• 동의생리병리학회지
• /
• 제19권2호
• /
• pp.370-374
• /
• 2005

Streptococcus mutans (S. mutans) is known as the causative bacterial playing the most important role informing plaque and it is being noticed as major causative bacteria of dental caries. Therefore, development of more effective, substantial and safe preventive agent against dental caries and periodontal disease is honestly required. The present study was designed to investigate the effect of Cyperus rotundus (Cyperaceae) methanol extracts on the growth, acid production, adhesion, and insoluble glucan synthesis of S. mutans. The methanol extract of C. rotundus showed concentration dependent inhibitory activity against the growth and acid production of S. mutans, and produced significant inhibition at the concentration of 0.5, 1, 2 and 4 mg/ml compared to the control group. The extracts markedly inhibited S. mutans adherence to HA treated with saliva, and cell adherence was repressed by more than 50% at the concentration of 0.5 mg/ml and complete inhibition was observed at the concentration of 4 mg/ml. On the activity of glucosyltransferase which synthesizes water insoluble glucan from sucrose, methanol extract of C. rotundus showed more than 10% inhibition over the concentration of 2 mg/ml. Thus, the application of C. rotundus can be considered a useful and a practical method for the prevention of dental caries.

• 치위생과학회지
• /
• 제19권3호
• /
• pp.198-204
• /
• 2019

Background: Oxidative stress is a known to be associated with in the pathogenesis of many inflammatory diseases, including periodontitis. Ursolic acid is a pentacyclic triterpenoid with has antimicrobial, antioxidative, and anticancer properties. However, the role of ursolic acid in the regulating of osteogenesis remains undetermined. This study was aimed to elucidate the crucial osteogenic effects of ursolic acid and its ability to inhibit oxidative stress by targeting the immediate early response 3 (IER3)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Methods: Cell proliferation was determined using water-soluble tetrazolium salt assay, cell differentiation was evaluated by alkaline phosphatase (ALP) activity, and formation of calcium nodules was detected using alizarin red S stain. Generation of reactive oxygen species (ROS) was determined using by DCFH-DA fluorescence dye in hydrogen peroxide ($H_2O_2$)-treated MG-63 cells. Expression levels of IER3, Nrf2, and heme oxygenase-1 (HO-1) were analyzed using western blot analysis. Results: Our results showed that ursolic acid up-regulated the proliferation of osteoblasts without any cytotoxic effects, and promoted ALP activity and mineralization. $H_2O_2$-induced ROS generation was found to be significantly inhibited on treatment with ursolic acid. Furthermore, in $H_2O_2$-treated cells, the expression of the early response genes: IER3, Nrf2, and Nrf2-related phase II enzyme (HO-1) was enhanced in the presence of ursolic acid. Conclusion: The key findings of the present study elucidate the protective effects of ursolic acid against oxidative stress conditions in osteoblasts via the IER3/Nrf2 pathway. Thus, ursolic acid may be developed as a preventative and therapeutic agent for mineral homeostasis and inflammatory diseases caused due to oxidative injury.

• 구강회복응용과학지
• /
• 제38권2호
• /
• pp.90-96
• /
• 2022

목적: 이 연구의 목적은 표고 버섯 추출물의 치주 병원체에 대한 항균 활성과 인간 치은 섬유아세포에 대한 세포 독성을 조사하는 것입니다. 연구 재료 및 방법: 표고버섯을 물과 아세톤에 닮근 후, 상등액을 건조하여 추출물을 채취하였다. 추출물에 대한 치주 병원체의 감수성을 조사하였다. 인간 치은 섬유아세포에 추출물을 처리하고 CCK-8 용액을 이용하여 세포 생존율을 측정하였다. 결과: 표고버섯 물추출물은 2.5 mg/ml에서 치주병원체의 증식을 유의하게 감소시켰다(P < 0.05). 아세톤 추출물은 0.32 mg/ml에서 Porphyromonas gingivalis와 Tannerella forsythia의 성장을 유의하게 억제하였고, 0.64 mg/ml에서 Treponema denticola의 성장을 유의하게 억제하였다(P < 0.05). 추출물의 세포독성은 2.5 mg/ml의 농도에서 나타났으며, 1.25 mg/ml 농도의 추출물이 4시간 후부터 세포 생존률을 감소시키는 것으로 나타났다. 결론: 표고버섯 추출물은 치주염 유발 세균에 대한 항균 활성과 염증 완화 효과를 보였다. 따라서 추출물은 치주질환 예방 및 치료에 대한 후보물질이 될 수 있다.

