93 research outputs found

    AGEs increase lipocalin 2 expression

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    Background and Objectives: Diabetes mellitus (DM), a risk factor of periodontal diseases, exacerbates the pathological condition of periodontitis. A major factor for DM complications is advanced glycation end-products (AGEs) that accumulate in periodontal tissues and cause inflammatory events. Lipocalin 2 (LCN2) is an antimicrobial peptide and inflammation-related factor, and LCN2 levels increase in DM. In the present study, the effects of AGEs and lipopolysaccharide of Porphyromonas gingivalis (P.g-LPS) on LCN2 expression in human oral epithelial cells (TR146 cells) and the role of secreted LCN2 in periodontitis with DM were investigated. Material and Methods: TR146 cells were cultured with AGEs (AGE2) and control BSA and cell viability was estimated, or with P.g-LPS. Conditioned medium and cell lysates were prepared from cultures of epithelial cells and used for western blotting and ELISA to analyze LCN2, RAGE, IL-6, MAPK and NF-κB. RNA was isolated from AGE-treated TR146 cells and differentiated HL-60 (D-HL-60) cells and used for quantitative real-time PCR to examine the expression of LCN2 and interleukin-6 (IL-6) mRNAs. RAGE- and LCN2-siRNAs (siRAGE, siLCN2) were transfected into epithelial cells, and AGE-induced LCN2 expression was investigated. D-HL-60 cells were co-cultured with TR146 cells that were transfected with siLCN2 and treated with AGEs, IL-6 mRNA expression in D-HL-60 cells and cell migration were investigated. Results: AGEs increased the expression levels of LCN2 and IL-6 in oral epithelial cells. siRAGE and a neutralizing antibody for RAGE inhibited AGE-induced LCN2 expression. AGEs stimulated the phosphorylation of ERK, p38 and NF-kB in epithelial cells, and their inhibitors suppressed AGE-induced LCN2 expression. In contrast, P.g-LPS did not show a significant increase on LCN2 level in TR146 cells that expressed toll-like receptor 2. In co-culture experiments, AGE-induced LCN2 inhibited IL-6 mRNA expression in D-HL-60 cells, and LCN2 knockdown in epithelial cells suppressed HL-60 cell migration. Conclusion: These results suggested that AGEs increase LCN2 expression via RAGE, MAPK, and NF-κB signaling pathways in oral epithelial cells, and secreted LCN2 may influence the pathological condition of periodontitis with DM

    β-defensin 2 synthesized by a cell-free protein synthesis system and encapsulated in liposomes inhibits adhesion of Porphyromonas gingivalis to oral epithelial cells

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    β-defensin 2 (BD-2), an antimicrobial peptide (AMP), is expressed by oral epithelial cells and plays an important role in innate immunity of the oral cavity. Cell-free protein synthesis (CFPS) systems have been studied for the synthesis of various proteins, however, the synthesis of BD-2 by a CFPS system has not been extensively explored. Liposomes have been developed as tools for drug delivery. A delivery of liposome-encapsulated AMP to oral epithelium may be useful to prevent oral infectious diseases. In the present study, we investigated the antimicrobial activity of the BD-2 protein, artificially synthesized using a CFPS system and encapsulated in liposomes. BD-2 protein was artificially synthesized using template DNA and a reconstituted CFPS system and was identified by western blotting. Bilayer liposomes were prepared using 1,2-dioleoyl-sn-glycero-3-phospho-choline and 3-sn-phosphatidylcholine from egg yolk. The artificially synthesized BD-2 was encapsulated in liposomes, collected by ultrafiltration, and detected by western blotting. Human oral epithelial cells were cultured with the liposome-encapsulated BD-2 and the concentration of BD-2 in the cell lysate of the culture with the synthesized BD-2 was higher than that of the control cultures. The antimicrobial activity of the synthesized BD-2 was investigated by an adhesion assay of Porphyromonas gingivalis to oral epithelial cells. The artificially synthesized BD-2 and its liposome significantly inhibited adhesion of P. gingivalis to oral epithelial cells. These results suggest that artificially synthesized BD-2 and liposome-encapsulated BD-2 shows antimicrobial activity and can potentially play a role in oral healthcare for periodontal diseases

