• Molecules and Cells
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• v.27 no.2
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• pp.257-261
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• 2009

Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) is an important pathogen casuing aggressive periodontitis. The present study was designed to investigate the chemokines expression regulated by A. actinomycetemcomitans lipopolysaccharide (LPS). Chemokines genes expression profiling was performed in Raw 264.7 cells by analyses of microarray and reverse transcription-polymerase chain reaction (RT-PCR). Microarray results showed that the induction of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-$1{\alpha}$ (MIP-$1{\alpha}$), MIP-$1{\beta}$, MIP-$1{\gamma}$, regulated upon activation, normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein-2 (MIP-2), and interferon-${\gamma}$ inducible protein 10 (IP 10) by A. actinomycetemcomitans LPS was increased to 12.5, 1.53, 9.09, 17.3, 2.82, 16.1, and 18.1 folds at 18 h, respectively. To check these chemokines expression by A. actinomycetemcomitans LPS, we examined gene expressions by RT-PCR, and found that the expression of MIP-$1{\beta}$, MIP-$1{\gamma}$, RANTES, MIP-2, and IP 10 was increased 107.1, 93.6, 106.8, 86.5, and 162.0 folds at 18 h, respectively. These results indicate that A. actinomycetemcomitans LPS stimulates the several chemokines expressions (MIP-$1{\alpha}$, MIP-$1{\beta}$, MIP-$1{\gamma}$, RANTES, MIP-2, and IP 10) in Raw 264.7 cells.

• International Journal of Oral Biology
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• v.39 no.1
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• pp.15-22
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• 2014

Aggregatibacter actinomycetemcomitans is an important pathogen in the development of localized aggressive periodontitis. Lipopolysaccharide (LPS) is a virulent factor of periodontal pathogens that contributes to alveolar bone loss and connective tissue degradation in periodontal disease. Our present study was designed to investigate the cytokine expression and signaling pathways regulated by A. actinomycetemcomitans LPS (Aa LPS). Cytokine gene expression profiling in RAW 264.7 cells was performed by microarray analyses. The cytokine mRNA and protein levels and related signaling pathways induced by Aa LPS were measured by RT-PCR, ELISA and western blotting. Microarray results showed that Aa LPS strongly induced the expression of NF-${\kappa}B$, NF-${\kappa}B$-related genes, inflammatory cytokines, TNF-${\alpha}$ and IL-$1{\beta}$ in RAW 264.7 cells. NF-${\kappa}B$ inhibitor pretreatment significantly reduced the levels of TNF-${\alpha}$ and IL-$1{\beta}$ mRNA and protein. In addition, the Aa LPS-induced TNF-${\alpha}$ and IL-$1{\beta}$ expression was inhibited by p38/JNK MAP kinase inhibitor pretreatment. These results show that Aa LPS stimulates TNF-${\alpha}$ and IL-$1{\beta}$ expression through NF-${\kappa}B$ and p38/JNK activation in RAW 264.7 cells, suggesting the essential role of this pathway in the pathogenesis of localized aggressive periodontitis.

• Microbiology and Biotechnology Letters
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• v.44 no.4
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• pp.557-562
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• 2016

Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis are gram-negative bacteria frequently found in lesions from patients with periodontitis manifesting alveolar bone loss. Lipopolysaccharides are a major virulence factor of gram-negative bacteria. Bone resorption is known to be regulated by bacteria and their virulence factors. In the present study, we investigated the effects of A. actinomycetemcomitans and P. gingivalis on bone resorption. Heat-killed A. actinomycetemcomitans (HKAa) and heatkilled P. gingivalis (HKPg) induced bone loss in the femurs of mice after intraperitoneal administration. HKAa and HKPg augmented the differentiation of committed osteoclast precursors into osteoclasts, while they inhibited the differentiation of bone marrow-derived macrophages into osteoclasts. Concordant with the effects of the heat-killed whole cells, LPS purified from A. actinomycetemcomitans and P. gingivalis also augmented osteoclast differentiation from committed osteoclast precursors but attenuated it from bone marrow-derived macrophages. Taken together, these results suggest that the whole cells and lipopolysaccharides of A. actinomycetemcomitans and P. gingivalis induce the differentiation of committed osteoclast precursors into osteoclasts, potentially contributing to bone resorption in vivo.

