Cinnamaldehyde Protects against P. gingivalis Induced Intestinal Epithelial Barrier Dysfunction in IEC-6 Cells via the PI3K/Akt-Mediated NO/Nrf2 Signaling Pathway.

Porphyromonas gingivalis (Pg), a Gram-negative oral pathogen, promotes and accelerates periodontitis-associated gut disorders. Intestinal epithelial barrier dysfunction is crucial in the pathogenesis of intestinal and systemic diseases. In this study, we sought to elucidate the protective role of cinnamaldehyde (CNM, an activator of Nrf2) against P. gingivalis (W83) and Pg-derived lipopolysaccharide (Pg-LPS) induced intestinal epithelial barrier dysfunction via antioxidative mechanisms in IEC-6 cells. IEC-6 (ATCC, CRL-1592) cells were pretreated with or without CNM (100 µM), in the presence or absence of P. gingivalis (strain W83, 109 MOI) or Pg-LPS (1, 10, and 100 µg/mL), respectively, between 0-72 h time points by adopting a co-culture method. Intestinal barrier function, cytokine secretion, and intestinal oxidative stress protein markers were analyzed. P. gingivalis or Pg-LPS significantly (p < 0.05) increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels expressing oxidative stress damage. Pg-LPS, as well as Pg alone, induces inflammatory cytokines via TLR-4 signaling. Furthermore, infection reduced Nrf2 and NAD(P)H quinone dehydrogenase 1 (NQO1). Interestingly, inducible nitric oxide synthase (iNOS) protein expression significantly (p < 0.05) increased with Pg-LPS or Pg infection, with elevated levels of nitric oxide (NO). CNM treatment suppressed both Pg- and Pg-LPS-induced intestinal oxidative stress damage by reducing ROS, MDA, and NO production. Furthermore, CNM treatment significantly upregulated the expression of tight junction proteins via increasing the phosphorylation levels of PI3K/Akt/Nrf2 suppressing inflammatory cytokines. CNM protected against Pg infection-induced intestinal epithelial barrier dysfunction by activating the PI3K/Akt-mediated Nrf2 signaling pathway in IEC-6 cells.

Cystatin C: immunoregulation role in macrophages infected with Porphyromonas gingivalis.

Background: Periodontitis is a chronic infectious disease, characterized by an exacerbated inflammatory response and a progressive loss of the supporting tissues of the teeth. Porphyromonas gingivalis is a key etiologic agent in periodontitis. Cystatin C is an antimicrobial salivary peptide that inhibits the growth of P. gingivalis. This study aimed to evaluate the antimicrobial activity of this peptide and its effect on cytokine production, nitric oxide (NO) release, reactive oxygen species (ROS) production, and programmed cell death in human macrophages infected with P. gingivalis. Methods: Monocyte-derived macrophages generated from peripheral blood were infected with P. gingivalis (MOI 1:10) and stimulated with cystatin C (2.75 µg/ml) for 24 h. The intracellular localization of P. gingivalis and cystatin C was determined by immunofluorescence and transmission electron microscopy (TEM). The intracellular antimicrobial activity of cystatin C in macrophages was assessed by counting Colony Forming Units (CFU). ELISA assay was performed to assess inflammatory (TNFα, IL-1ß) and anti-inflammatory (IL-10) cytokines. The production of nitrites and ROS was analyzed by Griess reaction and incubation with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. Programmed cell death was assessed with the TUNEL assay, Annexin-V, and caspase activity was also determined. Results: Our results showed that cystatin C inhibits the extracellular growth of P. gingivalis. In addition, this peptide is internalized in the infected macrophage, decreases the intracellular bacterial load, and reduces the production of inflammatory cytokines and NO. Interestingly, peptide treatment increased ROS production and substantially decreased bacterial-induced macrophage apoptosis. Conclusions: Cystatin C has antimicrobial and immuno-regulatory activity in macrophages infected with P. gingivalis. These findings highlight the importance of understanding the properties of cystatin C for its possible therapeutic use against oral infections such as periodontitis.

Elevated subgingival temperature infers high bacterial pathogen counts in severe periodontitis.

