Porphyromonas Gingivalis Lipopolysaccharide Research Articles

Positive regulation of Porphyromonas gingivalis lipopolysaccharide-stimulated osteoblast functions by strontium modification of an SLA titanium implant surface.

This study aimed to investigate whether strontium (Sr) ion delivery to the surface of microstructured titanium (Ti) oral implants drives favorable osteoblast functions of mesenchymal stem cells (MSCs) under a Porphyromonas gingivalis lipopolysaccharide (LPS)-stimulated inflammatory condition. The goal is to gain insight for future surface engineering of Ti implants with increased resistance to peri-implantitis and better bone recovery capacity from peri-implant bone destruction. The Sr ions were introduced into a clinically available sandblasted/acid-etched (SLA) Ti implant surface by wet chemical treatment. The results showed that Sr ion incorporation enhances the osteogenesis-related cell functions of bipotent ST2 stem cells in the microstructured SLA type implant surface. Early spreading and osteogenic differentiation (osteogenesis-related mRNA expression, ALP activity and osteocalcin protein secretion) were notably increased in Sr-incorporated SLA implant surface under 1 µg of P. gingivalis LPS stimulation. In addition, Sr ion modification increased early osteogenic differentiation (ALP mRNA expression and ALP activity) of ST2 cells, again under the more severe inflammatory condition induced by a high dose of LPS (10 µg/mL). These results indicate that surface chemistry modification using Sr ions provides microstructured Ti oral implants with greater resistance to the development and progression of inflammatory bone tissue destruction, and increased bone recovery capacity in the bacterial LPS-contaminated Ti oral implant surface by enhancing the early osteogenic differentiation of MSCs.

LncRNA MALAT1 regulates inflammatory cytokine production in lipopolysaccharide-stimulated human gingival fibroblasts through sponging miR-20a and activating TLR4 pathway.

It has been reported that long non-coding RNAs (lncRNAs), such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), act as key regulators of the development of inflammatory diseases. However, it is unclear whether MALAT1 regulates the function of human gingival fibroblasts (HGFs) in periodontitis. This study is to explore the role of MALAT1 on inflammatory cytokine production of HGFs. Primary HGFs were harvested from human gingiva. MALAT1 was detected in inflammatory and healthy gingival tissues via quantitative real-time PCR (qRT-PCR). Bioinformatics analysis, dual-luciferase reporter assay, and RNA-binding protein immunoprecipitation (RIP) were used to detect the relationship among MALAT1, toll-like receptor 4 (TLR4), and microRNA (miR) -20a. After transfection LPS-treated HGFs with MALAT1 siRNA (si-MALAT1), miR-20a mimic or overexpression MALAT1 plasmid (sno-MALAT1), the levels of MALAT1, miR-20a, TLR4, IL-6 and IL-8 were analyzed by qRT-PCR, enzyme-linked immunosorbent assay, or western blot assay. MALAT1 up-regulated in inflammatory gingival tissues of chronic periodontitis. MiR-20a was bound with MALAT1 and TLR4 3'-UTR in RNA-protein complex with Ago2, respectively. Moreover, MALAT1, TLR4, IL-6, and IL-8 increased while miR-20a decreased after 1μg/mL Porphyromonas gingivalis lipopolysaccharide (LPS) or Escherichia coli LPS stimulation. MiR-20a inhibited the expression of proinflammatory cytokines via binding to TLR4 3'-UTR. In addition, MALAT1 increased TLR4 level and the secretion of inflammatory cytokines. MALAT1 enhances inflammatory cytokine production through sponging miR-20a and releasing TLR4, indicating a regulatory role of MALAT1 in periodontal inflammation.

Quercetin Inhibits Inflammatory Response Induced by LPS from Porphyromonas gingivalis in Human Gingival Fibroblasts via Suppressing NF-κB Signaling Pathway.

