Investigation of peri-implant diseases prevalence and related risk indicators in patients with treated severe periodontitis over 4 years after restoration.

Background/purpose: History of periodontitis is a well-documented risk indicator of peri-implantitis. However, the influence of severity of periodontitis is still unclear, especially for severe periodontitis. This study was aimed to investigate the prevalence of peri-implant disease and analyze the risk indicators in patients with treated severe periodontitis. Materials and methods: A total of 182 implants from 88 patients (44 males and 44 females) with severe periodontitis with a mean fellow-up period of 76.5 months were enrolled in this study. Patient and implant information, and periodontal and peri-implant conditions were collected to evaluate the prevalence of peri-implant disease and risk indicators. Results: The prevalence of peri-implantitis was 9.1% and 6.6% at the patient-level and implant-level. The prevalence of peri-implant mucositis was 76.1% and 51.1% at the patient-level and implant-level. Risk indicators of peri-implantitis included older age (OR: 1.132), poor proximal cleaning habits (OR: 14.218), implants in anterior area (OR: 10.36), poor periodontal disease control (OR: 12.76), high peri-implant plaque index (OR: 4.27), and keratinized tissue width (KTW)＜2 mm (OR: 19.203). Conclusion: Implants in patients with severe periodontitis after periodontal treatment and maintenance show a low prevalence (9.1%) of peri-implantitis and a relatively high prevalence (76.2%) of peri-implant mucositis. Patient age, peri-implant proximal cleaning habits, implant position, periodontal disease control, peri-implant plaque index, and KTW are associated with prevalence of peri-implantitis.

The expression levels of chemotaxis-related molecules CXC chemokine receptor 1, interleukin-8, and pro-platelet basic protein in gingival tissues.

Background/purpose: Excessive host immune response is thought to be an important cause of periodontal tissue damage during periodontitis. The potent chemotaxis produced by locally released chemokines is the key signal to trigger this response. Here, we aimed to investigate the expression of CXC chemokine receptor 1 (CXCR1), and chemokines interleukin-8 (IL-8) and pro-platelet basic protein (PPBP) in human inflammatory gingival tissues compared with healthy tissues. Materials and methods: A total of 54 human gingival tissues, 27 healthy and 27 inflammatory samples, were collected. Fifteen specimens of each group were employed for quantitative reverse transcription polymerase chain reaction to determine the mRNA levels of CXCR1, IL-8, and PPBP. Six samples of each group were used for Western blotting to investigate the protein expression of CXCR1 and for enzyme-linked immunosorbent assay to evaluate the protein levels of IL-8 and PPBP, respectively. Results: The mRNA levels of chemokine receptor CXCR1, chemokine IL-8, and PPBP in inflammatory gingival tissues were significantly higher than those in healthy controls (P < 0.05). The protein levels of CXCR1, IL-8, and PPBP in inflammatory gingival tissues were also significantly higher than those in healthy gingival tissues (P < 0.05). Conclusion: When compared to healthy gingival tissues, the expression of CXCR1, IL-8, and PPBP in inflammatory gingival tissues is higher.

Activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 6 by Porphyromonas gingivalis regulates programmed cell death in epithelium.

Background/purpose: Gingival epithelial cells form a physiological barrier against bacterial invasion. Programmed cell death (PCD) regulated by pathogen precognition receptors (PRRs) lead to tissue destruction and is closely related to inflammatory diseases. The purpose of this study was to investigate whether nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 6 (NLRP6) expresses in periodontal epithelium and induces PCD of epithelial cells infected by Porphyromonas gingivalis (P. gingivalis), therefore involves in periodontitis. Material and methods: The expression of NLRP6 was detected in periodontal epithelium from human gingival sections and HaCaT cells stimulated by P. gingivalis. NLRP6 was over-expressed by adenovirus infection in HaCaT or knocked down by siRNA in P. gingivalis infected HaCaT, and the cell death was observed by transmission electron microscopy and flow cytometry analysis. In addition, qPCR and Western blot were performed to determine the expression of NLRP6 and the pyroptosis excutors, caspase-1 and gasdermin D. Enzyme-linked immunosorbent assay were performed to detect the secretion of IL-1ß and IL-18. Results: NLRP6 was up-regulated in both gingival epithelium of patients with periodontitis and P. gingivalis infected HaCaT. Over-expression of NLRP6 in HaCaT led to caspase-1 dependent pyroptosis. Interestingly, knockdown of NLRP6 with siRNA followed by P. gingivalis stimulation inhibited pyroptosis and induced apoptosis. Conclusion: Up-regulation of NLRP6 by P. gingivalis in HaCaT led to pyroptosis, while knocking down NLRP6 inhibited pyroptosis and induced apoptosis, which indicated this PRR may play a crucial role in periodontitis by regulating PCD in periodontal epithelium.