    Gan-Lu-Yin (Kanroin), Traditional Chinese Herbal Extracts, Reduces Osteoclast Differentiation In Vitro and Prevents Alveolar Bone Resorption in Rat Experimental Periodontitis

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    Gan-Lu-Yin (GLY), a traditional Chinese herbal medicine, shows therapeutic effects on periodontitis, but that mechanism is not well known. This study aims to clarify the precise mechanism by investigating the inhibitory effects of GLY extracts on osteoclastogenesis in vitro and on bone resorption in periodontitis in vivo. RAW264.7 cells are cultured with soluble receptor activator of nuclear factor-kappa B (sRANKL) and GLY extracts (0.01–1.0 mg/mL), and stained for tartrate-resistant acid phosphatase (TRAP) to evaluate osteoclast differentiation. Experimental periodontitis is induced by placing a nylon ligature around the second maxillary molar in rats, and rats are administered GLY extracts (60 mg/kg) daily for 20 days. Their maxillae are collected on day 4 and 20, and the levels of alveolar bone resorption and osteoclast differentiation are estimated using micro-computed tomography (CT) and histological analysis, respectively. In RAW264.7 cells, GLY extracts significantly inhibit sRANKL-induced osteoclast differentiation at a concentration of more than 0.05 mg/mL. In experimental periodontitis, administering GLY extracts significantly decreases the number of TRAP-positive osteoclasts in the alveolar bone on day 4, and significantly inhibits the ligature-induced bone resorption on day 20. These results show that GLY extracts suppress bone resorption by inhibiting osteoclast differentiation in experimental periodontitis, suggesting that GLY extracts are potentially useful for oral care in periodontitis

    Calprotectin and cross-linked N-telopeptides of type I collagen levels in crevicular fluid from implant sites with peri-implant diseases : a pilot study

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    Background: Peri-implant crevicular fluid (PICF) contains calprotectin and NTx, which are markers for inflammation and bone resorption, respectively. The aims of this pilot study were to compare calprotectin and NTx levels in PICF from implant sites with or without peri-implant diseases and to evaluate the usefulness of calprotectin and NTx as diagnostic markers for peri-implant diseases. Methods: Thirty-five patients with dental implants participated in this pilot study. PICF samples were collected from peri-implant disease sites (n = 40) and non-diseased (healthy) sites (n = 34) after clinical indicators including probing depth (PD), bleeding on probing (BOP), gingival index (GI), and bone loss (BL) rate were investigated. Calprotectin and NTx amounts in PICF were measured using their respective ELISA kits and then compared between diseased and healthy samples. The relationship between PICF calprotectin or NTx levels and clinical indicator levels was investigated. A receiver operating characteristic (ROC) curve analysis of calprotectin and NTx was performed to predict peri-implant diseases. Results: Calprotectin and NTx levels in PICF were significantly higher from peri-implant disease sites than from healthy sites. PICF calprotectin amounts correlated with PD, and its levels were significantly higher in the GI-1 and GI-2 groups than in the GI-0 group. PICF NTx amounts correlated with PD and the BL rate. ROC curves indicated that PICF calprotectin and NTx are useful biomarkers for peri-implant diseases. Conclusions: Calprotectin and NTx in PICF have potential as biomarkers for the diagnosis of peri-implant diseases

    Lipocalin 2, synthesized using a cell-free protein synthesis system and encapsulated into liposomes, inhibits the adhesion of Porphyromonas gingivalis to human oral epithelial cells

