Hidemi Kurihara

Hiroshima University, Hirosima, Hiroshima, Japan

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Publications (182)377.51 Total impact

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    ABSTRACT: Apoptosis is thought to contribute to the progression of periodontitis. It has been suggested that the apoptosis of epithelial cells may contribute to the loss of epithelial barrier function. Smad2, a downstream signaling molecule of TGF-β receptors (TGF-βRs), is critically involved in apoptosis in several cell types. However, the relationship between smad2 and bacteria-induced apoptosis has not yet been elucidated. It is possible that the regulation of apoptosis induced by periodontopathic bacteria may lead to novel preventive therapies for periodontitis. Therefore, in the present study, we investigated the involvement of smad2 phosphorylation in apoptosis of human gingival epithelial cells induced by Aggregatibacter actinomycetemcomitans (Aa). Aa apparently induced the phosphorylation of smad2 in primary human gingival epithelial cells (HGECs) or the human gingival epithelial cell line, OBA9 cells. In addition, Aa induced phosphorylation of the serine residue of the TGF-β type I receptor (TGF-βRI) in OBA9 cells. SB431542 (a TGF-βRI inhibitor) and siRNA transfection for TGF-βRI, which reduced both TGF-βRI mRNA and protein levels, markedly attenuated the Aa-induced phosphorylation of smad2. Furthermore, the disruption of TGF-βRI signaling cascade by SB431542 and siRNA transfection for TGF-βRI abrogated the activation of cleaved caspase-3 expression and repressed apoptosis in OBA9 cells treated with Aa. Thus, Aa induced apoptosis in gingival epithelial cells by activating the TGF-βRI-smad2-caspase-3 signaling pathway. The results of the present study may suggest that the periodontopathic bacteria, Aa, activates the TGF-βR/smad2 signaling pathway in human gingival epithelial cells and induces apoptosis in epithelial cells, which may lead to new therapeutic strategies that modulate the initiation of periodontitis.
    Journal of dental research. 09/2014;
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    ABSTRACT: Background and Objective Brain-derived neurotrophic factor (BDNF) promotes the regeneration of periodontal tissue. Although a local inflammatory step is required to initiate the subsequent process of tissue regeneration, excessive inflammation may inhibit or delay tissue regeneration. Therefore, the regulation of inflammation is essential for periodontal tissue regeneration. In the present study, we examined the influence of BDNF on the human microvascular endothelial cell (HMVEC) barrier dysfunction induced by interleukin (IL)-1β or tumor necrosis factor (TNF)-α to determine the effects of BDNF on the regulation of local inflammation in periodontal tissue regeneration.Material and Methods Endothelial permeability was analyzed using a Dextran transport assay with transwell plates. The expression of vascular endothelial (VE)-cadherin was assessed by immunoblotting and immunofluorescence microscopy.ResultsBDNF (25 ng/mL) inhibited increase induced in endothelial permeability by IL-1β and TNF-α. IL-1β and TNF-α decreased VE-cadherin protein levels, while BDNF recovered the reduction in HMVECs. BDNF protected the increase induced in endothelial permeability by IL-1β and TNF-α through TrkB. The single addition of BDNF into the culture increased the expression of VE-cadherin in HMVECs.ConclusionBDNF played an important role in inhibiting endothelial barrier dysfunction, which suggests that it may assist in enhancing periodontal tissue regeneration.
    Journal of Periodontal Research 09/2014; · 1.99 Impact Factor
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    ABSTRACT: Background and Objective Migration of the junctional epithelium occurs in association with the formation of a periodontal pocket. Although the migration of junctional epithelium is known to be related to the proliferation and migration of gingival junctional epithelial cells, the mechanism has not been clarified. In patients with periodontitis, the levels of interleukin-8 (IL-8) in both gingival tissue and gingival crevicular fluid are dramatically increased. IL-8 has broad bioactive functions. In this study, we examined the role of IL-8 in DNA synthesis, migration and protection against apoptosis in cultured human gingival epithelial cells (HGEC).Material and MethodsDNA synthesis was estimated by measuring the incorporation of bromodeoxyuridine. The migration of gingival epithelial cells was assessed in a wound-healing assay. The expression of integrin beta-1 was analyzed using immunofluorescence confocal microscopy and western blotting. Cleaved caspase-3 was detected using western blotting and a Caspase-Glo assay kit.ResultsIL-8 increased the synthesis of DNA in HGEC, and the maximal effect was seen at 25 or 50 ng/mL of IL-8. In addition, 50 ng/mL of IL-8 induced cell migration, and a neutralizing antibody of integrin beta-1 inhibited the migration. IL-8 also activated expression of integrin beta-1. Furthermore, IL-8 reduced the Aggregatibacter actinomycetemcomitans-induced increase in caspase-3 expression in HGEC.ConclusionIL-8 may facilitate the migration of gingival junctional epithelium by enhancing DNA synthesis, migration and preventing apoptosis of gingival epithelial cells.