OBJECTIVES: Periodontal inflammation may be assessed by bleeding on probing and subgingival temperature. This pilot study evaluated the intrapatient relationship between subgingival temperature and selected bacterial groups/species in deep periodontal pockets with bleeding on probing. MATERIALS AND METHODS: In each of eight adults, an electronic temperature probe identified three "hot" pockets with elevated subgingival temperature and three "cool" pockets with normal subgingival temperature among premolars/molars with 6‒10 mm probing depths and bleeding on probing. Microbial samples collected separately from the hot and cool periodontal pockets were cultured for selected periodontal pathogens. RESULTS: Hot compared to cool periodontal pockets revealed significantly higher absolute and normalized subgingival temperatures and yielded higher mean proportions of Porphyromonas gingivalis (10.2% for hot vs. 2.5% for cool, p = 0.030) and total red/orange complex periodontal pathogens (48.0% for hot vs. 24.6% for cool, p = 0.012). CONCLUSIONS: Hot versus cool deep periodontal pockets harbored significantly higher levels of major periodontal pathogens. Subgingival temperature measurements may potentially be useful to assess risk of periodontitis progression and the efficacy of periodontal therapy.

Extracellular vesicles from Aggregatibacter actinomycetemcomitans exhibit potential antitumorigenic effects in oral cancer: a comparative in vitro study.

Aggregatibacter actinomycetemcomitans is an opportunistic Gram-negative periodontopathogen strongly associated with periodontitis and infective endocarditis. Recent evidence suggests that periodontopathogens can influence the initiation and progression of oral squamous cell carcinoma (OSCC). Herein we aimed to investigate the effect of A. actinomycetemcomitans-derived extracellular vesicles (EVs) on OSCC cell behavior compared with EVs from periodontopathogens known to associate with carcinogenesis. EVs were isolated from: A. actinomycetemcomitans and its mutant strains lacking the cytolethal distending toxin (CDT) or lipopolysaccharide (LPS) O-antigen; Porphyromonas gingivalis; Fusobacterium nucleatum; and Parvimonas micra. The effect of EVs on primary and metastatic OSCC cells was assessed using cell proliferation, apoptosis, migration, invasion, and tubulogenesis assays. A. actinomycetemcomitans-derived EVs reduced the metastatic cancer cell proliferation, invasion, tubulogenesis, and increased apoptosis, mostly in CDT- and LPS O-antigen-dependent manner. EVs from F. nucleatum impaired the metastatic cancer cell proliferation and induced the apoptosis rates in all OSCC cell lines. EVs enhanced cancer cell migration regardless of bacterial species. In sum, this is the first study demonstrating the influence of A. actinomycetemcomitans-derived EVs on oral cancer in comparison with other periodontopathogens. Our findings revealed a potential antitumorigenic effect of these EVs on metastatic OSCC cells, which warrants further in vivo investigations.

Efficacy of a mouthwash containing ε-poly-L-lysine, funme peptides and domiphen in reducing halitosis and supragingival plaque: a randomized clinical trial.

OBJECTIVE: To evaluate the antibacterial effectiveness of a combination of ε-poly-L-lysine (ε-PL), funme peptide (FP) as well as domiphen against oral pathogens, and assess the efficacy of a BOP® mouthwash supplemented with this combination in reducing halitosis and supragingival plaque in a clinical trial. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the compound against Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, and Aggregatibacter actinomycetemcomitans were determined by the gradient dilution method. Subsequently, the CCK-8 assay was used to detect the toxicity of mouthwash on human gingival fibroblastst, and the effectiveness in reducing halitosis and supragingival plaque of the mouthwash supplemented with the combination was analyzed by a randomized, double-blind, parallel-controlled clinical trial. RESULTS: The combination exhibited significant inhibitory effects on tested oral pathogens with the MIC < 1.56% (v/v) and the MBC < 3.13% (v/v), and the mouthwash containing this combination did not inhibit the viability of human gingival fibroblasts at the test concentrations. The clinical trial showed that the test group displayed notably lower volatile sulfur compounds (VSCs) at 0, 10, 24 h, and 7 d post-mouthwash (P < 0.05), compared with the baseline. After 7 days, the VSC levels of the and control groups were reduced by 50.27% and 32.12%, respectively, and notably cutting severe halitosis by 57.03% in the test group. Additionally, the Plaque Index (PLI) of the test and control group decreased by 54.55% and 8.38%, respectively, and there was a significant difference in PLI between the two groups after 7 days (P < 0.01). CONCLUSIONS: The combination of ε-PL, FP and domiphen demonstrated potent inhibitory and bactericidal effects against the tested oral pathogens, and the newly formulated mouthwash added with the combination exhibited anti-dental plaque and anti-halitosis properties in a clinical trial and was safe. TRIAL REGISTRATION: The randomized controlled clinical trial was registered on Chinese Clinical Trial Registry (No. ChiCTR2300073816, Date: 21/07/2023).

