Gingivalis-induced Periodontitis Research Articles

A cleaved adhesin DNA vaccine targeting dendritic cell against Porphyromonas gingivalis-induced periodontal disease.

Arg-gingipain A (RgpA) is the primary virulence factor of Porphyromonas gingivalis and contains hemagglutinin adhesin (HA), which helps bacteria adhere to cells and proteins. Hemagglutinin's functional domains include cleaved adhesin (CA), which acts as a hemagglutination and hemoglobin-binding actor. Here, we confirmed that the HA and CA genes are immunogenic, and using adjuvant chemokine to target dendritic cells (DCs) enhanced protective autoimmunity against P. gingivalis-induced periodontal disease. C57 mice were immunized prophylactically with pVAX1-CA, pVAX1-HA, pVAX1, and phosphate-buffered saline (PBS) through intramuscular injection every 2 weeks for a total of three administrations before P. gingivalis-induced periodontitis. The DCs were analyzed using flow cytometry and ribonucleic acid sequencing (RNA-seq) transcriptomic assays following transfection with CA lentivirus. The efficacy of the co-delivered molecular adjuvant CA DNA vaccine was evaluated in vivo using flow cytometry, immunofluorescence techniques, and micro-computed tomography. After the immunization, both the pVAX1-CA and pVAX1-HA groups exhibited significantly elevated P. gingivalis-specific IgG and IgG1, as well as a reduction in bone loss around periodontitis-affected teeth, compared to the pVAX1 and PBS groups (p<0.05). The expression of CA promoted the secretion of HLA, CD86, CD83, and DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) in DCs. Furthermore, the RNA-seq analysis revealed a significant increase in the chemokine (C-C motif) ligand 19 (p<0.05). A notable elevation in the quantities of DCs co-labeled with CD11c and major histocompatibility complex class II, along with an increase in interferon-gamma (IFN-γ) cells, was observed in the inguinal lymph nodes of mice subjected to CCL19-CA immunization. This outcome effectively illustrated the preservation of peri-implant bone mass in rats afflicted with P. gingivalis-induced peri-implantitis (p<0.05). The co-administration of a CCL19-conjugated CA DNA vaccine holds promise as an innovative and targeted immunization strategy against P. gingivalis-induced periodontitis and peri-implantitis.

RgpA-Engineered/Functionalized DNA Vaccine as a Novel Prophylactic Vaccination to Prevent Porphyromonas gingivalis-Induced Periodontitis: An in Vivo Study.

Arg-gingipain A (rgpA) and Arg-gingipain B (rgpB) are crucial virulence factors associated with Porphyromonas gingivalis (P. gingivalis) and have been recognized as promising targets for antibacterial vaccines. Although vaccines containing rgpA have shown efficacy, the incorporation of rgpB, which lacks the haemagglutinin adhesin (HA) domain, diminishes the vaccine's effectiveness. This study aims to assess the immunogenicity of the functional HA domain of rgpA in mouse periodontitis models. A total of 24 mice were randomly divided into four groups, each receiving different immune injections: group A received phosphate-buffered saline (PBS) as an empty control; group B received pVAX1 as a negative control (NC); group C received pVAX1-HA; and group D received pVAX1-rgpA. The mice were subjected to intramuscular injections every two weeks for a total of three administrations. Prior to each immunization, blood samples were collected for antibody detection under isoflurane anesthesia. Following the final immunization, periodontitis was induced two weeks later by using sutures soaked in a P. gingivalis solution. The mice were euthanized after an additional two-week period. To assess the safety of the procedure, major organs were examined through hematoxylin-eosin (HE) staining. Subsequently, the levels of IgG, IgG1, and IgG2a in the serum were quantified via enzyme-linked immunosorbent assay (ELISA). Additionally, the expression of inflammatory factors in the gingiva, including interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor alpha (TNF-α), was determined using quantitative real-time reverse transcript PCR (qRT-PCR). The extent of bone loss in periodontal tissues was evaluated using micro-computed tomography (micro-CT) and HE staining. HE staining of the organs confirmed the absence of vaccine-induced toxicity in vivo. After the second immunization, both the rgpA and HA groups displayed significantly higher specific IgG titers in comparison to the NC and PBS groups (p < 0.05). Furthermore, the rgpA and HA groups exhibited a noteworthy predominance of IgG1 antibodies after three immunization doses, while there was a noticeable reduction in IgG2a levels observed following ligation with P. gingivalis sutures, as opposed to the NC and PBS groups (p < 0.05). Additionally, both the HA and rgpA groups showed a significant decrease in the expression of inflammatory factors such as IL-6, IL-1β, and TNF-α, as well as a reduction in bone loss around periodontitis-affected teeth, when compared to the NC and PBS groups (p < 0.05). The results of this study demonstrate that the rgpA-engineered/functionalized HA gene vaccine is capable of eliciting a potent prophylactic immune response against P. gingivalis-induced periodontitis, effectively serving as an immunogenic and protective agent in vivo.

