Gingivalis LPS Research Articles

Gingipains may be one of the key virulence factors of Porphyromonas gingivalis to impair cognition and enhance blood-brain barrier permeability: An animal study.

Blood-brain barrier (BBB) disorder is one of the early findings in cognitive impairments. We have recently found that Porphyromonas gingivalis bacteraemia can cause cognitive impairment and increased BBB permeability. This study aimed to find out the possible key virulence factors of P. gingivalis contributing to the pathological process. C57/BL6 mice were infected with P. gingivalis or gingipains or P. gingivalis lipopolysaccharide (P. gingivalis LPS group) by tail vein injection for 8 weeks. The cognitive behaviour changes in mice, the histopathological changes in the hippocampus and cerebral cortex, the alternations of BBB permeability, and the changes in Mfsd2a and Cav-1 levels were measured. The mechanisms of Ddx3x-induced regulation on Mfsd2a by arginine-specific gingipain A (RgpA) in BMECs were explored. P. gingivalis and gingipains significantly promoted mice cognitive impairment, pathological changes in the hippocampus and cerebral cortex, increased BBB permeability, inhibited Mfsd2a expression and up-regulated Cav-1 expression. After RgpA stimulation, the permeability of the BBB model in vitro increased, and the Ddx3x/Mfsd2a/Cav-1 regulatory axis was activated. Gingipains may be one of the key virulence factors of P. gingivalis to impair cognition and enhance BBB permeability by the Ddx3x/Mfsd2a/Cav-1 axis.

Human bone marrow mesenchymal stem cell-derived extracellular vesicles restore Th17/Treg homeostasis in periodontitis via miR-1246.

T-cell-mediated immunity is crucial in the immunopathology of periodontitis. The restoration of the homeostasis between the T helper cell 17 (Th17) and regulatory T cell (Treg) subsets by extracellular vesicles (EVs) obtained from human bone marrow stem cells (hBMSCs) promotes new bone formation and suppresses inflammation. Uncovering the functions of hBMSC-derived EVs in the immune microenvironment of periodontal tissue and their underlying regulatory mechanisms may shed new light on developing potential cell-free immunotherapies for periodontal regeneration. Here, we reported that the Th17/Treg ratio elevated in peripheral blood from periodontitis patients. Furthermore, we found that hBMSC-derived EVs could reduce the Th17/Treg ratio in CD4+ T cells from periodontitis patients in vitro and ameliorate conditions of experimental periodontitis in mice. Additionally, by investigating the differentially expressed miRNAs and target genes in EVs from hBMSCs stimulated with Porphyromonas gingivalis LPS using miRNA sequencing, we found that EV-miR-1246 is highly effective at downregulating the ratio of Th17/Treg in vitro. Mechanistically, EV-miR-1246 suppressed expression of its potential target angiotensin-converting enzyme 2 (ACE2) and increased the p-Yes-associated protein (YAP)1/YAP1 ratio in CD4+ T cells. Our results indicated that hBMSC-derived EVs improve periodontitis via miR-1246, consequently downregulating Th17/Treg ratio, and represented a promising therapeutic target for precision treatment in periodontitis.

Anti-Inflammatory Effects of β-Cryptoxanthin on 5-Fluorouracil-Induced Cytokine Expression in Human Oral Mucosal Keratinocytes

Oral mucositis is a typical adverse effect of chemotherapy, causing oral pain that significantly reduces the patient's quality of life. β-cryptoxanthin (β-cry) is a carotenoid abundant in citrus fruits with antioxidant and anti-inflammatory effects. However, the β-cry effect on oral mucositis remains unclear. We investigated the effects of 5-fluorouracil (5-FU) and β-cry on human normal oral mucosal keratinocytes (hOMK). hOMK was seeded on a culture plate and cultured with 5-FU and β-cry. The cell number, mRNA expression of inflammatory cytokines and matrix metalloproteinases (MMPs), and production of inflammatory cytokines in hOMK were evaluated. Additionally, the cell count and inflammatory cytokine production were analyzed when hOMK was co-stimulated with Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) in addition to 5-FU. The numbers of hOMK significantly reduced with 5-FU stimulation, whereas it increased with β-cry treatment. mRNA expression of interleukin (IL)-6, IL-8, metalloproteinase (MMP)-2, and MMP-9 and protein production of IL-6 and IL-8 in hOMK were augmented on 5-FU stimulation. Simultaneously, β-cry treatment significantly suppressed IL-8 and MMP-9 mRNA expression, and IL-8 production was induced on 5-FU stimulation. Co-stimulation with P. gingivalis LPS and 5-FU enhanced IL-6 and IL-8 production in hOMK. β-cry could enhance cell proliferation and suppress 5-FU-induced expression of inflammatory cytokines and MMP in hOMK. Thus, β-cry can alleviate the symptoms of chemotherapy-induced oral mucositis, and its combination with oral care is effective in managing oral mucositis.