Quercetin, a natural flavonol existing in many food resources, has been reported to be an effective antimicrobial and anti-inflammatory agent for restricting the inflammation in periodontitis. In this study, we aimed to investigate the anti-inflammatory effects of quercetin on Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide- (LPS-) stimulated human gingival fibroblasts (HGFs). HGFs were pretreated with quercetin prior to LPS stimulation. Cell viability was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), along with chemokine interleukin-8 (IL-8), were determined by enzyme-linked immunosorbent assay (ELISA). The mRNA levels of IL-1β, IL-6, IL-8, TNF-α, IκBα, p65 subunit of nuclear factor-kappa B (NF-κB), peroxisome proliferator-activated receptor-γ (PPAR-γ), liver X receptor α (LXRα), and Toll-like receptor 4 (TLR4) were measured by real-time quantitative PCR (RT-qPCR). The protein levels of IκBα, p-IκBα, p65, p-p65, PPAR-γ, LXRα, and TLR4 were characterized by Western blotting. Our results demonstrated that quercetin inhibited the LPS-induced production of IL-1β, IL-6, IL-8, and TNF-α in a dose-dependent manner. It also suppressed LPS-induced NF-κB activation mediated by TLR4. Moreover, the anti-inflammatory effects of quercetin were reversed by the PPAR-γ antagonist of GW9662. In conclusion, these results suggested that quercetin attenuated the production of IL-1β, IL-6, IL-8, and TNF-α in P. gingivalis LPS-treated HGFs by activating PPAR-γ which subsequently suppressed the activation of NF-κB.

Doxycycline inhibits NAcht Leucine-rich repeat Protein 3 inflammasome activation and interleukin-1β production induced by Porphyromonas gingivalis-lipopolysaccharide and adenosine triphosphate in human gingival fibroblasts

ObjectiveTo investigate the effect of adenosine triphosphate (ATP) on inflammasome activation by Porphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS) stimulation and the anti-inflammatory eff ;ect of doxycycline (Dox) in human gingival fibroblasts (HGFs). DesignThe optimal concentration of P. gingivalis-LPS (1.0 μg/mL) for cellular viability was determined by observing cell morphology and measuring the amount of formazan and the expression of pro-caspase-1. The expression of genes and proteins related to the NAcht Leucine-rich repeat Protein 3 (NLRP3) inflammasome, including NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), caspase-1 and its activated forms, and the inflammatory factor interleukin-1β (IL-1β) and its activated forms were measured. ResultsThe NLRP3 inflammasome (i.e., NLRP3, ASC, caspase-1) was not affected by stimulation with P. gingivalis-LPS or ATP. However, a combination of P. gingivalis-LPS and ATP significantly enhanced inflammasome activation and IL-1β production at the gene and protein levels as measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. Furthermore, doxycycline addition markedly inhibited inflammasome activation and IL-1β production induced by a combination of P. gingivalis-LPS and ATP. ConclusionsLPS, ATP, and doxycycline play critical roles in regulating host immune responses. This evidence provides guidance for the application of tetracycline drugs for the clinical treatment of periodontal disease.

Periodontitis-induced systemic inflammation exacerbates atherosclerosis partly via endothelial\u2013mesenchymal transition in mice

Growing evidence suggests close associations between periodontitis and atherosclerosis. To further understand the pathological relationships of these associations, we developed periodontitis with ligature placement around maxillary molars or ligature placement in conjunction with Porphyromonas gingivalis lipopolysaccharide injection at the ligature sites (ligature/P.g. LPS) in Apolipoprotein E knock out mice and studied the atherogenesis process in these animals. The mice were fed with high fat diet for 11 weeks and sacrificed for analyzing periodontitis, systemic inflammation, and atherosclerosis. Controls did not develop periodontitis or systemic inflammation and had minimal lipid deposition in the aortas, but mice receiving ligature or ligature/P.g. LPS showed severe periodontitis, systemic inflammation, and aortic plaque formation. The aortic plaque contained abundant macrophages and cells expressing both endothelial and mesenchymal cell markers. The severity of periodontitis was slightly higher in mice receiving ligature/P.g. LPS than ligature alone, and the magnitude of systemic inflammation and aortic plaque formation were also notably greater in the mice with ligature/P.g. LPS. These observations indicate that the development of atherosclerosis is due to systemic inflammation caused by severe periodontitis. In vitro, P.g. LPS enhanced the secretion of pro-inflammatory cytokines from macrophages and increased the adhesion of monocytes to endothelial cells by upregulating the expression of adhesion molecules from endothelial cells. Moreover, secretory proteins, such as TNF-α, from macrophages induced endothelial–mesenchymal transitions of the endothelial cells. Taken together, systemic inflammation induced by severe periodontitis might exacerbate atherosclerosis via, in part, causing aberrant functions of vascular endothelial cells and the activation of macrophages in mice.