    Journal of Periodontal Research 09/2014; · 1.99 Impact Factor
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    ABSTRACT: Background and Objective Periodontitis is an infectious disease caused by an interaction between the host and periodontopathogenic bacteria. Regulating the immune response in human gingival epithelial cells (HGEC) may contribute to the prevention of periodontitis. Irsogladine maleate (IM) has previously been shown to regulate inflammation and the cell–cell junctional barrier in HGEC. In addition to these functions, control of bacterial recognition is important for preventing inflammation in periodontal tissue. Innate immunity in gingival epithelium is the first line of defense and plays a crucial role against bacterial challenge. Therefore, the effect of IM on regulating toll-like receptor 2 (TLR2), which is part of the innate immunity, was determined in this study.Material and MethodsOBA-9, an immortalized human gingival epithelial cell line, and primary cultured HGEC were used in this study. Real-time PCR and western blotting were performed in OBA-9 or HGEC stimulated with whole cells of Porphyromonas gingivalis or with lipopolysaccharide (LPS) derived from P. gingivalis (PgLPS) in the presence or absence of IM to determine expression of TLR2 mRNA and production of TLR2 protein. Small interfering RNA (siRNA) against TLR2 was transfected into OBA-9 to clarify the association between the induction of TLR2 and interleukin-8 (IL-8) production.ResultsThe addition of IM into P. gingivalis or PgLPS-induced OBA-9 suppressed IL-8 production (p < 0.01). The addition of IM also abolished the induction of TLR2 by P. gingivalis or PgLPS in OBA-9 and primary cultured HGEC (p < 0.01). The suppressive effect of IM on the induction of TLR2 was also confirmed by immunohistostaining. Stimulation with peptidoglycan, a specific ligand for TLR2, suppressed the expression of toll-like receptor 4 (TLR4) mRNA in the presence of IM (p < 0.01). However, LPS derived from Escherichia coli, a ligand for TLR4, did not induce the expression of TLR2 mRNA. The PgLPS-induced expression of TLR4 mRNA was abolished by IM. Knockdown of TLR2 by siRNA transfection resulted in a weaker response of induction of IL8 mRNA in P. gingivalis or PgLPS-stimulated OBA-9.Conclusion These results suggest that IM suppresses the induction of IL-8 production by regulating increased levels of TLR2.
    Journal of Periodontal Research 09/2014; · 1.99 Impact Factor
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    ABSTRACT: AimTo examine the in vitro effects of LL37 on the expression of vascular endothelial growth factor (VEGF) in human pulp cells, and to identify the intracellular signaling pathway involved.MethodologyPulp cells at passage 6 were treated with 10 μg mL−1 synthesized LL37, and an inhibition assay was performed with MAPK or NF-κB inhibitors to determine the possible signaling pathway. VEGF mRNA, VEGF protein and phosphorylated ERK1/2 levels were determined by real-time PCR, ELISA and Western blot, respectively. Data were analyzed using t-tests.ResultsLL37 significantly increased both the mRNA and protein levels of VEGF in pulp cells (p < 0.01). However, the pretreatment with an ERK kinase inhibitor suppressed these increases. Furthermore, the inhibitor blocked LL37-induced ERK1/2 phosphorylation.ConclusionsLL37 activated the ERK pathway to boost VEGF secretion from human pulp cells. Because of this angiogenic effect and its reported induction of pulp cell migration and antimicrobial activity against cariogenic bacteria, LL37 may be applicable as a pulp capping material.This article is protected by copyright. All rights reserved.