Adenosine A2A Receptor Antagonist Improves Cognitive Impairment by Inhibiting Neuroinflammation and Excitatory Neurotoxicity in Chronic Periodontitis Mice.

The adenosine A2A receptor antagonist SCH58261 has been reported to have anti-inflammatory effects. However, its role in chronic periodontitis (CP)-induced cognitive impairment, which is associated with Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS), remains unclear. This study investigated the role of SCH58261 in mice with CP-induced cognitive impairment. C57BL/6J mice were used to develop CP model by injecting 0.5 mg/kg P. gingivalis LPS into the palatal gingival sulcus of maxillary first molars twice a week for four weeks. The mice were divided into control, P. gingivalis LPS (P-LPS), P-LPS + SCH58261, and SCH58261 groups. The passive avoidance test (PAT) and Morris water maze (MWM) were used to assess cognition in mice. Furthermore, CD73/adenosine, neuroinflammation, glutamate transporters, and glutamate were assessed. Compared with the P-LPS group, 0.1 and 0.5 mg/kg SCH58261 increased latency and decreased error times in PAT, but increased platform crossing number in MWM. SCH58261 inhibited microglial activation, and decreased pro-inflammatory cytokines and glutamate levels, but increased GLT-1 and PSD95 expression in the hippocampus. This was the first report of SCH58261 treatment for CP-induced cognitive impairment, which may be related to its anti-inflammatory activities and anti-glutamate excitatory neurotoxicity. This suggests that SCH58261 can be used as a novel agent to treat cognitive impairment.

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.

Chlorogenic Acid as a Positive Regulator in LPS-PG-Induced Inflammation via TLR4/MyD88-Mediated NF-κB and PI3K/MAPK Signaling Cascades in Human Gingival Fibroblasts.

Gingival inflammation is one of the main causes that can be related to various periodontal diseases. Human gingival fibroblast (HGF) is the major constituent in periodontal connective tissue and secretes various inflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), upon lipopolysaccharide stimulation. This study is aimed at investigating the anti-inflammatory mechanism of chlorogenic acid (CGA) on Porphyromonas gingivalis LPS- (LPS-PG-) stimulated HGF-1 cells. The concentration of NO and PGE2, as well as their responsible enzymes, inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), was analyzed by Griess reaction, ELISA, and western blot analysis. LPS-PG sharply elevated the production and protein expression of inflammatory mediators, which were significantly attenuated by CGA treatment in a dose-dependent manner. CGA treatment also suppressed activation of Toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (MyD88) and nuclear factor- (NF-) κB in LPS-PG-stimulated HGF-1 cells. Furthermore, LPS-PG-induced phosphorylation of extracellular regulated kinase (ERK) and Akt was abolished by CGA treatment, while c-Jun N-terminal kinase (JNK) and p38 did not have any effect. Consequently, these results suggest that CGA ameliorates LPS-PG-induced inflammatory responses by attenuating TLR4/MyD88-mediated NF-κB, phosphoinositide-3-kinase (PI3K)/Akt, and MAPK signaling pathways in HGF-1 cells.

Immunomodulatory effects of Nd:YAG (1064 nm) and diode laser (810 nm) wavelengths to LPS-challenged human gingival fibroblasts