A novel LncRNA SPIRE1/miR-181a-5p/PRLR axis in mandibular bone marrow-derived mesenchymal stem cells regulates the Th17/Treg immune balance through the JAK/STAT3 pathway in periodontitis.

Periodontitis is a microbial-related chronic inflammatory disease associated with imbalanced differentiation of Th17 cells and Treg cells. Bone marrow-derived mesenchymal stem cells (BM-MSCs) possess wide immunoregulatory properties. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) contribute to the immunomodulation in the pathological mechanisms of inflammatory diseases. However, critical lncRNAs/miRNAs involved in immunomodulation of mandibular BM-MSCs largely remain to be identified. Here, we explored the molecular mechanisms behind the defective immunomodulatory ability of mandibular BM-MSCs under the periodontitis settings. We found that mandibular BM-MSCs from P. gingivalis-induced periodontitis mice had significantly reduced expression of LncRNA SPIRE1 than that from normal control mice. LncRNA SPIRE1 knockdown in normal BM-MSCs caused Th17/Treg cell differentiation imbalance during the coculturing of BM-MSCs and CD4 T cells. In addition, LncRNA SPIRE1 was identified as a competitive endogenous RNA that sponges miR-181a-5p in BM-MSCs. Moreover, miR-181a-5p inhibition attenuated the impact of LncRNA SPIRE1 knockdown on the ability of BM-MSCs in modulating Th17/Treg balance. Prolactin receptor (PRLR) was validated as a downstream target of miR-181a-5p. Notably, targeted knockdown of LncRNA SPIRE1 or PRLR or transfection of miR-181a-5p mimics activated the JAK/STAT3 signaling in normal BM-MSCs, while treatment with STAT3 inhibitor C188-9 restored the immunomodulatory properties of periodontitis-associated BM-MSCs. Furthermore, BM-MSCs with miR-181a-5p inhibition or PRLR-overexpression showed enhanced in vivo immunosuppressive properties in the periodontitis mouse model. Our results indicate that the JAK/STAT3 pathway is involved in the immunoregulation of BM-MSCs, and provide critical insights into the development of novel targeted therapies against periodontitis.

Porphyromonas gingivalis-induced periodontitis could contribute to cognitive impairment in Sprague-Dawley rats via the P38 MAPK signaling pathway.

Periodontitis is one of the most common oral diseases and has been shown to be a risk factor for systemic diseases. Our aim was to investigate the relationship between periodontitis and cognitive impairment and to explore the role of the P38 MAPK signaling pathway in this process. We established a periodontitis model by ligating the first molars of SD rats with silk thread and injecting Porphyromonas gingivalis (P. gingivalis) or P. gingivalis plus the P38 MAPK inhibitor SB203580 at the same time for ten weeks. We assessed alveolar bone resorption and spatial learning and memory using microcomputed tomography and the Morris water maze test, respectively. We used transcriptome sequencing to explore the genetic differences between the groups. The gingival tissue, peripheral blood and hippocampal tissue were assessed for the cytokines TNF-α, IL-1β, IL-6, IL-8 and C reactive protein (CRP) with enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). We observed the presence of P. gingivalis in the hippocampus of rats by paraffin-fluorescence in situ hybridization (FISH). We determined the activation of microglia by immunofluorescence. Finally, Western blot analysis was employed to determine the expression of amyloid precursor protein (APP), β-site APP-cleaving enzyme 1 (BACE1) and P38MAPK pathway activation. We demonstrated that silk ligature-induced periodontitis plus injection of P. gingivalis into subgingival tissue could lead to memory and cognitive impairment. Transcriptome sequencing results suggested that there were neurodegenerative diseases in the P. gingivalis group, and the MWM test showed that periodontitis reduced the spatial learning and memory ability of mild cognitive impairment (MCI) model rats. We found high levels of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8) and CRP in the gingiva, peripheral blood and hippocampus, and the expression of APP and BACE1 was upregulated, as was the P38 MAPK pathway activation. Activated microglia and the presence of P. gingivalis were also found in the hippocampus. P38 MAPK inhibitors mitigated all of these changes. Our findings strongly suggest that topical application of P. gingivalis increases the inflammatory burden in the peripheral and central nervous systems (CNS) and that neuroinflammation induced by activation of P38 MAPK leads to impaired learning and memory in SD rats. It can also modulate APP processing. Therefore, P38 MAPK may serve as a linking pathway between periodontitis and cognitive impairment.

Anti-Inflammatory Effects of Geniposidic Acid on Porphyromonas gingivalis-Induced Periodontitis in Mice.