• Journal of Periodontal and Implant Science
• /
• 제47권4호
• /
• pp.219-230
• /
• 2017

Purpose: The purpose of this study was to compare the characteristics of single- and dualspecies in vitro oral biofilms made by static and dynamic methods. Methods: Hydroxyapatite (HA) disks, 12.7 mm in diameter and 3 mm thick, were coated with processed saliva for 4 hours. The disks were divided into a static method group and a dynamic method group. The disks treated with a static method were cultured in 12-well plates, and the disks in the dynamic method group were cultured in a Center for Disease Control and Prevention (CDC) biofilm reactor for 72 hours. In the single- and dual-species biofilms, Fusobacterium nucleatum and Porphyromonas gingivalis were used, and the amount of adhering bacteria, proportions of species, and bacterial reduction of chlorhexidine were examined. Bacterial adhesion was examined with scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Results: Compared with the biofilms made using the static method, the biofilms made using the dynamic method had significantly lower amounts of adhering and looser bacterial accumulation in SEM and CLSM images. The proportion of P. gingivalis was higher in the dynamic method group than in the static method group; however, the difference was not statistically significant. Furthermore, the biofilm thickness and bacterial reduction by chlorhexidine showed no significant differences between the 2 methods. Conclusions: When used to reproduce periodontal biofilms composed of F. nucleatum and P. gingivalis, the dynamic method (CDC biofilm reactor) formed looser biofilms containing fewer bacteria than the well plate. However, this difference did not influence the thickness of the biofilms or the activity of chlorhexidine. Therefore, both methods are useful for mimicking periodontitis-associated oral biofilms.

• Journal of Periodontal and Implant Science
• /
• 제33권2호
• /
• pp.277-288
• /
• 2003

It has been suggested that increased number and activity of phagocytes in periodontitis lesion results in a high degree of reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, nitric oxide and peroxynitrite. There are few reports on the relationship between ROS and MMPs expressions in gingival fibroblast. We studied to elucidate whether and how ROS, especially nitric oxide affects the MMP expression. Human gingival fibroblasts and HTl080 cells (human fibrosarcoma sell line as reference) were grown in DMEM supplemented with 10 mM HEPES, 50 mg/L gentamicin, and 10% heat inactivated fetal bovine serum with addition of various reactive oxygen species (ROS). Culture media conditioned by cells were examined by gelatin zymography. HT1080 cells expressed proMMP-2 and proMMP-9, but human gingival fibroblasts (HGF) produced only proMMP-2. Hydrogen peroxide upregulated MMP-9 expression in HT1080 cells, whereas in human gingival fibroblast SNP treatment showed marked increase in MMP-2 level compared to other ROS. These results suggest that the effects of ROS on MMPs expressions are cell-type specific. RT-PCR for MMP-2 and TIMP-2 m-RNA were performed using total RNA from cultured cells under the influence various kinase inhibitors. In HT1080 cells, treatment with FPTI III (Ras processing inhibitor) and LY294002 (PI3-kinase inhibitor) resulted in inhibition of MMP-2 and MMP-9 expressions, suggesting that Ras/P13-kinase pathway is important for MMPs expression in HT1080 cells. In gingival fibroblasts, treatment with FPTI III and PDTC (NF-kB inhibitor) showed marked decrease in MMP-2 regardless of the of SNP , suggesting that Ras/NF-kB could be the key pathway for NO-induced MMP-2 expression in gingival fibroblasts. This study showed that ROS, especially nitric oxide, could be the critical mediator of periodontal disease progression through control of MMP-2 expression in gingival fibroblasts possibly via Ras/NF-kB pathway.