    International Endodontic Journal 08/2014; · 2.05 Impact Factor
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    ABSTRACT: Objective: In periodontitis, gingival epithelium represents the first line of defense against pathological microbial invasion or challenge and plays a crucial role as a mechanical barrier. Transforming growth factor β1 (TGF-β1) is important cytokines with pleiotrophic properties to regulate a broad spectrum of cellular processes including cell growth, differentiation, and apoptosis. Interestingly, TGF-β1 levelis elevated in gingival crevicular fluid obtained from periodontal pockets, however, the role of TGF-β1 on gingival epithelium is still unclear. Accordingly, in this study, we aimed to investigate whether TGF-β1 affects gingival epithelial cells viability and to reveal its molecular mechanism. Method: OBA9 cells (an immortalized human gingival epithelial cell line, kindly gifted by Prof. Murakami, Osaka University) were cultured with Humedia-KB2 medium. OBA9 cells were treated with TGF-β1, then, to assess the cell viability and apoptotic cells, MTS assay and TUNEL staining were conducted. The signal transduction molecules relating to the cell death were examined by Western blotting. Furthermore, to investigate the role of TGF-β type I receptor(TβRI)on the cell viability and apoptosis in the signaling event, siRNA transfection was performed. Result: TGF-β1 reduced the cell viability and increased apoptotic cells. Consistent with this result, the level of cleaved caspase3 and caspase9 were remarkably elevated. TGF-β1 up-regulated the expression of mitochondria-dependent apoptosis related proteins, such as Bak, Bax, Bim, and Bad. Otherwise, anti-apoptotic molecules, Bcl-2, Bcl-x(L) were obviously decreased in OBA9 cells by TGF-β1 treatment. TGF-β1 treatment facilitated the phosphorylation of smad2, which is downstream target of TβRI. The disruption of the TβRI by TβRI siRNA abrogated its phosphorylation and cleaved caspase3/9 expression induced by TGF-β1. TβRI siRNA significantly attenuated TGF-β1-induced cell apoptosis. Conclusion: These findings suggested that TGF-β1 activates mitochondria-dependent apoptosis in gingival epithelial cells via TβRI -smad2 pathway. TGF-β1 may play a role in the onset or progression of periodontitis.
    IADR General Session and Exhibition 2014; 06/2014
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    ABSTRACT: This study aimed to observe the regenerative effect of brain-derived neurotrophic factor (BDNF) in a non-human primate furcation defect model. Class II furcation defects were created in the first and second molars of 8 non-human primates to simulate a clinical situation. The defect was filled with either, Group A: BDNF (500 µg/ml) in high-molecular weight-hyaluronic acid (HMW-HA), Group B: BDNF (50 µg/ml) in HMW-HA, Group C: HMW-HA acid only, Group D: empty defect, or Group E: BDNF (500 µg/ml) in saline. The healing status for all groups was observed at different time-points with micro computed tomography. The animals were euthanized after 11 weeks, and the tooth-bone specimens were subjected to histologic processing. The results showed that all groups seemed to successfully regenerate the alveolar buccal bone, however, only Group A regenerated the entire periodontal tissue, i.e., alveolar bone, cementum and periodontal ligament. It is suggested that the use of BDNF in combination with a scaffold such as the hyaluronic acid in periodontal furcation defects may be an effective treatment option.
    PLoS ONE 01/2014; 9(1):e84845. · 3.53 Impact Factor
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    ABSTRACT: Brain-derived neurotrophic factor (BDNF) activates its receptor TrkB, and promotes neuronal survival, differentiation, and synaptic functions. Furthermore, we have revealed that BDNF can also regulate cementoblast differentiation and cellular survival via TrkB–ERK/Akt signaling cascade, which, in turn, results in the induction of periodontal tissue regeneration. Recently, using in silico screening with a BDNF loop-domain pharmacophore, a small molecule BDNF mimetic, called LM22A-4 that can facilitate TrkB signaling in hippocampal neurons to prevent cell death, was identified. Therefore, this study aimed to investigate the effect of LM22A-4 on cementoblast differentiation and its molecular mechanism. LM22A-4 and BDNF stimulation was found to enhance OPN, ALPase, and OC mRNA expression in immortalized human cementoblast-like (HCEM) cells, indicating cementoblast differentiation. In addition, similar to this result, both LM22A-4 and BDNF treatment facilitated TrkB phosphorylation and TrkB binding to adaptor proteins, such as Shc, GRB2, and SOS1, indicating TrkB activation. Importantly, the downstream target ERK and Akt was also phosphorylated by LM22A-4 and BDNF stimulation. Moreover, BDNF mimetic stimulation transactivated ERK from the cytoplasm into the nuclei in HCEM cells. It is noteworthy that a tyrosine kinase receptor inhibitor, K252a, an MEK–ERK inhibitor (U0126), and a PI3Kinase–Akt inhibitor (LY294002) remarkably attenuated TrkB, ERK, and Akt phosphorylation as well as increase of OPN mRNA expression in the HCEM cells, respectively. These findings suggest that the small molecule BDNF mimetic LM22A-4 regulates cementoblast differentiation via the TrkB–ERK/Akt signaling cascade. Therefore, this small compound may lead to the development of a novel therapeutic approach for periodontal tissue regeneration.