Periodontal disease is predominantly caused by the pathogenic bacterium Porphyromonas gingivalis that produces inflammation-inducing factors in the host. Eucommia ulmoides is a plant native to China that has been reported to reduce blood pressure, promote weight loss, and exhibit anti-inflammatory effects. Geniposidic acid (GPA) is the major component of E. ulmoides. Herein, we investigated the effects of GPA on P. gingivalis-induced periodontitis by measuring the inflammatory responses in human gingival epithelial cells (HGECs) after P. gingivalis stimulation and GPA addition in a P. gingivalis-induced periodontitis mouse model. We found that GPA addition suppressed interleukin (IL)-6 mRNA induction (33.8% suppression), IL-6 production (69.2% suppression), toll-like receptor (TLR) 2 induction, and mitogen-activated protein kinase (MAPK) phosphorylation in HGECs stimulated by P. gingivalis. Inoculation of mice with GPA inhibited P. gingivalis-induced alveolar bone resorption (25.6% suppression) by suppressing IL-6 and TLR2 production in the serum and gingiva. GPA suppressed osteoclast differentiation of bone marrow cells induced by M-CSF and sRANKL in mice (56.7% suppression). GPA also suppressed the mRNA expression of OSCAR, NFATc1, c-Fos, cathepsin K, and DC-STAMP. In summary, GPA exerts an anti-inflammatory effect on periodontal tissue and may be effective in preventing periodontal disease.

Dec2 negatively regulates bone resorption in periodontitis.

The potential role of the transcription factor Differentiated embryo-chondrocyte 2 (Dec2) in the progression of inflammatory diseases such as periodontitis has been unclear. Here, the effect of Dec2 on the expression of RANKL and on osteoclastogenesis was determined. Wild-type (WT) and Dec2 knockout (KO) mice as a model for periodontitis were used to assess alveolar bone resorption by microcomputed tomography (CT). Western blot, flow cytometry, quantitative real-time PCR, and immunohistochemical analyses were utilized to detect inflammation and osteoclasts. Luciferase reporter and Chromatin immunoprecipitation (ChIP) assays examined the interaction between Dec2 and RANKL. Micro-CT showed that the alveolar bone resorption of Dec2KO mice was more severe than WT mice after treatment with P.gingivalis. Immunohistochemistry and Tartrate-resistant acid phosphatase staining showed active osteoclast differentiation in Dec2KO mice. There was an increase in CD11b+ F4/80+ and CD4+ RANKL+ T cells in Dec2KO mice treated with P.gingivalis. Moreover, inflammatory and immune markers were expressed at significantly higher levels in gingival mononuclear cells in Dec2KO mice. Furthermore, luciferase reporter and ChIP assays confirmed the direct binding of Dec2 protein to the RANKL gene. Dec2 has an immune regulation ability that modulates P.gingivalis-induced periodontitis via RANKL.

Nano-emulsion of mangosteen rind extract in a mucoadhesive patch for periodontitis regenerative treatment: An in vivo study

ObjectiveTo investigate the therapeutic potential of nano-emulsion of mangosteen rind extract in a mucoadhesive gingival patch on periodontitis, and its effect on tumor necrosis factor alpha (TNF-α), receptor activator of nuclear factor kappa Β ligand (RANKL), and interleukin 10 (IL-10) expression. MethodsSixty Wistar rats were divided into four groups: positive control group (mucoadhesive patch with doxycycline), negative control group (mucoadhesive patch), treatment group I (mucoadhesive patch with mangosteen rind extract), and treatment group II (mucoadhesive patch with nano-emulsion of mangosteen rind extract). An experimental model of Porphyromonas gingivalis-induced periodontitis was established in rats by treatment with 0.03 mL bacteria locally (1 × 1010 colony-forming units) seven times at 2-day intervals in the gingival sulcus of mandibular anterior teeth. Treatment was 1 h/day for 3 days. On days 3, 5, and 7, five rats from each group were killed. TNF-α, IL-10, and RANKL expression was determined by dissecting the lower jaw for immunohistochemistry. ResultsThe mucoadhesive patch with nano-emulsion mangosteen rind extract significantly decreased TNF-α and RANKL expression and increased IL-10 expression (p < 0.05) compared to the treatment I, positive and negative control groups. ConclusionA mucoadhesive gingival patch with nano-emulsion of mangosteen rind extract has the potential to treat periodontitis by decreasing TNF-α, RANKL, and increasing IL-10 expression.

Effects of aloe-emodin on alveolar bone in Porphyromonas gingivalis-induced periodontitis rat model: a pilot study.