Cryo-EM Structure of Porphyromonas gingivalis RNA Polymerase.

Porphyromonas gingivalis, an anaerobic CFB (Cytophaga, Fusobacterium, and Bacteroides) group bacterium, is the keystone pathogen of periodontitis and has been implicated in various systemic diseases. Increased antibiotic resistance and lack of effective antibiotics necessitate a search for new intervention strategies. Here we report a 3.5 Å resolution cryo-EM structure of P. gingivalis RNA polymerase (RNAP). The structure displays new structural features in its ω subunit and multiple domains in ß and ß' subunits, which differ from their counterparts in other bacterial RNAPs. Superimpositions with E. coli RNAP holoenzyme and initiation complex further suggest that its ω subunit may contact the σ4 domain, thereby possibly contributing to the assembly and stabilization of initiation complexes. In addition to revealing the unique features of P. gingivalis RNAP, our work offers a framework for future studies of transcription regulation in this important pathogen, as well as for structure-based drug development.

The impact of Thiopeptide antibiotics on inflammatory responses in periodontal tissues through the regulation of the MAPK pathway.

Periodontitis is a bacteria-induced inflammatory disease that damages the tissues supporting the teeth, gums, periodontal ligaments, and alveolar bone. Conventional treatments such as surgical procedures, anti-inflammatory drugs, and antibiotics, are somewhat effective; however, these may lead to discomfort and adverse events, thereby affecting patient outcomes. Therefore, this study aimed to find an effective method to prevent the onset of periodontal disease and explore the specific mechanisms of their action.The impact of thiostrepton on Porphyromonas gingivalis and periodontal ligament stem cells was evaluated in an inflammatory microenvironment. In vivo experiments were performed using a mouse periodontitis model to assess the effectiveness of locally applied thiostrepton combined with a silk fibroin hydrogel in impeding periodontitis progression. Thiostrepton exhibited significant antimicrobial effects against Porphyromonas gingivalis and anti-inflammatory properties by regulating the MAPK pathway through DUSP2. Locally applied thiostrepton effectively impeded the progression of periodontitis and reduced tissue damage. Thiostrepton treatment is a promising and tolerable preventive strategy for periodontitis, offering antimicrobial and anti-inflammatory benefits. These findings suggest the potential of thiostrepton as a valuable addition to periodontitis management, warranting further research and clinical exploration to improve patient outcomes.

Microbiological Effects of Sodium Hypochlorite/-Amino Acids and Cross-linked Hyaluronic Acid Adjunctive to Non-surgical Periodontal Treatment.

PURPOSE: To investigate the microbiological outcomes obtained with either subgingival debridement (SD) in conjunction with a gel containing sodium hypochlorite and amino acids followed by subsequent application of a cross-linked hyaluronic acid gel (xHyA) gel, or with SD alone. MATERIALS AND METHODS: Forty-eight patients diagnosed with stages II-III (grades A/B) generalised periodontitis were randomly treated with either SD (control) or SD plus adjunctive sodium hypochlorite/amino acids and xHyA gel (test). Subgingival plaque samples were collected from the deepest site per quadrant in each patient at baseline and after 3 and 6 months. Pooled sample analysis was performed using a multiplex polymerase chain reaction (PCR)-based method for the identification of detection frequencies and changes in numbers of the following bacteria: Aggregatibacter actinomycetemcomitans (A.a), Porphyromonas gingivalis (P.g), Tannerella forsythia (T.f), Treponema denticola (T.d), and Prevotella intermedia (P.i). RESULTS: In terms of detection frequency, in the test group, statistically significant reductions were found for P.g, T.f, T.d and P.i (p < 0.05) after 6 months. In the control group, the detection frequencies of all investigated bacterial species at 6 months were comparable to the baseline values (p > 0.05). The comparison of the test and control groups revealed statistically significant differences in detection frequency for P.g (p = 0.034), T.d (p < 0.01) and P.i (p = 0.02) after 6 months, favouring the test group. Regarding reduction in detection frequency scores, at 6 months, statistically significant differences in favour of the test group were observed for all investigated bacterial species: A.a (p = 0.028), P.g (p = 0.028), T.f (p = 0.004), T.d (p <0.001), and P.i (p = 0.003). CONCLUSIONS: The present microbiological results, which are related to short-term outcomes up to 6 months post-treatment, support the adjunctive subgingival application of sodium hypochlorite/amino acids and xHyA to subgingival debridement in the treatment of periodontitis.