• International Journal of Oral Biology
• /
• 제34권2호
• /
• pp.87-95
• /
• 2009

Porphyromonas gingivalis is a major etiologic agent of chronic periodontitis and cytokines produced by macrophages play important roles in the pathogenesis of periodontal diseases. In this study we investigated the cytokine response of phorbol myristate acetatedifferentiated THP-1 cells exposed to P. gingivalis. Compared with the prominent cell wall components of P. gingivalis (lipopolysaccharide and the major fimbrial protein FimA), live P. gingivalis stimulated much higher levels of cytokine production. In addition, whereas low multiplicity of infection challenges (MOI=10) of P. gingivalis 381 stimulated high levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), interleukin-6 (IL-6), and IL-1${\beta}$, high dose challenges with this bacterium (MOI = 100) resulted in a substantially diminished production of MCP-1 and IL-6. Moreover, high MOI P. gingivalis challenges achieved only low levels of induction of MCP-1 and IL-6 mRNA. The decreased production of MCP-1 and IL-6 appeared to be mediated by P. gingivalis proteases, because high MOI challenges with congenic protease mutant strains of this microorganism (MT10 and MT10W) did not result in a diminished production of MCP-1 and IL-6. Similar to its protease mutant strains, leupeptin (a protease inhibitor)- treated P. gingivalis at high doses induced high levels of MCP-1 production. To examine the mechanisms underlying the diminished production of MCP-1 by P. gingivalis proteases, the activation of mitogen-activated protein (MAP) kinases and NF-${\kappa}$B was compared between the 381 and MT10W strains. Whilst high doses of both 381 and MT10W similarly activated the three members of the MAP kinase family, the DNA binding activity of NF-${\kappa}$B, as revealed by gel shift assays, was greatly increased only by MT10W. Taken together, our data indicate that P. gingivalis stimulates the production of high levels of TNF-${\alpha}$, IL-1${\beta}$, IL-6, and MCP-1 but that high dose challenges with this bacterium result in a diminished production of MCP-1 and IL-6 via the protease-mediated suppression of NF-${\kappa}$B activation in THP-1 macrophagic cells.

• Journal of Periodontal and Implant Science
• /
• 제38권sup2호
• /
• pp.317-324
• /
• 2008

Purpose: Aggregatibacter actinomycetemcomitans was associated with localized aggressive periodontitis, endocarditis, meningitis, and osteomyelitis. The cytolethal distending toxin (CDT) of A. actinomycetemcomitans was considered as a key factor of these diseases is composed of five open reading frames (ORFs). Among of them, An enzymatic subunit of the CDT, CdtB has been known to be internalized into the host cell in order to induce its genotoxic effect. However, CdtB can not be localized in host cytoplasm without the help of a heterodimeric complex consisting of CdtA and CdtC. So, some studies suggested that CdtC functions as a ligand to interact with GM3 ganglioside of host cell surface. The precise role of the CdtC protein in the mechanism of action of the holotoxin is unknown at the present time. The aim of this study was to generate recombinant CdtC proteins expression from A. actinomycetemcomitans, through gene cloning and protein used to investigate the function of Cdt C protein in the bacterial pathogenesis. Materials and Methods: The genomic DNA of A. actinomycetemcomitans Y4 (ATCC29522) was isolated using the genomic DNA extraction kit and used as template to yield cdtC genes by PCR. The amplifed cdtC genes were cloned into T-vector and cloned cdt C gene was then subcloned to pET28a expression vector. The pET28a-cdtC plasmid expressed in BL21 (DE3) Escherichia coli system. Diverse conditons were tested to opitimize the expression and purification of functional CdtC protein in E. coli. Results: In this study we reconstructed CdtC subunit of A. actinomycetemcomitans Y4 and comfirmed the recombinant CdtC expression by SDS-PAGE and Western Blotting. The expression level of the recombinant CdtC was about 2% of total bacterial proteins. Conclusion: The lab condition of procedure for the purification of functionally active recombinant CdtC protein is established. The active recombinant CdtC protein will serve to examine the role of CdtC proteins in the host recognition and enzyme activity of CDT and investigate the pathological process of A. actinomycetemcomitans in periodontal disease.

