BACKGROUND: Periodontitis is characterized by alveolar bone resorption, aggravated by osteoblast dysfunction, and associated with intracellular oxidative stress linked to the nuclear factor erythroid 2-related factor 2 (NRF2) level. We evaluated the molecular mechanism of periodontitis onset and development and the role of NRF2 in osteogenic differentiation. METHODS: Primary murine mandibular osteoblasts were extracted and exposed to Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) or other stimuli. Reactive oxygen species (ROS) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining were used to detect intracellular oxidative stress. Alkaline phosphatase staining and alizarin red S staining were used to detect the osteogenic differentiation of osteoblasts. Immunofluorescence and western blotting were used to determine the changes in the mitogen-activated protein kinase (MAPK) pathway and related molecule activities. Immunofluorescence colocalization and co-immunoprecipitation were performed to examine the nuclear translocation of NRF2 and its interaction with dual-specific phosphatase 1 (DUSP1) in cells. RESULTS: Ligated tissue samples showed higher alveolar bone resorption rate and lower NRF2 level than healthy periodontal tissue samples. Pg-LPS increased intracellular oxidative stress levels and inhibited osteogenic differentiation, whereas changes in NRF2 expression were correlated with changes in the oxidative stress and osteogenesis rate. NRF2 promoted the dephosphorylation of the MAPK pathway by nuclear translocation and the upregulation of DUSP1 expression, thus enhancing the osteogenic differentiation capacity of mandibular osteoblasts. The interaction between NRF2 and DUSP1 was observed. CONCLUSIONS: NRF2 and its nuclear translocation can regulate the osteogenic differentiation of mandibular osteoblasts under Pg-LPS conditions by interacting with DUSP1 in a process linked to the MAPK pathway. These findings form the basis of periodontitis treatment.
Animals ; NF-E2-Related Factor 2/physiology* ; Lipopolysaccharides/pharmacology* ; Osteoblasts/drug effects* ; Mice ; Porphyromonas gingivalis/chemistry* ; Cell Differentiation ; Osteogenesis ; Dual Specificity Phosphatase 1/metabolism* ; Mandible/cytology* ; Reactive Oxygen Species/metabolism* ; Oxidative Stress ; Periodontitis/metabolism* ; Cells, Cultured ; Male ; Cell Nucleus/metabolism*

OBJECTIVES: To investigate the efficacy of Ag₂Se nanoparticles for eliminating intracellular Porphyromonas gingivalis (P. gingivalis) in esophageal cancer and examine the effect of P. gingivalis clearance on progression of esophageal cancer. METHODS: Ag₂Se nanoparticles were synthesized via a chemical synthesis method. The effects of Ag₂Se nanoparticles on P. gingivalis viability and colony-forming ability were assessed using fluorescence staining and colony formation assays. In a mouse model bearing subcutaneous murine esophageal cancer cell allograft with P. gingivalis infection, the effect of treatment with Ag₂Se nanoparticles on the abundance of P. gingivalis in the tumor tissues was quantified using RNAscope in situ hybridization and quantitative polymerase chain reaction (qPCR), and the changes in tumor volume were monitored. The biosafety of Ag₂Se nanoparticles was assessed by examining liver and kidney functions and pathological changes in the major organs of the mice. RESULTS: Transmission electron microscopy revealed that the synthesized Ag₂Se nanoparticles were uniformly dispersed spherical particles with a diameter around 50 nm. In vitro experiments demonstrated that exposure to Ag₂Se nanoparticles significantly reduced the viability and clonal proliferation capacity of P. gingivalis in a dose-dependent manner. In the tumor-bearing mice, treatment with Ag₂Se nanoparticles significantly reduced the abundance of P. gingivalis in tumor tissues and suppressed tumor cell proliferation. No significant damages to the liver and kidney functions or the major organs were observed in Ag₂Se nanoparticle-treated mice, demonstrating good biocompatibility of Ag₂Se nanoparticles. CONCLUSIONS Ag₂Se nanoparticles exhibit significant bactericidal and inhibitory effects against P. gingivalis, and can effectively eliminate intracellular P. gingivalis to suppress the growth of esophageal cancer allograft in mice, suggesting the potential of Ag₂Se nanoparticles in the treatment of esophageal cancer.
Animals ; Porphyromonas gingivalis/drug effects* ; Mice ; Esophageal Neoplasms/pathology* ; Nanoparticles ; Metal Nanoparticles ; Bacteroidaceae Infections ; Cell Line, Tumor

