Publications by authors named "Miki Yamada-Hara"

9 Publications

  • Page 1 of 1

Rice bran-derived protein fractions enhance sulforaphane-induced anti-oxidative activity in gingival epithelial cells.

Arch Oral Biol 2021 Sep 24;129:105215. Epub 2021 Jul 24.

Division of Periodontology, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan. Electronic address:

Objective: Food-derived bioactive peptides have been reported to exhibit various beneficial effects, including anti-microbial, anti-inflammatory, and anti-oxidant properties. Oxidative stress has been implicated in the development of several inflammatory diseases such as periodontal disease. However, the anti-oxidative effect of food-derived bioactive peptides in gingival epithelial cells (GECs) is unknown. Therefore, we examined the bioactivity of the peptides in GECs.

Design: Food-derived peptide fractionations derived from rice bran, rice endosperm, corn, and soy were screened for anti-oxidative effects using anti-oxidant response element (ARE)-luciferase-transfected HEK 293 cells. The induction of anti-oxidation-related genes and proteins in GECs by the fractions were examined by quantitative PCR and Western blotting, respectively. Then, the fraction-mediated anti-oxidative effects were examined by measuring intracellular reactive oxygen species (ROS) levels using flow cytometry. Furthermore, the anti-oxidative response-related cellular signaling pathways were analyzed via Western blotting.

Results: Although treatment with the food-derived peptides alone did not activate anti-oxidative responses, co-treatment with sulforaphane (SFN; a potent anti-oxidant) and certain food-derived peptides enhanced anti-oxidative responses in ARE-luciferase-transfected HEK 293 cells. The fractions augmented heme oxygenase-1 mRNA and protein expression in GECs. The percentage of ROS-positive cells was significantly decreased by co-treatment with SFN and peptide fractions derived from rice bran. Furthermore, the involvement of both nuclear factor erythroid 2-related factor 2 (Nrf2) and extracellular signal-regulated kinase (ERK) in the enhancement of anti-oxidative responses was demonstrated by Western blotting.

Conclusions: Peptides derived from rice bran enhances SFN-induced anti-oxidative responses in GECs through ERK-Nrf2-ARE signaling.
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http://dx.doi.org/10.1016/j.archoralbio.2021.105215DOI Listing
September 2021

Obesity-Related Gut Microbiota Aggravates Alveolar Bone Destruction in Experimental Periodontitis through Elevation of Uric Acid.

mBio 2021 06 1;12(3):e0077121. Epub 2021 Jun 1.

Research Unit for Oral-Systemic Connection, Division of Oral Science for Health Promotion, Niigata University Graduate School of Medical and Dental Sciences, Chuo-ku, Niigata, Japan.

Obesity is a risk factor for periodontal disease (PD). Initiation and progression of PD are modulated by complex interactions between oral dysbiosis and host responses. Although obesity is associated with increased susceptibility to bacterial infection, the detailed mechanisms that connect obesity and susceptibility to PD remain elusive. Using fecal microbiota transplantation and a ligature-induced PD model, we demonstrated that gut dysbiosis-associated metabolites from high-fat diet (HFD)-fed mice worsen alveolar bone destruction. Fecal metabolomics revealed elevated purine degradation pathway activity in HFD-fed mice, and recipient mice had elevated levels of serum uric acid upon PD induction. Furthermore, PD induction caused more severe bone destruction in hyperuricemic than normouricemic mice, and the worsened bone destruction was completely abrogated by allopurinol, a xanthine oxidase inhibitor. Thus, obesity increases the risk of PD by increasing production of uric acid mediated by gut dysbiosis. Obesity is an epidemic health issue with a rapid increase worldwide. It increases the risk of various diseases, including periodontal disease, an oral chronic infectious disease. Although obesity increases susceptibility to bacterial infection, the precise biological mechanisms that link obesity and susceptibility to periodontal disease remain elusive. Using fecal microbial transplantation, experimental periodontitis, and metabolomics, our study demonstrates uric acid as a causative substance for greater aggravation of alveolar bone destruction in obesity-related periodontal disease. Gut microbiota from obese mice upregulated the purine degradation pathway, and the resulting elevation of serum uric acid promoted alveolar bone destruction. The effect of uric acid was confirmed by administration of allopurinol, an inhibitor of xanthine oxidase. Overall, our study provides new insights into the pathogenic mechanisms of obesity-associated periodontal disease and the development of new therapeutic options for the disease.
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http://dx.doi.org/10.1128/mBio.00771-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262938PMC
June 2021

Ingestion of Porphyromonas gingivalis exacerbates colitis via intestinal epithelial barrier disruption in mice.

