Effect Of Porphyromonas Gingivalis Lipopolysaccharide Research Articles

Porphyromonas gingivalis lipopolysaccharide affects the angiogenic function of endothelial progenitor cells via Akt/FoxO1 signaling

Endothelial progenitor cells (EPCs) function as the angiogenic switch of many physiological and pathological conditions. We aimed to investigate the effects of Porphyromonas gingivalis lipopolysaccharide on the angiogenic capacity of EPCs and delineate the underlying mechanisms. EPCs were isolated from human umbilical blood. CCK-8 assay was undertaken to analyze the cell viability. The migration and tube formation capacity were assessed by wound healing and tube formation, respectively. The protein expression of Akt/p-Akt, endothelial nitric oxide synthase (eNOS)/p-eNOS, and Forkhead box O1 (FoxO1)/p-FoxO1 was determined by Western blot. The intracellular localization of FoxO1 was evaluated by immunofluorescent staining. P. gingivalis LPS at 10μg/ml significantly increased the viability (10.9 ± 2.9%), migration (16.3 ± 3.1%), and tube formation (38.6 ± 5.5%) of EPCs, along with increased phosphorylation of Akt, eNOS, and FoxO1. Mechanistically, Akt inhibition by specific inhibitor wortmannin and FoxO1 forced expression by adenovirus transfection in EPCs markedly attenuated the P. gingivalis LPS-induced eNOS activation, tube formation, and migration. Moreover, P. gingivalis LPS-induced phosphorylation and nuclear exclusion of FoxO1 were blunted by Akt inhibition. The present study suggests that P. gingivalis LPS could affect the angiogenic function of EPCs through the Akt/FoxO1 signaling. The current findings may shed light on the clinical association of periodontitis with aberrant angiogenesis seen in atherosclerotic plaque rupture.

Porphyromonas gingivalis lipopolysaccharide enhances the proliferation of human periodontal ligament cells via upregulation of cyclin D1, cyclin A and cyclin B1.

Human periodontal ligament cells (hPDLCs) play a notable role in periodontal tissue homeostasis and regeneration. However, the effect of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the proliferation of hPDLCs remains unclear. The present study investigated the effects of Pg-LPS on the proliferation profile of hPDLCs, and the involvement of cyclins and cyclin-dependent kinases in the process. hPDLCs were treated with Pg-LPS, and cell proliferation and cycle were detected using Cell Counting Kit-8 assays and flow cytometry. The mRNA expression levels of the cyclins and cyclin-dependent kinases (CDKs), including cyclins A, B1, D1 and D2 and CDK1, 2 and 4, were detected using reverse transcription-quantitative PCR. The protein expression levels of cyclins A, B1 and D1 were analysed using western blotting. The proliferation of hPDLCs was significantly increased after treatment with Pg-LPS at the concentrations of 0.001, 0.01, 0.1, 1 and 10 µg/ml for 24, 36 and 48 h compared with the cells cultured without LPS (P<0.01). The proliferation index of hPDLCs was significantly enhanced after treatment with Pg-LPS (0.0001, 0.001, 0.01, 0.1, 1 and 10 µg/ml) for 24 h (P<0.01). However, the S-phase fraction (SPF) only significantly increased after treatment with Pg-LPS at 0.01 µg/ml for 24 h (P<0.05), while the G2/M-phase fraction increased (P<0.01) and the G0/G1-phase fraction decreased (P<0.01) compared with the controls. The proliferation index and SPF increased, peaked at 24 h and then decreased at 48 h in both Pg-LPS-stimulated and control groups. Notably, Pg-LPS significantly upregulated the expression levels of cyclins D1, A and B1 after 24 h compared with those in the controls. Overall, the present study indicated that Pg-LPS may enhance the proliferation of hPDLCs, potentially through upregulation of cyclins D1, A and B1.