Objective: To study the effect of microRNA-126 (miR-126) on the polarization of human monocyte-derived macrophages stimulated by Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS). Methods: Macrophages derived from human myeloid leukemia mononuclear cells were stimulated by Pg-LPS (5 mg/L) and by Pg-LPS (5 mg/L) after 24 h-transfection of miR-126 mimic or negative control RNA for 48 h, respectively. Real-time quantitative-PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western blotting were conducted to detect the changes in miR-126, pro-inflammatory factor tumor necrosis factor-α (TNF-α), anti-inflammatory factors interleukin-10 (IL-10), inducible nitric oxide synthase (iNOS), arginase-1 (Arg-1) and M1 polarization-related pathways such as nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Results: Compared with non-LPS stimulation group (TNF-α: 1.000±0.020, iNOS: 1.125±0.064, miR-126: 1.004±0.113, IL-10: 1.003±0.053, Arg-1: 1.130±0.061), the mRNA levels of TNF-α (3.105±0.278) and iNOS (4.296±0.003) increased significantly (t=6.53, P=0.003; t=42.63, P<0.001, respectively), while miR-126, IL-10 and Arg-1 expressions (0.451±0.038, 0.545±0.004 and 0.253±0.017) decreased significantly (t=7.95, P=0.001; t=7.36, P=0.002; t=11.94, P<0.001, respectively) after Pg-LPS stimulated by human-derived macrophages for 48 h. The protein expression of iNOS, TNF-α, Arg-1 and IL-10 were consistent at mRNA levels. Meanwhile, the expressions of phospho-NF-κB p65 (p-p65), phospho-extracellular signal-regulated kinase (p-ERK) and phospho-p38 MAPK (p-p38) increased significantly, while the expression of Arg-1 decreased significantly. Compared with the negative controls (scramble RNA) (TNF-α: 1.141±0.197, iNOS: 1.173±0.115, IL-10: 1.032±0.138, Arg-1: 0.933±0.044), the mRNA levels of TNF-α (0.342±0.022) and iNOS (0.588±0.085) expressions significantly decreased (t=5.35, P=0.006; t=5.05, P=0.007), while IL-10 (1.786±0.221) and Arg-1 expressions (2.152±0.229) significantly increased (t=3.71, P=0.021; t=6.21, P=0.003) after Pg-LPS stimulation with miR-126 mimic transfection. The relative protein expressions of iNOS, p-p65, p-ERK and p-p38 significantly decreased (t=13.00, P<0.001; t=6.98, P=0.002; t=10.86, P<0.001; t=8.32, P=0.001), while the protein level of Arg-1 significantly increased (t=12.08, P<0.001). Conclusions: Pg-LPS could promote M1 polarization of macrophages. miR-126 might inhibit the effect of Pg-LPS on the M1 polarization of macrophages through down-regulating NF-κB and MAPK signaling pathways.
Cell Polarity ; Humans ; Interleukin-10/metabolism* ; Lipopolysaccharides/pharmacology* ; Macrophage Activation ; Macrophages/drug effects* ; MicroRNAs/metabolism* ; NF-kappa B/metabolism* ; Porphyromonas gingivalis ; RNA, Messenger/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

In the present study, two new limonoids, 1α, 7α-dihydroxyl-3α-acetoxyl-12α-ethoxylnimbolinin (1) and 1α-tigloyloxy-3α-acetoxyl-7α-hydroxyl-12β-ethoxylnimbolinin (2), together with other four known limonoids (3-6), were isolated from the fruits of Melia toosendan. Their structures were elucidated by means of extensive spectroscopic analyses (NMR and ESI-MS) and comparisons with the data reported in the literature. The isolated compounds were evaluated for their antibacterial activities. Compound 4 exhibited significant antibacterial activity against an oral pathogen, Porphyromonas gingivalis ATCC 33277, with an MIC value of 15.2 μg·mL(-1). Compound 2 was also active against P. gingivalis ATCC 33277, with an MIC value of 31.25 μg·mL(-1). In conlcusion, our resutls indicate that these compounds may provide a basis for future development of novel antibiotics.
Anti-Bacterial Agents ; chemistry ; isolation & purification ; pharmacology ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Fruit ; chemistry ; Limonins ; chemistry ; isolation & purification ; Magnetic Resonance Spectroscopy ; Melia ; chemistry ; Molecular Structure ; Porphyromonas gingivalis ; drug effects ; growth & development ; Spectrometry, Mass, Electrospray Ionization

OBJECTIVEThis aimed to investigate the effect of specific sequence oligodeoxynucleotide MT01 on the biological properties of osteoblasts invaded by Porphyromonas gingivalis (P. gingivalis ) by evaluating proliferation, cell cycle, and apoptosis.