Objective: Human gingival fibroblasts (hGFs) are involved in inflammatory responses to bacteria by recognizing pathogen-associated molecular patterns. In search of host modulation strategies to increase LPS tolerance, Low level laser therapy (LLLT) has been suggested as an alternative treatment that reduces periodontal tissue inflammation. In this study, we investigate whether 810 nm (diode) and 1064 nm (Nd:YAG) laser wavelengths, modulate pro-inflammatory responses to LPS challenges in hGFs. Design: Primary hGFs were challenged with Porphyromonas gingivalis LPS and irradiated with either Diode (810 nm) or with Nd:YAG (1064 nm) lasers. Cell cultures were examined for cell proliferation by MTT assay and IL-6 and IL-8 expression by qPCR at 24, 48 and 72 h. IL-6 and IL-8 protein levels were detected via ELISA. Results: Naïve hGF populations irradiated with both Diode 810 nm and Nd:YAG 1064 nm lasers demonstrated cellular proliferation (p < 0.05), but LLLT did not affect cellular viability in LPS-challenged cells. IL-6 and IL-8 gene expression levels revealed significant anti-inflammatory effects of irradiation with both examined wave- lengths on hGFs challenged with P. gingivalis LPS. Protein levels of these cytokines were increased by LPS challenge. Treatment with LLLT inhibited this increase for both wavelengths evaluated in the study at a statistically significant level particularly for the first 48 h. Conclusions: The present study demonstrates a modulatory effect of LLLT using both 810 nm diode and Nd:YAG 1064 nm lasers in gingival fibroblasts by decreasing the production of IL-6, IL-8 in response to LPS.

GDF15 Supports the Inflammatory Response of PdL Fibroblasts Stimulated by P. gingivalis LPS and Concurrent Compression.

Periodontitis is characterized by bacterially induced inflammatory destruction of periodontal tissue. This also affects fibroblasts of the human periodontal ligaments (HPdLF), which play a coordinating role in force-induced tissue and alveolar bone remodeling. Excessive inflammation in the oral tissues has been observed with simultaneous stimulation by pathogens and mechanical forces. Recently, elevated levels of growth differentiation factor 15 (GDF15), an immuno-modulatory member of the transforming growth factor (TGFB) superfamily, were detected under periodontitis-like conditions and in force-stressed PdL cells. In view of the pleiotropic effects of GDF15 in various tissues, this study aims to investigate the role of GDF15 in P. gingivalis-related inflammation of HPdLF and its effect on the excessive inflammatory response to concurrent compressive stress. To this end, the expression and secretion of cytokines (IL6, IL8, COX2/PGE2, TNFα) and the activation of THP1 monocytic cells were analyzed in GDF15 siRNA-treated HPdLF stimulated with P. gingivalis lipopolysaccharides alone and in combination with compressive force. GDF15 knockdown significantly reduced cytokine levels and THP1 activation in LPS-stimulated HPdLF, which was less pronounced with additional compressive stress. Overall, our data suggest a pro-inflammatory role for GDF15 in periodontal disease and demonstrate that GDF15 partially modulates the force-induced excessive inflammatory response of PdLF under these conditions.

STAT1/SOCS1/3 Are Involved in the Inflammation-Regulating Effect of GAS6/AXL in Periodontal Ligament Cells Induced by Porphyromonas gingivalis Lipopolysaccharide In Vitro.

Periodontitis involves chronic inflammation of the tissues around the teeth caused by plaque and the corresponding immune response. Growth arrest-specific protein 6 (GAS6) and AXL receptor tyrosine kinase (AXL) are known to be involved in inflammatory diseases, while signal transducer and activator of transcription-1 (STAT1) and suppressor of cytokine signaling (SOCS) are related to inflammatory processes. Moreover, miRNA34a directly targets AXL to regulate the AXL expression. However, the specific roles of GAS6 and AXL in periodontitis remain unclear. This study was designed to explore the effect and mechanism of AXL on the expression of inflammatory cytokines induced by Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) in human periodontal ligament cells (hPDLCs). The effects of different concentrations of P. gingivalis LPS on the expression of GAS6/AXL in hPDLCs were observed. Additionally, the effect of LPS on AXL was investigated by transfection of the miRNA34a inhibitor. AXL was knocked down or overexpressed to observe the release of inflammatory cytokines interleukin- (IL-) 8 and IL-6. The results showed that the expression levels of GAS6 and AXL decreased after P. gingivalis LPS infection. Transfection of a miR-34a inhibitor to hPDLCs demonstrated a role of miR-34a in the downregulation of AXL expression induced by LPS. Moreover, AXL knockdown or overexpression influencing the expression of IL-8 and IL-6 was investigated under LPS stimulation. AXL knockdown decreased the expression of STAT1 and SOCS1/3. Overall, these results demonstrate that AXL inhibits the expression of LPS-induced inflammatory cytokines in hPDLCs and that STAT1 and SOCS1/3 are involved in the regulation of inflammation by GAS6/AXL.