Periodontal ligament fibroblasts regulate osteoblasts by exosome secretion induced by inflammatory stimuli

ObjectivesThis study evaluated the role of human periodontal ligament fibroblasts (hPDLFs)-derived exosomes in periodontitis progression and discovered whether hPDLFs influence bone remodeling activity via exosome secretion. Materials and MethodsExosomes were isolated and quantified from Porphyromonas gingivalis lipopolysaccharide (LPS)-treated primary hPDLFs and evaluated by western blotting, dynamic light scattering, and transmission electron microscopy. GW4869 was used to block exosome secretion in conditioned medium (CM). hPDLFs-derived CM, CM containing GW4869 (CM + GW4869) and exosomes were used to stimulate MG-63 cell lines. The expression levels of proinflammatory mediators, osteogenic genes, and osteoclastogenesis-related genes were measured by quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, western blotting, and alkaline phosphatase staining. ResultsExosome-enriched protein and total exosomal protein levels were higher in the LPS-treated group than in the vehicle controls. hPDLFs-derived exosomes were incorporated into MG-63 osteoblasts and slightly upregulated the expression of Interleukin-6 and tumor necrosis factor-alpha. CM and exosomes inhibited alkaline phosphatase, Collagen-I, Runt-related transcription factor 2, and Osteoprotegerin expression as well as ALP activity, and blocking exosome secretion by GW4869 eliminated the inhibitory effects. ConclusionThese results indicate that LPS-pretreated hPDLFs induce inflammation and inhibit osteogenic activity of osteoblasts through secreting exosomes. This study provides a potential mechanism by which localized periodontal inflammation may influence bone remodeling by release exosomes.

MicroRNA-200c Attenuates Periodontitis by Modulating Proinflammatory and Osteoclastogenic Mediators.

This study tested whether microRNA (miR)-200c can attenuate the inflammation and alveolar bone resorption in periodontitis by using an in vitro and a rat model. Polyethylenimine (PEI) was used to facilitate the transfection of plasmid DNA encoding miR-200c into primary human gingival fibroblasts (HGFs) and gingival tissues of rats. We first analyzed how proinflammatory and osteoclastogenic mediators in HGFs with overexpression of miR-200c responded to Porphyromonas gingivalis lipopolysaccharide (LPS-PG) challenge in vitro. We observed that overexpression of miR-200c significantly reduced interleukin (IL)-6 and 8 and repressed interferon-related developmental regulator-1 (IFRD1) in HGFs. miR-200c also downregulated p65 and p50. In a rat model of periodontitis induced by an LPS injection at the gingival sulcus of the second maxillary molar (M2), we analyzed how the mediators in rat gingiva and alveolar bone resorption responded to miR-200c treatment by a local injection of PEI-plasmid miR-200 nanoplexes. We observed that the local injection of miR-200c significantly upregulated miR-200c expression in gingiva and reduced IL-6, IL-8, IFRD1, and the ratio of receptor activator of nuclear factor kappa-B ligand/osteoprotegerin. Using micro-computed tomography analysis and histomorphometry, we further confirmed that local treatment with miR-200c effectively protected alveolar bone resorption in the rat model of periodontitis by reducing the distance between the cemento-enamel junction and the alveolar bone crest and the inter-radicular space in the upper maxilla at M2. These findings imply that miR-200c may serve as a unique means to prevent periodontitis and associated bone loss.

Morus alba Stem Extract Suppresses Matrix Metalloproteinases (MMP)-1, MMP-9, and Tissue Inhibitors of Metalloproteinase (TIMP)-1 Expression via Inhibition of IκBα Degradation Induced by Porphyromonas gingivalis LPS Signal in THP-1 Cells.

Objectives The aim of this study is to evaluate the inhibitory effects of M. alba stem extract (MSE) on the expression of matrix metalloproteinases (MMP)-1, MMP-9, and tissue inhibitors of metalloproteinase (TIMP)-1 in Porphyromonas gingivalis lipopolysaccharide (LPS)-activated-acute monocytic leukemia cell line (THP-1). Materials and Methods THP-1 cells were treated with noncytotoxic concentrations of MSE combined with 1 µg/mL of P. gingivalis LPS. The mRNA levels of MMP-1, MMP-9, and TIMP-1 were evaluated via quantitative real-time polymerase chain reaction. The secreted proteins in the culture media were detected by enzyme-linked immunosorbent assay. The degradation of inhibitor of kappa B-alpha (IκBα) protein was tracked by Western blotting. Statistical Analysis Comparisons in experiments were analyzed with analysis of variance followed by Tukey honestly significant difference comparison test. Results Twenty and 40 µg/mL of MSE significantly downregulated MMP-1 and MMP-9 genes and protein expression but upregulated the gene expression of TIMP-1 ( p < 0.05). P. gingivalis LPS induced degradation of IκBα, while addition of MSE (20 and 40 µg/mL) increased IκBα cytosolic levels. MSE was able to suppress the P. gingivalis LPS-induced MMPs expression and also increased the gene expression of TIMP-1 via the inhibition of the cytoplasmic IκBα degradation in THP-1 cells. Conclusions The present observations suggest that MSE exerted a positive effect on the regulatory mechanism between MMPs and TIMP, which is an important implication for the therapeutic potential of MSE in periodontitis.