Aloe-emodin (AE), a natural anthraquinone abundant in aloe plants and rhubarb (Rheum rhabarbarum), has long been used to treat chronic inflammatory diseases. However, AE's underlying mechanisms in periodontal inflammation have not been fully elucidated. Acidic mammalian chitinase (AMCase) is a potential biomarker involved in bone remodeling. This study aimed to evaluate AE's effect on periodontitis in rats and investigate AMCase expression. Eighteen Sprague-Dawley rats were separated into the following groups: healthy (group 1), disease (group 2), vehicle (group 3), AE high-dose (group 4), and AE low-dose (group 5). Porphyromonas gingivalis ligatures were placed in rats (groups 2-5) for 7 days. Groups 4 and 5 were then treated with AE for an additional 14 days. Saliva was collected from all groups, and probing pocket depth was measured in succession. Periodontal pocket tissues were subjected to histomorphometric analysis after the rats were sacrificed. Bone marrow-derived macrophages and murine macrophages were stimulated with receptor activator of nuclear factor-κB ligand (RANKL) and treated with different concentrations of AE. AMCase expression was detected from the analysis of saliva, periodontal pocket tissues, and differentiated osteoclasts. Among rats with P. gingivalis-induced periodontitis, the alveolar bone resorption levels and periodontal pocket depth were significantly reduced after treatment with AE. AMCase protein expression was significantly higher in the disease group than in the healthy control (P<0.05). However, AE inhibited periodontal inflammation by downregulating AMCase expression in saliva and periodontal pocket tissue. AE significantly reduced RANKL-stimulated osteoclastogenesis by modulating AMCase (P<0.05). AE decreases alveolar bone loss and periodontal inflammation, suggesting that this natural anthraquinone has potential value as a novel therapeutic agent against periodontal disease.

Mouse Model of Loeys-Dietz Syndrome Shows Elevated Susceptibility to Periodontitis via Alterations in Transforming Growth Factor-Beta Signaling.

Loeys–Dietz syndrome (LDS) is a syndromic connective tissue disorder caused by a heterozygous missense mutation in genes that encode transforming growth factor (TGF)-β receptor (TGFBR) 1 and 2. We encountered a patient with LDS, who had severe periodontal tissue destruction indicative of aggressive periodontitis. The patient had a missense mutation in the glycine and serine-rich domain of TGFBR1 exon 3. This G-to-T mutation at base 563 converted glycine to valine. We established an LDS model knock-in mouse that recapitulated the LDS phenotype. Homozygosity of the mutation caused embryonic lethality and heterozygous knock-in mice showed distorted and ruptured elastic fibers in the aorta at 24 weeks of age and died earlier than wildtype (WT) mice. We stimulated mouse embryonic fibroblasts (MEFs) from the knock-in mouse with TGF-β and examined their responses. The knock-in MEFs showed downregulated Serpine 1 mRNA expression and phosphorylation of Smad2 to TGF-β compared with WT MEFs. To clarify the influence of TGF-β signaling abnormalities on the pathogenesis or progression of periodontitis, we performed pathomolecular analysis of the knock-in mouse. There were no structural differences in periodontal tissues between WT and LDS model mice at 6 or 24 weeks of age. Micro-computed tomography revealed no significant difference in alveolar bone resorption between WT and knock-in mice at 6 or 24 weeks of age. However, TGF-β-related gene expression was increased significantly in periodontal tissues of the knock-in mouse compared with WT mice. Next, we assessed a mouse periodontitis model in which periodontal bone loss was induced by oral inoculation with the bacterial strain Porphyromonas gingivalis W83. After inoculation, we collected alveolar bone and carried out morphometric analysis. P. gingivalis-induced alveolar bone loss was significantly greater in LDS model mice than in WT mice. Peritoneal macrophages isolated from Tgfbr1G188V/+ mice showed upregulation of inflammatory cytokine mRNA expression induced by P. gingivalis lipopolysaccharide compared with WT macrophages. In this study, we established an LDS mouse model and demonstrated that LDS model mice had elevated susceptibility to P. gingivalis-induced periodontitis, probably through TGF-β signal dysfunction. This suggests that TGF-β signaling abnormalities accelerate the pathogenesis or progression of periodontitis.

The Effects of Rice Husk Liquid Smoke in Porphyromonas gingivalis-Induced Periodontitis.

Objectives The purpose of this study was to analyze the effects of rice husk liquid smoke in Porphyromonas gingivalis -induced periodontitis in the inflammatory and proliferation marker such as nuclear factor kappa β (NF-kB), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), transforming growth factor-β (TGF-β), fibroblast growth factor 2 (FGF2), collagen type 1 (COL-1) expression, and the number of macrophages, lymphocytes, and fibroblasts. Materials and Methods Rice husk liquid smoke is obtained by the pyrolysis process. Porphyromonas gingivalis -induced periodontitis in 20 μL phosphate-buffered saline containing 1 × 10 9 CFU was injected into the lower anterior gingival sulcus of Wistar rats. The periodontitis was then treated with 20 μL/20 g body weight of rice husk liquid smoke once a day for 2 and 7 days, respectively. After treatment, the bone and lower anterior gingival sulcus were analyzed with immunohistochemistry and hematoxylin–eosin staining. Results The treatment of periodontitis with rice husk liquid smoke showed a lower NF-kB, TNF-α, and IL-6 expression and a higher TGF-β, FGF2, and COL-1 expression than the control after treatment for 2 and 7 days ( p < 0.05), respectively. The number of macrophages and fibroblasts was also higher when compared with the control group ( p < 0.05), but the number of lymphocytes was lower than the control ( p < 0.05). Conclusion Rice husk liquid smoke showed its effects on Porphyromonas gingivalis -induced periodontitis with a decrease in inflammatory markers and an increase in proliferation markers. The development of a rice husk liquid smoke periodontitis treatment is promising.