    International immunopharmacology 01/2014; · 2.21 Impact Factor
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    ABSTRACT: Gingival epithelium is the primary barrier against microorganism invasion and produces inflammatory cytokines. Amphotericin B, a major antifungal drug, binds to cholesterol in the mammalian cell membrane in addition to fungal ergosterol. Amphotericin B has been shown to regulate inflammatory cytokines in host cells. To investigate the suppressive effect of amphotericin B on the gingival epithelium, we examined the expression of interleukin (IL)-8 and IL-6 and involvement of MAP kinase in human gingival epithelial cells (HGEC) stimulated by Aggregatibacter actinomycetemcomitans. Amphotericin B and the p38 MAP kinase inhibitor down-regulated the A. actinomycetemcomitans-induced increase in the expression of IL-8 and IL-6 at the mRNA. The ERK inhibitor suppressed the A. actinomycetemcomitans-induced IL-8 mRNA expression. Amphotericin B inhibited the A. actinomycetemcomitans-induced phosphorylation of ERK and p38 MAP kinase. Furthermore, amphotericin B inhibited the A. actinomycetemcomitans-induced production of prostaglandin E2. These results suggest that amphotericin B regulate inflammatory responses in HGEC.
    Cellular Immunology. 01/2014;
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    ABSTRACT: Background/purpose Periodontitis is an infectious inflammatory disease. Gingival epithelium is the primary barrier against invasion of microorganisms and produces inflammatory cytokines. Our previous studies showed that regulating the function on the gingival epithelium was useful in suppressing the onset of periodontal disease. Houttuynia cordata is commonly used in traditional oriental medicine formulations and has been associated with a broad range of pharmacological activities. To investigate the potential of H. cordata as a preventive medicine, we examined the effect of H. cordata on the expression of inflammatory-related genes in human gingival epithelial cells (HGECs) exposed to Aggregatibacter actinomycetemcomitans. Materials and methods The messenger RNA (mRNA) levels of matrix metalloproteinase-3 (MMP-3), interleukin (IL)-8, IL-6, and intercellular adhesion molecule-1 (ICAM-1) were determined by real-time polymerase chain reaction. Results A. actinomycetemcomitans facilitated the mRNA expression of MMP-3, IL-8, IL-6, and ICAM-1 in HGECs, whereas these mRNA levels were attenuated by the H. cordata treatment. In addition, the extracellular signal-regulated kinase (ERK) signaling cascade, which was reported to be involved in A. actinomycetemcomitans-induced increases in MMP-3, IL-8, or ICAM-1, was disturbed by the H. cordata treatment in HGECs. Furthermore, H. cordata also inhibited the tumor necrosis factor-α-induced enhancement in these genes in HGECs. Conclusion These results suggest that H. cordata suppresses the expression of inflammatory-related genes in gingival epithelial cells by inhibiting the ERK signaling cascade. This might result in the suppression of inflammatory response in gingival epithelium, thereby contributing to the prevention of periodontitis.