The anti-nociceptive effects of Porphyromonas gingivalis lipopolysaccharide

ObjectiveThe objective of this study was to assess the effect of Porphyromonas gingivalis lipopolysaccharide (PG LPS) on acute pain-related behaviour induced in rats and to measure its impact on the levels of pro-inflammatory cytokines (IL-1β, IL-6) and anti-inflammatory (IL-10) cytokines. DesignThe Brennan model was used to induce acute pain like signs in rats' hind paw. Twenty-four hours following the surgery the rats were divided into 5 groups and the affected paws were injected with 0.2 m l of one of three commercialized forms PG LPS doses (high - 1 mg/ml, medium - 0.6 mg/m l and low - 0.2 mg/m l), diclofenac sodium (1 mg/kg) or saline. Tactile allodynia, mechanical hyperalgesia, body temperature and paw swelling were assessed at baseline, 24 h postoperatively and 2 h after the paw injection. The affected and contra-lateral paw tissue was assessed for the mentioned above cytokines levels employing enzyme-linked immunosorbent assay. ResultsThis study may suggest that PG LPS can reduce pain like behaviour via increased levels of anti-inflammatory cytokine IL-10 (5900 ± 748, p < 0.05). The high PG LPS dose and diclofenac reduced the tactile allodynia and mechanical hyperalgesia significantly (42.2 ± 4 and1.6 ± 0.3, p < 0.05). PG LPS high dose increase IL-10 levels while diclofenac reduces IL-1β levels significantly (5900 ± 748 and 1760 ± 271.2). The LPS administration had no effect on paw swelling and did not increase rat’s body temperature. ConclusionThe results demonstrated that PG LPS local application could possess anti- nociceptive properties, which at least in part is mediated by an increase in IL-10 levels.

Exosomal microRNA-155-5p from PDLSCs regulated Th17/Treg balance by targeting sirtuin-1 in chronic periodontitis.

The mechanism of local inflammation and systemic injury in chronic periodontitis is complicated, in which and exosomes play an important role. In our study, we found that T helper cell 17 (Th17)/regulatory T cell (Treg) balance is destabilized in the peripheral blood of patients with periodontitis, with upregulated Th17 or downregulated Treg, respectively. Porphyromonas gingivalis lipopolysaccharide (LPS) was used to simulate the inflammatory microenvironment of chronic periodontitis. The exosomes were extracted from periodontal ligament stem cells (PDLSCs) in LPS-induced periodontitis environment, which inversely effected on CD4+ T cells under normal and inflammatory conditions. Furthermore, compared with exosomes from normal PDLSCs, lower expression of microRNA-155-5p (miR-155-5p) and higher expression of Sirtuin-1 (SIRT1) were observed in exosomes from LPS-stimulated PDLSCs. Exosomes from PDLSCs alleviated inflammatory microenvironment through Th17/Treg/miR-155-5p/SIRT1 regulatory network. This study aimed to find the "switching" factors that affected the further deterioration of periodontitis to maximally control the multiple downstream damage signal factors to further understand periodontitis and find new targets for its treatment.

Matrix Metalloproteinase‐9 Release from Porphyromonas gingivalis Lipopolysaccharide (LPS)‐treated Rat Brain Microglia In Vitro