METHODSMG63 osteoblasts were recovered and incubated with MT01, CpG ODN, metronidazole (MNZ), and gentamicin (GEN) for 3 h. P. gingivalis (the multiplicity of infection was 100:1) was added subsequently and cocultured for another 24 and 48 h. Cells with PBS comprised the blank group, whereas cells with P. gingivalis comprised the negative controls. Six experimental groups were established: PBS group, P. gingivalis group, MT01+P. gingivalis group, CpG ODN+ P. gingivalis group, MNZ+P. gingivalis group, and GEN+P. gingivalis group. The proliferative ability was measured by methyl thiazolyl tetrazolium assay, and the percentages of apoptosis and cell cycle were examined by flow cytometry.

RESULTSCompared with the blank group, proliferation increased significantly in the MT01+P. gingivalis group (P < 0.05). The ratio of cells was lower at the G₁ phase and higher at the S phase in the MT01+P. gingivalis group compared with the results in the P. gingivalis group (P < 0.05). Early cell apoptosis in the MT01+P. gingivalis group was significantly lower than that in the P. gingivalis group (P < 0.05).

CONCLUSIONMT01 can promote the proliferation, reduce the ratio of the G₁phase, increase the ratio of the S phase, and inhibit the early apoptosis of osteoblasts invaded by P. gingivalis.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Division ; Cell Proliferation ; drug effects ; Flow Cytometry ; Gentamicins ; pharmacology ; Humans ; Metronidazole ; pharmacology ; Oligodeoxyribonucleotides ; pharmacology ; Osteoblasts ; cytology ; drug effects ; Porphyromonas gingivalis ; pathogenicity

OBJECTIVEThis study measures the glutaredoxin (Grx) gene and protein expression in umbilical vein endothelial cells upon exposure to Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). The involvement of the Akt-signaling pathway is also determined.

METHODSEA-hy926 cells were pretreated with 1,000 ng · mL⁻¹ P. gingivalis LPS for 4, 12, 18, and 24 h, and then real-time reverse transcription polymerase chain reaction was employed to detect Grx1 expression. The effect of Grx on Akt activity was investigated using Western blot for the control, LPS (1,000 ng · mL⁻¹ LPS), and carmus- tine (BCNU) groups (1,000 ng · mL⁻¹ LPS, and the EA-hy926 cells were pretreated with 25 μmol · ml⁻¹ BCNU for 30 min).

RESULTSGene expression of Grx1 significantly increased in LPS group compared with that in the control group. The Grx1 expression reached the peak level in 12 h, and the variation between the expression in 4 and 12 h was significant (P < 0.05). After 12 h, the protein levels of Grx and phosphorylated-Akt (p-Akt) significantly increased in the LPS group (P < 0.05), whereas the BCNU group showed a considerable decrease in both Grx and p-Akt expression levels (P < 0.05). Moreover, a slight difference was observed in the total Akt protein levels in the three groups (P > 0.05).

CONCLUSIONGrx expression increased upon exposure of EA-hy926 cells to the LPS. Akt activity could be inhibited by BCNU (a Grx inhibitor), which indicated that Akt might act as a downstream regulator of Grx.

Endothelial Cells ; Glutaredoxins ; genetics ; Humans ; Lipopolysaccharides ; pharmacology ; Oxidative Stress ; drug effects ; Phosphorylation ; Porphyromonas gingivalis ; pathogenicity ; Proto-Oncogene Proteins c-akt ; drug effects ; Signal Transduction ; drug effects ; Umbilical Veins

Osteoclasts are bone-specific multinucleated cells generated by the differentiation of monocyte/macrophage lineage precursors. Regulation of osteoclast differentiation is considered an effective therapeutic approach to the treatment of bone-lytic diseases. Periodontitis is an inflammatory disease characterized by extensive bone resorption. In this study, we investigated the effects of sodium fluoride (NaF) on osteoclastogenesis induced by Porphyromonas gingivalis, an important colonizer of the oral cavity that has been implicated in periodontitis. NaF strongly inhibited the P. gingivalis-induced alveolar bone loss. That effect was accompanied by decreased levels of cathepsin K, interleukin (IL)-1β, matrix metalloproteinase 9 (MMP9), and tartrate-resistant acid phosphatase, which were up-regulated during P. gingivalis-induced osteoclastogenesis. Consistent with the in vivo anti-osteoclastogenic effect, NaF inhibited osteoclast formation caused by the differentiation factor RANKL (receptor activator of nuclear factor κB ligand) and macrophage colony-stimulating factor (M-CSF). The RANKL-stimulated induction of the transcription factor nuclear factor of activated T cells (NFAT) c1 was also abrogated by NaF. Taken together, our data demonstrate that NaF inhibits RANKL-induced osteoclastogenesis by reducing the induction of NFATc1, ultimately leading to the suppressed expression of cathepsin K and MMP9. The in vivo effect of NaF on the inhibition of P. gingivalis-induced osteoclastogenesis strengthens the potential usefulness of NaF for treating periodontal diseases.
Acid Phosphatase ; drug effects ; Alveolar Bone Loss ; microbiology ; prevention & control ; Animals ; Anti-Bacterial Agents ; therapeutic use ; Anti-Inflammatory Agents ; therapeutic use ; Bacteroidaceae Infections ; microbiology ; prevention & control ; Bone Density Conservation Agents ; therapeutic use ; Cathepsin K ; drug effects ; Interleukin-1beta ; drug effects ; Interleukin-6 ; analysis ; Interleukin-8 ; drug effects ; Isoenzymes ; drug effects ; Macrophage Colony-Stimulating Factor ; drug effects ; Male ; Matrix Metalloproteinase 9 ; drug effects ; Osteoclasts ; drug effects ; Periodontitis ; microbiology ; prevention & control ; Porphyromonas gingivalis ; drug effects ; RANK Ligand ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sodium Fluoride ; therapeutic use ; Tartrate-Resistant Acid Phosphatase ; Transcription Factors ; drug effects ; X-Ray Microtomography ; methods