Comparative analysis of antibodies to four major periodontal bacteria in respiratory diseases: a cohort study.

OBJECTIVES: To make a descriptive comparison of antibodies to four major periodontal bacteria and their relation to the respiratory diseases asthma and bronchitis/emphysema, and to cancer incidence. METHODS: The serum of a random sample of men with no history of cancer incidence (n=621) was analysed by the ELISA method for antibody levels of four periodontal bacteria; the anaerobes of the so-called red complex Tannerella forsythia (TF), Porphyromonas gingivalis (PG), and Treponema denticola (TD), and the facultative anaerobe Aggregatibacter actinomycetemcomitans (AA). The antibody readings were divided into quartiles and the distribution of cases of the relevant diseases as compared with the non-cases. Comparisons of the quartile distributions were by the Pearson χ2 test. Data and serum from the Oslo II study of Norwegian men from 2000 were used. The ELISA analyses were performed on thawed frozen serum. Cancer data from 17.5 years of follow-up were provided by the Norwegian Cancer Registry. RESULTS: In all, 52 men had reported asthma and 23 men had bronchitis/emphysema at the health screening. Results on cancer incidence are given for all respiratory cancers, n=23, and bronchi and lung cancers separately, n=18. Stratified analyses were performed for the four endpoints showing significant association with low levels of TD antibodies for bronchitis; p=0.035. Both TF and TD were significant for low levels of antibodies among daily smokers; p=0.030 for TF and p<0.001 for TD in the analysis of the full study sample. For PG and AA, no such associations were observed. An association with respiratory cancers was not observed. CONCLUSION: A low level of TD was associated with bronchitis/emphysema compared with the rest of the cohort. In the total study sample, low levels of antibodies to both TF and TD were associated with daily smoking.

Association of DOK3 and infiltrated tumor-associated macrophages with risk for the prognosis of Porphyromonas gingivalis-infected oral cancer: a 12-year data analysis of 200 patients from a tertiary teaching hospital, Urumqi, China.

BACKGROUND: While there is an understanding of the association between the expression of Porphyromonas gingivalis (P. gingivalis) and prognosis of oral squamous cell carcinoma (OSCC), significance specially to address the relevance between different immunohistochemical intensities of P. gingivalis and tumor-associated macrophages (TAMs) in OSCC tissue and related clinicopathologic characteristics has not been well investigated. The present study aimed to investigate the pathological features related to M2-TAM in P. gingivalis-infected OSCC and ascertain its clinical relevance with patients' prognosis. METHODS: A prospective cohort study was designed to comparatively analyze 200 patients from June 2008 to June 2020. Bioinformatics analyses were implemented to identify DOK3 as a key molecule and to appraise immunocyte infiltration using Gene Expression Omnibus and The Cancer Genome Atlas databases. Immunohistochemical evaluation was performed to analyze the association between the expression levels of P. gingivalis, DOK3, and M2-TAM and clinicopathological variables using Fisher's exact test or Pearson's chi-square test. Cox analysis was used to calculate hazard ratios (HR) with corresponding 95% confidence interval (CI) for various clinicopathological features. The Kaplan-Meier approach and log-rank test were used to plot the survival curves. RESULTS: The expression level of P. gingivalis was positively associated with DOK3 and M2-TAMs expression level (P < 0.001). Parameters, including body mass index, clinical stage, recurrence, tumor differentiation, and P. gingivalis, DOK3, and M2-TAM immunoexpression levels, affected the prognosis of patients with OSCC (all P < 0.05). In addition, P. gingivalis (HR = 1.674, 95%CI 1.216-4.142, P = 0.012), DOK3 (HR = 1.881, 95%CI 1.433-3.457, P = 0.042), and M2-TAM (HR = 1.649, 95%CI 0.824-3.082, P = 0.034) were significantly associated with the 10-year cumulative survival rate. CONCLUSIONS: Elevated expression of P. gingivalis and DOK3 indicates M2-TAM infiltration and unfavorable prognosis of OSCC, and could be considered as three novel independent risk factors for predicting the prognosis of OSCC.

Oral Microbially-Induced Small Extracellular Vesicles Cross the Blood-Brain Barrier.