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    Background and objective: Lipocalin 2 (LCN2), a glycoprotein expressed in epithelial cells and leukocytes, has an antibacterial effect and plays a role in innate immunity. The delivery of LCN2 encapsulated in liposomes to oral epithelium may be useful to prevent oral infectious diseases. This study aimed to investigate the inhibitory effect of LCN2, artificially synthesized using a cell-free protein synthesis (CFPS) system, on the adhesion of Porphyromonas gingivalis to oral epithelial cells in order to approach oral healthcare using LCN2. Methods: LCN 2 was synthesized using a CFPS system and assayed by Western blotting, mass spectrometry and enzyme-linked immunosorbent assay (ELISA). The bilayer liposomes were prepared by the spontaneous transfer method using 1,2-dioleoyl-sn-glycero-3 phosphocholine (DOPC), 3-sn-phosphatidylcholine from Egg Yolk (Egg-PC), and 1,2-dioleoyl-sn-glycero-3 phosphoethanolamine (DOPE). The cellular and medium fractions derived from the culture of oral epithelial cells with liposome-encapsulated LCN2 were assayed by Western blotting and ELISA. The effect of the synthesized LCN2 on adhesion of the labeled P. gingivalis to oral epithelial cells was investigated as an evaluation of its antibacterial activity. Results: The synthesized LCN2 protein was identified by Western blotting; its amino acid sequence was similar to that of recombinant LCN2 protein. The additions of DOPE and octa-arginine in the outer lipid-layer components of liposome significantly increased the delivery of liposomes to epithelial cells.When oral epithelial cells were cultured with the synthesized and liposome-encapsulated LCN2, LCN2 was identified in the cellular and medium fractions by Western blotting and its concentration in the cellular fraction from the culture with the synthesized LCN2 was significantly higher than that of a template DNA-free protein. The synthesized LCN2 and liposome-encapsulated LCN2 significantly inhibited the adhesion of P. gingivalis to oral epithelial cells compared with template DNA-free protein. Conclusion: LCN2 was artificially synthesized by a CFPS system, encapsulated in liposomes and delivered to oral epithelial cells, and demonstrated an antibacterial action against P. gingivalis. This approach may become a useful model for oral healthcare

    S100A9は、骨細胞様細胞においてMAPKsおよびSTAT3シグナル伝達経路を介してIL-6およびRANKLの発現を増加させる

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    Objective: Calprotectin is hetero-complex of S100A8 and S100A9 and mainly secreted from neutrophils, monocytes and chondrocytes in inflammatory condition. Calprotectin binds to RAGE and TLR4, and induces the expression of pro-inflammatory chemokines and cytokines in various cells. Periodontitis is chronic inflammatory disease to lead gingival inflammation and alveolar bone resorption. Calprotectin levels in gingival crevicular fluid of periodontitis patients are higher than healthy patients. In the present study, the effects of S100A8 and S100A9 on the expressions of pro-inflammatory cytokines and bone metabolism related factor in mouse osteocyte like cells (MLO-Y4-A2) were investigated. Design: MLO-Y4-A2 cells were treated with S100A8 and S100A9, and the expressions of RAGE, TLR4, RANKL and several inflammatory cytokines were analyzed by PCR and Western blotting or ELISA methods. To investigate the intracellular signaling pathways, phosphorylation of MAPK and STAT3 was determined by Western blotting, and chemical specific inhibitors and siRNAs were used. Results: Expressions of IL-6 and RANKL were increased by treatment with S100A9 but not S100A8. However, both S100A8 and S100A9 did not changed expression of IL-1β, IL-8 and TNF-α. Although RAGE and TLR4 expressions were not up-regulated by S100A9 treatment, transfection of siRNA for RAGE and TLR4 significantly decreased IL-6 and RANKL expressions. In addition, S100A9 activated p38, ERK and STAT3 signaling pathways, and inhibitors for these factors significantly decreased S100A9 induced IL-6 and RANKL expressions. Conclusions: These results indicated that S100A9 induces IL-6 and RANKL production via engagement with RAGE and TLR4 signalings in osteocytes and suggested that S100A9 may play important roles in the periodontal alveolar bone destruction