Effects of Porphyromonas gingivalis lipopolysaccharide on the proliferation and migration of human umbilical artery smooth muscle cells under co-culture conditions

Objective: To investigate the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) of type Ⅰand Ⅳ fimA on the proliferation and migration of human umbilical artery smooth muscle cells (HUASMC) under co-culture conditions and to explore the biological basis and possible mechanisms of the relationship between periodontitis and atherosclerosis (As). Methods: Type Ⅰ and Ⅳ fimA Pg were anaerobically cultured and the Pg-LPS was extracted, purified and identified. Human umbilical vein endothelial cells (HUVEC) and HUASMC were cultured in vitro and the HUVEC-HUASMC co-cultured cell model was established using rat tail type Ⅰ collagen. The experiment was divided into three groups: group T1 (co-cultured cells were stimulated with type Ⅰ fimA Pg-LPS at the mass concentrations of 0.5, 1.0, 2.0, 5.0, 10.0 mg/L), group T2 (co-cultured cells were stimulated with type Ⅳ fimA Pg-LPS at the mass concentrations of 0.5, 1.0, 2.0, 5.0, 10.0 mg/L), and negative control group (without LPS). Cell counting kit-8 (CCK-8) was used to detect the proliferation ability of HUASMC and the migration ability of HUASMC was observed by using the Transwell migration chamber. Comparasons of the changes in the proliferation and migration ability of HUASMC after 2, 8, 24 and 48 h of Pg-LPS stimulated co-cultured cells with various mass concentrations of Pg-LPS were conducted. Results: For the co-cultured cells under the action of type Ⅰ and Ⅳ fimA Pg-LPS, at 24 and 48 h, in each mass concentration group of the two types of Pg-LPS (0.5, 1.0, 2.0, 5.0, 10.0 mg/L), HUASMC's A values were significantly up-regulated compared to the negative control group (P<0.05) and for the co-cultured cells after the stimulation of type Ⅳ fimA Pg-LPS at concentrations of 5 and 10 mg/L at 48 h, the A values (1.386±0.044, 1.455±0.058) of HUASMC were significantly higher than that of HUASMC (1.168±0.064, 1.204±0.088) in the same concentrations of type Ⅰ fimA Pg-LPS (P<0.05). In addition, the A values of HUASMC in stimulated co-cultured cells under concentrations of 5.0 and 10.0 mg/L of type Ⅳ fimA Pg-LPS at 48 h were significantly higher than that in stimulated co-culture cells under the concentrations of 0.5 and 1.0 mg/L of type Ⅳ fimA Pg-LPS (1.170±0.082, 1.239±0.089) (P<0.05). The migration results of HUASMC showed that at 8, 24, and 48 h, the numbers of migration of HUASMC in each mass concentration group of type Ⅰ and Ⅳ fimA Pgas-LPS were significantly higher than that of HUASMC under the same Pg-LPS mass concentration at 2 h in the same group (P<0.05). The migration quantities of HUASMC in the other Pg-LPS mass concentration groups at 48 h were significantly higher than that of HUASMC under the same Pg-LPS mass concentration at 24 h in the same group (P<0.05), except for 10.0 mg/L type Ⅳ fimA Pg-LPS. The numbers of HUASMC migration in 2.0 mg/L type Ⅳ fimA Pg-LPS stimulated co-cultured cells at 48 h (204.00±20.98) were significantly higher than that in type Ⅰ fimA Pg-LPS stimulated co-culture cells at the same concentration (141.89±18.28) (P<0.05). Further more, at the same observation time point, the higher the Pg-LPS concentration, the greater the number of HUASMC migration (P<0.05). Conclusions: Both Ⅰ and Ⅳ fimA Pg-LPS could enhance the proliferation and migration ability of HUASMC in the co-culture system, and the virulence of Pg-LPS might be related to the fimA genotype. Ⅳ fimA Pg-LPS showed a more significant stimulating effect and more likely to cause HUASMC dysfunction than Ⅰ fimA Pg-LPS, which provided part of the basis for the progression of As in severe periodontitis.

Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) influences adipocytes injuries through triggering XBP1 and activating mitochondria-mediated apoptosis

ABSTRACT Obesity is an important public-health problem worldwide. This study aimed to determine effects of porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on adipocytes injuries and explore associated mechanisms. Adipocytes were isolated from SD rats. pLVX-XBP1 (XBP1 over-expression) and pLVX-XBP1-RNAi (silencing XBP1) were structured and transfected into adipocytes. All adipocytes were divided into pLVX-NC, pLVX-XBP1, pLVX-NC+Pg-LPS and pLVX-XBP1+ Pg-LPS group. Oil-Red O staining was employed to identify isolated adipocytes. Quantitative real-time PCR (qRT-PCR) was used to examine gene transcription of IL-6, TNF-α, leptin, adiponectin. Western blotting was used to detect Bax and caspase-3 expression. Adipocytes were successfully isolated and identified with Oil-Red O staining. Both XBP1 mimic and XBP1 RNAi were effectively transfected into adipocytes with higher expressing efficacy. XBP1 over-expression significantly aggravated Pg-LPS induced inflammatory response compared to adipocytes without Pg-LPS treatment (p<0.05). Pg-LPS significantly enhanced leptin and inhibited adiponectin expression by up-regulating XBP1 expression (p<0.05). XBP1 silence significantly alleviated Pg-LPS induced inflammatory response and reduced leptin, enhanced adiponectin expression in Pg-LPS treated adipocytes compared to adipocytes without Pg-LPS treatment (p<0.05). Pg-LPS induced apoptosis of adipocytes by enhancing XBP1 expression and modulating Bcl-2/Bax pathway associated molecules. In conclusion, Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) induces adipocytes injuries through modulating XBP1 expression and initialling mitochondria-mediated apoptosis.

Porphyromonas gingivalis-Lipopolysaccharide induces cytokines and enzymes of the mouse cementoblasts

Lipopolysaccharide is a potent virulence factor of Porphyromonas gingivalis and has been implicated predominant pathogen in the development and progression of periodontal diseases. The aim of this study was to determine the effect of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on cementoblasts.Cementoblast (OCCM-30) were evaluated proliferation using real-time cell analyzer. In addition, total RNA was isolated at 8, 16, 24 and 72 h from 1000 ng/mL Pg-LPS treated OCCM-30 cells and mRNA expressions of pro/anti-inflammatory cytokine mediators, extracellular matrix enzymes and their tissue inhibitors and of oxidative stress enzymes were studied by real-time polymerase chain reaction. Proliferation analysis indicated that Pg-LPS slightly decreased proliferation of OCCM-30. Pg-LPS had a time-dependent impact on the expression of cytokines and enzymes. There was statistically significant up-regulation of IL-1β and IL-10 in response to Pg-LPS at 8, 16, 24, 72 h but IL-6 expression was reduced compared to control at 8 h. While IL-8 and IL-17 expressions were determined higher than control group at 16 and 24 h, their expressions were decreased compared to control groups at 72 h (p < 0.01). While MMP-1, MMP-2, MMP-3, TIMP-1, TIMP-2 expressions increased, MMP-9 expression reduced at time-points. Also, a time-dependent up-regulation in mRNA levels for oxidative stress enzymes was detected. These results indicated that up-regulation in the transcripts of inflammation-associated cytokines and degradation enzymes were noted in the cementoblasts exposed to Pg-LPS. Cementoblasts infected with the virulence factors of periodontopathogens might also involve to the induction of inflammation and degradation of the periodontal tissues.