Porphyromonas gingivalis (Pg) and its gingipain proteases contribute to Alzheimer's disease (AD) pathogenesis through yet unclear mechanisms. Cellular secretion of small extracellular vesicles or exosomes (EXO) increases with aging as part of the senescence-associated secretory phenotype (SASP). We have shown that EXO isolated from Pg-infected dendritic cells contain gingipains and other Pg antigens and transmit senescence to bystander gingival cells, inducing alveolar bone loss in mice in vivo. Here, EXO were isolated from the gingiva of mice and humans with/without periodontitis (PD) to determine their ability to penetrate the blood-brain barrier (BBB) in vitro and in vivo. PD was induced by Pg oral gavage for 6 weeks in C57B6 mice. EXO isolated from the gingiva or brain of donor Pg-infected (PD EXO) or control animals (Con EXO) were characterized by NTA, Western blot, and TEM. Gingival PD EXO or Con EXO were labeled and injected into the gingiva of uninfected WT mouse model. EXO biodistribution in brains was tracked by an in vivo imaging system (IVIS) and confocal microscopy. The effect of human PD EXO on BBB integrity and permeability was examined using TEER and FITC dextran assays in a human in vitro 3D model of the BBB. Pg antigens (RGP and Mfa-1) were detected in EXO derived from gingival and brain tissues of donor Pg-infected mice. Orally injected PD EXO from donor mice penetrated the brains of recipient uninfected mice and colocalized with hippocampal microglial cells. IL-1ß and IL-6 were expressed in human PD EXO and not in Con EXO. Human PD EXO promoted BBB permeability and penetrated the BBB in vitro. This is the first demonstration that microbial-induced EXO in the oral cavity can disseminate, cross the BBB, and may contribute to AD pathogenesis.

Differential Response of Human Dendritic Cells upon Stimulation with Encapsulated or Non-Encapsulated Isogenic Strains of Porphyromonas gingivalis.

During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1ß, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1ß, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.

Identification of a new genetic variant (G231N, E232T, N235D) of peptidylarginine deiminase from P. gingivalis in advanced periodontitis.

Chronic periodontitis (CP), an inflammatory disease of periodontal tissues driven by a dysbiotic subgingival bacterial biofilm, is also associated with several systemic diseases, including rheumatoid arthritis (RA). Porphyromonas gingivalis, one of the bacterial species implicated in CP as a keystone pathogen produces peptidyl arginine deiminase (PPAD) that citrullinates C-terminal arginine residues in proteins and peptides. Autoimmunity to citrullinated epitopes is crucial in RA, hence PPAD activity is considered a possible mechanistic link between CP and RA. Here we determined the PPAD enzymatic activity produced by clinical isolates of P. gingivalis, sequenced the ppad gene, and correlated the results with clinical determinants of CP in patients from whom the bacteria were isolated. The analysis revealed variations in PPAD activity and genetic diversity of the ppad gene in clinical P. gingivalis isolates. Interestingly, the severity of CP was correlated with a higher level of PPAD activity that was associated with the presence of a triple mutation (G231N, E232T, N235D) in PPAD in comparison to W83 and ATCC 33277 type strains. The relation between mutations and enhanced activity was verified by directed mutagenesis which showed that all three amino acid residue substitutions must be introduced into PPAD expressed by the type strains to obtain the super-active enzyme. Cumulatively, these results may lead to the development of novel prognostic tools to assess the progress of CP in the context of associated RA by analyzing the ppad genotype in CP patients infected with P. gingivalis.

[Study on the antimicrobial effect of new bioactive glass against common bacteria in apical periodontitis of deciduous teeth].

PURPOSE: To study the antimicrobial effect of different concentrations of new bioactive glass(BG) on common bacteria in apical periodontitis of deciduous teeth. METHODS: The diameter (mm) of the inhibitory rings formed after treatment of Enterococcus faecalis, Porphyromonas gingivalis and Clostridium nucleatum with the new bioactive glass was detected and observed by paper diffusion method, and the minimal inhibitory concentration(MIC), minimal bactericidal concentration (MBC) and minimal biofilm eradication concentration (MBEC) of E. faecalis, P. gingivalis and C. pseudomallei were determined. The mixed plaques of the three bacteria were treated with 20, 40, 60 and 80 mg/mL of the new bioactive glass for 24 h. The results were analyzed by laser confocal microscopy. The antibacterial effect of the new bioactive glass on the mixed plaque was observed by confocal laser scanning microscopy (CLSM). Statistical analysis was performed with GraphPad Prism 10.0 software. RESULTS: The new bioactive glass showed strong antibacterial potential against the common bacteria of apical periodontitis; the MBEC of the new bioactive glass on the plaque was significantly greater than MIC and MBC of Enterococcus faecalis, Porphyromonas gingivalis and Clostridium nucleatum, and as the concentration of the new bioactive glass increased, the number of dead bacteria in the mixed plaque increased, and there was significant difference from that of the blank control group (P＜0.05). CONCLUSIONS: The novel bioactive glass shows significant antibacterial efficacy against Enterococcus faecalis, Porphyromonas gingivalis and Clostridium nucleatum, which are the common bacteria in apical periodontitis of deciduous teeth.