• Journal of Periodontal and Implant Science
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• v.37 no.sup2
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• pp.297-310
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• 2007

치조골 흡수는 파골세포와 matrix metalloproteinases (MMPs)에 의한 골의 무기질과 유기질의 파괴로 일어나는 과정이다. 세균성 산물, 주로 내독소는 치은조직 내에서 염증세포의 유주, 사이토카인 생산, 조직파괴 효소 분비 및 파골세포 활성 등의 국소 면역반응을 유도한다. A. actinomycetemcomitans는 급진성 치주염의 원인 균주중 하나로 그 내독소는 치조골의 흡수와 관련된다. MMP-13은 세균성 산물이나 염증성 사이토카인의 자극에 의해 분비되며, 최근의 연구 결과들은 MMP-13이 치주질환의 진행과 골 흡수 과정에서 일정한 역할을 담당하는 것으로 보고하고 있으나, A. actinomycetemcomitans 내독소와 MMP-13과의 관련성에 대한 연구는 미미하다. 이에 이번 연구에서는 A. actinomycetemcomitans 내독소에 의한 MMP-13의 발현과 파골세포 형성을 세포배양을 통하여 관찰하였고, 백서 구개부 치은에 A. actinomycetemcomitans 내독소를 주입하여 흡수가 진행되고 있는 치조골에서 파골세포의 분화와 MMP-13의 발현을 TRAP 염색, 면역조직화학적 방법 등을 통해 관찰하여 다음과 같은 결과를 얻었다. MMP-13 mPNA의 발현은 A. actinomycetemcomitans 내독소 (1ug/ml)로 24시간 자극한 마우스 치주인대 섬유모세포에서 생리식염수로 자극한 세포에 비하여 약 2.6배 증가하였으며 마우스 대식세포에서는 TRAP 양성 세포가 대조군보다 더 많이 나타났다. A. actinomycetemcomitans 내독소를 주입한 백서 치주조직에서는 대조군보다 더 심한 골소실을 보였다. TRAP-양성 다핵 파골세포 유사세포는 치주염군과 대조군 모두 치조골에서 관찰되었다. TRAP-양성 다핵 파골세포 유사세포는 치주염군에서 대조군보다 유의하게 많은 숫자가 관찰되었으며, 치주염군에서 대조군보다 유의하게 많은 숫자가 관찰되었다. MMP-13 면역양성 반응은 치주염군에서 거친 골연을 갖는 치조골상에 배열된 조골세포와 그 인접한 치주인대에서 관찰되었으며 대조군에서는 MMP-13 면역 양성 반응이 치조골 표면에서만 일부 관찰되었다. 이상의 결과는 A. actinomycetemcomitans 내독속가 MMP-13의 발현을 증가시키며 파골세포의 활성을 통하여 치조골의 흡수를 유도함을 시사한다. 또한 A. actinomycetemcomitans 내독소 투여에 의한 실험적 모델은 백서에서 중등도의 골 소실을 동반한 만성 치주염 모델로 향후 치주질환 치료제의 효과를 평가하는데 유용하게 사용될 수 있으리라 기대된다.