Effect of Porphyromonas gingivalis lipopolysaccharide on innate immune responses in bone-marrow derived macrophages and osteoclasts

Objective To analyze the immune responses of bone-marrow derived macrophages and osteoclasts to lipopolysaccharide from Porphyromonas gingivalis (Pg-LPS) so as to provide reference for host immunomodulatory therapy of periodontitis. Methods Bone marrow mononuclear cells from C57BL/6 mice were obtained and induced into bone marrow macrophages (BMM) and osteoclasts (OC) by conditioned medium. The BMMs and OCs were divided into blank control group and Pg-LPS treated experimental group. Pg-LPS (10 μg/ml) was used to stimulate BMM and OC. The mRNA and protein expression of TLR-2 and TLR-4 and secretion of inflammatory cytokines were detected, respectively. Furthermore, the effect of Pg-LPS on the differentiation of BMM into OC was evaluated. Results Mouse bone marrow mononuclear cells were successfully induced into BMM and mature OC. In the presence of Pg-LPS, the gene level of TLR-2 in BMM was significantly up-regulated by (41.41±13.07) folds (P 0.05). Moreover, the productions of tumor necrosis factor-α and interleukin-6 in response to LPS in BMM and OC remarkably increased (P<0.01). Lower cytokine expression was observed in OC than that in BMM (P<0.01). In addition, Pg-LPS obviously inhibited the number and size of OC formation, with a reduction in area percentage of approximately 47%. Conclusions Pg-LPS generated a robust immune inflammatory response to BMM, while OC had a relatively weaker immune response to the Pg-LPS. Pg-LPS might inhibite the differentiation of BMM into OC. Key words: Macrophages; Osteoclasts; Immune response

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.

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.

Porphyromonas gingivalis lipopolysaccharide-induced macrophages modulate proliferation and invasion of head and neck cancer cell lines

AimThe aim of this study was to investigate the effect of Porphyromonas gingivalis lipopolysaccharide (LPS)-induced macrophages on head and neck squamous cell carcinoma (HNSCC) cell line proliferation and invasion. Main methodsTHP-1 monocytes were differentiated toward macrophages using 12.5 ng/ml phorbol 12-myristate 13-acetate treatment for 48 h. The expression of interleukin-6 (IL-6) mRNA and cytokine by monocytes and macrophages was determined using real time PCR and ELISA, respectively. The cells were analyzed for CD14 expression using immunofluorescent labeling. The macrophages were induced using 1 μg/ml P. gingivalis LPS for 24 h, and the conditioned medium (CM) was collected. The monocyte, macrophage, and LPS-induced macrophage CM were evaluated for IL-6 and tumor necrosis factor-alpha (TNF-α), and nitric oxide (NO) content using ELISA and the Griess Reagent System, respectively. Human primary (HN18, HN30, and HN4) and metastatic (HN17, HN31, and HN12) HNSCC cell lines were treated with the monocyte, macrophage, and LPS-induced macrophage CM. The proliferation and invasion of the HNSCC cell lines were evaluated using MTT and modified Boyden chamber assays, respectively. Key findingsMacrophages demonstrated increased IL-6 and CD14 expression. The P. gingivalis LPS significantly induced macrophage NO secretion, however, that of TNF-α decreased. The LPS-induced macrophages CM inhibited HN4 proliferation. Interestingly, the LPS-induced macrophage CM promoted invasion of all HNSCC cell lines. SignificanceOur data demonstrate that P. gingivalis LPS-induced macrophages increased NO secretion. The activated macrophage CM inhibited HN4 cell proliferation and promoted invasion of all HNSCC cell lines.

Changes in Expression of the Membrane Receptors CD14, MHC-II, SR-A, and TLR4 in Tissue-Specific Monocytes/Macrophages Following Porphyromonas gingivalis-LPS Stimulation.