Mechanism and functional verification of genes by virulence factors of P. gingivalis in ferroptosis.

OBJECTIVE: Porphyromonas gingivalis (P. gingivalis) is a key etiological agent in periodontitis and functions as a facultative intracellular microorganism and involves many virulence factors. These virulence factors participate in multiple intracellular processes, like ferroptosis, the mechanistic underpinnings remain to be elucidated. Aim of this study was to investigate the effects of virulence factors on the host cells. DESIGN: Human umbilical vein endothelial cells (HUVECs) were treated with 4% paraformaldehyde-fixed P. gingivalis, and subsequent alterations in gene expression were profiled via RNA-seq. Further, the molecules associated with ferroptosis were quantitatively analyzed using qRT-PCR and Western blot. RESULTS: A total of 1125 differentially expressed genes (DEGs) were identified, encompassing 225 upregulated and 900 downregulated. Ferroptosis was conspicuously represented in the kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, with notable upregulation of Heme oxygenase 1 (HMOX1), Ferritin light chain (FTL), and Solute carrier family 3 member 2 (SLC3A2) and downregulation of Scavenger receptor class A member 5 (SCARA5) and glutaminase (GLS). Random selection of DEGs for validation through qRT-PCR corroborated the RNA-Seq data (R2 = 0.93). Kelch like ECH associated protein 1 (Keap1) protein expression decreased after 4 and 8 h, while NFE2 like bZIP transcription factor 2 (Nrf2) and HMOX1 were elevated, with significant nuclear translocation of Nrf2. CONCLUSIONS: The virulence factors of P. gingivalis may potentially instigating ferroptosis through activation of the Keap1-Nrf2-HMOX1 signaling cascade, in conjunction with modulating the expression of other ferroptosis-associated elements. Further research is necessary to achieve a thorough comprehension of these complex molecular interactions.

[A preliminary in vivo and in vitro study of endothelial cell pyroptosis in the periodontal inflammatory environment].

Objective: To observe whether endothelial cells undergo pyroptosis in the inflammatory periodontal environment by using a model in vivo and in vitro, providing an experimental basis for indepth understanding of the underlying pathogenesis of periodontitis. Methods: According to the classification of periodontal diseases of 2018, gingival tissues were collected from periodontally healthy subjects and patients with stage Ⅲ-Ⅳ, grade C periodontitis, who presented Department of Oral and Maxillofacial Surgery and Department of Periodontology, School of Stomatology, The Fourth Military Medical University from April to May 2022. Immunohistochemical staining was performed to detect the expression level and distribution of gasdermin D (GSDMD), a hallmark protein of cell pyroptosis, in gingival tissues. Periodontitis models were established in each group by ligating the maxillary second molar teeth of three mice for 2 weeks (ligation group). The alveolar bone resorption was determined by micro-CT (mice without ligation treatment were used as the control group), and the colocalization of GSDMD and CD31 were quantitatively analyzed by immunofluorescence staining in gingival tissues of healthy and inflammatory mice. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg) combined with adenosine triphosphate (ATP) at various concentrations of 0.5, 1.0, 2.5, 5.0, and 10.0 mg/L, respectively, and the 0 mg/L group was set as the control group at the same time. Scanning electron microscopy was used to observe the morphology of HUVECs. Western blotting was used to detect the expression of gasdermin D-N terminal domains (GSDMD-N) protein and immunofluorescence cell staining was used to detect the expression and distribution of GSDMD. Cell counting kit-8 (CCK-8) was used to detect the proliferative ability of HUVECs, and propidium iodide (PI) staining was used to detect the integrity of cell membrane of HUVECs. Results: Immunohistochemistry showed that GSDMD in gingival tissues of periodontitis was mainly distributed around blood vessels and its expression level was higher than that in healthy tissues. Micro-CT showed that alveolar bone resorption around the maxillary second molar significantly increased in ligation group mice compared with control subjects (t=8.88, P<0.001). Immunofluorescence staining showed significant colocalization of GSDMD with CD31 in the gingival vascular endothelial cells in mice of ligation group. The results of scanning electron microscopy showed that there were pores of different sizes, the typical morphology of pyroptosis, on HUVECs cell membranes in the inflammatory environment simulated by ATP combined with different concentrations of LPS, and 2.5 mg/L group showed the most dilated and fused pores on cell membranes, with the cells tended to lyse and die. Western blotting showed that the expression of GSDMD-N, the hallmark protein of cell pyroptosis, was significantly higher in 2.5 and 5.0 mg/L groups than that in the control group (F=3.86, P<0.01). Immunofluorescence cell staining showed that the average fluorescence intensity of GSDMD in 2.5 mg/L group elevated the most significantly in comparison with that in the control group (F=35.25, P<0.001). The CCK-8 proliferation assay showed that compared to the control group (1.00±0.02), 0.5 mg/L (0.52±0.07), 1.0 mg/L (0.57±0.10), 2.5 mg/L (0.58±0.04), 5.0 mg/L (0.55±0.04), 10.0 mg/L (0.61±0.03) groups inhibited cell proliferation (F=39.95, P<0.001). PI staining showed that the proportion of positive stained cells was highest [(56.07±3.22)%] in 2.5 mg/L group (F=88.24, P<0.001). Conclusions: Endothelial cells undergo significant pyroptosis in both in vivo and in vitro periodontal inflammatory environments, suggesting that endothelial cell pyroptosis may be an important pathogenic factor contributing to the pathogenesis of periodontitis.