• Journal of Periodontal and Implant Science
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• v.53 no.2
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• pp.110-119
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• 2023

Purpose: This study aimed to investigate the proper wavelengths for safe levels of light-emitting diode (LED) irradiation with bactericidal and photobiomodulation effects in vitro. Methods: Cell viability tests of fibroblasts and osteoblasts after LED irradiation at 470, 525, 590, 630, and 850 nm were performed using the thiazolyl blue tetrazolium bromide assay. The bactericidal effect of 470-nm LED irradiation was analyzed with Streptococcus gordonii, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, and Tannerella forsythia. Levels of nitric oxide, a proinflammatory mediator, were measured to identify the anti-inflammatory effect of LED irradiation on lipopolysaccharide-stimulated inflammation in RAW 264.7 macrophages. Results: LED irradiation at wavelengths of 470, 525, 590, 630, and 850 nm showed no cytotoxic effect on fibroblasts and osteoblasts. LED irradiation at 630 and 850 nm led to fibroblast proliferation compared to no LED irradiation. LED irradiation at 470 nm resulted in bactericidal effects on S. gordonii, A. actinomycetemcomitans, F. nucleatum, P. gingivalis, and T. forsythia. Lipopolysaccharide (LPS)-induced RAW 264.7 inflammation was reduced by irradiation with 525-nm LED before LPS treatment and irradiation with 630-nm LED after LPS treatment; however, the effects were limited. Conclusions: LED irradiation at 470 nm showed bactericidal effects, while LED irradiation at 525 and 630 nm showed preventive and treatment effects on LPS-induced RAW 264.7 inflammation. The application of LED irradiation has potential as an adjuvant in periodontal therapy, although further investigations should be performed in vivo.

• International Journal of Oral Biology
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• v.36 no.3
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• pp.109-116
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• 2011

Periodontitis results from the activation of host immune and inflammatory defense responses to subgingival plaque bacteria, most of which are gram-negative rods with lipopoly-saccharides (LPSs) in their cell walls. LPSs have been known to induce proinflammatory responses and recently it was reported also that they induce the expression of microRNAs (miRNAs) in host cells. In our current study therefore, we aimed to examine and compare the miRNA expression patterns induced by the LPSs of major periodontopathogens in the human gingival epithelial cell line, Ca9-22. The cells were treated with 1 ${\mu}g$/ml of E. coli (Ec) LPS or 5 ${\mu}g$/ml of an LPS preparations from four periodontopathogens Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Aggregatibacter actinomycetemcomitans (Aa), and Fusobacterium nucleatum (Fn) for 24 h. After small RNA extraction from the treated cells, miRNA microarray analysis was carried out and characteristic expression profiles were observed. Fn LPS most actively induced miRNAs related to inflammation, followed by Aa LPS, Pi LPS, and Ec LPS. In contrast, Pg LPS only weakly activated miRNAs related to inflammation. Among the miRNAs induced by each LPS, miR-875-3p, miR-449b, and miR-520d-3p were found to be commonly up-regulated by all five LPS preparations, although at different levels. When we further compared the miRNA expression patterns induced by each LPS, Ec LPS and Pi LPS were the most similar although Fn LPS and Aa LPS also induced a similar miRNA expression pattern. In contrast, the miRNA profile induced by Pg LPS was quite distinctive compared with the other bacteria. In conclusion, miR-875-3p, miR-449b, and miR-520d-3p miRNAs are potential targets for the diagnosis and treatment of periodontal inflammation induced by subgingival plaque biofilms. Furthermore, the observations in our current study provide new insights into the inflammatory miRNA response to periodontitis.

• Journal of dental hygiene science
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• v.20 no.4
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• pp.221-229
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• 2020