Spirulina maxima reduces inflammation and alveolar bone loss in Porphyromonas gingivalis-induced periodontitis

BackgroundPeriodontitis is a common oral disease characterized as inflammation on gingival tissue and alveolar bone resorption. Spirulina maxima has been reported to have anti-oxidative and anti-inflammatory effects on gastric ulcers. However, its effects on gingival inflammation and alveolar bone resorption of periodontitis have not been studied. PurposeThis study was designed to investigate the effects of S. maxima on the P. gingivalis-induced periodontitis and to elucidate its mechanism. MethodsThe phycocyanin contents in S. maxima were identified by high-performance liquid chromatography. 8-week old SD rats were induced periodontitis by inoculation with P. gingivalis for 14 days. The rats were then orally treated with S. maxima 100, 200, 400 mg/kg, or indomethacin (IND, positive control) 5 mg/kg for an additional 14 days. Inflammatory responses, expressions of collagenases in gingival tissue, osteoclast formation and activation, alveolar bone resorption, osteogenesis-related markers, and BMP2/Smad signaling in alveolar bone were measured. ResultsPro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and inflammatory transcription factor NF-κB were decreased in gingival tissue by S. maxima administration. Also, myeloperoxidase (MPO) activity and matrix metalloproteinase (MMPs) expression were decreased by S. maxima administration. Conversely, S. maxima increased IL-4, anti-inflammatory cytokine from Th2 cells. The osteoprotegerin (OPG) / receptor activator of NF-κB ligand (RANKL) expression ratio, which represents osteoclast-osteoblast balance, was increased in S. maxima-treated groups. The alveolar bone loss and the number of TRAP-positive osteoclast cells were also declined in S. maxima-treated groups while the osteoblasts count was increased. Besides, in S. maxima-treated groups, the osteogenesis-related factors were promoted and BMP-2/Smad pathway was up-regulated in a periodontitis condition. ConclusionS. maxima reduces periodontitis induced by P. gingivalis through anti-inflammatory effect and resultant reduction in bone loss, suggesting that S. maxima might be a potential agent for treating periodontitis.

The sialidase inhibitor, DANA, reduces Porphyromonas gingivalis pathogenicity and exerts anti-inflammatory effects: An in vitro and in vivo experiment.

Sialidase has an important role in the pathogenesis of periodontitis and Porphyromonas gingivalis is a sialidase-producing organism implicated in periodontitis development. The aim of this study was to evaluate the anti-virulence and anti-inflammatory properties of the sialidase inhibitor, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA), in vitro and in vivo. The effects of DANA on P. gingivalis sialidase and cell viability were determined, and the effects of DANA on P. gingivalis virulence were evaluated by assessment of growth curves, cell morphology, biofilm formation, fimbriae gene expression, and gingipains and lipopolysaccharide (LPS) activity. Anti-inflammatory effects of DANA on LPS-induced macrophages were assessed by measurement of tumor necrosis factor-alpha (TNF-α), interleukin (IL-1β), inducible nitric oxide synthase (iNOS) secretions. The effect of DANA on P. gingivalis-induced periodontitis in rats was analyzed by radiography, stereoscopic microscopy, histopathology, and immunohistochemistry. Sialidase inhibition rate of 1mM DANA was 72.01%. Compared with untreated controls, treatment with DANA inhibited P. gingivalis growth and biofilm formation, and significantly decreased expression of the fimA, fimR, and fimS genes, as well as gingipains activity. DANA did not influence macrophage viability, but significantly inhibited TNF-α, IL-1β, and iNOS production in LPS-stimulated macrophages. In the periodontitis rat model, DANA prevented alveolar bone absorption and inhibited TNF-α and IL-1β production. DANA can reduce the growth, the biofilm formation and the virulence of P. gingivalis and exhibits anti-inflammatory effects, as well as effects against rat periodontitis, suggesting that DANA should be considered for development as a new adjunctive treatment for periodontitis.

Catechin ameliorates Porphyromonas gingivalis-induced inflammation via the regulation of TLR2/4 and inflammasome signaling.