Structure-function analysis of PorXFj, the PorX homolog from Flavobacterium johnsioniae, suggests a role of the CheY-like domain in type IX secretion motor activity.

The type IX secretion system (T9SS) is a large multi-protein transenvelope complex distributed into the Bacteroidetes phylum and responsible for the secretion of proteins involved in pathogenesis, carbohydrate utilization or gliding motility. In Porphyromonas gingivalis, the two-component system PorY sensor and response regulator PorX participate to T9SS gene regulation. Here, we present the crystal structure of PorXFj, the Flavobacterium johnsoniae PorX homolog. As for PorX, the PorXFj structure is comprised of a CheY-like N-terminal domain and an alkaline phosphatase-like C-terminal domain separated by a three-helix bundle central domain. While not activated and monomeric in solution, PorXFj crystallized as a dimer identical to active PorX. The CheY-like domain of PorXFj is in an active-like conformation, and PorXFj possesses phosphodiesterase activity, in agreement with the observation that the active site of its phosphatase-like domain is highly conserved with PorX.

Assessment of the Antibacterial Effect of Vitamin D3 against Red Complex Periodontal Pathogens: A Microbiological Assay.

AIM: The study aims is to evaluate the antibacterial effect of vitamin D3 against the red complex bacteria, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia in chronic periodontitis patients. MATERIALS AND METHODS: The study comprised 98 participants with chronic periodontitis. All clinical parameters including plaque index (PI), gingival bleeding index (GBI), probing pocket depth (PPD), clinical attachment level (CAL), and a microbiological assay of P. gingivalis, T. denticola, T. forsythia were assessed at the baseline. All study participants who underwent scaling and root planning were divided into two groups, A and B, each with 49 patients and only group B patients were advised to take vitamin D supplementation of 60,000 IU granules, once daily for 2 months. All the patients of both the groups were recalled at the end of 2nd month and all the clinical and microbiological parameters were reassessed. RESULTS: After two months, there was a reduction in all the clinical markers in both groups, but the group B patients showed more improvement following non-surgical treatment vitamin D intake. There was also a statistical reduction in P. gingivalis, T. denticola, and T. forsythia following administration of vitamin D in group B patients compared to group A. CONCLUSION: These discoveries proposed that vitamin D has a superb antimicrobial impact against red complex periodontal microbes and might be considered a promising compound in the counteraction of periodontal disease. CLINICAL SIGNIFICANCE: Vitamin D is considered to possess anti-inflammatory and antimicrobial activity, which may help to delay the progression of periodontitis. So, vitamin D3 can be used as a potential supplement that could be employed to stop the advancement of periodontal disease. How to cite this article: Govindharajulu R, Syed NK, Sukumaran B, et al. Assessment of the Antibacterial Effect of Vitamin D3 against Red Complex Periodontal Pathogens: A Microbiological Assay. J Contemp Dent Pract 2024;25(2):114-117.