    ショウガオールはヒト歯肉線維芽細胞において酸化ストレス反応の調節を介してAGEs誘導性のIL-6およびICAM-1産生を抑制する

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    Advanced glycation end-products (AGEs) cause diabetes mellitus (DM) complications and accumulate more highly in periodontal tissues of patients with periodontitis and DM. AGEs aggravate periodontitis with DM by increasing the expression of inflammation-related factors in periodontal tissues. 6-Shogaol, a major compound in ginger, has anti-inflammatory and anti-oxidative activities. However, the influence of shogaol on DM-associated periodontitis is not well known. In this study, the effects of 6-shogaol on AGEs-induced oxidative and anti-oxidative responses, and IL-6 and ICAM-1 expression in human gingival fibroblasts (HGFs) were investigated. When HGFs were cultured with 6-shogaol and AGEs, the activities of reactive oxygen species (ROS) and antioxidant enzymes (heme oxygenase-1 [HO-1] and NAD(P)H quinone dehydrogenase 1 [NQO1]), and IL-6 and ICAM-1 expressions were investigated. RAGE expression and phosphorylation of MAPKs and NF-κB were examined by western blotting. 6-Shogaol significantly inhibited AGEs-induced ROS activity, and increased HO-1 and NQO1 levels compared with the AGEs-treated cells. The AGEs-stimulated expression levels of receptor of AGE (RAGE), IL-6 and ICAM-1 and the phosphorylation of p38, ERK and p65 were attenuated by 6-shogaol. These results suggested that 6-shogaol inhibits AGEs-induced inflammatory responses by regulating oxidative and anti-oxidative activities and may have protective effects on periodontitis with DM

    High Glucose Induces Severe Periodontitis.

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    Background/Aims: Diabetic patients are susceptible to severe periodontitis, but the precise mechanism is not fully understood. Aim of this study was to explore the biological pathogenesis of severe periodontitis in diabetic patients focusing on the crosstalk of human gingival fibroblasts (HGFs) and macrophages. Methods: A total of 70 periodontitis patients with or without diabetes mellitus (DM) were enrolled, and the statistical relationships of diabetic conditions to the periodontal inflammatory parameters were examined by cross-sectional study. In in vitro study, HGFs cell line CRL-2014® (ATCC) and differentiated THP-1 macrophages were cultured with normal glucose (NG: 5.5 mM) or high glucose (HG: 25 mM) condition, and treated with indicated inflammatory factors such as calprotectin (CPT), interleukin (IL)-1β and IL-6. To examine the effects of HG on soluble IL-6 receptor (sIL-6R) production in THP-1 macrophages, the supernatants were collected and the sIL-6R levels were measured by ELISA. To examine the effects of HG on IL-1β or IL-6-induced matrix metalloproteinase (MMPs) production in HGFs, the supernatants were collected. Levels of MMP-1 and tissue inhibitor of MMP-1 (TIMP-1) were measured by ELISA. Finally, after conditioned medium (CM) from THP-1 macrophages cultured with NG or HG conditions was collected, HGFs were treated with the CM. The supernatants were collected 24 hours later and the levels of MMP-1 and TIMP-1 were measured. To examine the specific effects of IL-1β contained in CM on MMP-1 and TIMP-1 production in HGFs, IL-1 receptor antagonist (IL-1ra) was used. Results: There were statistical correlation between IL-1β and sIL-6R levels in gingival crevicular fluid (GCF) and HbA1c in periodontitis patients with DM (IL-1β: P=0.035, sIL-6R: P=0.040). HG and CPT significantly induced sIL-6R production in THP-1 macrophages. HG significantly enhanced IL-1β or IL-6/sIL-6R-induced MMP-1 production in HGFs. The increase of MMP-1 by both IL-1β and IL-6/sIL-6R was significantly inhibited by specific ERK or IκB inhibitors. Corresponding to the regulation of MMP-1 production, HG condition increased the phosphorylation of p44/42 MAPK and IκBα in HGFs treated with IL-1β or IL-6/sIL-6R. Finally, MMP-1 production in HGFs cultured with HG increased significantly by CM from THP-1 macrophages cultured with HG. The induction of MMP-1 by the CM from THP-1 macrophages cultured with HG was significantly inhibited by dose dependent of IL-1ra in HGFs cultured with HG. Conclusion: Diabetic conditions such as HG induce IL-1β and sIL-6R production from macrophages in inflammatory periodontal tissues and may exacerbate the periodontitis synergistically via MMP-1 production from HGFs