Role of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 6 in activation of inflammation in human umbilical vein endothelial cells stimulated by Porphyromonas gingivalis-an in vitro study.

Background/purpose: Porphyromonas gingivalis (P. gingivalis) could induce the activation of vascular endothelial cells and promote the formation of atherosclerosis. Nucleotide-binding oligomerization domain-like receptor family pyrin domain containing (NLRP) 6 could recognize P. gingivalis, but its role in atherosclerosis was unknown. The purpose of this study is to investigate the role of NLRP6 in the activation of inflammation in human umbilical vein endothelial cells (HUVECs) stimulated by P. gingivalis. Materials and methods: The expression level of NLRP6 in HUVECs with or without P. gingivalis-challenge was observed. Down-regulating the expression of NLRP6 in HUVECs, the expression levels of interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein (MCP)-1 were detected. Then, the HUVECs with NLRP6-overexpressed were stimulated by P. gingivalis, the levels of inflammatory cytokines above were examined and compared with those in HUVECs triggered by P. gingivalis only. To evaluate the effect of NLRP6 on bacterial immune escape, the NLRP6 was overexpressed, and the colonies of P. gingivalis that survived in HUVECs were calculated. Results: NLRP6 was expressed in HUVECs and decreased after P. gingivalis stimulation. Downregulation of NLRP6 decreased the expression levels of IL-1ß, IL-6, IL-8, TNF-α and MCP-1 in HUVECs. Those cytokines above in NLRP6-overexpressed HUVECs with P. gingivalis-stimulation significantly increased than in the cells with P. gingivalis-stimulation only. Furthermore, over-expression of NLRP6 decreased the colonies of P. gingivalis survival in HUVECs. Conclusion: NLRP6 regulated the activation of inflammation in HUVECs triggered by P. gingivalis and played an important role in P. gingivalis survival in endothelial cells.

Role of immune-related lncRNAs--PRKCQ-AS1 and EGOT in the regulation of IL-1ß, IL-6 and IL-8 expression in human gingival fibroblasts with TNF-α stimulation.

Background/purpose: It was reported that lncRNAs have an effect on immune-related diseases, however, their roles in periodontitis remain to be investigated. The aim of this study was to look for immune-related lncRNAs in periodontitis, and to preliminarily explore their function in vitro. Materials and methods: CIBERSORT was used to analyze abundance of immune cell in the periodontal tissue. Correlation between the expression profile of lncRNAs and abundance of immune cell was calculated and immune-related lncRNAs were identified. The expressions of immune-related lncRNAs identified were validated by RT-qPCR with 15 periodontitis and 15 healthy gingival tissues. The expressions of PRKCQ-AS1 and EGOT in HGFs were detected under the stimulation of different concentrations of TNF-α (0, 10, 15, 20, 30 ng/mL) and different duration (0, 12, 24 and 48 h). Then, siRNA was used to silence PRKCQ-AS1 and EGOT in HGFs. The expression level of IL-1ß, IL-6, IL-8 of the HGFs after stimulated by 15 ng/mL TNF-α, and the activation of NF-κB pathway was observed. Results: PRKCQ-AS1 and EGOT were identified as top 2 immune-related lncRNAs in periodontal tissues. The expressions of PRKCQ-AS1 and EGOT were significantly up-regulated in inflamed periodontal tissue and in HGFs under TNF-α stimulation. After knock-down of PRKCQ-AS1 and EGOT, expression level of IL-1ß, IL-6, and IL-8 in HGFs with TNF-α stimulation were decreased, and activation of NF-κB pathway was inhibited. Conclusion: PRKCQ-AS1 and EGOT were firstly identified as immune-related lncRNAs in periodontal tissue, and they regulate the expression of IL-1ß, IL-6, and IL-8 of HGFs through the NF-κB pathway.