Porphyromonas gingivalis is a major periodontopathogen found in patients with chronic periodontitis that can lead to alveolar bone or tooth loss. Interleukin-1β (IL-1β), a proinflammatory cytokine, is most relevant to the pathogenesis of periodontitis. Catechin is one of the main polyphenol compounds found in green tea and possesses a range of health benefits. This study examined the anti-inflammatory effects of catechin in THP-1-derived macrophages infected with P. gingivalis as well as its effects on P. gingivalis-induced periodontitis in a mouse model. The cytokine levels and relevant protein expression in THP-1 cells were measured using an enzyme-linked immunosorbent assay and Western blot analysis, respectively. An apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) pyroptosome formation was measured by confocal laser scanning microscopy. Micro-computed tomography was used to determine the level of bone loss induced by a P. gingivalis oral infection. Catechin attenuated the production of IL-1β by inhibiting pro-IL-1β expression via the downregulation of nuclear factor-κB, p38 mitogen-activated protein kinase, and Toll-like receptor signaling. In addition, catechin inhibited the activation of inflammasomes induced by P. gingivalis, but did not affect the growth of P. gingivalis. Catechin reduced the level of alveolar bone loss in a P. gingivalis-induced periodontitis mouse model. Catechin possesses anti-inflammatory properties by reducing the level of IL-1β production, suggesting that it can potentially be used for the prevention and treatment of periodontal inflammation caused by P. gingivalis.

Effects of omega-3 fatty acids and aspirin on Porphyromonas gingivalis-induced periodontitis in rats.

Periodontitis is a common chronic inflammatory disease caused by bacteria which can result in periodontal tissue inflammation, as well as alveolar bone resorption. The purpose of this study was to evaluate the effects of omega-3 fatty acids plus aspirin (ASA) on ligature-induced periodontitis in rats. Ninety-six male Sprague-Dawley (SD) rats (age 6 weeks) were randomly divided into eight groups (n=12 each) and had ligatures placed for 7days, followed by daily treatment with specific drug regimens for 14days. The rats were sacrificed 20days after drug treatment, and their maxillary were subjected to histomorphometric analysis. RAW264.7 cells were cultured with lipopolysaccharide (LPS) or receptor activator (NF)-κB ligand (RANKL), and treated with various concentrations of omega-3 and ASA. Then, cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS) protein expression and receptor activator of nuclear factor κ B (RANK), tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP-9), MMP-2, and Cathepsin-K gene expression were detected. The administration of omega-3 fatty acids and aspirin significantly inhibited tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in serum of rats. Histomorphometric analysis showed omega-3 fatty acids plus aspirin promoted alveolar bone increase. Omega-3 fatty acids only, aspirin only, or omega-3 fatty acids plus aspirin also inhibited the protein expressions of COX-2 and iNOS in LPS-stimulated RAW264.7 cells. In addition, omega-3 combined with ASA also inhibited the RANKL-induced gene expressions of MMPs in dose-dependent manners. These results demonstrate that omega-3 fatty acids plus aspirin could decrease alveolar bone loss, while simultaneously increasing the protection against periodontal inflammation.

Ameliorating Effects of Jixueteng in a Mouse Model of Porphyromonas gingivalis-Induced Periodontitis: Analysis Based on Gingival Microcirculatory System

Jixueteng, the dried stem of Spatholobus suberectus Dunn (Leguminosae), is a traditional Chinese herbal medicine that promotes blood circulation and can be used to treat blood stasis. In this study, we aimed to investigate the potential of Jixueteng as a preventive and therapeutic drug for periodontitis. We investigated the inhibitory effects of Jixueteng on Porphyromonas gingivalis ( P. gingivalis)-induced gingival circulatory disturbances in mice. Seventy-two male C57BL/6N mice (4-week-old) were randomly divided into the following four groups of 12 mice each. Group 1 served as the P. gingivalis noninfected control (control group). Group 2 was administered Jixueteng extract in drinking water to P. gingivalis noninfected control mice. Group 3 was infected orally with P. gingivalis; and group 4 was administered Jixueteng extract in drinking water and then infected with P. gingivalis. To evaluate the effect of Jixueteng on gingival microcirculation systems, we examined gingival blood flow (GBF) in oral microcirculation in vivo in a mouse model of periodontitis. Gingival reactive hyperemia (GRH) was determined using laser Doppler flowmetry. GRH was elicited by the release of occlusive gingival compression by the laser Doppler probe (diameter 1.0 mm) for 1 min. GRH was estimated by basal blood flow, maximum response (Peak), the time taken for the maximum response to fall to one half (T1/2) and increased total amount of blood flow (Mass). Furthermore, to determine the effect of an oral application of P. gingivalis and/or Jixueteng on GBF and gingival microvessel ultrastructure, morphological analysis of gingival microvessels was performed by using a vascular resin cast model. Administration of Jixueteng to P. gingivalis-infected animals significantly reduced GRH, especially T1/2 and Mass, compared to that in P. gingivalis-infected animals. Alternatively, in the morphological analysis, reduction of the gingival capillary network which resulted from P. gingivalis-infection was improved by Jixueteng administration. Since Jixueteng ameliorates P. gingivalis infection-induced gingival circulatory disturbance, it may be useful in the treatment of P. gingivalis-induced periodontitis.