The aim of the study was to provide a theoretical foundation for understanding the relationship between periodontal diseases and systemic diseases by examining the inflammatory effect of Porphyromonas gingivalis lipopolysaccharide (LPS) on monocytes/macrophages isolated from tissues distinct from the oral cavity in normal and hyperlipidemic New Zealand white rabbits. Macrophages were isolated from four separate tissues (mononuclear cells from blood, alveolar macrophages, peritoneal macrophages, and Kupffer cells) from both normal and hyperlipidemic New Zealand white rabbits. Cells were either stimulated for 24h in vitro with P. gingivalis-LPS or Escherichia coli-LPS, or were pre-treated with IL-10 before P. gingivalis-LPS treatment. RNA was isolated and the expression of SR-A, TLR4, CD14, and MHC-II measured by RT-PCR. For MHC-II, the suppression effects of P. gingivalis-LPS were similar to the effects of E. coli-LPS in all macrophages examined. In general, the magnitude of the effects of P. gingivalis-LPS on gene expression was lower than that of E. coli-LPS, and there were differences in the relative membrane receptors between the two, implying that the two LPSs stimulate different responses. IL-10 increased the expression of the defensive receptor SR-A and decreased the expression of CD14, TLR4, and the antigen-presenting molecule MHC-II in all types of macrophages examined, regardless of hyperlipidemic state. These data are consistent with an anti-inflammatory effect of IL-10. P. gingivalis-LPS is an activator of gene expression in macrophages isolated from tissues distinct from the oral cavity.

Effects of Porphyromonas gingivalis lipopolysaccharide on the expression of key genes involved in cholesterol metabolism in macrophages.

IntroductionCardiovascular diseases are positively correlated with periodontal disease. However, the molecular mechanisms linking atherosclerosis and periodontal infection are not clear. This study aimed to determine whether Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) altered the expression of genes regulating cholesterol metabolism in macrophages in the presence of low-density lipoprotein (LDL).Material and methodsTHP-1-derived macrophages were exposed to different concentrations (0.1, 1, 10 µg/ml) of LPS in the presence of 50 µg/ml native LDL. Macrophages were also incubated with 1 µg/ml LPS for varying times (0, 24, 48, or 72 h) in the presence of native LDL. Foam cell formation was determined by oil red O staining and cholesterol content quantification. CD36, lectin-like oxidized LDL receptor-1 (LOX-1), ATP-binding cassette G1 (ABCG1), and acetyl CoA acyltransferase 1 (ACAT1) expression levels were measured by western blot and qRT-PCR.ResultsFoam cell formation was induced in a time- and concentration-dependent manner as assessed by both morphological and biochemical criteria. Pg-LPS caused downregulation of CD36 and ABCG1 but upregulation of ACAT1, while LOX-1 expression was not affected (p = 0.137).ConclusionsPg-LPS appears to be an important link in the development of atherosclerosis by mechanisms targeting cholesterol homeostasis, namely, excess cholesterol ester formation via ACAT1 and reduced cellular cholesterol efflux via ABCG1.

Female sex hormones modulate Porphyromonas gingivalis lipopolysaccharide-induced Toll-like receptor signaling in primary human monocytes.

Female sex hormones are elevated and are potential host response modifiers during pregnancy. Modulation of immune responses by estrogen and progesterone may be responsible for periodontal inflammation. Therefore, we aimed to investigate the role of β-estradiol and progesterone in human monocyte immune responses, at cellular and molecular levels, to identify their role as a possible immunological link between pregnancy and periodontal disease. Primary human monocytes were purified from human peripheral blood mononuclear cells by adherent method. Expression of Toll-like receptor (TLR) 2, 4 and CD14 was analyzed by flow cytometry. TLR2, TLR4, cyclooxygenase-2 (COX2), nuclear factor-kappa B (NF-κB) and NF-κB inhibitor-alpha mRNA expressions were measured using real-time reverse transcriptase-polymerase chain reaction and prostaglandin E2 secretion was assayed by enzyme-linked immunosorbent assay. NF-κB expression was also examined by immunofluorescence. Western blotting was performed to determine the activation of mitogen-activated protein kinase pathway. We report herein that both β-estradiol and progesterone significantly reduced TLR2 expression at both protein and mRNA levels but had less of an effect on TLR4 expression in primary human monocytes. We also found that the hormones decreased monocyte cell surface protein expression of CD14. Significantly, β-estradiol and progesterone dose-dependently downregulated monocyte expression of COX2 mRNA. Pretreatment monocytes with β-estradiol or progesterone reduced effects of Porphyromonas gingivalis lipopolysaccharide (LPS) on COX2 mRNA expression and decreased prostaglandin E2 secretion by the monocytes. Furthermore, we demonstrated that both β-estradiol and progesterone inhibited P. gingivalis LPS-induced NF-κB signaling pathway through the upregulation of NF-κB inhibitor-alpha expression. However, neither β-estradiol nor progesterone altered the phosphorylation of the p38, the extracellular signal-regulated kinase 1/2 and the c-Jun N-terminal activated kinase in P. gingivalis LPS-stimulated monocytes. Thus, the inhibitory effects of these hormones on the response of human monocytes to P. gingivalis LPS appear to be independent on mitogen-activated protein kinase signaling pathway. The results of the present study suggest that β-estradiol and progesterone could influence the immune response of human monocytes to periodontal pathogens and this process may have a role in the clinical manifestations of periodontal disease associated with pregnancy.