Subgingival microbial changes in Down Syndrome adults with periodontitis after chlorhexidine adjunct non-surgical therapy and monthly recalls-A 12-month case series study.

OBJECTIVES: Down Syndrome (DS) adults are at risk for periodontitis. Previous reports indicated difficulties in periodontopathogen reduction or eradication in DS individuals after periodontal treatment. This case series follows the subgingival microbial changes in adult DS individuals with periodontitis who received chlorhexidine adjunct non-surgical therapy plus 12-month recalls. METHODS: Twenty periodontitis DS participants (7 females; 25.5 ± 5.6 years of age; 3 with generalized periodontitis) partook in a study involving non-surgical mechanical periodontal therapy, twice daily chlorhexidine gel toothbrushing, chlorhexidine mouthwash, and monthly recalls. The subgingival microbiota profile was followed at baseline, 6-, and 12-months post-operation. RESULTS: Desulfobulbus, Saccharibacteria (TM7), Tannerella, and Porphyromonas were the major subgingival genera in this DS cohort. Favorable chlorhexidine adjunct non-surgical treatment outcomes were observed, with the relative abundance of Desulfobulbus sp. HMT 041, Saccharibacteria (TM7) [G-1] bacterium HMT 346 or 349, and Tannerella forsythia significantly reduced at the end of the study, but no significant reduction of Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans could be observed. Relative abundance of Desulfobulbus sp. HMT 041 and T. forsythia were also found to be significantly associated with plaque, bleeding on probing, and probing pocket depth (PPD, in mm) at a site level, while the relative abundance of Halomonas pacifica was negatively associated with PPD. CONCLUSIONS: Successful chlorhexidine adjunct non-surgical treatment with hygiene care was accompanied by a subgingival microbial shift involving certain periodontopathogenic species, except P. gingivalis and A. actinomycetemcomitans. Further investigations are required to clarify the mechanism underpinning the unchanged relative abundance of the above two pathogens despite favorable clinical responses. CLINICAL SIGNIFICANCE: DS adults face challenges achieving optimal home care or hygiene for periodontal healing and disease prevention. Chemical adjunct mechanical periodontal therapy plus regular recalls appeared promising clinically and microbiologically, with subgingival periodontopathogenic species reduction. The persistence of A. actinomycetemcomitans and P. gingivalis in subgingival niches post-treatment warrants further investigation.

Imbalanced EphB4/EphrinB2 Signaling Modulates Bone Resorption in Periodontitis Induced by Porphyromonas gingivalis.

Periodontitis, a chronic infectious disease in periodontal tissues, is characterized by an imbalance of alveolar bone resorption and remodeling, which eventually results in tooth loosening and even tooth loss. The etiology of periodontitis is polymicrobial synergy and dysbiosis, in which Porphyromonas gingivalis (P. gingivalis) is one of the primary pathogens responsible for periodontitis progression. The interplay of EphrinB2/EphB4 is crucial for osteoblast-osteoclast communication during bone remodeling and healing. This study investigates the mechanism of EphB4/EphrinB2 transduction modulating osteogenesis inhibition and bone resorption in periodontitis induced by P. gingivalis. An in vivo model of chronic periodontitis provoked by P. gingivalis was constructed, the inflammation and bone resorption were evaluated. The expression of EphB4 and EphrinB2 proteins in periodontal tissues was detected, which was also evaluated, respectively, in osteoblasts and osteoclasts infected with P. gingivalis in vitro. Then, a simulated coculture model of osteoblasts and osteoclasts was established to activate the forward and reverse pathways of EphB4/EphrinB2 with P. gingivalis infection. This study showed that P. gingivalis infection promoted alveolar bone resorption in rats and enhanced EphB4 and EphrinB2 expression in periodontal tissues. EphB4 and molecules associated with osteogenesis in osteoblasts infected with P. gingivalis were inhibited, while EphrinB2 and osteoclast differentiation-related markers in osteoclasts were activated. In conclusion, this study suggested that EphB4/EphrinB2 proteins were involved in alveolar bone remodeling in the process of periodontitis induced by P. gingivalis infection. Moreover, attenuated EphB4/EphrinB2 with P. gingivalis infection weakened osteoblast activity and enhanced osteoclast activity.