Whole-transcriptome analysis of periodontal tissue and construction of immune-related competitive endogenous RNA network.

BACKGROUND: In periodontitis, noncoding RNAs may play a regulatory role in the immune microenvironment through competitive endogenous RNA. We aimed to profile noncoding RNA expression and construct immune-related ceRNA network in periodontitis. METHODS: Five inflamed periodontal tissue and five healthy gingivae were collected for whole-transcriptome sequencing. Differential gene, functional enrichment, and protein-protein interaction network analysis were performed to explore the function of differentially expressed genes. CIBERSORTx was used to analyze level of immune cell infiltration in the periodontal tissue. An immune-related competitive endogenous RNA network was constructed and expression of key regulators in the network was validated. RESULTS: Compared with healthy gingiva, 200 mRNAs, 90 long noncoding RNAs, 65 microRNAs, and 518 circular RNAs were differentially expressed, and cell chemotaxis was significantly enhanced in inflamed periodontal tissue. Immune cell infiltration analysis showed that neutrophils, macrophages M1, T follicular helper cells, and naive B cells were significantly increased in periodontitis. Key regulators including JUN, FOS, THBS1, KLF2, WIF1, were identified and their expression was then validated. CONCLUSION: We constructed an immune-related competitive endogenous RNA network in periodontal tissue, which provided new insights into immune homeostasis in periodontitis and laid a foundation for further study of noncoding RNAs. Key regulators in this network may be promising targets for future periodontitis treatment.

NLRC5 modulates bone metabolism and plays a role in periodontitis.

INTRODUCTION: NOD-like receptor C5 (NLRC5) plays a significant role in the immune system, and is one of the largest members of the pattern recognition receptor family. Previous studies have found that NLRC5 might be involved in the regulation of various diseases, such as fibrotic diseases and cancers; however, its effect on bone metabolism-related diseases has not been reported. METHODS: Skeletons of Nlrc5-/- mice generated by CRISPR/Cas9 and wild-type (WT) mice were compared using X-ray, micro-computed tomography, double labeling, and histological examination. Tartrate-resistant acid phosphatase and pit-absorption assays were performed to evaluate the effect of NLRC5 on osteoclasts differentiation and osteoclastic capacity. The influence of NLRC5 on osteoblasts differentiation and bone formation were studied using alkaline phosphatase and alizarin red staining, respectively. Experimental periodontitis was induced by Porphyromonas gingivalis infection and ligature to investigate the role of NLRC5 in inflammatory periodontal bone loss. RESULTS: Adenovirus-mediated NLRC5 overexpression in human bone marrow mesenchymal stem cells regulated osteogenesis positively. The femoral osteogenesis ability was significantly weakened in Nlrc5-/- mice. Histology showed that the area of the femoral trabeculae in the Nlrc5-/- mice was less than that in the WT mice, and radiology suggested that the Nlrc5-/- mice had fewer trabeculae and a thinner bone cortex than those of the WT mice. Nlrc5 knockout decreased osteoblast mineralization and increased osteoclastogenesis in vitro. NLRC5 was downregulated in periodontitis and P. gingivalis infection. In the experimental periodontitis model, the alveolar bone loss, inflammatory cell infiltration, and inflammatory cytokines secretion (interleukin [IL]-1ß, IL-6, and tumor necrosis factor alpha [TNF-α]) in the Nlrc5-/- mice were significantly enhanced compared to WT mice. CONCLUSION: We verified a novel role of NLRC5 in bone metabolism by regulating both osteoclasts activity and osteoblasts activity. Our results revealed a protective effect of NLRC5 against periodontal inflammation and alveolar bone destruction. NLRC5 could be a novel treatment target to prevent periodontal bone destruction.

Efficacy of periodontal minimally invasive surgery with and without regenerative materials for treatment of intrabony defect: a randomized clinical trial.