Background: The purpose of this study was to investigate the anti-oral microbial activity and anti-inflammatory effects of rosmarinic acid (RA) in lipopolysaccharide (LPS)-stimulated MC3T3-E1 osteoblastic cells on a titanium (Ti) surface during osseointegration, and to confirm the possibility of using RA as a safe natural substance for the control of peri-implantitis (PI) in Ti-based dental implants. Methods: A disk diffusion test was conducted to confirm the antimicrobial activity of RA against oral microorganisms. In order to confirm the anti-inflammatory effects of RA, inflammatory conditions were induced with 100 ng/ml of LPS in MC3T3-E1 osteoblastic cells on the Ti surface treated with or without 14 ㎍/ml of RA. The production of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated MC3T3-E1 osteoblastic cells on the Ti surface was confirmed using an NO assay kit and PGE2 enzyme-linked immunosorbent assay kit. Reverse transcription polymerase chain reaction and western blot analysis were performed to confirm the expression of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in total RNA and protein. Results: RA showed weak antimicrobial effects against Streptococcus mutans and Escherichia coli, but no antimicrobial activity against the bacteria Aggregatibacter actinomycetemcomitans and the fungus Candida albicans. RA reduced the production of pro-inflammatory mediators, NO and PGE2, and proinflammatory cytokines, TNF-α and IL-1β, in LPS-stimulated MC3T3-E1 osteoblastic cells on the Ti surface at the protein and mRNA levels. Conclusion: RA not only has anti-oral microbial activity, but also anti-inflammatory effects in LPS-stimulated MC3T3-E1 osteoblasts on the Ti surface, therefore, it can be used as a safe functional substance derived from plants for the prevention and control of PI for successful Ti-based implants.

• Journal of Periodontal and Implant Science
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• v.34 no.1
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• pp.71-81
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• 2004

Peri-implantitis could be the result of biomechanical and occlusal overload as well as microbiologic invasion. The dental implant may be more susceptible to dental plaque than the natural tooth, as the predictability of a stable soft tissue attachment complex has not yet been confirmed. With the development of peri-implantitis, the implant surface would be exposed to the oral environment and becomes coated with bacteria. The objective of therapy for this condition is to regain integration of the implant with bone. Since fibroblast adherence to surfaces is impeded by endotoxin, it would seem that decontamination would be desirable to obtain maximum osseointegration. The purpose of this study was to determine whether various chemotherapeutic and mechanical treatments(distilled water, air-powder abrasive, hypersaturated citric acid, tetracycline) can detoxify contaminated titanium implant surface by means of kinetic LAL test. Experimental rough surface titanium disks were fabricated. All of them were divided into two groups(A.a group and P.g group) and each contaminated by A. actinomycetemcomitans and P. gingivalis suspension. Contaminated disks were treated with distilled water, air-powder abrasive, citric acid and tetracycline, and then all disks were placed into LPS-free water for elution. The results were as follows : 1. In A.a group, LPS elute level of all test groups were significantly lower than control group(p<0.05). 2. In A.a group, LPS elute level of test 2, test 3 and test4 groups were significantly lower than that of control group(p<0.05). But, among the test 2, test 3, test4 groups, the significant differences were not detected. 3. In P.g group, LPS elute level of test 2, test 3 and test 4 groups were lower than that of control group(p<0.05). But, among the test groups, the significant differences were not detected. From the result of this study, it would be concluded that air-powder abrasive, hypersaturated citric acid and tetracycline treatments may be effective at reducing endotoxin level on rough titanium implant surfaces, and can be clinically used. But the treatments in peri-implantitis differentially impact osseointegration making one method clinically superior. To gain this knowledges, further molecular biologic and histopathologic studies should be developed.

• Journal of Periodontal and Implant Science
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• v.36 no.3
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• pp.579-590
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• 2006