J Periodontal Res 2021 Apr 29;56(2):275-288. Epub 2021 Jan 29.

Research Unit for Oral-Systemic Connection, Division of Oral Science for Health Promotion, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.

Objective: This study aimed to evaluate the effects of ingested periodontal pathogens on experimental colitis in mice and to elucidate its underlying mechanisms.

Background: Inflammatory bowel disease (IBD) is defined as a chronic intestinal inflammation that results in damage to the gastrointestinal tract. Epidemiological studies have shown an association between IBD and periodontitis. Although a large number of ingested oral bacteria reach gastrointestinal tract constantly, the effect of ingested periodontal pathogens on intestinal inflammation is still unknown.

Methods: Experimental colitis was induced by inclusion of dextran sodium sulfate solution in drinking water of the mice. Major periodontal pathogens (Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum) were administered orally every day during the experiment. The severity of colitis between the groups was compared. In vitro studies of the intestinal epithelial cell line were conducted to explore the molecular mechanisms by which periodontal pathogens affect the development of colitis.

Results: The oral administration of P. gingivalis significantly increased the severity of colitis when compared to other pathogens in the DSS-induced colitis model. The ingested P. gingivalis disrupted the colonic epithelial barrier by decreasing the expression of tight junction proteins in vivo. In vitro permeability assays using the intestinal epithelial cell line suggested the P. gingivalis-specific epithelial barrier disruption. The possible involvement of gingipains in the exacerbation of colitis was implied by using P. gingivalis lacking gingipains.

Conclusion: Porphyromonas gingivalis exacerbates gastrointestinal inflammation by directly interacting with the intestinal epithelial barrier in a susceptible host.
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http://dx.doi.org/10.1111/jre.12816DOI Listing
April 2021

Rice peptide with amino acid substitution inhibits biofilm formation by Porphyromonas gingivalis and Fusobacterium nucleatum.

Arch Oral Biol 2021 Jan 30;121:104956. Epub 2020 Oct 30.

Division of Periodontology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, 2-5274 Gakkocho-dori, Chuo-ku, Niigata, 951-8514, Japan. Electronic address:

Objective: Rice peptide has antibacterial properties that have been tested in planktonic bacterial culture. However, bacteria form biofilm at disease sites and are resistant to antibacterial agents. The aim of this study was to clarify the mechanisms of action of rice peptide and its amino acid substitution against periodontopathic bacteria and their antibiofilm effects.

Design: Porphyromonas gingivalis and Fusobacterium nucleatum were treated with AmyI-1-18 rice peptide or its arginine-substituted analog, G12R, under anaerobic conditions. The amount of biofilm was evaluated by crystal violet staining. The integrity of the bacteria cytoplasmic membrane was studied in a propidium iodide (PI) stain assay and transmission electron microscopy (TEM).

Results: Both AmyI-1-18 and G12R inhibited biofilm formation of P. gingivalis and F. nucleatum; in particular, G12R inhibited F. nucleatum at lower concentrations. However, neither peptide eradicated established biofilms significantly. According to the minimum inhibitory concentration and minimum bactericidal concentration against P. gingivalis, AmyI-1-18 has bacteriostatic properties and G12R has bactericidal activity, and both peptides showed bactericidal activity against F. nucleatum. PI staining and TEM analysis indicated that membrane disruption by G12R was enhanced, which suggests that the replacement amino acid reinforced the electostatic interaction between the peptide and bacteria by increase of cationic charge and α-helix content.

Conclusions: Rice peptide inhibited biofilm formation of P. gingivalis and F. nucleatum, and bactericidal activity via membrane destruction was enhanced by amino acid substitution.
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http://dx.doi.org/10.1016/j.archoralbio.2020.104956DOI Listing
January 2021

Epithelial TRPV1 channels: Expression, function, and pathogenicity in the oral cavity.

J Oral Biosci 2020 09 13;62(3):235-241. Epub 2020 Jun 13.

Division of Periodontology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, 951-8514, Japan.