    Journal of Dental Sciences. 01/2014;
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    ABSTRACT: Background: MicroRNA (miRNA) are short non-coding RNAs that are involved in post-transcriptional regulation of gene expression. The differential miRNA expression in innate and acquired immunity has been shown to regulate immune cell development and function. miRNA expression has been demonstrated to affect pathophysiology of inflammatory disease, such as rheumatoid arthritis and lupus. As such, this study explores the role of miRNA in the context of pathophysiology of destructive periodontitis. Specifically, this investigation profiles the differentially expressed miRNA of Porphyronomas gingivalis stimulated human gingival epithelial cells (HGEC). Methods: The specific miRNA differentially expressed in Pg-stimulated OBA-9, immortalized HGEC, were analyzed using microarray. Real-time PCR and Western blotting were performed to confirm the level of miRNA expression and to determine target production of miRNA in OBA-9. The production of interleukin (IL)-8 was measured to determine the bioactivity of target protein regulated by miRNA. Results: miR-584, which targets lactoferrin receptor (LfR), was 3.39-fold up-regulated by Pg-stimulation. This up-regulation of miR-584 was confirmed by real-time PCR. Pg- stimulation resulted in the suppression of LfR at mRNA and protein levels. The transfection of the miR inhibitor for miR-584 in OBA-9 recovered Pg-induced suppression of LfR. The addition of human lactoferrin (hLf) had a suppressive effect on IL-8 production in Pg-stimulated OBA-9. However, hLf also decreased IL-8 production strongly in Pg-stimulated OBA-9 in the presence of the miR inhibitor for miR-584. Conclusion: These findings suggest that the up-regulation of miR-584 by Pg in OBA-9 inhibits the anti-inflammatory effects of hLf via the suppression of LfR.
    Journal of Periodontology 11/2013; · 2.40 Impact Factor
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    ABSTRACT: Brain-derived neurotrophic factor (BDNF) enhances periodontal tissue regeneration. Tissue regeneration is characterized by inflammation that directs the quality of tissue repair. In this study, we investigated the anti-apoptotic effect of BDNF against the toxicity of tumor necrosis factor α (TNFα), which is known for its pro-apoptotic action in human microvascular endothelial cells (HMVECs). We demonstrate that BDNF attenuates TNFα-increased Annexin V-positive cells, lactic dehydrogenase (LDH) release, and intercellular adhesion molecule 1 (ICAM-1) mRNA and cleaved caspase-3 expression. In addition, biochemical analyses indicate that TNFα increases phosphatase and tensin homolog (PTEN) expression; however, it decreases phosphorylated PTEN. BDNF did not affect PTEN expression, but it did increase the phosphorylation of PTEN. BDNF-induced Akt phosphorylation was inhibited by TNFα. Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay showed that the PTEN inhibitor bpV(pic) rescues HMVECs from TNFα-induced apoptosis. In conclusion, BDNF protects HMVECs from toxicity of TNFα through the regulation of the PTEN/Akt pathway.
    Biochemistry and Cell Biology 10/2013; 91(5):341-9. · 2.92 Impact Factor
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    ABSTRACT: Human bone marrow mesenchymal stromal cells are useful in regenerative medicine for various diseases, but it remains unclear whether the aging of donors alters the multipotency of these cells. In this study, we examined age-related changes in the chondrogenic, osteogenic and adipogenic potential of mesenchymal stromal cells from 17 donors (25-81 years old), including patients with or without systemic vascular diseases. All stem cell lines were expanded with fibroblast growth factor-2 and then exposed to differentiation induction media. The chondrogenic potential was determined from the glycosaminoglycan content and the SOX9, collagen type 2 alpha 1 (COL2A1) and aggrecan (AGG) messenger RNA levels. The osteogenic potential was determined by monitoring the alkaline phosphatase activity and calcium content, and the adipogenic potential was determined from the glycerol-3-phosphate dehydrogenase activity and oil red O staining. Systemic vascular diseases, including arteriosclerosis obliterans and Buerger disease, did not significantly affect the trilineage differentiation potential of the cells. Under these conditions, all chondrocyte markers examined, including the SOX9 messenger RNA level, showed age-related decline, whereas none of the osteoblast or adipocyte markers showed age-dependent changes. The aging of donors from young adult to elderly selectively decreased the chondrogenic potential of mesenchymal stromal cells. This information will be useful in stromal cell-based therapy for cartilage-related diseases.