BackgroundThe putative association between periodontal disease (PD), a chronic inflammatory disease and neuroinflammation, a condition which involves brain microglia, remains under investigation. We have reported that Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS), a bacterium implicated in periodontitis, activates both classical (M1‐type) and alternative (M2‐type) activation of neonatal rat microglia (BMG) with concomitant release of superoxide anion, thromboxane B2, cytokines, and chemokines (Faseb Journal 32.1 Supplement: 702.2, 2018). We hypothesized that matrix metalloproteinase 9 (MMP‐9) release by Pg‐treated microglia might be detectable by both ELISA and visualized by confocal fluorescence imaging.MethodsP. gingivalis LPS (Pg LPS) 105 ng/mL from InvivoGen (San Diego, CA) and Escherichia coli LPS (Ec LPS) 026:B6 1 ng/mL from Difco Lab, Detroit, MI (positive control) were used to treat rat BMG for 18 hours at 35.9°C in vitro. MMP‐9 release was assessed with a rat MMP‐9 ELISA kit (R&amp;D Systems). Confocal fluorescence imaging with a Nikon A1R laser confocal microscope was used to identify BMGs using a primary mouse anti‐rat CD11b/c antibody (Ab) (AbD SeroTec, Raleigh, NC), and secondary donkey anti‐mouse Ab (Thermo Fischer, Waltman, MA). MMP‐9 was visualized with a primary rabbit anti‐rat MMP‐9 polyclonal Ab, and secondary goat anti‐rabbit Ab, both from Abcam, Cambridge, MA.ResultsMMP‐9 presence in tissue culture supernates was detectable by ELISA in both Ec LPS and Pg LPS‐treated BMG. In contrast, MMP‐9 was observed by confocal fluorescence imaging only within Ec LPS‐treated BMG but not in Pg LPS‐treated BMG.ConclusionsBoth ELISA and confocal fluorescence imaging provide additional support for our working hypothesis, because Pg LPS stimulated BMGs to release MMP‐9. Thus, our current observations extend our studies on the effect of Pg LPS on rat brain microglia activation and contribute to further characterize the putative role of Pg LPS in both periodontitis, neuroinflammation and neurodegeneration.Support or Funding InformationSupport by College of Dental Medicine, College of Graduate Studies, and Chicago College of Osteopathic Medicine, Midwestern University are gratefully acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of chronic Porphyromonas gingivalis lipopolysaccharide infusion on skeletal muscles in mice.

Periodontitis, which is caused by various oral organisms, predominantly affects adults, and is one of the main causes of tooth loss, as well as leading to progression of numerous systemic diseases. However, its relationship to sarcopenia (aging-associated degenerative loss of skeletal muscle mass and function) remains unclear. The aim of this study was to investigate the effects of Porphyromonas gingivalis lipopolysaccharide (PG-LPS) on skeletal muscle in mice, and to establish the underlying mechanisms. Mice (C57BL/6) were injected with PG-LPS (0.8mg/kg/day) for 4weeks. This treatment significantly decreased the weight of fast-twitch skeletal muscles (masseter and tibialis anterior muscles), but not that of slow-twitch skeletal muscle (soleus muscle). The area of fibrosis was significantly increased in masseter muscle, but remained unchanged in the other two muscles. The number of apoptotic myocytes was significantly increased (approximately eightfold) in masseter muscle. These data suggest that persistent subclinical exposure to PG-LPS might reduce the size of fast-twitch skeletal muscle, but not slow-twitch skeletal muscle. Masseter muscle appears to be especially susceptible to the adverse effects of PG-LPS, because muscle remodeling (muscle fibrosis and myocyte apoptosis) was induced solely in masseter muscle. Thus, periodontitis might be one of the major causes of oral sarcopenia.

Continuous intracerebroventricular injection of Porphyromonas gingivalis lipopolysaccharide induces systemic organ dysfunction in a mouse model of Alzheimer's disease.

Systemic organ dysfunction is one of the important issues for the patients with Alzheimer's disease (AD) and their caregivers. Recent evidences suggest that periodontitis is a possible risk factor for progression of AD and lipopolysaccharide derived from Porphyromonas gingivalis (Pg-LPS) which is a major periodontopathic bacteria induces cognitive impairment in mice. However, the precise relationships between the brain exposure of Pg-LPS and systemic organ dysfunction in AD patients are still undetermined. In this study, we investigated whether brain exposure of Pg-LPS induced systemic organ dysfunction in a model of AD mouse. We employed 6 (young) and 13 (middle-aged) months-old 5XFAD mice and 6 months-old littermate (LT) mice, and treated with intracerebroventricular (ICV) injection of 2 μg Pg-LPS or saline (vehicle). The animals were monitored cognitive functions (Y maze, nest building, and Morris water maze tests), motor functions (wire hang and rotarod tests), physical condition (symptom score), and blood pressure (BP). Twenty-eight days later, their organs were weighted and the organ damages were examined. Continuous ICV injection of 2 μg/day Pg-LPS increased ionized calcium binding adapter molecule-1 (Iba-1) and cluster of differentiation 3 (CD3) positive cells in periventricular area of 5XFAD mice without enhancement of cognitive impairment, amyloid β protein deposition, expressions of phosphorylated nuclear factor-kappa B (NF-κB) and cyclooxygenase-2 (COX-2). In addition, the Pg-LPS lowered the latency of rotarod test in young 5XAD mice and also reduced symptom score and weight of gastrocnemius muscle in the middle-aged animals. Moreover, the Pg-LPS induced cardiac atrophy in both young and middle-aged 5XFAD mice, and increased Iba-1 positive cells in left ventricle of the young animals. On the other hand, single ICV injection of 2 μg Pg-LPS in 5XFAD and continuous injection of 2 μg/day Pg-LPS in LT mice did not show any positive findings. Our present results demonstrated that continuous brain exposure of Pg-LPS started sarcopenia and cardiac injury without enhancing cognitive impairment in AD model mice.