The therapeutic potential of regulatory T lymphocytes in periodontitis: A systematic review

This systematic review aimed to: (a) generate a descriptive synthesis of preclinical studies assessing the therapeutic potential of regulatory T lymphocytes (Tregs) to arrest periodontitis, (b) evaluate the methodological heterogeneity of the reviewed animal studies and (c) assess the risk of bias (RoB) of the included studies. The electronic search for animal studies included the MEDLINE, EMBASE, Web of Science and LILACS databases. In addition, a manual search assessed the high-ranked scientific journals in "periodontics/immunology" and the references listed in the included studies. There were no language, year or publication status restrictions. Two independent reviewers selected and extracted the data, and Cohen's Kappa coefficient was calculated to determine the inter-examiner agreement. The Systematic Review Center for Laboratory Animal Experimentation's (SYRCLE) tool was used to assess the RoB. A total of 21 of the 425 studies obtained from the database search were included. Treg function was mainly described in Porphyromonas gingivalis-induced periodontitis (57.1%) in mice (76.2%), where Treg suppression was strongly related to disease progression and Treg induction was strongly related to immuno-inflammatory response reduction. Of those 21 studies, eight included eight animal experiments using three distinct therapeutic approaches, including: P.gingivalis-driven immunization (n=3), retinoic acid inoculation (n=2) and anti-inflammatory molecules in polymeric carriers (n=3), which could modulate the Treg activity through cytokine production (interleukin-10 and transforming growth factor-β1), CC-chemokine- and CC-chemokine receptor-mediated chemoattraction (CCL22 and CCR4) or Th17-associated receptor activator of nuclear factor κB ligand (RANKL) downregulation. However, the studies with animal experiments did not specify the randomization sequences and housing conditions that were used, and therefore, 42.11% of the entries were rated as unclear RoB. Distinct therapeutic strategies involving Tregs could potentially suppress the immuno-inflammatory response and restore alveolar bone homeostasis during periodontitis. Nevertheless, important methodological variability, poor reporting of treatment effect estimates and unclear RoB suggest using caution when assessing the results of these studies.

Transcription factor DEC1 is required for maximal experimentally induced periodontal inflammation.

Disruption of transcriptional regulation is a confounding factor associated with a wide range of human inflammatory diseases. To investigate mechanistic links between transcription factor DEC1 and pathways underlying inflammation, wild-type and DEC1 knockout (KO) C57BL/6 mice were treated with Porphyromonas gingivalis (or carboxymethyl cellulose as a control) to induce periodontal inflammation. It provoked an inflammatory response within the oral environment, which showed robust variation in alveolar bone resorption and expression of inflammatory cytokines. Male DEC1KO mice and their wild-type littermates were used for the experimental periodontitis model. Measurement of alveolar bone resorption, micro-computed tomography, isolation of gingival mononuclear cells (GMCs), flow cytometry and immunohistochemical analysis were used in this study. Human gingival fibroblast cells (HGF-1) were used for DEC1 over-expression and short interference RNA (siRNA) studies and quantitative real-time polymerase chain reaction and western blot analysis were performed. Micro-computed tomography analysis demonstrated that P.gingivalis caused a decrease in bone area of wild-type mice compared with DEC1KO mice. Expression of inflammatory and immune markers in GMCs was significantly decreased in DEC1KO mice after treatment with P.gingivalis. Conversely, interleukin (IL)-4 and IL-10 mRNAs were significantly increased in GMCs isolated from DEC1KO mice. The results show that treatment of DEC1KO mice with P.gingivalis decreased the numbers of CD11b+ F4/80+ and CD4+ RANKL+ T cells. Moreover, expression of CD4, F4/80, RANKL and cathepsin K in inflammatory cell infiltrates was significantly reduced in DEC1KO mice treated with P.gingivalis compared with controls. Furthermore, over-expression of DEC1 in HGF-1 cells increased the expression of IL-1β and tumor necrosis factor-α mRNAs and their expression levels reached a maximum in response to treatment with lipopolysaccharide. Inhibition of DEC1 by short interference RNA interference suppressed the P.gingivalis-derived lipopolysaccharide-induced expression of IL-1β, tumor necrosis factor-α and toll-like receptor4. These results suggest that transcription factor DEC1 can modulate P.gingivalis-induced periodontitis in the oral mucosa.

Preventive effects of the novel antimicrobial peptide Nal-P-113 in a rat Periodontitis model by limiting the growth of Porphyromonas gingivalis and modulating IL-1\u03b2 and TNF-\u03b1 production