    Cytotherapy 06/2013; · 3.06 Impact Factor
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    ABSTRACT: LL37, an antimicrobial peptide, exhibits multiple bio-functions in various types of cells, including migration, cytokine production, apoptosis, and angiogenesis. Neovascularization and the subsequent recruitment of stem cells are essential for tissue engineering therapy, including bone regeneration. We hypothesized that LL37 can facilitate successful bone regeneration. To prove this hypothesis, the present study tested the effects of LL37 on bone formation in a rat calvarial bone defect model. Synthesized LL37 markedly induced newly formed bone. Interestingly, morphologically fibroblastic cells were observed in animals treated with LL37 on day 7, the early stage of tissue regeneration, which were positive for STRO-1, a marker of mesenchymal stem cells (MSCs), and accumulated in the bone defect area where cells positive for CD34, a marker of endothelial cells, were also localized. In addition, LL37 stimulated tube formation by endothelial cells and the proliferation of MSCs in vitro. These findings demonstrated for the first time that LL37 can regulate angiogenesis and the recruitment of stem cells to promote bone regeneration.
    Peptides 06/2013; · 2.52 Impact Factor
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    ABSTRACT: Objective: Transplanted human mesenchymal stem cells (hMSCs) have potential to regenerate periodontal tissue defect. The neighboring periodontal cells might affect the proliferation and differentiation of the transplanted hMSCs through direct cell-cell contact or humoral factors in the site. In this study, we focused on humoral factors secreted by human gingival fibroblast (HGF) or human periodontal ligament cells (HPL cells) in osteogenic differentiation of hMSCs. Method: hMSCs were stimulated by co-culturing with HGF or HPL cells in Transwell®. For osteogenesis, the cells were co-cultured in osteogenic induction medium for 3 weeks. Total RNA was isolated from hMSCs at 3 days, 1 week and 2 weeks, and mRNA expression of osteogenic differentiation markers was examined by real-time PCR. To examine expression of undifferentiationed MSC markers, transcription factors, ETV1, ETV5, KLF12, GATA6, SOX11 and SIM2, and expression of microRNA, hMSCs were co-cultured for 7 days. Total RNA was isolated from hMSCs at 1 day, 3 days and 7 days, and mRNA and microRNA expression were examined by real-time PCR. Result: Humoral factors secreted by HGF or HPL cells suppressed calcification and the mRNA expression for osteocalcin, BMP2 and Runx2. ETV1, ETV5 and KLF12 in co-cultured hMSCs with HGF were increased, and GATA6 was decreased. In addition, the co-cultured hMSCs with HPL cells increased SOX11 and SIM2, and decreased GATA6. Moreover, humoral factors secreted by them upregulated eight microRNAs and downregulated eight microRNAs of 377 microRNAs. Conclusion: These data suggested the humoral factors secreted by HGF and HPL cells play important roles on the suppression of osteogenic differentiation of hMSCs through the regulation of transcription factors and microRNAs.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
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    ABSTRACT: Objective: Amphotericin B is a polyene anti-fungal agent and has a high affinity for ergosterol. Amphotericin B has been used for periodontal therapy by some dentists, although there is no evidence. Periodontitis is inflammatory disease induced by periodontal bacteria. Since fungus is not periodontal pathogenesis, amphotericin B may regulate host cell functions in inflammatory condition. Gingival epithelium is primary barrier against microorganism invasion, and produces inflammatory cytokines such as interleukin (IL)-6 and IL-8. Thus, the interaction between epithelial cells and bacteria plays an important role in the initial stage of inflammation. In this study, we investigated the effect of amphotericin B on the expression of inflammatory cytokines in human gingival epithelial cells (HGEC) stimulated by Aggregatibactor actinomycetemcomitans (A.a ). Method: HGEC were prepared from healthy gingival tissue(D47-2). HGEC was stimulated by amphotericin B, or A.a in the presence of amphotericin B. ERK inhibitor (PD98059) or p38 MAPK inhibitor (SB203580) was added to analyze the signaling pathway. To investigate anti-inflammatory effects of amphotericin B, IL-6 and IL-8 expressions at mRNA and protein levels were examined by realtimePCR and ELISA, respectively. The phosphorylation of ERK and p38 MAPK were determined by Western blotting. Result: Amphotericin B or A.a increased IL-6 and IL-8, and the increase by A.a is more than amphotericin B. However, amphotericin B suppressed A.a induced increase in IL-6 and IL-8 at the mRNA and protein levels in HGEC. SB203580 suppressed A.a-induced up-regulation on IL-6 mRNA, and PD98059 suppressed up-regulation on IL-8 mRNA. In addition, amphotericin B or A.a induced phosphorylation of ERK and p38 MAPK, although the pretreatment of amphotericin B inhibited the A.a-induced phosphorylation. Conclusion: These findings suggested that amphotericin B down-regulates inflammatory responses of gingival epithelium as a partial agonist and may relief clinical symptoms of periodontitis.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