Background: The oral cavity serves as an entrance to the body and is therefore exposed to various exogenous stimuli, including mechanical forces, chemical agents, and bacterial components. The oral mucosa responds to these stimuli to maintain homeostasis and good oral health. The transient receptor potential vanilloid 1 (TRPV1) ion channel functions as an environment-sensing protein and is involved in a wide variety of cellular responses. Recent studies have revealed that epithelial TRPV1 ion channels in the oral cavity play pivotal roles in several pathophysiological conditions. In this review, we summarize the features of epithelial TRPV1 channels in the oral cavity and focus on their cellular function and pathogenicity with reference to related findings in other organs and tissues.

Highlight: TRPV1 channels are widely expressed in epithelial cells in the oral cavity and play pivotal roles in fundamental cellular processes and disease progression.

Conclusion: This review suggests that oral epithelial TRPV1 contributes to several cellular functions such as cell proliferation, barrier function, and inflammation. Further understanding of the characteristics of epithelial TRPV1 in the oral cavity may provide new insights into the prevention or treatment of diseases.
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http://dx.doi.org/10.1016/j.job.2020.05.005DOI Listing
September 2020

A bacterial metabolite induces Nrf2-mediated anti-oxidative responses in gingival epithelial cells by activating the MAPK signaling pathway.

Arch Oral Biol 2020 Feb 5;110:104602. Epub 2019 Nov 5.

Research Unit for Oral-Systemic Connection, Division of Oral Science for Health Promotion, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan. Electronic address:

Objective: Oxidative stress, which is defined as an imbalance between pro-oxidant and antioxidant systems, has been implicated in the development and/or progression of several inflammatory diseases, including periodontal disease. The reactive oxygen species (ROS) are the primary inducers of oxidative stress. In the induction of cytoprotective enzymes, the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling in antioxidant systems takes a main role. Notably, 10-oxo-trans-11-octadecenoic acid (KetoC), known as a bioactive metabolite generated by intestinal microorganisms, has been reported to have beneficial effects on several biological responses. Therefore, we investigated the antioxidant effect of KetoC on gingival epithelial cells (GECs) in this present study.

Methods: An SV40-T antigen-transformed human gingival epithelial cell line (Epi4) was used for experiments. The alteration of anti-oxidative stress related genes was analyzed by qPCR. The cellular ROS levels were evaluated by flow cytometry. To explore its molecular mechanisms, ARE promotor activity was analyzed by luciferase assay; the involvement of mitogen-activated protein kinase (MAPK) and G protein-coupled receptor 120 (GPR120) were evaluated by Western blotting and luciferase assay, respectively.

Results: KetoC significantly increased the expression of antioxidant-related genes in GECs. The level of ROS was significantly inhibited by the pretreatment of KetoC. Extracellular signal-regulated kinase (ERK) phosphorylation by KetoC promoted both the nuclear translocation of Nrf2 and its binding to the ARE in GECs. Further, GPR120 regulated the activation of KetoC induced-Nrf2-ARE signaling.

Conclusion: KetoC exerts a protective function against the oxidative stress in GECs through GPR120-dependent ERK-Nrf2-ARE signaling.
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http://dx.doi.org/10.1016/j.archoralbio.2019.104602DOI Listing
February 2020

Gingival epithelial barrier: regulation by beneficial and harmful microbes.

Tissue Barriers 2019 7;7(3):e1651158. Epub 2019 Aug 7.

Research Unit for Oral-Systemic Connection, Division of Oral Science for Health Promotion, Graduate School of Medical and Dental Sciences, Niigata University , Niigata , Japan.

The gingival epithelium acts as a physical barrier to separate the biofilm from the gingival tissue, providing the first line of defense against bacterial invasion in periodontal disease. Disruption of the gingival epithelial barrier, and the subsequent penetration of exogenous pathogens into the host tissues, triggers an inflammatory response, establishing chronic infection. Currently, more than 700 different bacterial species have been identified in the oral cavity, some of which are known to be periodontopathic. These bacteria contribute to epithelial barrier dysfunction in the gingiva by producing several virulence factors. However, some bacteria in the oral cavity appear to be beneficial, helping gingival epithelial cells maintain their integrity and barrier function. This review aims to discuss current findings regarding microorganism interactions and epithelial barrier function in the oral cavity, with reference to investigations in the gut, where this interaction has been extensively studied.
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http://dx.doi.org/10.1080/21688370.2019.1651158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748373PMC
July 2020

Antimicrobial function of the polyunsaturated fatty acid KetoC in an experimental model of periodontitis.

J Periodontol 2019 12 12;90(12):1470-1480. Epub 2019 Sep 12.