B cell activating factor regulates periodontitis development by suppressing inflammatory responses in macrophages

BackgroundB cell activating factor (BAFF) is a member of the tumor necrosis factor (TNF) superfamily with immunomodulatory effects on both innate and adaptive immune responses. Periodontitis is an inflammatory disease characterized by periodontal soft tissue inflammation and the progressive loss of periodontal ligament and alveolar bone. Macrophages are closely related to periodontitis progression. However, the role of BAFF in periodontitis development and macrophage polarization and the underlying mechanism remain unknown.MethodsIn vivo, a ligation-induced mouse model of periodontitis for BAFF blockade was established to investigate the expression of inducible nitric oxide synthase (iNOS) through real-time PCR (RT-PCR) and immunohistochemistry. In addition, the level of TNF-α in the periodontium, the number of osteoclasts, and alveolar bone resorption were observed. In vitro, RAW 264.7 macrophage cells were treated with 100 ng/mL Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) in either the presence or absence of 50 nM small interfering RNA (siRNA) targeting BAFF, followed by further incubation for 24 h. These cells and supernatants were collected and stored for RT-PCR, enzyme-linked immunosorbent assay, western blotting and immunofluorescence microscopy.ResultsIn vivo, BAFF blockade decreased the levels of TNF-α in the periodontium in a ligature-induced mouse periodontitis model. Reduced osteoclast formation and lower alveolar bone loss were also observed. In addition, BAFF blockade was related to the expression of polarization signature molecules in macrophages. In vitro, BAFF knockdown notably suppressed the production of TNF-α in RAW 264.7 cells stimulated by P. gingivalis LPS. Moreover, BAFF knockdown attenuated the polarization of RAW 264.7 cells into classically activated macrophages (M1), with reduced expression of iNOS.ConclusionsBased on our limited evidence, we showed BAFF blockade exhibits potent anti-inflammatory properties in mice experimental periodontitis in vivo and in P. gingivalis LPS-treated RAW 264.7 cells in vitro, and macrophage polarization may be responsible for this effect.

Therapeutic potential of a novel semi-synthetic-sulfated-polysaccharide to suppress inflammatory mediators in P. gingivalis LPS stimulated human monocytes/macrophages

BackgroundChronic periodontitis is associated with an increased risk for systemic conditions such as cardiovascular disease, diabetes, and osteoporosis. During chronic periodontitis, endotoxin (lipopolysaccharide, LPS) produced by P. gingivalis provokes monocyte accumulation and differentiation into macrophages and increased secretion of pro-inflammatory cytokines and matrix metalloproteases (MMPs). While normal levels of MMPs are important in cellular function, increased levels of cytokines and MMPs can cause connective tissue destruction.ResultsIn the current study, we investigated the therapeutic capability of a novel semi-synthetic sulfated polysaccharide (SAGE) on the production of cytokines and MMPs by cultured human mononuclear cells and macrophages stimulated with endotoxin LPS produced by P. gingivalis, a periodontally-relevant cell culture model. Our research demonstrated SAGE inhibited the LPS induced synthesis of inflammatory mediators including TNF-α, IL-1β, PGE2, and MMP-9 in this periodontal-relevant cell culture model. In addition, TLR-2 and TLR-4 levels were also reduced with the SAGE treatment.ConclusionsThe therapeutic potential of this novel semi-synthetic sulfated polysaccharide compound may help to prevent tissue damage and bone loss in patients with periodontal disease or other inflammatory diseases.

Overexpression of SGLT2 in the kidney of a P. gingivalis LPS-induced diabetic nephropathy mouse model