OBJECTIVES: The minimally invasive surgical technique was modified in suture (MISTms) in this study. The trial was to determine the efficacy of MISTms with and without regenerative materials for the treatment of intrabony defect and to identify factors influencing 1-year clinical attachment level (CAL) gain. METHODS: Thirty-six patients with interdental intrabony defects were randomly assigned to MISTms (MISTms alone, 18) or MISTms plus deproteinized bovine bone mineral and collagen membrane (MISTms combined, 18). Wound healing was evaluated by early healing index (EHI) at 1, 2, 3, and 6 weeks. Probing depth (PD), CAL, gingival recession, radiographic defect depth, and distance from the base of defect to the cementoenamel junction were recorded at baseline and 1 year postoperatively. A one-year composite outcome measure based on the combination of CAL gain and post-surgery PD was evaluated. Factors influencing 1-year CAL gain were analyzed. RESULTS: Fifteen patients in MISTms-alone and 16 in the MISTms-combined group finished the study. The MISTms-alone group showed significantly better wound healing at 1 week. CAL significantly gained in the MISTms-alone and MISTms-combined group, with 2.53 ± 1.80 mm and 2.00 ± 1.38 mm respectively. The radiographic bone gain was 3.00 ± 1.56 mm and 3.85 ± 1.69 mm respectively. However, there were no significant differences between the two groups about 1-year outcomes. Lower EHI (optimal wound healing) and more baseline CAL positively influenced 1-year CAL gain. CONCLUSIONS: MISTms is an effective treatment for intrabony defects. The regenerative materials do not show an additional effect on 1-year outcomes. Early wound healing and baseline CAL are factors influencing 1-year CAL gain. CLINICAL RELEVANCE: MISTms with and without regenerative materials are both effective treatments for intrabony defect. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: ChiCTR2100043272.

Effect of Pudilan Keyanning antibacterial mouthwash on dental plaque and gingival inflammation in patients during periodontal maintenance phase: study protocol for double-blind, randomised clinical trial.

INTRODUCTION: Plaque control plays a critical role in the prevention and treatment of periodontitis. Antibacterial mouthwash is one of the most important tools for plaque control. Pudilan, including extracts of Scutellaria baicalensis root, Taraxacum mongolicum, Bunge corydalis herb and Isatis indigotica, was reported playing the role of anti-inflammatory and anti-bacterial. However, its effect on dental plaque and periodontal inflammation remains unknown. We aimed to assess the efficacy of Pudilan Keyanning antibacterial mouthwash which contains the active essence of Pudilan and 0.03%-0.06% cetylpyridinium chloride, as well as Pudilan active essence for plaque control and gingival anti-inflammation in patients during periodontal maintenance phase. METHODS AND ANALYSIS: In this double-blind, randomised, placebo-controlled clinical trial, a total of 120 participants during periodontal maintenance phase will be enrolled. After supragingival scaling, they will be randomly assigned into three groups in a 1:1:1 ratio: the Pudilan Keyanning antibacterial mouthwash group, a chlorhexidine acetate mouthwash (0.12%) group or a placebo group with mouthwash containing the same components as the Pudilan Keyanning mouthwash except for Pudilan active ingredients. They will rinse with mouthwash, respectively, two times per day for 6 weeks. Clinical parameters (such as plaque index, bleeding index) and the level of volatile sulfide in the breath will be measured and analysed. The subgingival plaque will be collected and analysed microbiologically. Questionnaire feedback will be analysed. ETHICS AND DISSEMINATION: The study protocol (V.4) was reviewed and approved by the Medical Ethical Committee of Peking University School and Hospital of Stomatology (Ethics Approval No. PKUSSIRB-201950153b). All participants signed a written consent form. TRIAL REGISTRATION NUMBER: ChiCTR2000041253.

Natural progression of periodontal diseases in Chinese villagers based on the 2018 classification.