Research Unit for Oral-Systemic Connection, Division of Oral Science for Health Promotion, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.

Background: The bioactive metabolite KetoC, generated by intestinal bacteria, exerts various beneficial effects. Nevertheless, its function in the pathogenesis of periodontitis remains unclear. Here, we investigated the effect of KetoC in a mouse model of periodontitis and explored the underlying mechanism.

Methods: Thirty-one 8-week-old male C57BL/6N mice were randomly divided into four groups (non-ligation, non-ligation + KetoC, ligation + Porphyromonas gingivalis, and ligation + P. gingivalis + KetoC) (n = 7/8 mice/group) and given a daily oral gavage of KetoC (15 mg/mL) or vehicle for 2 weeks. To induce periodontitis, a 5-0 silk ligature was placed on the maxillary left second molar on day 7, and P. gingivalis W83 (10 colony-forming unit [CFU]) was administered orally every 3 days. On day 14, all mice were euthanized. Alveolar bone destruction was determined from the level of the cemento-enamel junction to the alveolar bone crest. Moreover, bone loss level was confirmed from gingival tissue sections stained with hematoxylin and eosin. The presence of P. gingivalis was quantified using real-time polymerase chain reaction. In vitro, the bacteriostatic and bactericidal effects of KetoC were assessed by analyzing its suppressive activity on the proliferation of P. gingivalis and using a live/dead bacterial staining kit, respectively. A double-bond-deficient metabolite (KetoB) was then used to investigate the importance of double-bond structure in the antimicrobial activity of KetoC on P. gingivalis.

Results: In vivo, KetoC attenuated alveolar bone destruction and suppressed P. gingivalis in the periodontitis group. In vitro, KetoC (but not KetoB) downregulated the proliferation and viability of P. gingivalis in a dose-dependent manner.

Conclusions: KetoC reduced alveolar bone destruction in a periodontitis model via its antimicrobial function. Therefore, this bioactive metabolite may be valuable in clinical applications to support periodontal therapy.
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http://dx.doi.org/10.1002/JPER.19-0130DOI Listing
December 2019

-Microglobulin and Neutrophil Gelatinase-Associated Lipocalin, Potential Novel Urine Biomarkers in Periodontitis: A Cross-Sectional Study in Japanese.

Int J Dent 2019 20;2019:1394678. Epub 2019 Mar 20.

Division of Periodontology, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Science, 2-5274 Gakko-cho, Chuo-ku, Niigata 951-8514, Japan.

Objectives: Several serum biomarkers have been reported to increase in periodontitis patients as possible mediators linking periodontal inflammation to systemic diseases. However, the relationship between periodontitis and urine biomarkers is still unclear. The aim of this cross-sectional study was to investigate potential urine biomarkers of periodontitis in a Japanese population.

Materials And Methods: This study included 108 male subjects, and microbiological and clinical parameters were evaluated as a periodontitis marker. The correlation between nine urine biomarkers (typically used to diagnose kidney disease) and periodontal parameters was analyzed. Based on the findings, -microglobulin ( -MG) and neutrophil gelatinase-associated lipocalin (NGAL) were selected for comparison and multivariate regression analysis, and the Kruskal-Wallis test followed by Bonferroni correction was used to identify differences in their concentrations between the three periodontitis groups (severe, moderate, and no/mild periodontitis).

Results: -MG and NGAL exhibited a significant correlation with clinical parameters of periodontitis. The prevalence of clinical parameters such as bleeding on probing and number of sites with probing depth (PD) ≥ 6 mm were greater in the -MG high group (≥300 g/g creatinine) than in the normal group (=0.017 and 0.019, respectively). Multivariate regression analysis indicated that the number of sites with PD ≥ 6 mm was independently associated with urine -MG. Moreover, the number of sites with the clinical attachment level (CAL) ≥ 6 mm was greater in the NGAL high group (highest quartile) (=0.041). Multivariate regression analysis showed that the number of sites with CAL ≥ 6 mm was associated independently with urine NGAL. Finally, -MG was significantly higher in the severe periodontitis subjects compared to the no/mild periodontitis subjects.

Conclusion: The significant association between urine -MG or NGAL and periodontitis was revealed. These biomarkers can potentially be used to screen for or diagnose periodontitis. This trial is registered with the UMIN Clinical Trials Registry UMIN000013485.
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http://dx.doi.org/10.1155/2019/1394678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446109PMC
March 2019
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