BackgroundThe overexpression of sodium-glucose cotransporter 2 (SGLT2) in diabetic kidneys has been reported. It has also been established that the diabetic glomerular endothelium expresses the toll-like receptors TLR2 and TLR4. The present study aims to examine the renal SGLT2 induction by the TLR2/4 ligand Porphyromonas (P.) gingivalis lipopolysaccharide (Pg-LPS) in mouse diabetic nephropathy.MethodsImmunohistochemical study and tissue RT-PCR analyses were performed on mouse kidneys in streptozotocin (STZ)-induced diabetic ICR mice (STZ-ICR), in healthy ICR mice administered Pg-LPS (LPS-ICR), and in diabetic ICR mouse kidneys with Pg-LPS-induced nephropathy (LPS-STZ).ResultsIn the quantitative analysis of blood sugar levels, the mean time to reach 600 mg/dl was shorter in the LPS-STZ than in the STZ-ICR kidneys. The rise in blood glucose levels was significantly steeper in the LPS-STZ than in the STZ-ICR kidneys. According to these data the LPS-STZ model suggests a marked glucose intolerance. The expression of SGLT2 was significantly stronger in the whole of the renal parenchyma of the LPS-STZ than in the LPS-ICR or in the STZ-ICR. The expression of SGLT2 was observed both in the renal tubules and around the renal tubules, and in the glomeruli of the LPS-STZ kidneys. In the analysis by tissue real-time PCR and cell ELISA, the expression of the SGLT2 gene and protein was significantly stronger in the LPS-STZ than in the LPS-ICR or in the STZ-ICR. There were no differences in the renal SGLT2 production in the LPS-ICR and the STZ-ICR kidneys.ConclusionsAbnormally high renal expression of SGLT2 occurs in diabetic kidneys with P. gingivalis LPS. Periodontitis may be an exacerbating factor in diabetic nephropathy as well as in diabetes.

The imbalanceof Th17/Tregvia STAT3 activation modulatescognitive impairment in P. gingivalisLPS-induced periodontitis mice.

Periodontitis is one of the most common oral diseases worldwide, and it is associated with various systemic diseases, including cognitive diseases. STAT3 regulates the inflammatory cascade and influences adaptive immunity by modulating Th17/Treg cell differentiation. In this study, we aimed to explore the effect of adaptive immunity inside and outside the brain on the association between periodontitis and cognitive impairment and understand the role of the STAT3 signaling pathway. We established Porphyromonas gingivalis LPS-induced periodontitis mice models by injecting P. gingivalis LPS into the gingival sulcus of mice. Behavioral tests showed that learning and memory abilities were impaired. The flow cytometry data showed an imbalance in the Th17/Treg ratio in the blood and brain samples of the mice. The expression of Th17-related cytokines (IL-1β, IL-17A, IL-21, and IL-22) increased, whereas that of Treg-related cytokines (IL-2 and IL-10) decreased in both the blood and the brain. The level of LPS increased and the STAT3 signaling pathway was activated during this process. These effects were reversed by C188-9, a STAT3 inhibitor. In conclusion, P. gingivalis LPS-induced periodontitis may promote the occurrence and progression of cognitive impairment by modulating the Th17/Treg balance inside and outside the brain. The STAT3 signaling pathway may have immunoregulatory effects on the mouth-to-brain axis.

Targeting TSP-1 decreased periodontitis by attenuating extracellular matrix degradation and alveolar bone destruction

An important factor in periodontitis pathogenesis relates to a network of interactions of various cytokines. Thrombospondin-1 (TSP-1) is upregulated in several inflammatory diseases. We previously found that Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS)-induced TSP-1 production, and that TSP-1 simultaneously and effectively elevated inflammatory cytokines in THP-1 macrophages. This suggests that TSP-1 plays an important role in the pathology of periodontitis. However, the function of TSP-1 on oral cells is largely unknown. This study aimed to elucidate the underlying molecular mechanisms of TSP-1 in human periodontal fibroblasts (hPDLFs). We demonstrated that TSP-1 is highly expressed in the gingival crevicular fluid of patients with chronic periodontitis and in the inflammatory gingival tissues of rats. TSP-1 overexpression or treatment with recombinant human TSP-1(rTSP-1) promoted the expression of MMP-2, MMP-9 and RANKL/OPG in hPDLFs, while anti-TSP-1 inhibited cytokines production from P. gingivalis LPS-treated hPDLFs. Additional experiments showed that SB203580 (a special p38MAPK inhibitor) inhibited MMP-2, MMP-9 and RANKL/OPG expression induced by rTSP-1. Thus, TSP-1 effectively promoted P. gingivalis LPS-induced periodontal tissue (extracellular matrix (ECM) and alveolar bone) destruction by the p38MAPK signalling pathway, indicating that it may be a potential therapeutic target against periodontitis.

An activated-zinc oral rinse reduces pro-inflammatory cytokine secretion and promotes proliferation in Porphyromonas gingivalis LPS-challenged gingival tissues - A pilot study.