BACKGROUND: Based on the 2018 classification, we aimed to determine the prevalence, distribution, and progression of periodontitis in the rural Chinese population without access to dental care. METHODS: In all, 404 subjects (28.7 ± 8.9 years, M:F = 182:222) were randomly enrolled in 1992 and re-called in 1996. With the new classification, the prevalence and distribution of stage, grade, and extent were characterized. Stage progression was compared with the progression of clinical attachment loss (CAL) and radiographic bone loss (RBL). RESULTS: At baseline, 94.1% villagers suffered from periodontitis, of whom 53.7% were in Stage III/IV. The prevalence of Stage III/IV increased from 18.2% in the age group of 15 to 24 years to 60.9% in 25 to 34-year-old group and 88.7% in the 35 to 44-year-old group. Significantly more Stage III/IV, generalized, and Grade C periodontitis were found in male villagers than female villagers. In 1996, the prevalence rate of periodontitis increased to 98.5%, with 80.0% in Stage III/IV. Further, 84.2% villagers presented with Grade C periodontitis based on longitudinal ΔCAL. The rate of progression (≥1 site with ΔCAL ≥3 mm) was 63.7%. Stage progression correlated significantly with CAL and RBL progression in Stage I/II, but this association was not found in Stage III/IV. Among subjects with disease progression in Stage III/IV, 90.4% shifted from localized to generalized cases. Furthermore, ceiling effects were observed in Stage III/IV. CONCLUSIONS: In villagers without access to dental care, 94.1% suffered from periodontitis, with more than half having Stage III/IV disease based on the 2018 classification. The majority cases presented with rapid periodontal progression. Although stage progression correlated significantly with CAL and RBL progression in Stage I/II, ceiling effects existed in Stage III/IV.

LOX-1 Regulates P. gingivalis-Induced Monocyte Migration and Adhesion to Human Umbilical Vein Endothelial Cells.

Porphyromonas gingivalis (P. gingivalis) is one of the main periodontal bacteria. This pathogen was reported to enhance monocyte migration and adhesion to endothelial cells in atherosclerosis. The scavenger receptor lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) plays a pivotal role in atherogenesis. The aim of this study was to investigate whether LOX-1 modulates P. gingivalis-mediated monocyte migration and adhesion to endothelial cells and how it works. The results showed that the migration and adhesion of monocytic THP-1 cells to human umbilical vein endothelial cells (HUVECs) were significantly enhanced when HUVECs or THP-1 cells were challenged with P. gingivalis. Meanwhile, the expression level of LOX-1 in both HUVECs and THP-1 cells were also significantly increased by P. gingivalis stimulation. It is well known that ligand/receptor pairs monocyte chemoattractant protein-1 (MCP-1)/CC chemokine receptor 2 (CCR2), selectins/Integrins, and cell adhesion molecules (CAMs)/Integrins mediate monocyte migration and adhesion to endothelial cells. In this study, LOX-1 was demonstrated to be crucially involved in P. gingivalis-induced THP-1 cell migration and adhesion to HUVECs, by regulating expression of ligands MCP-1, intercellular adhesion molecule-1 (ICAM-1) and E-selectin in HUVECs and that of their receptors CCR2 and Integrin αMß2 in THP-1 cells. The nuclear factor-kappa B (NF-κB) signaling pathway was proved to be involved in this process. In conclusion, LOX-1 plays a crucial role in P. gingivalis-induced monocyte migration and adhesion to endothelial cells. This result implies LOX-1 may act as a bridge in linking periodontitis to atherosclerosis.

Subgingival microbiome in Chinese patients with generalized aggressive periodontitis compared to healthy controls.

OBJECTIVE: The aim of the study was to profile the subgingival microbiome of Chinese adults with generalized aggressive periodontitis (GAgP) using human oral microbe identification microarray (HOMIM), and to compare the results with matched periodontal healthy controls. DESIGN: 15 subjects with GAgP and 15 age- and gender- matched periodontal healthy controls were included. Subgingival plaque samples were collected from the deepest pockets of patients with GAgP and matched sites in controls and then analyzed by 16S rRNA-based microarrays. Student's paired t-test was used to compare clinical parameters and mean number of bacterial taxa detected between the two groups. Fisher's exact probability test and Wilcoxon Rank Sum were used to compare bacterial species between all samples. A multiple linear regression model was used for correlations among age, gender and bacterial with clinical parameters. RESULTS: From a total sum of 379 strains tested, 171 bacterial strains were detected from subgingival plaques of the GAgP patients, more than the 157 strains detected in control group. Mean number of subgingival bacterial taxa detected in GAgP group was 68 (SD = 21.06) while in control group was 45 (SD = 21.60). 47 bacterial taxa were detected more frequently in GAgP group while 12 taxa were more prevalent in control group. The significantly more prevalent and abundant taxa of bacteria in GAgP group included Filifactor alocis, Desulfobulbus sp., Fretibacterium sp., Porphyromonas gingivalis, Tannerella forsythia, Porphyromon as endodontalis, Peptostreptococcaceae spp., Parvimonas micra, Eubacterium nodatum and Eubacterium saphenum. Meanwhile the more abundant taxa in control group were Streptococcus spp. and Pseudomonas aeruginosa. CONCLUSIONS: There are more taxa of bacteria in subgingival plaques of Chinese patients with GAgP than in healthy controls. F. alocis, Desulfobulbus sp., Fretibacterium sp., P. gingivalis and T. forsythia are strongly associated with GAgP. High-throughout microbiological results may help dentists have a better understanding of subgingival microbiome of GAgP.