1. 연구배경 교원질 분해작용을 하는 호중구의 세포질 효소인 기질금속단백분해효소-8은 치주질환, 류마티스 관절염, 그리고 궤양결장염과 같은 염증성 질환에서 농도가 증가한다고 알려져 있다. 최근에는 A. actinomycetemcomitans의 leukotoxin이 사람호중구에서 기질금속단백분해효소-8의 분비를 유도하는 것이 보고되었다. 이 연구의 목적은 선천면역 체계에서 세포표면 항원무리14, Toll-like 수용기, 그리고 $NF-{\kappa}$ B경로를 통하여 A. actinomycetemcomitans의 지질다당질로 유도된 기질금속단백분해효소-8의 분비 여부와 세포기전을 알아보고자 하였다. 2. 연구재료 및 방법 건강한 개인 제공자(남자 13명, 여자 3명)로부터 얻은 개개인의 20ml 말초혈액을 제조사의 지침에 따라 호중구를 추출한 후 항세포표면 항원무리14와 함께 $4^{\circ}C$에서 30분간 전배양 한 후, $37^{\circ}C$에서 9시간 동안 배양시켰다. 추출한 호중구에 Toll-like 수용기 억제제 또는 $NF-{\kappa}$ B억제제인 TPCK를 첨가한 후 $37^{\circ}C$에서 1시간 동안 전배양하고 $37^{\circ}C$에서 9시간 동안 배양시켰다. 호중구에 세포뼈대 억제제인 cholchicine, nocodazole, demecolcine, 그리고 cytochalasin B를 A. actinomycetemcomitans의 지질다당질과 함께 $37^{\circ}C$에서 9시간 동안 배양시켰다. 기질금속단백분해효소-8 분비량은 효소면역측정법을 통해 결정하였다. 통계처리는 일원배치 분산분석법을 이용하였다(p<0.05). 3. 결과 A. actinomycetemcomitans 지질다당질은 기질금속단백분해효소-8의 분비를 증가시켰다. 기질금속단백분해효소-8의 분비는 항세포표면 항원무리14에 의해서 억제되었지만, 항 Toll-like 수용기2, 항 Toll-like 수용기4 항체는 억제시키지 못했다. $NF-{\kappa}$ B 억제제는 A. actinomycetemcomitans의 지질다당질로 유도된 $NF-{\kappa}$ B 결합 활성도와 기질금속단백분해효소-8 분비를 억제하였다. 미세섬유 중합반응 억제제는 A. actinomycetemcomitans의 지질다당질로 유도된 기질금속단백분해효소-8의 분비를 억제시켰으나, 미세관 중합반응억제제는 억제시키지 못했다. 4. 결론 위의 연구결과를 종합하여 볼 때, 기질금속단백분해효소-8은 A. actinomycetemcomitans의 지질다당질로 유도되며, 세포표면 항원무리-$NF-{\kappa}$ B 경로를 통하여 분비되고, 이 분비 과정은 미세섬유 계통이 관여하는 것으로 보인다.

• Journal of Periodontal and Implant Science
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• v.23 no.1
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• pp.145-158
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• 1993

The oral microbiota such as P. gingivalis, P. intermedia and A. actinomycetemcomitans play a primary role in the initiation and progression of the periodontal disease. The purpose of this study was to evaluate the antimicrobial effects and inhibitory effects of honokiol and magnolol on the bacterial collagenase activity, cytotoxicity and cytokine production of periodontopathic microorganisms. The antimicrobial activities of honokiol and magnolol was evaluted with minimum inhibition concentration. Honokiol was more active than magnolol, but less than chlorhexidine on antimicrobial activity. The inhibitory effects of magnolol and honokiol on the collagenolytic activity and cytotoxicity were evaluated using a Collagenokit CLN-100 and rapid colorimetric assay (MTT method) for cellular growth and survival of gingival fibroblast and periodontalligament cell and $[^3H]-thymidine$ incorporation for the gingival epithelial cell. The inhibitory effects on the collagenolytic activity was the highest in chlorhexidine, and the lowest in magnolol. Magnolol had the lowest cytotoxic effect and chlorhexidine had the highest. The inhibitory effects on cytokine production was evaluated using $interleukin-1{\beta}$ ELISA kit (Cistron Biotech.), IL-6, $TNF-{\alpha}$ ELISA kit (Genzyme) and inhibitory effects were higher than bacterial LPS and there is no difference among the honokiol, magnolol and chlorhexidine. From these results, the antimicrobial and antienzymatic activities of honokiol and magnolol were seemed to inhibit bacterial growth and enzyme activities with lesser cytotoxic activities. Therefore, it was suggested that honokiol and magnolol are very effective antimicrobial agents on periodontal pathogens.