The Pros1/Tyro3 axis protects against periodontitis by modulating STAT/SOCS signalling.

Periodontitis, an oral inflammatory disease caused by periodontal pathogen infection, is the most prevalent chronic inflammatory disease and a major burden on healthcare. The TAM receptor tyrosine kinases (Tyro3, Axl and Mertk) and their ligands (Gas6 and Pros1) play a pivotal role in the resolution of inflammation and have been associated with chronic inflammatory and autoimmune diseases. In this study, we evaluated the effects of exogenous Pros1 in in vitro and in vivo models of periodontitis. We detected higher Pros1 but lower Tyro3 levels in inflamed gingival specimens of periodontitis patients compared with healthy controls. Moreover, Pros1 was mostly localized in the gingival epithelium of all specimens. In cultured human gingival epithelial cells (hGECs), Porphyromonas gingivalis LPS (p.g‐LPS) stimulation down‐regulated Pros1 and Tyro3. Exogenous Pros1 inhibited p.g‐LPS–induced production of TNF‐α, IL‐6, IL‐1β, MMP9/2 and RANKL in a Tyro3‐dependent manner as revealed by PCR, Western blot analysis, ELISA and gelatin zymography. Pros1 also restored Tyro3 expression down‐regulated by p.g‐LPS in hGECs. In rats treated with ligature and p.g‐LPS, administration of Pros1 attenuated periodontitis‐associated gingival inflammation and alveolar bone loss. Our mechanistic studies implicated SOCS1/3 and STAT1/3 as mediators of the in vitro and in vivo anti‐inflammatory effects of Pros1. Collectively, the findings from this work supported Pros1 as a novel anti‐inflammatory therapy for periodontitis.

Advanced glycation end-product 2 and Porphyromonas gingivalis lipopolysaccharide increase sclerostin expression in mouse osteocyte-like cells

Sclerostin is a secreted glycoprotein that is mainly expressed in osteocytes, exerts negative effects on bone formation, and is present at elevated levels in diabetes mellitus (DM). Periodontitis is an infectious disease caused by periodontopathic bacteria, a complication of DM, and sometimes associated with severe inflammation and alveolar bone resorption. Advanced glycation end-products (AGEs) are a major pathogen in DM complications and adversely influence periodontitis in DM patients. In the present study, the effects of AGE2 and Porphyromonas gingivalis lipopolysaccharide (P-LPS) on the expression of sclerostin in mouse osteocyte-like cells (MLO-Y4-A2 cells) and its function in osteoblast differentiation were investigated. AGE2 and P-LPS up-regulated the expressions of receptor of AGE (RAGE) and Toll-like receptor 2 (TLR2), respectively, and significantly up-regulated that of sclerostin and interleukin 6 (IL-6) in osteocytes. Sclerostin, RAGE and TLR2 levels were synergistically increased by AGE2 and P-LPS. The siRNAs of RAGE and TLR2 significantly inhibited AGE2- and P-LPS-induced sclerostin expression. AGE2 up-regulated sclerostin expression in osteocyte-like cells via the RAGE, ERK and JNK, and NF-κB signal pathways. On the other hand, P-LPS elevated sclerostin levels via the TLR2, JNK and p38, and NF-κB signal pathways. When osteocytes pre-treated with AGE2 and P-LPS and osteoblastic cells (MC3T3-E1) were co-cultured in the medium with a sclerostin-neutralizing antibody, AGE2- and P-LPS-induced decreases in alkaline phosphatase activity and Runx2 expression in osteoblastic cells were significantly inhibited by the sclerostin-neutralizing antibody. These results suggest that AGE2 and P-LPS influence bone metabolism and inflammation through the regulation of sclerostin expression, and may aggravate periodontitis with DM.