Lipopolysaccharide-regulated production of bone sialoprotein and interleukin-8 in human periodontal ligament fibroblasts: the role of toll-like receptors 2 and 4 and the MAPK pathway.

Lipopolysaccharide (LPS) on the cell wall of periodontal pathogens is a major mediator of the inflammatory response and can enhance alveolar bone resorption in periodontitis. Bone sialoprotein is an early marker of osteoblast differentiation. The proinflammatory cytokine, interleukin-8 (IL-8), induces osteoclast differentiation, maturation and maintenance of bone resorption activity. However, the effects of LPS from periodontal pathogens on the expression of bone sialoprotein and IL-8 in human osteoblasts and the mechanism of periodontal bone metabolism regulation are rather unclear. The objectives of this study were to determine the effects of Porphyromonas gingivalis LPS on the production of bone sialoprotein and IL-8 in human periodontal ligament fibroblasts (hPDLFs), and to investigate whether toll-like receptor (TLR) 2, TLR4 and MAPKs pathways are involved in the regulation of production of bone sialoprotein and IL-8 by P.gingivalis LPS. The third-generation of hPDLFs were cultured with mineralization-inducing culture medium. After hPDLFs were treated with P.gingivalis LPS, bone sialoprotein and IL-8 mRNA expression were detected using Real time PCR. Then hPDLFs were transiently transfected with siTLR2 or siTLR4 (20nm) or inhibited by MAPK signaling pathways inhibitors, and then bone sialoprotein and IL-8 mRNA and protein expression were also detected using Real time PCR and western blotting. Treatments with 0.01 and 0.1mg/L of P.gingivalis LPS for 8h up-regulated bone sialoprotein mRNA expression, whereas 10 and 100mg/L of P.gingivalis LPS induced a significant decrease in the expression of bone sialoprotein mRNA. In contrast, IL8 mRNA levels were increased significantly by 10mg/L of P.gingivalis LPS. Interestingly, small interfering RNA (siRNA) knock down of the TLR2 and ERK1/2 inhibitor, PD98059, abolished the effects of P.gingivalis LPS on the bone sialoprotein mRNA level, whereas siRNA knock down of the TLR2 and p38 MAPK inhibitor, SB203580, blocked the effect of P.gingivalis LPS on IL-8 in hPDLFs. This study suggests that in hPDLFs, P.gingivalis LPS suppresses bone sialoprotein and enhances IL-8 gene and protein expression via TLR2 and ERK1/2 or the p38 MAPK signaling pathway, respectively.

Different effects of Porphyromonas gingivalis lipopolysaccharide and TLR2 agonist Pam3CSK4 on the adhesion molecules expression in endothelial cells