BackgroundP-113 (AKRHHGYKRKFH-NH2) is a 12-amino-acid histidine-rich peptide derived from histatin 5 that is highly degradable in high salt concentrations and biological fluids such as serum, plasma and saliva. Nal-P-113, a novel antimicrobial peptide whose histidine residues are replaced by the bulky amino acids β-naphthylalanine, causes the antimicrobial peptide to retain its bactericidal activity even in physiological environments. This study evaluated the effect of the novel antimicrobial peptide Nal-P-113 in a rat periodontitis model and the mechanisms of action of Nal-P-113 for suppressing periodontitis.MethodsPeriodontitis was induced in mandibular first molars in rats receiving a ligature and infected with Porphyromonas gingivalis. Animals were randomly divided into six groups: a, P. gingivalis W83 alone; b, P. gingivalis W83 with 6.25 μg/mL of Nal-P-113; c, P. gingivalis W83 with 25 μg/mL of Nal-P-113; d, P. gingivalis W83 with 100 μg/mL of Nal-P-113; e, P. gingivalis W83 with 400 μg/mL of Nal-P-113; and f, control without P. gingivalis W83 or Nal-P-113. Morphometric analysis was used to evaluate alveolar bone loss. Microbiological assessment of the presence of Porphyromonas gingivalis and total bacteria was performed using absolute quantitative real-time PCR and scanning electron microscopy. Gingival tissue was collected for western blot and immunohistochemical assays of IL-1β and TNF-α levels.ResultsAlveolar bone loss was inhibited by 100 μg/mL or 400 μg/mL of Nal-P-113 compared to the control group (P < 0.05). Lower amounts of P. gingivalis and total bacteria were found in groups d and e compared with group a (P < 0.05). A decrease in the levels of IL-1β and TNF-α was detected in group d and group e compared to the control group (P < 0.05). The amount of P. gingivalis was positively correlated with IL-1β and TNF-α expression in periodontal tissue (P < 0.05).ConclusionsNal-P-113 exhibited protective effects on Porphyromonas gingivalis-induced periodontitis in rats by limiting the amount of bacteria and modulating IL-1β and TNF-α production. The use of Nal-P-113 in vivo might serve as a beneficial preventive or therapeutic approach for periodontitis.

The aggravation of arthritis by periodontitis is dependent of IL-17 receptor A activation.

To evaluate whether Porphyromonas gingivalis-induced periodontitis aggravates the antigen-induced arthritis (AIA) model, and whether this effect is dependent on the Th17/IL-17 signalling pathway. Antigen-induced arthritis was triggered by local injection of methylated bovine serum albumin into the knee joint of previously immunized C57BL/6 wild-type (WT) and IL-17 receptor A (IL-17RA)-knockout mice. Periodontal disease in naïve or arthritic mice was induced by oral infection with P. gingivalis. Animals were sacrificed 7, 15 and 30days after infection. Alveolar bone loss, joint histopathology, articular hyperalgesia and joint cytokine production were assessed, in addition to the proportion of Th17 and Treg cells isolated from the inguinal lymph nodes. No influence of experimentally-induced arthritis was found on the alveolar bone resorption induced by P. gingivalis. However, mice with experimentally-induced arthritis that were exposed to P. gingivalis presented higher joint damage and Th17 frequencies when compared to non-infected mice. The aggravation of arthritis by periodontitis was accompanied by increased TNF and IL-17 production and articular neutrophil infiltration, whereas arthritis aggravation and changes in neutrophil infiltration were absent in IL-17RA-deficient mice. The effects of P. gingivalis-induced periodontitis on arthritis are dependent on Th17 expansion and IL-17RA signalling, which lead to increased neutrophil infiltration into the joints.

CXCR4 signaling in macrophages contributes to periodontal mechanical hypersensitivity in Porphyromonas gingivalis-induced periodontitis in mice.

BackgroundPeriodontitis is an inflammatory disease accompanied by alveolar bone loss and progressive inflammation without pain. However, the potential contributors eliminating pain associated with gingival inflammation are unknown.Resultswe examined the involvement of CXC chemokine receptor type 4 (CXCR4) on the mechanical sensitivity of inflamed periodontal tissue, using a mouse model of periodontitis established by the ligation of the tooth cervix of a maxillary second molar and inoculation with Porphyromonas gingivalis (P. gingivalis). Infiltration of inflammatory cells into gingival tissue was not observed following the inoculation. Under light anesthesia, the mechanical head withdrawal threshold (MHWT) on the buccal gingiva was measured using an electronic von Frey anesthesiometer. No significant changes in MHWT were observed in the mice with P. gingivalis-induced periodontitis during the experimental period. Continuous administration of CXCR4 neutralizing antibody to the gingival tissue significantly decreased MHWT and increased the number of gingival CXCR4 immunoreactive macrophages in the periodontitis group. Nitric oxide metabolites in the gingival tissue were significantly increased after the inoculation of P. gingivalis and were reduced by gingival CXCR4 neutralization. Gingival L-arginine administration induced gingival mechanical allodynia in naive animals. Moreover, the decrease in MHWT after treatment with P. gingivalis and CXCR4 neutralization was partially reversed by nitric oxide synthase inhibition in the gingival tissue. Nuclear factor-kappa B was expressed in infiltrating macrophages after inoculation of P. gingivalis and administration of the nuclear factor-kappa B activator betulinic acid induced gingival mechanical allodynia in naive mice.ConclusionsThese findings suggest that CXCR4 signaling inhibits nitric oxide release from infiltrating macrophages and is involved in modulation of the mechanical sensitivity in the periodontal tissue in P. gingivalis-induced periodontitis.