in periapical granulomas were also detected. The expression levels of BZLF-1 mRNA were similar to the numbers of P. endodontalis cells in each specimen. CONCLUSIONS: n-butyric acid produced by P. endodontalis reactivated latent EBV.

iRoot FM exerts an antibacterial effect on Porphyromonas endodontalis and improves the properties of stem cells from the apical papilla.

AIM: To investigate the antibacterial activity of a novel intracanal medicament, iRoot FM, against Porphyromonas endodontalis and its effects on the proliferation and osteo-/odontogenic differentiation of stem cells from apical papilla (SCAP). METHODOLOGY: The agar diffusion test was used to compare the antimicrobial efficacy of iRoot FM with two traditional intracanal medicaments, calcium hydroxide [Ca(OH)2 ] and triple antibiotic paste (TAP). The CCK-8 assay was used to assess the proliferation rate of SCAP when exposed to the three intracanal medicaments. The expression levels of ALP and DMP-1 and the capacity to form mineralized nodules were used to evaluate the osteo-/odontogenic differentiation of SCAP, as assessed by real-time PCR, Western blotting and alizarin red S staining. Data were statistically analysed with one-way analysis of variance (anova), and comparisons between each of two groups were analysed by the least significance difference method. P values less than 0.05 were considered statistically significant. RESULTS: The zone of inhibition against P. endodontalis produced by iRoot FM was 20.74 ± 4.35 mm, whilst the zones of inhibition of Ca(OH)2 and TAP were 24.89 ± 3.84 mm and 34.51 ± 1.20 mm. The antibacterial capacity of iRoot FM was similar to that of Ca(OH)2 (P > 0.05). SCAP, cultured in conditioned medium with iRoot FM, was associated with greater proliferation and osteo-/odontogenic differentiation capacity than those cultured in conditioned medium with Ca(OH)2 and TAP (P < 0.05). Moreover, iRoot FM had no negative effects on the proliferation rate of SCAP. CONCLUSIONS: iRoot FM exhibited excellent antibacterial activity against P. endodontalis and could improve the proliferation and differentiation of SCAP. The findings provide evidence that iRoot FM has potential as an intracanal medicament for endodontic procedures in immature permanent teeth.

[Effects of lipopolysaccharides from Porphyromonas endodontalis on the expression of MIP-1α in mouse osteoblasts].

PURPOSE: To investigate the effects of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) on the expression of macrophageinflammatoryprotein-1α (MIP-1α) mRNA and protein levels in MC3T3-E1 cells and the influence of curcumin in the process. METHODS: MC3T3-E1 cells were treated with 20 mg/L P.e-LPS for different times (0-48 h). The expression of MIP-1α mRNA and protein was detected by real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) and enzyme linked immunosorbent assay(ELISA). MC3T3-E1 cells were pretreated with inhibitor of (curcumin) for 1 h, and then treated with 20 mg/L P.e-LPS. The expression of MIP-1α was also detected by real-time RT-PCR and ELISA.Statistical analysis was performed using one-way ANOVA and Dunnett's t test with SPSS 13.0 software package. RESULTS: In the observation time (0-48 h), the impact of 20 P.e-LPS mg/L on induction of MIP-1α in MC3T3-El cells exhibited a time-dependent manner. The expression of MIP-1α mRNA and protein decreased significantly after pretreatment with 10 µmol/L curcumin for 1 h. CONCLUSIONS: The results suggest that P.e-LPS may mediate MIP-1α expression in MC3T3-E1 cells, and curcumin has a significant inhibitory effect on this process.

[Effects of Porphyromonas endodontalis lipopolysaccharides on the expression of matrix metalloproteinase-9 in mouse osteoblasts].

Objective: To evaluate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on the expression of matrix metalloproteinase-9 (MMP-9) mRNA and protein as well as enzyme activity in MC3T3-E1 cells and the role of nuclear factor-κB (NF-κB) in the process, so as to investigate the expression of MMP-9 dependent signaling pathways in mouse osteoblasts induced by Pe LPS. Methods: The experiment was conducted in 3 sessions: MC3T3-E1 cells were treated with various concentrations of Pe LPS (0-20 mg/L) and 10 mg/L Pe LPS for different time intervals (0-48 h). The expression of MMP-9 mRNA and protein were detected by real-time reverse transcription-PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), while the enzyme activity was detected by gelatin zymography method. The expression of MMP-9 mRNA was also detected in 10 mg/L Pe LPS treated MC3T3-El cells after pretreated with specific NF-κB inhibitor BAY 11-7082 for l h. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. Results: The levels of MMP-9 mRNA and protein increased significantly after the treatment with various concentrations of Pe LPS (0-20 mg/L), which indicated that Pe LPS induced osteoblasts to express MMP-9 in dose dependent manners. The expression of MMP-9 protein increased from (5 395±362) ng/L (blank control group) to (12 684±375) ng/L (20 mg/L group). Maximal induction of MMP-9 mRNA expression was found in the MC3T3-E1 cells treated with 10 mg/L Pe LPS for 24 h. The expression of MMP-9 mRNA in the 20 mg/L group was about 7 times than that in the blank control group. After 24 h, the expression of MMP-9 mRNA decreased. Maximal expression of MMP-9 protein was found in the MC3T3-E1 cells treated with 10 mg/L Pe LPS for 48 h ([35 055±2 346] ng/L) showing the highest enzyme activity. The mRNA of MMP-9 decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h. Conclusions: Pe LPS might induce the expression of MMP-9 in MC3T3-E1 cells through the signaling of NF-κB.