Recent researches suggest an association between periodontitis and cardiovascular disease. Periodontopathic bacteria and/or their component might play a role in the development of atherosclerotic lesions. In the present study, we investigated in vitro the effect of Porphyromonas gingivalis lipopolysaccharide (LPS) on the expression of adhesion molecules in human umbilical vein endothelial cells (HUVECs) as well as its effect on Escherichia coli LPS-induced response. The effect of P. gingivalis LPS was compared with that of toll-like receptor 2 agonist synthetic triacylated lipoprotein Pam3-Cys-Ser-(Lys)4 (Pam3CSK4). Gene and protein expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin were measured using RT-PCR and flow cytometry, respectively. P. gingivalis LPS stimulated the expression levels of all adhesion molecules in a dose-dependent manner. However, the response of HUVECs to P. gingivalis LPS was markedly lower than that to E. coli LPS. Moreover, P. gingivalis LPS attenuated E. coli LPS-induced responses when HUVECs were simultaneously stimulated with both kinds of LPS. Treatment with Pam3CSK4 resulted in a minor increase of adhesion molecule expression and did not diminish E. coli LPS-induced responses. Our data suggest that P. gingivalis LPS induces in vitro the expression of adhesion molecules in endothelial cells, which might promote atherogenesis. Qualitatively different responses of HUVECs to P. gingivalis LPS and Pam3CSK4 suggest that besides TLR2 other signaling pathways might be involved in the effects of P. gingivalis LPS.

Effects of Porphyromonas gingivalis lipopolysaccharide on osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling

In bone remodeling, the Eph family is involved in regulating the process of osteoclast and osteoblast coordination in order to maintain bone homeostasis. In this study, the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling were investigated. An osteoblast-osteoclast co-culture system was achieved successfully. Hence, direct contact and communication between osteoblasts and osteoclasts was permitted. Regarding the protein expression and gene expression of EphB4 and EphrinB2, it was shown that Pg-LPS increased the expression of EphB4 while inhibiting the expression of EphrinB2. Therefore, the results indicate that, when treated with Pg-LPS, the EphB4 receptor on osteoblasts and the EphrinB2 ligand on osteoclasts may generate bidirectional anti-osteoclastogenic and pro-osteoblastogenic signaling into respective cells and potentially facilitate the transition from bone resorption to bone formation. This study may contribute to the control of osteoblast differentiation and bone formation at remodeling, and possibly also modeling, sites.

Effects of Porphyromonas gingivalis lipopolysaccharide on apoptotic genes in foam cells

To investigate the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on apoptotic genes in foam cells. Macrophages from THP-1 monocytes and foam cells from macrophages by oxLDL inducement were treated with oxidized low density lipoprotein (oxLDL) or oxLDL+ Pg-LPS. Cell apoptosis was detected by acridine orange-ethidium bromide (AO-EB) staining. Eleven atherosclerotic related apoptotic genes were examined with polymerase chain reaction (PCR) array, and apoptotic gene p53, c-Myc and caspase-3 were evaluated with real-time PCR. Pg-LPS enhanced cell apoptosis rate during and after foam cells formation [(5.47+/-0.93)% vs. (7.50+/-0.54)%]. PCR array demonstrated that it increased B-cell CLL-lymphoma 2 (BCL2) related protein A1 (BCL2A1) transcription during foam cells formation (>2 fold), and promoted BCL2 and BCL2A1 transcription after foam cells formation (>2 fold). It promoted p53 and caspase-3 transcription level (4.50x10(-3)+/-4.02x10(-4) vs. 5.30x10(-2)+/-4.58x10(-3)), whereas inhibited c-Myc transcription level (1.53x10(-2)+/-5.77x10(-4)) during foam cells formation. It promoted caspase-3 transcription (6.00x10(-2)+/-6.08x10(-3)), and inhibited p53 transcription (4.23x10(-3)+/-5.85x10(-4)) after foam cells formation. Pg-LPS affected apoptotic gene transcription during and after foam cells formation and enhanced cell apoptosis.

