Ryosuke Koshi

The effect of butyric acid on adhesion molecule expression by human gingival epithelial cells

BACKGROUND AND OBJECTIVE Short-chain fatty acids, such as butyric acid, are detected in periodontal pockets and are thought to be involved in the initiation and progression of periodontal disease. In the present study, we examined the effects of butyric acid on adhesion molecule expression by human gingival epithelial cells. MATERIAL AND METHODS The human gingival carcinoma cell line, Ca9-22, was cultured in media that contained different concentrations of butyric acid. RESULTS Cell numbers were significantly decreased in a dose-dependent manner by butyric acid at concentrations of > or = 0.2 mM. The expression of intercellular adhesion molecule-1 mRNA was significantly increased 6 h after stimulation. By contrast, the expression levels of integrins alpha 6 and beta 4 were decreased. Similar results were obtained by flow cytometry. CONCLUSION The results of the present study indicate that butyric acid alters the expression of adhesion molecules by Ca9-22 cells. The elucidation of the mechanism of action of butyric acid on the periodontium may help to clarify several aspects of the onset and progression of periodontal disease.

Hepatocytes produce tumor necrosis factor-α and interleukin-6 in response to Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE The liver plays a major role in clearing systemic bacterial infections. In addition, inflammatory cytokines produced in the liver play a critical role in systemic cytokine levels. The aim of this study was to investigate the production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by hepatocytes in response to periodontal pathogens. MATERIAL AND METHODS The mouse hepatic carcinoma cell line Hepa-1.6 and the mouse macrophage-like cell line RAW 264 were co-cultured in Transwell insert plates. Cells were stimulated with bacterial extracts prepared from Porphyromonas gingivalis and the induction of TNF-α and IL-6 was measured using real-time PCR and ELISA. RESULTS After stimulation with bacteria, the induction of TNF-α and IL-6 was observed in RAW 264 cells and Hepa-1.6 cells. Significant reduction of TNF-α mRNA expression in Hepa-1.6 cells was observed after treatment with antibody to TNF-α. CONCLUSION The results obtained in the present study show that P. gingivalis extract induces TNF-α and IL-6 in an in vitro liver model and that macrophage-derived TNF-α mediates the induction of TNF-α in hepatocytes.

Nicotine-Mediated Ca(2+)-Influx Induces IL-8 Secretion in Oral Squamous Cell Carcinoma Cell.

Cigarette smoking is one of the most important risk factors for the development of various diseases. Nicotine is the most extensively investigated component of cigarette smoke, and a comprehensive analysis of the genes induced by nicotine stimulation revealed that interleukin‐8 (IL‐8) was induced in oral squamous cell carcinoma cell (OSCC). Based on this background, the signaling mechanisms of nicotine‐mediated IL‐8 induction in OSCC was investigated. Augmented IL‐8 secretion by Ca9‐22 cells was blocked by the NF‐κB inhibitor L‐1‐4′‐tosylamino‐phenylethyl‐chloromethyl ketone (TPCK) and the nicotinic acetylcholine receptor (nAChR)‐specific inhibitor α‐bungarotoxin (αBtx). The downstream signaling pathway was further examined by pre‐incubating the cells with inhibitors against mitogen‐activated protein kinase (MEK), protein kinase C (PKC), and Ca2+/calmodulin‐dependent kinase II (CaMK II). Only the CaMK II inhibitor was found to exert an inhibitory effect on nicotine‐mediated IL‐8 secretion. Pre‐treatment of the Ca9‐22 cells with the Ca2+ chelator BAPTA‐AM drastically inhibited IL‐8 secretion. Although nicotine stimulation induced the phosphorylation of the NF‐κB p65 subunit, pre‐treatment with BAPTA‐AM was found to inhibit this activity significantly. CaMK II‐dependent p65 phosphorylation was confirmed by pre‐incubation of the cells with CaMK II inhibitor. The results from this study indicate that the binding of nicotine to nAChR induces Ca2+ influx, which results in the activation and phosphorylation of CaMK II and NF‐κB p65, respectively. Nicotine‐mediated IL‐8 induction should be a trigger for the initiation of various diseases. J. Cell. Biochem. 117: 1009–1015, 2016.

Nicotine-induced expression of low-density lipoprotein receptor in oral epithelial cells.