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    ABSTRACT: Objective: Gingival crevicular fluid (GCF) is one of the most robust biological samples for the study of periodontal disease (PD). Thus far, however, no method for monitoring biomarkers of mouse GCF has been reported. Therefore, this study aims to establish a new method of detecting GCF in a mouse PD model. Method: To induce PD, a sterilized 5-0 silk suture ligature was placed around the maxillary second molar on the left side (inflammatory site) of wild-type mice (C57BL/6 6- to 8-week-old males), while the right side was left untreated (healthy site). After placement of ligature for 1, 3, 5, and 7 days, the original suture was removed, and a GCF-sampling suture was placed at left side as well as right side for 10 min, respectively. The GCF-sampling sutures were submerged in PBS containing 0.05% Tween 20 (100 ml) for the detection of cytokines using ELISA. The levels of alveolar bone resorption were evaluated by surgical microscope and μCT. Result: The levels of IL-1β, TNF-β and IL-6 in the GCF-sampling suture of inflammatory site were significantly higher than those of the healthy site throughout all detection times, peaking at 1 day (*P <0.05). However, in contrast to GCF, cytokines, as detected in saliva and serum, were remarkably lower at all sampling times, indicating the absence of saliva/serum contamination. Based on the microscopic and μCT analyses, inflammation site also showed significantly elevated level of bone resorption (P<0.05) at 7 days, together with elevated sRANKL in GCF (P<0.05), compared to the healthy site. Conclusion: Using a silk suture, we were able to induce PD and monitor cytokines secreted in GCF of mice, thus expanding the capability of non-invasive longitudinal monitoring of host-derived biomarkers in GCF using a mouse PD model. Supported by NIDCR grants, DE-003420, DE-018499, DE-021873 and DE-019917.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
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    ABSTRACT: Objective: Brain-derived neurotrophic factor (BDNF) activates its receptor trkB, promoting neuronal survival, differentiation, and synaptic function. Moreover, we have revealed that BDNF can also regulate cementoblast differentiation via trkB-ERK signaling cascade, which, in turn, results in the induction of periodontal tissue regeneration. Otherwise, very recently, using in silico screening with a BDNF loop-domain pharmacophore, small molecule BDNF mimetic, LM22A-4, which can facilitate trkB signaling in hippocampal neurons to prevent cell death, was identified. Therefore, this study aimed to investigate the effect of BDNF mimetic small compound, LM22A-4 on cementoblasts differentiation and its molecular mechanism. Method: LM22A-4 was chemically synthesized. Immortalized human cmentoblast-like cells (HCEMs), established with transfection of hTERT gene were used in the following experiments. HCEMs were exposed to LM22A-4 in the presence or absence of K252a, which is a tyrosine kinase receptor specific inhibitor. The mRNA expressions of osteopontin (OPN), alkaline phosphatase (ALP) and osteocalcin (OC) were analyzed by real-time PCR. The phosphorylation of ERK was measured by Western blotting. Result: LM22A-4, as well as BDNF stimulation, enhanced the mRNA expression of OPN, ALP, and OC in HCEMs. In addition, similar to this result, both LM22A-4 and BDNF treatment elevated the phosphorylation of ERK. It is noteworthy that a tyrosine kinase receptor inhibitor, K252a remarkably abrogated the increase of the mRNA levels and ERK phosphorylation. Conclusion: These findings suggested that small molecule BDNF mimetic, LM22A-4, regulates cementoblast differentiation through trkB-ERK signaling pathway. Therefore, this small compound may lead to the development of therapeutic novel approach for the periodontal tissue regeneration.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