ObjectivesThe use of adjunct post‐treatment mouth rinses containing chlorhexidine (CHX) for periodontitis patients is associated with side effects that reduce patient compliance. Our aim was to evaluate the proinflammatory and cell proliferation effects of an activated‐zinc mouth rinse (SM) that has been suggested as an alternative post‐treatment therapeutic.Materials and MethodsTissue models of gingival epithelium were used to simulate periodontal disease and compare inflammatory reactions after treatment with CHX or SM. Tissues were exposed to Porphyromonas gingivalis LPS and wounded to simulate periodontal disease. Tissues were treated and incubated for 6, 12, or 24 h. Inflammatory cytokines were measured in culture medium by ELISA and local expression of Toll‐like receptor (TLR)‐4 and proliferation marker Ki‐67 was visualized by immunohistochemistry.ResultsSM and CHX treatments decreased secretion of IL‐1β and IL‐8 into culture media at all time points. IL‐1β secretion levels were further decreased by SM compared to CHX treatment at all time points. TLR‐4 expression appeared significantly increased 12 h post‐treatment in the CHX tissues but remained relatively low in SM tissues at all time points. Ki‐67 results suggest that cell proliferation was increased in the SM tissues earlier than CHX tissues.ConclusionsOur data suggest that SM may reduce inflammation in gingival tissues.

Anti-inflammatory activity of psoralen in human periodontal ligament cells via estrogen receptor signaling pathway

Psoralen is one of the most effective ingredients extracted from the Chinese herb, Psoralea corylifolia L. Studies have found that psoralen has anti-inflammatory and estrogen-like effects; however, little research has been conducted to elucidate the mechanisms underlying these effects. Through the molecule docking assay, psoralen was found to have a better combination with ERα than ERβ. In human periodontal ligament cells, psoralen was found to upregulate the estrogen target genes (e.g., CTSD, PGR, TFF1) and down-regulate the expression of inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) stimulated by P. gingivalis LPS, as well as TLR4-IRAK4-NF-κb signaling pathway proteins. These effects were reversed by the ER antagonist ICI 182780. These results indicated that psoralen may exert anti-inflammatory effects as an agonist to ER, which could provide a theoretical basis for the use of psoralen for adjuvant therapy and prevention of periodontitis.

Intergenerational effects of pre-pregnancy chronic lipopolysaccharide from Porphyromonas gingivalis on the learning, memory and seizure susceptibility of offspring

ObjectiveThe aim of this study was to investigate the effects of pre-pregnancy chronic exposure to Porphyromonas gingivalis LPS (Pg LPS) on the learning, memory, and seizure susceptibility of the offspring. DesignTo achieve periodontitis, Pg LPS (5 μg/kg) was injected into the gingival of five female rats every 48 h for three weeks. Five control female rats received saline (0.9 %) and five female were kept intact. The concentrations of TNF-α and IL-6 were measured in the blood samples. One week after the final injection, females were mated with intact males. Following birth and weaning, two male and two female offspring were randomly selected from each mother, and new groups of male and female offspring were defined for behavioral assessments. Morris water maze was used to evaluate spatial memory, shuttle box was used to investigate avoidance memory and a pentylenetetrazole-induced seizure was used to evaluate seizure susceptibility in the offspring. ResultsSpatial learning and avoidance memory significantly decreased in both male and female offspring of Pg LPS-exposed female rats, compared to the control offspring. Latency to reach seizure stages 1 and 2 significantly increased in the male offspring, but not the female offspring of Pg LPS-exposed female, compared to the control offspring. However, no significant difference was found in latency to reach stages 3–5. ConclusionPre-pregnancy exposure to Pg LPS could affect some behavioral functions in both male and female offspring intergenerationally.

NLRP6-caspase 4 inflammasome activation in response to cariogenic bacterial lipoteichoic acid in human dental pulp inflammation.