Porphyromonas gingivalis lipopolysaccharide-induced periodontitis and serum amyloid-beta peptides

ObjectiveThe aim of this investigation was to determine the circulating levels of amyloid beta (Aβ) peptides using the Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) model to induce periodontitis. MethodsExperimental periodontitis was induced in 6 male Sprague-Dawley rats. Alveolar bone loss was measure by micro computed tomography. Serum concentrations of Aβ1–40 and Aβ1–42 prior to periodontal induction, at 24 h, 7, 14, and 21 days the last injection of Pg-LPS. ResultsThe distance between the cemento-enamel junction and the bone crest (i.e., alveolar bone loss) was significantly higher at the end of periodontal induction compared to baseline (2.92 ± 0.29 mm vs. 3.8 ± 0.28 mm, P < 0.001). Periodontitis evoked a slight acute elevation of Aβ1–40 serum levels that were maintained during the whole experiment. Aβ1–42 peptide levels peak at the end of the study. A positive strong correlation was observed between alveolar bone loss and Aβ1–40 serum levels at 7 days (r = 0.695, P = 0.012) and as well as with serum Aβ1–42 concentrations at 21 days (r = 0.968, P = 0.002). ConclusionsPeriodontitis induced Pg-LPS produced increased serum levels of Aβ peptides. Further studies are needed to confirm our results and to investigate the mechanisms by which periodontitis could be associated with an overexpression of Aβ.

Oral lichen-planus-associated fibroblasts acquire myofibroblast characteristics and secrete pro-inflammatory cytokines in response to Porphyromonas gingivalis lipopolysaccharide stimulation

BackgroundOral lichen planus (OLP) is a chronic inflammatory oral mucosal disease in which comprehensive inflammation-related cytokines are involved. These cytokines are commonly produced by immune cells and specific nonimmune cells including keratinocytes, endothelial cells and fibroblasts. This raises the question of whether fibroblasts in OLP lesions contribute to the inflammatory process upon inflammatory simulation.MethodsPrimary cultured Oral lichen-planus-associated fibroblasts (OLP AFs, n = 5) and normal buccal mucosal fibroblasts (NFs, n = 5) were examined by immunohistochemistry, Western blotting and reverse transcription-polymerase chain reactions (RT-PCR). Various inflammatory mediators were evaluated with a multiplex assay. Differences among groups were assessed using a Student’s test or repeated measures one-way ANOVA, as appropriate.ResultsOLP AFs express significantly higher levels of α-smooth muscle actin (α-SMA) than NFs, indicating the presence of myofibroblasts. Myofibroblasts secrete Interleukin (IL)-6, IL-8, and tumor necrosis factor-α (TNF-α) in response to Porphyromonas gingivalis lipopolysaccharide (pg. LPS).ConclusionOLP AFs demonstrated α-SMA expression and secreted pro-inflammatory cytokines in response to pg. LPS stimulation.

Regulation of gingival epithelial cytokine response by bacterial cyclic dinucleotides

ABSTRACTBackground: Cyclic dinucleotides (cyclic di-guanosine monophosphate (c-di-GMP) and cyclic di-adenosine monophosphate (c-di-AMP)) and lipopolysaccharides (LPS) are pathogen-associated molecular patterns (PAMPs). Individual impacts of PAMPs on immune system have been evaluated, but simultaneous actions of multiple PAMPs have not been studied. Objective: Examination the effects of cyclic dinucleotides and Porphyromonas gingivalis LPS on gingival epithelial cytokine response. Methods: Human gingival keratinocytes (HMK) were incubated with 1, 10, and 100 µM concentrations of c-di-GMP and c-di-AMP, either in the presence or absence of P. gingivalis LPS. Intra- and extracellular levels of interleukin (IL)-1β, IL-8, IL-1Ra, monocyte chemoattractant protein (MCP)-1, and vascular endothelial growth factor (VEGF), were measured using the Luminex technique. Results: LPS decreased extracellular IL-8 levels, while the presence of c-di-AMP inhibited this effect. Incubating HMK cells with c-di-AMP (alone or with LPS) elevated the extracellular level of MCP-1. Extracellular VEGF level increased when cells were incubated with LPS and c-di-GMP together, or with c-di-AMP alone. LPS and c-di-AMP suppressed intracellular IL-1β levels. The c-di-AMP elevated intracellular levels of IL-1Ra. Conclusion: c-di-AMP and, to a lesser extent, c-di-GMP regulate keratinocyte cytokine response, either as an aggregator or as a suppressor of LPS, depending on the cytokine type.