This study used in vivo microdialysis to examine the effects of intragingival application of lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (Pg-LPS) on gingival tumour necrosis factor (TNF)-α and interleukin (IL)-6 levels in rats. A microdialysis probe with an injection needle attached to the surface of the dialysis membrane was implanted into the gingiva of the upper incisor. For comparison, the effects of LPS derived from Escherichia coli (Ec-LPS) on IL-6 and TNF-α levels were also analysed. Pg-LPS (1 μg/1 μL) or Ec-LPS (1 or 6 μg/1 μL) was applied by microsyringe, with gingival dialysates collected every hour. Enzyme-linked immunosorbent assay (ELISA) revealed that gingival dialysates contained approximately 389 pg·mL⁻¹ of IL-6 basally; basal TNF-α levels were lower than the detection limit of the ELISA. Pg-LPS failed to alter IL-6 levels but markedly increased TNF-α levels, which remained elevated for 2 h after treatment. Neither IL-6 nor TNF-α were affected by Ec-LPS. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the gingiva expresses Toll-like receptor (TLR) 2 and TLR4 mRNA. Immunohistochemical examination showed that TLR2 and TLR4 are expressed by gingival epithelial cells. The present study provides in vivo evidence that locally applied Pg-LPS, but not Ec-LPS, into the gingiva transiently increases gingival TNF-α without affecting IL-6. The present results suggest that TLR2 but not TLR4 expressed on gingival epithelial cells may mediate the Pg-LPS-induced increase in gingival TNF-α in rats.
Animals ; Gingiva ; drug effects ; metabolism ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; administration & dosage ; Male ; Porphyromonas gingivalis ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Toll-Like Receptor 2 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the effect of cetylpyridinium chloride buccal tablets on halitosis induced by oral conditions.

METHODSWith Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum as the testing bacteria, the minimal inhibitory concentration (MIC) of cetylpyridinium chloride buccal tablets was determined using minute amount serial dilution test. The production of volatile sulfur compounds (VSCs) was measured using sulfide detector halimeter in the anaerobic bacteria culture at 4 and 8 h after addition of the tablets. The effect of the tablets in suppressing odor production by mouth-borne halitosis bacteria was assessed using cysteine challenge test in healthy volunteers, and the effectiveness was evaluated by measuring the reduction in VSCs production and the duration of the effect.

RESULTSCetylpyridinium chloride buccal tablets inhibited the growth of all the 3 bacteria. The tablets obviously inhibited VSCs production by the 3 bacteria with a effect similar to chlorhexidine. Compared with distilled water gargle, the buccal tablets significantly reduced cysteine-induced VSCs production level in the healthy volunteers (P<0.05), and the effect lasted for 230 min.

CONCLUSIONCetylpyridinium chloride tablets can obviously suppress bacteria responsible for oral halitosis and produce good effects in the treatment of halitosis induced by oral conditions.

Cetylpyridinium ; therapeutic use ; Fusobacterium nucleatum ; drug effects ; Halitosis ; drug therapy ; Humans ; Microbial Sensitivity Tests ; Porphyromonas gingivalis ; drug effects ; Prevotella intermedia ; drug effects ; Sulfur Compounds ; analysis ; Tablets ; Volatile Organic Compounds ; analysis

Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria; however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces.
Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; drug effects ; Colony Count, Microbial ; Fatty Acids ; pharmacology ; Humans ; Lipids ; pharmacology ; Microscopy, Electron ; Mouth Mucosa ; chemistry ; immunology ; microbiology ; Porphyromonas gingivalis ; chemistry ; drug effects ; ultrastructure ; Saliva ; chemistry ; microbiology ; Sphingolipids ; pharmacology ; Virulence ; drug effects