PPARγ affects nitric oxide in human umbilical vein endothelial cells exposed to Porphyromonas gingivalis.

OBJECTIVE: Porphyromonas gingivalis induces nitric oxide (NO) synthesis in human umbilical vein endothelial cells (HUVECs). Peroxisome proliferator-activated receptor (PPARγ) has an anti-inflammation function, and its involvement in this NO induction process requires elucidation. Here, we focused on PPARγ expression in HUVECs exposed to P. gingivalis, and investigated its effects on NO synthesis. MATERIALS AND METHODS: HUVECs were time-dependently stimulated by P. gingivalis W83 for 0-24h. PPARγ expression was assessed at the mRNA and protein levels, and PPARγ activation was measured using dual-luciferase reporter assays. NO synthesis and NO synthase (NOS) expression in response to P. gingivalis were examined in HUVECs pretreated with representative PPARγ agonist (15-deoxy-Δ12,14-prostaglandin J2 10µM) or antagonist (GW9662 10µM). In addition, NO synthesis and NOS expression in the P. gingivalis infected and control groups were detected. RESULTS: The PPARγ mRNA level in HUVECs increased after exposure to P. gingivalis for 1h and its protein level increased at 2h. Luciferase-induced PPARγ increased in P. gingivalis-exposed HUVECs. NO synthesis in the infected group at 4h, and in the PPARγ-activated group at 8h, was higher than that in controls. Inducible NOS increased in the infected and PPARγ-activated groups at 4 and 8h. The total endothelial NOS (eNOS) and phospho-eNOS levels were lower in the infected group than controls, but did not change in the PPARγ-activated group. CONCLUSIONS: Activated PPARγ induces NO generation through the NOS pathway in HUVECs exposed to P. gingivalis.

Follicular dendritic cell-secreted protein may enhance osteoclastogenesis in periodontal disease.

PURPOSE OF THE STUDY: Follicular dendritic cell-secreted protein (FDC-SP) has been found to be expressed in periodontal ligament (PDL), a layer of soft connective tissue between tooth root and alveolar bone, and involved in immunoreaction. This study was performed to explore the potential role of FDC-SP in periodontal disease. MATERIALS AND METHODS: The human periodontal ligament cells (hPDLCs) were stimulated with Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) and FDC-SP expression was examined by real-time PCR and western blot. Then this molecule was overexpressed or silenced in hPDLCs by transfection of FDC-SP expression plasmids or its small-interfering (si) RNA, respectively, and the effects of FDC-SP on expression of osteogenesis- and osteoclastogenesis-related genes in hPDLCs were analyzed by real-time PCR and western blot. RESULTS: Our results showed that P. gingivalis LPS upregulated FDC-SP expression in hPDLCs. Overexpression of FDC-SP could decrease the expression of osteogenesis-related genes, increase the expression of osteoclastogenesis-related genes and RANKL/OPG ratio in hPDLCs. Meanwhile, silence of FDC-SP expression in hPDLCs remarkably inversed the above results. CONCLUSIONS: LPS-induced upregulation of FDC-SP expression in hPDLCs may enhance osteoclastogenesis in periodontal disease.

[Effect of peroxisome proliferator-activated receptor-γ on endothelial cells oxidative stress induced by Porphyromonas gingivalis].