• Journal of Oral Medicine and Pain
• /
• 제34권2호
• /
• pp.169-177
• /
• 2009

천연 식물 추출물을 구강 질환에 활용하는 방안을 모색하기 위하여, 본 연구는 인진쑥에서 추출한 쑥추출액을 치의학분야에 활용하고자 S. gordonii Challis, S. gordoii G9B, S. mutans GS5, S. sobriuns 6715, E. faecalis ATCC 4083, A. actinomycetem Y4, P. gingivalis A7A1-28, P. gingivalis W83, Pr. intermedia ATCC 25611, F. nucleatum KTCT 2488, C. albicans ATCC 18804에 대한 항균효과를 미생물학적으로 실험하여, 생균수 검사를 통해 다음과 같은 결과를 얻을 수 있었다. 1. 인진쑥추출액에 의한 최소억제농도(minimum inhibitory concentration; MIC)와 최소살균농도(minimum bactericidal concentration; MBC)가 모두 쑥추출액 농도 2.0%이하에서 관찰된 세균은 치주질환 원인균인 P. gingivalis A7A1-28와 P. gingivalis W83, Pr. intermedia ATCC 25611로 나타났다. 2. P. gingivalis A7A1-28의 MIC는 쑥추출액 농도 1.2%, MBC는 2.0%로 관찰되었다. 3. P. gingivalis W83의 MIC는 쑥추출액 농도 1.4%, MBC는 2.0%로 관찰되었다. 4. Pr. intermedia ATCC 25611의 MIC는 쑥추출액 농도 1.2%, MBC는 2.0%로 관찰되었다. 천연 자연물질인 인진쑥이 구강질환을 일으키는 대표적인 균주에 대한 효과를 연구한 본 실험의 결과, 인진쑥은 구강 세균, 특히 P. gingivalis A7A1-28와 P. gingivalis W83, Pr. intermedia ATCC 25611에 대한 항균효과가 있음이 증명되었다. 따라서 이 천연물질이 부작용이 없는 한계 내에서 사용된다면, 구강질환자의 구강 환경 개선을 위한 치약, 구강세척제 등의 구강용품들을 통해 임상에 적극적으로 사용될 수 있을 것으로 생각된다.

• Molecules and Cells
• /
• 제26권5호
• /
• pp.436-442
• /
• 2008

Osteoclasts are multinucleated cells with the unique ability to resorb bone. Elevated activity of these cells under pathologic conditions leads to the progression of bone erosion that occurs in osteoporosis, periodontal disease, and rheumatoid arthritis. Thus, the regulation of osteoclast apoptosis is important for bone homeostasis. In this study, we examined the effects of the Janus tyrosine kinase 2 specific inhibitor AG490 on osteoclast apoptosis. We found that AG490 greatly inhibited osteoclast apoptosis. AG490 stimulated the phosphorylation of Akt and ERK. Adenovirus-mediated expression of dominant negative (DN)-Akt and DN-Ras in osteoclasts inhibited the survival of osteoclasts despite the presence of AG490. Cytochrome c release during osteoclast apoptosis was inhibited by AG490 treatment, but this effect was inhibited in the presence of LY294002 or U0126. AG490 suppressed the pro-apoptotic proteins Bad and Bim, which was inhibited in osteoclasts infected with DN-Akt and DN-Ras adenovirus. In addition, constitutively active MEK and myristoylated-Akt adenovirus suppressed the cleavage of pro-caspase-9 and -3 and inhibited osteoclast apoptosis induced by etoposide. Taken together, our results suggest that AG490 inhibited cytochrome c release into the cytosol at least partly by inhibiting the pro-apoptotic proteins Bad and Bim, which in turn suppressed caspase-9 and -3 activation, thereby inhibiting osteoclast apoptosis.

• International Journal of Oral Biology
• /
• 제45권3호
• /
• pp.115-125
• /
• 2020

Resveratrol has been reported to exert anticancer activity via modulation of multiple pathways and genes. In this study, we examined the effect of resveratrol on YD-10B human oral squamous cell carcinoma cells and its molecular mechanisms of action. We found that resveratrol inhibited the proliferation of YD-10B cells in a dose- and time-dependent manner. The suppressive effect of resveratrol was accompanied by a reduction in Bmi-1 gene expression. We observed that silencing the Bmi-1 gene by small interfering RNA effectively downregulated the levels of GLUT1 mRNA and protein, which were also repressed by resveratrol. Bmi-1 silencing increased the number of YD-10B cells in S-phase arrest by approximately 2.3-fold compared with the control. In conclusion, the results of the present study demonstrate, for the first time, that resveratrol suppresses Bmi-1-mediated GLUT1 expression in human oral squamous cell carcinoma cells and suggest that the specific molecular targeting of Bmi-1 and/or GLUT1 expression can be combined with a chemotherapeutic strategy to improve the response of oral cancer cells to resveratrol.