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    ABSTRACT: BACKGROUND: We investigated the effects of dental infection with Porphyromonas gingivalis (P.g.), an important periodontal pathogen, on NASH progression, by feeding mice a high fat diet (HFD)and examining P.g. infection in the liver of NASH patients. METHODS: C57BL/6J mice were fed either chow-diet (CD) or HFD for 12 weeks, and then half of the mice in each group were infected with P.g. from the pulp chamber (HFD-P.g.(-), HFD-P.g.(+), CD-P.g.(-) and CD-P.g.(+)). Histological and immunohistochemical examinations, measurement of serum lipopolysaccharide (LPS) levels and ELISA for cytokines in the liver were performed. We then studied the effects of LPS from P.g. (P.g.-LPS) on palmitate-induced steatotic hepatocytes in vitro, and performed immunohistochemical detection of P.g. in liver biopsy specimens of NASH patients. RESULTS: Serum levels of LPS are upregulated in P.g.(+) groups. Steatosis of the liver developed in HFD groups, and foci of Mac2-positive macrophages were prominent in HFD-P.g.(+). P.g. was detected in Kupffer cells and hepatocytes. Interestingly, areas of fibrosis with proliferation of hepatic stellate cells and collagen formation were only observed in HFD-P.g.(+). In steatotic hepatocytes, expression of TLR2, one of the P.g.-LPS receptors, was upregulated. P.g.-LPS further increased mRNA levels of palmitate-induced inflammasome and proinflammatory cytokines in steatotic hepatocytes. We demonstrated for the first time that P.g. existed in the liver of NASH patients with advanced fibrosis. CONCLUSIONS: Dental infection of P.g. may play an important role in NASH progression through upregulation of the P.g.-LPS-TLR2 pathway and activation of inflammasomes. Therefore, preventing and/or eliminating P.g. infection by dental therapy may have a beneficial impact on management of NASH.
    Journal of Gastroenterology 01/2013; · 3.79 Impact Factor
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    ABSTRACT: Periodontitis is an infectious inflammatory disease. Our previous studies have revealed that irsogladine maleate (IM) regulates intercellular junctional function and chemokine secretion in gingival epithelium, resulting in the suppression of the onset of periodontal disease in a rat model. Therefore, it is plausible that IM is a promising preventive remedy for periodontal disease. In this study, to gain a better understanding of IM in gingival epithelial cells, we employed a DNA microarray analysis. More specifically, human gingival epithelial cells (HGEC) were exposed to Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) in the presence or absence of IM. Then, a human genome focus array was used. A. actinomycetemcomitans facilitated the expression of several inflammatory-related genes, including these for matrix metalloproteinase (MMP)-3, interleukin (IL)-6, and ICAM-1 in HGEC, while these mRNA levels were attenuated by IM treatment. Importantly, consistent with mRNA levels, immunoblotting, immunofluorescence staining and ELISA analysis indicated that IM also abrogated the A. actinomycetemcomitans-induced increase in MMP-3, IL-6, and ICAM-1 at the protein level. In addition, inhibition of the ERK or p38 MAP kinase signaling cascade, previously reported to be disturbed by IM treatment in HGEC, clearly blocked A. actinomycetemcomitans-induced MMP-3, IL-6, or ICAM-1 protein expression. Moreover, animal study revealed that IM-pretreatment inhibited the A. actinomycetemcomitans-induced increase of ICAM-1 in gingival junctional epithelium. Taken together, these results suggested that IM can regulate inflammatory responses in HGEC by inhibiting the ERK or p38 MAP kinase signaling cascade, which may result in suppression of inflammation in gingival tissue, thereby contributing to the prevention of periodontitis.
    International immunopharmacology 01/2013; · 2.21 Impact Factor

Publication Stats

3k Citations
377.51 Total Impact Points

Institutions

  • 2001–2014
    • Hiroshima University
      • • Department of Periodontal Medicine
      • • Department of Special Care Dentistry
      • • Department of Bacteriology
      • • Department of Dental and Medical Biochemistry
      • • Faculty of Dentistry
      Hirosima, Hiroshima, Japan
  • 1991–2009
    • Okayama University
      • Department of Microbiology
      Okayama, Okayama, Japan
  • 2000–2005
    • Boston University
      • Department of Periodontology and Oral Biology
      Boston, MA, United States
  • 2003
    • University of Louisville
      Louisville, Kentucky, United States
    • The Forsyth Institute
      Cambridge, Massachusetts, United States
  • 1995
    • Osaka Dental University
      Ōsaka, Ōsaka, Japan
  • 1986
    • Rikkyo University
      • Institute for Atomic Energy
      Edo, Tōkyō, Japan