    Glycated Albumin in Gingival Crevicular Fluid from Patients With Diabetes

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    Background: Diabetes mellitus (DM) patients have a high prevalence of periodontitis. DM-associated periodontitis (DM-P) is characterized by severe inflammation and tissue destruction. To diagnose DM-P is important for cures of periodontitis and DM. The purpose of this study was to investigate the levels of glycated albumin (GA), a DM marker, and calprotectin, an inflammatory marker, in gingival crevicular fluid (GCF) from patients with periodontitis and DM. Methods: Seventy-eight subjects participated in this study were the patients with DM, chronic periodontitis (CP), DM-P and healthy individuals (H). GCF and blood were collected from four groups. GA and calprotectin in GCF were analyzed using western blotting and ELISA, and their levels were compared among H, DM, CP, and DM-P groups. GA and glycated hemoglobin (HbA1c) in blood were determined, and the correlation between GCF GA level and blood HbA1c or GA level was investigated. ROC analysis for GCF GA level to predict DM was performed. Results: GA was identified in GCF, and its amount and concentration in GCF samples from DM and DM-P were significantly higher than those of non-DM groups (H and CP). Calprotectin amount in GCF from CP and DM-P was significantly higher than that in H and DM groups. GCF GA level was positively correlated to blood HbA1c and GA level. ROC analysis of GCF GA level showed an optimal cut-off value to predict DM. Conclusions: GA showed a high level in GCF from DM patients. GA and calprotectin in GCF may be useful markers to diagnose DM-associated periodontitis

    Effects of Candidalysin Derived from Candida albicans on the Expression of Pro-Inflammatory Mediators in Human Gingival Fibroblasts

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    Candida albicans (Ca) is frequently detected in the peri-implant sulcus with peri-implantitis, a major postoperative complication after oral implant therapy. However, the involvement of Ca in the pathogenesis of peri-implantitis remains unclear. In this study, we aimed to clarify Ca prevalence in the peri-implant sulcus and investigated the effects of candidalysin (Clys), a toxin produced by Ca, on human gingival fibroblasts (HGFs). Peri-implant crevicular fluid (PICF) was cultured using CHROMagar and Ca colonization rate and colony numbers were calculated. The levels of interleukin (IL)-1β and soluble IL-6 receptor (sIL-6R) in PICF were quantified by enzyme-linked immunosorbent assay (ELISA). Pro-inflammatory mediator production and intracellular signaling pathway (MAPK) activation in HGFs were measured by ELISA and Western blotting, respectively. The Ca colonization rate and the average number of colonies in the peri-implantitis group tended to be higher than those in the healthy group. IL-1β and sIL-6R levels in the PICF were significantly higher in the peri-implantitis group than in the healthy group. Clys significantly induced IL-6 and pro-matrix metalloproteinase (MMP)-1 productions in HGFs, and co-stimulation with Clys and sIL-6R increased IL-6, pro-MMP-1, and IL-8 production levels in HGFs compared with Clys stimulation alone. These findings suggest that Clys from Ca plays a role in the pathogenesis of peri-implantitis by inducing pro-inflammatory mediators
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