Background Nicotine use is one of the most important risk factors for the development of cardiovascular and periodontal diseases. Numerous reports have suggested the possible contribution of disturbed lipid metabolism for the development of both disease groups. Despite these observations, little is known about the relationship between tobacco smoking and the development of these diseases. Our previous microarray data revealed that nicotine induced low-density lipoprotein receptor (LDLR) expression in oral epithelial cells (OECs). The aim of the present study was to confirm nicotine-mediated LDLR induction and to elucidate the signaling mechanisms leading to the augmented expression of LDLR in OECs. Methods and Results LDLR and nicotinic acetylcholine receptor (nAChR) subunit expression was detected by real-time PCR. The production of LDLR was demonstrated by immunofluorescence staining. nAChR-mediated LDLR induction was examined by pre-incubation of the cells with its specific inhibitor, α-bungarotoxin (α-BTX). The functional importance of transcription factor specific protein 1 (Sp1) was examined by luciferase assay, mithramycin pre-incubation or by small interfering RNA (siRNA) transfection. The specific binding of Sp1 to R3 region of LDLR 5’-untranslated region was demonstrated with electrophoretic mobility shift assay (EMSA) and streptavidin-agarose precipitation assay followed by western blotting. The results confirmed that nicotine induced LDLR expression at the transcriptional level. Nicotine was sensed by nAChR and the signal was transduced by Sp1 which bound to the R3 region of LDLR gene. Augmented production of LDLR in the gingival epithelial cells was further demonstrated by immunofluorescence staining using the gingival tissues obtained from the smoking patients. Conclusions Taken together, the results suggested that nicotine might contribute to the development of both cardiovascular and periodontal diseases by inducing the LDLR in OECs thereby disturbing lipid metabolism.

Influences of mechanical barrier permeability on guided bone augmentation in the rat calvarium

We used radiological and histological analyses to evaluate the effects of mechanical barrier permeability in a rat model of calvarial guided bone augmentation (GBA). The calvaria of 20 rats were exposed, and one of four types of plastic caps (an occlusive cylindrical plastic cap; a plastic cap with no top; a plastic cap with three holes; and a plastic cap with four holes) was randomly placed on both sides. Newly generated bone in the plastic caps was evaluated with micro-computed tomography (micro-CT) and histological analysis. Micro-CT volumetric analysis and decalcified hematoxylin and eosin-stained sections showed that GBA barrier permeability was inversely associated with the quantity of augmented bone obtained. Massons trichrome staining showed that collagen in newly generated bony tissue was more mature in plastic caps with three holes than in those with more-permeable or more-occlusive barriers. Bone augmentation was inhibited in specimens exhibiting invasion of soft tissue through penetrating holes, and barrier permeability was associated with the quantity of augmented bone developed. In conclusion, moderate barrier permeability is optimal for development of mature augmented bone.

Comparison of the bone augmentation ability of absorbable collagen sponge with that of hydroxyapatite/collagen composite

The present study was designed to compare the bone augmentation ability of absorbable collagen sponge (ACS) with that of hydroxyapatite/collagen composite (HAP/Col) using a rat calvaria defect model. Bone defects were created artificially on the surface of the calvariae of 10-week-old male Fisher rats, and then cylindral plastic caps filled with ACS or HAP/Col were placed on the defects. This area was designated as the region of interest (ROI) and new bone formation was observed at 0, 4, 8, and 12 weeks after surgery using micro-CT. Histological examinations were performed using sections obtained from 12-week-old rats. Prominent new bone formation was observed in the HAP/Col group relative to the ACS group; onset of new bone augmentation was evident from 4 weeks after surgery in the HAP/Col group and from 8 weeks in the ACS group. Histological examination revealed that the entire area of the cap was filled with newly formed bone intermingled with the HAP/Col composite. Bone mineral density in the HAP/Col group was double that in the ACS group. These results indicate that the use of HAP/Col contributes significantly to new bone augmentation.

Periodontal regeneration with 0.3% basic fibroblast growth factor (FGF-2) for a patient with aggressive periodontitis: a case report

This case report describes the clinical efficacy of treatment with basic fibroblast growth factor (FGF-2) for periodontal regeneration. A patient with aggressive periodontitis participated in a clinical trial involving administration of 0.3% FGF-2 in comparison with a placebo control. To evaluate the efficacy of FGF-2, standardized radiographs were taken before surgery and at 12, 24, and 36 weeks after FGF-2 treatment. The rate of increase in alveolar bone height was 86.9% at 36 weeks. The 6-year postoperative radiograph showed significant development of alveolar bone in comparison with the first visit. FGF-2 treatment may be effective for periodontal regeneration in cases of aggressive periodontitis. (J Oral Sci 58, 137-140, 2016).

The effect of B vitamin supplementation on wound healing in type 2 diabetic mice

The aim of this study was to test the effects of B-group vitamin supplements on wound healing in diabetic mice. The mice in the experimental group were treated daily with 1 g/L B6, 1.25 mg/L B12, and 62.5 mg/L folic acid in their drinking water. Full-thickness excision wounds were created with 6-mm skin biopsy punches. Each wound closure was digitally photographed. Beginning on day 3 after wounding, the wound area in the diabetic mice was statistically larger than that of normal mice (p<0.05 vs diabetic mice). The diabetic mice treated with B vitamins displayed accelerated wound closure on day 3 (wound area 42.8 ± 11.3%, p<0.05). On day 9 after wounding, the wound area in the diabetic mice was also statistically larger than that of normal mice (p<0.05 vs diabetic mice). The diabetic mice treated with B vitamins displayed accelerated wound closure on day 3 (wound area 13.2 ± 16.8%, p<0.05). In addition, the high glucose level in the diabetic animals decreased significantly in response to B vitamin treatment. In conclusion, the results of this study indicate that B vitamin supplementation may improve wound healing in diabetic mice.