Effect of Porphyromonas gingivalis lipopolysaccharide on induced secretion of inflammatory cytokines by different cell lines

To determine the effect of Porphyromonas gingivalis lipopolysaccharide (LPS) on induced secretion of inflammatory cytokines by different cell lines. LPS of P. gingivalis strain ATCC33277 (Pg-LPS) was extracted with phenol-water method, and identified by Limulus test and infrared spectrum analysis. KB, HGF-1 and THP-1 cells were treated with Pg-LPS of different concentrations and time duration, a commercial LPS of E.coli strain O111:B4 (E-LPS) was used as the control. TNF-alpha, IL-1beta, IL-6 and IL-8 levels in culture supernatants were measured by quantitative ELISA. The minimal dosages of both Pg-LPS and E-LPS to solidify Limulus agents were 15 ng/ml and their infrared spectrums were similar. With the treatment of Pg-LPS or E-LPS, the TNF-alpha and IL-1beta levels secreted by HGF-1 cells were remarkably increased with a single perk (P<0.01) while a continuous enhancement of secretion by THP-1 cells was observed (P<0.01). Either Pg-LPS or E-LPS stimulated HGF-1 or THP-1 cells to continuously increase the secretion of IL-6 (P<0.01). Both Pg-LPS and E-LPS induced IL-8 secretion by THP-1 cells (P<0.01), but only Pg-LPS showed the similar effect on HGF-1 cells (P<0.01). Neither Pg-LPS nor E-LPS induced KB cells to secrete inflammatory cytokines. Pg-LPS can promote target cells to increase their secretion of inflammatory cytokines. KB cells can not be used as the target cell to determine inflammation-causing effect of LPS.

Regulation of Cementoblast Function by P. gingivalis Lipopolysaccharide via TLR2

Although cementoblasts express Toll-like receptors (TLR)-2 and -4, little is known regarding the possible participation of cementoblasts in the inflammatory response. We investigated the effects of Porphyromonas gingivalis lipopolysaccharide (LPS), tetra- and penta-acylated lipid A species (designated PgLPS(1435/1449) and PgLPS(1690), respectively), on gene expression of osteoclastogenesis-associated molecules in murine cementoblasts. Real-time quantitative RT-PCR analysis revealed that receptor activator of NF-kappaB ligand (RANKL), interleukin-6, Regulated on activation, normal T-cell expressed, and secreted (RANTES), macrophage inflammatory protein-1alpha, and monocyte chemoattractant protein-1 were rapidly and dramatically induced upon stimulation with PgLPS(1690), but only slightly induced with PgLPS(1435/1449). Osteoprotegerin, which was expressed constitutively, was not altered significantly. ELISA demonstrated synthesis of corresponding proteins. PgLPS(1690) significantly induced transcripts for NF-kappaB, and this activation was inhibited by pre-treatment with anti-TLR-2 but not with TLR-4 antibodies. These results suggest that cementoblasts participate in the recruitment of osteoclastic precursor cells by up-regulation of chemokines/cytokines.

Inflammatory cytokine gene expression in human periodontal ligament fibroblasts stimulated with bacterial lipopolysaccharides.

The effects of Porphyromonas gingivalis lipopolysaccharide (P-LPS) and Escherichia coli lipopolysaccharide (E-LPS) on the gene expression and production of inflammatory cytokines of human periodontal ligament fibroblasts (HPLF) were examined by a Northern (RNA blot) assay and enzyme-linked immunosorbent assay, respectively. mRNAs for interleukin-6 (IL-6), IL-8, and transforming growth factor beta (TGF-beta) were detected in HPLF cells, but IL-1 alpha, IL-1 beta, tumor necrosis factor alpha, transforming growth factor alpha, and granulocyte-macrophage colony-stimulating factor were not detected by reverse transcription-PCR. The expression of TGF-beta mRNA was not influenced by either LPS. P-LPS (1 to 10 micrograms/ml) and E-LPS (100 micrograms/ml) markedly stimulated the expression of IL-6 and IL-8 mRNAs compared with the control. The synthesis of IL-6 and IL-8 was also stimulated by 10 and 100 micrograms of both LPSs per ml, but IL-8 synthesis was not stimulated with E-LPS at 1 microgram/ml. Secretion of IL-6 and IL-8 into the culture medium was detected at 6 and 3 h, respectively, after exposure to P-LPS (10 micrograms/ml). These findings suggested that P. gingivalis leads to periodontal tissue destruction and alveolar bone resorption through IL-6 and IL-8 released from HPLF cells stimulated with its LPS.