To explore the presence and function of NLRP6-caspase 4 inflammasome in human pulp tissue and human dental pulp cells (HDPCs). Pulp tissue was collected from freshly extracted human caries-free third molars and third molars with irreversible pulpitis. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were performed to assess the expression of NLRP6-caspase 4 inflammasome. HDPCs were prepared from normal human pulp tissues and challenged with Porphyromonas gingivalis LPS. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR were performed to assess if LPS can upregulate NLRP6 and caspase-4. HDPCs were further challenged with LPS followed with cytosolic Streptococcus mutans lipoteichoic acid (LTA). SiRNA targeting NLRP6 and Casp4 and pharmacology inhibitor Ac-FLTD-CMK and MCC950 were used to assess if Streptococcus mutans LTA can activate the NLRP6 but not the NLRP3 inflammasome. Western blot and ELISA were performed to evaluate inflammasome activation. The Student's t-test and one-way anova were used for statistical analysis. NLRP6-caspase 4 inflammasome was upregulated and activated in inflamed human dental pulp tissue. In HDPCs, Porphyromonas gingivalis LPS upregulated the expression of NLRP6, CASP1 and CASP4 in a type I interferon dependent manner. After LPS priming, cytosolic Streptococcus mutans LTA triggered NLRP6-caspase 4 inflammasome activation. Knockdown of NLRP6 or CASP4 using siRNA or using pharmacology inhibitor Ac-FLTD-CMK but not MCC950 efficiently suppressed inflammasome activation by cytosolic LTA. NLRP6-caspase 4 inflammasome may play an important role in pulp inflammation and immune defence. Inflammatory caspases represent a pharmacological target to restrain pulpal inflammation.

Wnt antagonist secreted frizzled-related protein I (sFRP1) may be involved in the osteogenic differentiation of periodontal ligament cells in chronic apical periodontitis.

To explore the mechanism of secreted frizzled-related protein I (sFRP1) involvement in the osteogenic differentiation of human periodontal ligament cells (hPDLCs) under inflammatory conditions. hPDLCs were cultured in an osteogenic differentiation-inducing medium (odi) and subjected to the stimulation ofPorphyromonas gingivalis lipopolysaccharide (P.gingivalisLPS) with or without the inhibition of sFRP1. Quantitative real-time polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay were carried out to evaluate the expression of osteogenic markers as well as the classic Wnt signalling pathway. Periapical periodontitis was induced in Wistar rats to further confirm the effect of sFRP1 inhibitor on bone loss in vivo. After the Shapiro-Wilk normality test, data were analysed by Student's paired t-test or one-way Anova test with a P value less than 0.05 as the level of statistical significance. Significantly decreased mRNA and protein expression of osteogenic markers were detected in hPDLCs treated withP.gingivalisLPS during osteogenic induction (P<0.001). Increased expression of sFRP1 was observed (P<0.01), whilst Wnt/β-catenin signalling pathway was inhibited by the addition of P.gingivalisLPS (P<0.01). After the addition of the sFRP1 inhibitor, the decrease of osteogenic markers (P<0.05) and the inhibition of Wnt/β-catenin signalling pathway (P<0.05) were reversed significantly. The animal experiment further confirmed thatthe sFRP1 inhibitor significantly reduced bone loss of periapical lesions in vivo (P<0.0001). Wnt antagonist sFRP1 was involved in the osteogenic differentiation of hPDLCs under inflammation. Modulation of the Wnt/β-catenin signalling pathway through the inhibition of sFRP1 may offer a new perspective on the treatment of chronic apical periodontitis.

Lipopolysaccharides of Fusobacterium nucleatum and Porphyromonas gingivalis increase RANKL-expressing neutrophils in air pouches of mice

Increases of neutrophils and osteoclasts are pathological changes of periodontitis. RANKL is an osteoclast differentiation factor. The effect of periodontopathogen LPS on RANKL-expressing neutrophils has not been clarified yet. We evaluated numerical changes of RANKL-expressing neutrophils in air pouches of mice injected with LPSs of Fusobacterium nucleatum and Porphyromonas gingivalis. Mice with air pouches were assigned into saline (C)-, E. coli LPS- (Ec LPS)-, F. nucleatum LPS (Fn LPS)-, P. gingivalis LPS (Pg LPS)-, and Fn LPS and Pg LPS (Fn + Pg LPS)-injected groups. CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils in blood and air pouch exudates were determined by flow cytometry. In blood, compared to the C group, the Fn LPS group showed increases of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils whereas the Pg LPS group showed no significant differences. These increases in the Fn LPS group were not different to those in the Ec LPS group. In exudates, Fn LPS and Pg LPS groups showed increases of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils compared to the C group. Increased levels in the Fn LPS group were not different to those in the Ec LPS group, but Pg LPS group was lower than those in the Ec LPS group. In blood and exudates, the Fn + Pg LPS group showed no difference in levels of these neutrophils compared to the Ec LPS group. LPSs of F. nucleatum and P. gingivalis increased RANKL-expressing neutrophils although the degrees of increases were different. These suggest that periodontopathogen LPS can act as a stimulant to increase RANKL-expressing neutrophils.