OBJECTIVE: To detect the degree of oxidative stress in the process when Porphyromonas gingivalis (P. gingivalis) stimulates human vascular endothelium, And to investigate the effect of peroxisome proliferator-activated receptor(PPAR)γ on oxidative stress during this process. METHODS: Human vascular endothelial cells (HVECs) line EA.hy926 (American Type Culture Collection ,United States) was cultured in high glucose Dulbecco's modified eagle medium (DMEM). Four groups were designed: control group, P. gingivalis infected group, PPARγ activated group and PPARγ blocked group. In control group HVECs were cultured with only DMEM. In P. gingivalis infected group, HVECs were time-dependently stimulated by P. gingivalis W83 from 0 to 12 h. In PPARγ activated group or PPARγ blocked group, PPARγ was pre-activated or blocked by a representative PPARγ agonist(15d-PGJ2 10 µmol/L) or antagonist (GW966210 µmol/L) 30 minutes before the cells were stimulated by P. gingivalis. At 0, 0.5, 1, 1.5, 2, 4, 8, and 12 h, the culture medium was collected individually and centrifuged, and the supernatant was stored for assay. Glutathione peroxidase (GSH-PX) and malondialdehyde (MDA) were analysed by enzyme-linked immunosorbent assay. Cellular reactive oxygen species (ROS) were detected through 2',7'-dichlorofluorescin diacetate (DCFA-DA) fluorescent probe at various time points of the different groups. RESULTS: In P. gingivalis infected group, the levels of GSH-PX [(5.56±0.97) µmol/L] and MDA [(0.84±0.18) nmol/L] were significantly higher than those in control group [GSH-PX(4.71±0.64) µmol/L, MDA (0.59±0.18) nmol/L)]. The levels of GSH-PX and MDA in PPARγ activated group [GSH-PX (5.38±0.84) µmol/L, MDA (0.84±0.22) nmol/L] and in PPARγ blocked group [GSH-PX (5.37±0.76) µmol/L, MDA (0.85±0.14) nmol/L] were significantly higher than those in control group (P<0.05). In the PPARγ activated group, the levels of GSH-PX at 0.5 and 8 h were significantly higher than those from 1.5 h to 4 h (P<0.05), while no difference was observed on the MDA levels at different time points. There was no significant difference at various time points for the levels of GSH-PX and MDA in PPARγ blocked group. The level of cellular ROS detected by DCFH-DA in P. gingivalis infected group was significantly higher than that in control group (10 108.65 ± 1 805.18 vs. 6 049.06 ± 1 199.19,P<0.05). No difference was observed between PPARγ activated group (7 120.94±1 447.30) or PPARγ blocked group (6 727.35±1 483.68) and control group. CONCLUSION: Oxidative stress happens when P. gingivalis stimulates human vascular endothelium. PPARγ may involve in modulating oxidative stress during this process.

Adhesion of monocytes to periodontal fibroblasts requires activation of NOD1/2- and TLR4-mediated LFA-1 and VLA-4.

OBJECTIVE: The aim of this study was to investigate the roles of nucleotide-binding oligomerization domain-containing protein 1/2 (NOD1/2) and Toll-like receptor 4 (TLR4) in mediating the adhesion of monocytes to periodontal fibroblasts through leucocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4). DESIGN: The expression of NOD1, NOD2, and TLR4 was detected in the gingival tissue of patients with chronic periodontitis by immunohistochemistry. Then the adhesion of cells of human monocytic cell line U937 to human gingival fibroblasts (hGFs) and human periodontal ligament cells (hPDLCs) was investigated after U937 cells were treated with the agonists of NOD1, NOD2, and TLR4 for 24 h, or transfected with small interfering RNAs (siRNAs) targeting NOD1, NOD2, and TLR4 for 48 h. Meanwhile, the expression of LFA-1 and VLA-4 was examined in U937 cells through real-time polymerase chain reaction (PCR), Western blot, and flow cytometry. To confirm the roles of LFA-1 and VLA-4 involved in the process of adhesion, the adhesion blockade assay was performed using the corresponding blocking antibodies against these adhesion molecules. RESULTS: The immunostaining results showed that NOD1, NOD2, and TLR4 were highly expressed in the gingival tissue of patients with periodontitis, especially in the monocyte-infiltrated area. The activation of these receptors by agonists upregulated the expression of LFA-1 and VLA-4 in U937 cells, and it increased the affinity of U937 cells to hGFs or hPDLCs. On the other hand, knockdown of these receptors by specific siRNAs resulted in the opposite results. In addition, blocking either LFA-1 or VLA-4 in U937 cells significantly attenuated the agonist-triggered adhesion of U937 to periodontal fibroblasts (P<0.001). CONCLUSIONS: These results suggested that NOD1/2 and TLR4 mediated monocyte-periodontal fibroblast adhesion via the modulation of LFA-1 and VLA-4.