MicroRNA-146a reverses the inhibitory effects of Porphyromonas gingivalis lipopolysaccharide on osteogenesis of human periodontal ligament cells

Objective: To investigate the effects and mechanisms of microRNA-146a (miR-146a) on osteogenic differentiation of human periodontal ligament cells (hPDLC) stimulated by lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg). Methods: hPDLC were cultured in vitro and induced to the phase of osteogenic differentiation. These cells were divided into five groups: non-osteogenic differentiation cells, osteogenic differentiation cells, osteogenic differentiation cells treated with Pg LPS, osteogenic differentiation cells treated with Pg LPS and miR-146a mimic, osteogenic differentiation cells treated with Pg LPS and miR-146a negative control. Osteogenic markers and mineralization were detected via quantitative real-time PCR (qPCR) and alizarin red staining, respectively. Meanwhile, non-radioactive transcription factor assay was applied to explore the nuclear activity of nuclear factor kappa B (NF-κB) p65 in nuclear extracts of hPDLC. Results: Compared with cells of osteogenic differentiation in non-LPS-stimulated groups, Pg LPS could decrease the markers of osteogenic differentiation of hPDLC such as collagen Ⅰ (Col-Ⅰ), alkaline phosphatase (ALP), Runt-related transcription factor-2 (RUNX2) and osteocalcin (OCN) (P<0.05), inhibit mineralization, and stimulate NF-κB p65 nuclear activity expression (non-LPS stimulated group: 1.023±0.217, LPS stimulated group: 6.252±0.613, P=0.008). However, compared with cells in Pg LPS/miR-146a negative control group, miR-146a increased Col-Ⅰ (P=0.007) and OCN (P=0.049) mRNA expression, rather than ALP (P=0.167) and RUNX2 (P=0.580) at day 3; miR-146a also upregulated mRNA levels of Col-Ⅰ, ALP, RUNX2 and OCN (P<0.05) at day 7 and day 14, and enhance mineralization. Meanwhile, miR-146a mimic could decrease the nuclear activity of NF-κB p65 induced by Pg LPS in hPDLC (miR-146a: 2.427±0.354, negative control: 5.863±0.482, P=0.019). Conclusions: miR-146a could reverse the inhibitory effects of Pg LPS on osteogenic differentiation of hPDLC through enhancing the expression of osteogenic markers and decreasing inflammatory pathway in hPDLC.

Periodontal ligament fibroblasts migration injury via ROS/TXNIP/Nlrp3 inflammasome pathway with Porphyromonas gingivalis lipopolysaccharide

Inflammasomes serve as an intracellular machinery to initiate inflammatory response to various danger signals. However, the chronic periodontitis pathological relevance of this inflammasome activation, particularly in periodontal ligament fibroblasts, remains largely unknown. The present study demonstrated that Nlrp3 inflammasome components abundantly expressed in cultured mouse periodontal ligament fibroblasts (mPDLFs). In addition, our data demonstrated that P.g-LPS (Porphyromonas gingivalis Lipopolysaccharide), a major injurious factor during chronic periodontitis, could induce the mPDLFs migration dysfunction and the inhibition of Nlrp3 inflammasome by Isoliquiritigenin (ISO) markedly recovered the migration dysfunction in mPDLFs. And Nlrp3 inflammasome components could be aggregated to form an inflammasome complex on stimulation of P.g-LPS, as shown by fluorescence confocal microscopy. Correspondingly, P.g-LPS induced Nlrp3 inflammasome activation, caspase-1 activation, IL-1β and HMGB1 release, which were blocked by Nlrp3 inflammasome inhibitor (ISO). Interestingly, reactive oxygen species, TXNIP protein and TXNIP binding to Nlrp3 were markedly increased in mPDLFs with P.g-LPS. Furthermore, ROS generation inhibitor (Apocynin; APO) significantly reduced Nlrp3 inflammasome formation and IL-1β production in mPDLFs with P.g-LPS. And APO attenuated P.g-LPS-induced TXNIP protein expression and mPDLFs injury. In conclusion, our results demonstrate that ROS/TXNIP/Nlrp3 Inflammasome pathway is a key initiating mechanism necessary for P.g-LPS-induced subsequent mPDLFs inflammatory response leading to chronic periodontitis.