Beyond toll-like receptors: Porphyromonas gingivalis induces IL-6, IL-8, and VCAM-1 expression through NOD-mediated NF-κB and ERK signaling pathways in periodontal fibroblasts.

To investigate whether oligomerization domains (NODs) are involved in Porphyromonas gingivalis-induced interleukin (IL)-6, IL-8, and vascular cell adhesion molecule (VCAM)-1 expression beyond Toll-like receptors (TLRs), we investigated the role of NOD1/2 in P. gingivalis-induced IL-6, IL-8, and VCAM-1 expression in human gingival fibroblasts (hGFs) and periodontal ligament cells (hPDLCs). The mechanism was explored by activation and silence of NODs, electrophoretic mobility shift assay (EMSA), and pathway blockade assays. Results showed that P. gingivalis could induce NOD1, NOD2, IL-6, IL-8, and VCAM-1 expression in hGFs and hPDLs at mRNA and protein levels. Activation of NOD1/2 by agonists could clearly upregulate the expression of these genes, while silence of NOD1/2 could remarkably attenuate them. EMSA and blockade of NF-κB and extracellular-signal-regulated kinase (ERK)1/2 pathway assays also verified that the two pathways were involved in NOD1/2-mediated IL-6, IL-8, and VCAM-1 expression. In conclusion, our findings demonstrated that P. gingivalis induced IL-6, IL-8, and VCAM-1 expression in hGFs and hPDLCs through NOD1/2-mediated NF-κB and ERK1/2 signaling pathways beyond TLRs.

Intracellular adhesion molecule-1 is regulated by porphyromonas gingivalis through nucleotide binding oligomerization domain-containing proteins 1 and 2 molecules in periodontal fibroblasts.

BACKGROUND: The mechanism by which Porphyromonas gingivalis regulates intracellular adhesion molecule 1 (ICAM-1) expression in human periodontal ligament cells (hPDLCs) and human gingival fibroblasts (hGFs) is unknown. The aim of this study is to investigate whether nucleotide binding oligomerization domain-containing protein (NOD) 1 and NOD2 are involved in this process and the clinical significance of ICAM-1 in periodontitis. METHODS: hPDLCs and hGFs were treated with P. gingivalis, l-Ala-γ-d-glutamyl-mesodiaminopimelic acid (an agonist for NOD1), and muramyl dipeptide (an agonist for NOD2). Alternatively, cells transfected with small interfering RNA targeting NOD1and NOD2 were treated with P. gingivalis. ICAM-1, NOD1, and NOD2 were detected at mRNA and protein levels. In addition, clinical examinations were performed in 30 healthy controls and 40 patients with chronic periodontitis (CP) before and after treatment, and serum-soluble ICAM-1 (sICAM-1) levels in these individuals were detected by enzyme-linked immunosorbent assay. RESULTS: This study shows that P. gingivalis caused an increase in ICAM-1, NOD1, and NOD2 expression in periodontal fibroblasts. There was a linear correlation between ICAM-1 and NOD1 and NOD2 levels. Activation of NOD1 and NOD2 by the specific agonist led to the upregulation of ICAM-1, whereas knocking down NOD1 and NOD2 caused a reduction in P. gingivalis-induced ICAM-1 production. Furthermore, sICAM-1 levels were higher in patients with CP than in healthy controls and were positively related to the clinical periodontal parameters. After periodontal treatment, sICAM-1 levels decreased significantly. CONCLUSIONS: The present results indicate that sICAM-1 levels are correlated to the severity of periodontitis. NOD1 and NOD2 mediate P. gingivalis-induced ICAM-1 production in periodontal fibroblasts. NOD1 and NOD2 could be considered potential targets for periodontal therapy.