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Original article J Korean Acad Periodontol 29;39:331-337 치주인대세포에서 Aggregatibacter actinomycetemcomitans 의 IL-8 및활성산소종유도능 이양신 1, 박홍규 1, 김성환 2,3, 차정헌 1,2,3, 유윤정 1,2,3* 1. 연세대학교치과대학구강생물학교실 2. 연세대학교대학원응용생명과학과 3. 연세대학교치과대학 2단계 BK 21 사업단, 구강과학연구소및구강악안면경조직재생연구센터 Induction of IL-8 and reactive oxygen species in periodontal ligament cells by Aggregatibacter actinomycetemcomitans Yang-Sin Lee 1, Hong Gyu Park 1, Sung Whan Kim 2,3, Jeong-Heon Cha 1,2,3, Yun-Jung Yoo 1,2,3* 1. Department of Oral Biology, College of Dentistry, Yonsei University 2. Department of Applied Life Science, The Graduate School, Yonsei University 3. BK21 project, Oral Science Research Center, and Research Center for Orofacial Hard Tissue Regeneration, College of Dentistry, Yonsei University ABSTRACT Purpose: Interleukin (IL)-8 is one of pro-inflammatory cytokines. Reactive oxygen species (ROS) are reduced metabolites of O 2. Aggregatibacter actinomycetemcomitans is one of representative periodontopathogens. To investigate the role of A. actinomycetemcomitans in IL-8 expression of periodontal ligament (PDL) cells, we estimated the production of IL-8 and ROS in A. actinomycetemcomitans treated PDL cells. Methods: The IL-8 production was determined by enzyme-linked immunosorbent assay. The ROS production was estimated using H 2DCFDA and FACS. Results: A. actinomycetemcomitans increased the production of IL-8 and ROS at 1, 1, and 5 multiplicity of infection. N-acetylcysteine, an antioxidant of ROS, down-regulated the production of IL-8 induced by A. actinomycetemcomitans. Conclusions: These results suggest that A. actinomycetemcomitans induces IL-8 production and ROS may act as a mediator in this process. (J Korean Acad Periodontol 29;39:331-337) KEY WORDS: Aggregatibacter actinomycetemcomitans; interleukin-8; periodontal ligament; reactive oxygen species. 서론 치주염은치태내세균에의하여치아지지조직이파괴되는염증성질환으로치은염증및치조골흡수가유도되는질 Correspondence: Yun-Jung Yoo Department of Oral Biology, College of Dentistry, Yonsei University 134 Sinchon-dong, Seodaemoon-gu, Seoul, 12-752, Korea E-mail: yu618@yuhs.ac, Tel: 2-2228-36, Fax: 2-2227-793 Received: Jun 13, 29; Accepted: Aug 1, 29 본논문은 26 년도정부재원 ( 교육인적자원부학술연구조성사업비 ) 으로한국학술진흥재단의지원을받아연구되었음 (KRF-26-531-E 78). 환이다. 치주염에의한치주조직파괴는세균의직접적인작용또는세균에대한방어, 즉염증및면역반응에의하여유도된다 1). 따라서염증유도 cytokine 은치주염에의한치주조직파괴에있어서중요한역할을하는것으로생각되고있다. Interleukin (IL)-8 은 CXC chemokine 이다. IL-8 은염증유도 cytokine 으로염증세포인호중구의부착분자발현, 화학주성및활성화를유도한다 2). 또한 IL-8 은이러한염증유도능뿐만아니라골흡수유도능도있는것으로보고되었다. Receptor activator of NF-κB ligand(rankl) 는파골세포의

분화를유도하는 cytokine 으로 IL-8 은 RANKL 의존또는비의존경로를통해파골세포형성을유도하는것으로보고되었다 3-5). 치주염시치은열구액및치주조직의 IL-8 이증가하며치주염과의상관관계가있는것으로보고되어치주염의병인기전에있어서 IL-8 의중요성이제시되었다 6-8). 치주염의병인기전에있어서치주인대세포가생성하는염증유도 cytokine 의중요성이강조되어왔다. Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans 및 Treponema species는치주염관련세균이다 9). 이들세균중 P. gingivalis 및 Treponema species 가치주인대세포의 IL-8 발현을유도하는것으로보고되었다 1-12). 치주인대세포에서 A. actinomycetemcomitans 의 IL-6 발현유도능이보고되었으나 13), A. actinomycetemcomitans 가 IL-8 의발현을유도하는지보고된것이없으며치주염관련세균에의한 IL-8 발현에관련된신호전달체계도명확히밝혀져있지않다. 활성산소종 (reactive oxygen species, ROS) 은 O 2 의환원대사물로 superoxide anion, hydrogen peroxide 및 hydroxyl radical 이이에해당된다 14). Superoxide anion 은 NADPH oxidase, xanthin oxidase 또는 mitochondrial electron transport chain oxidase 을통해형성되며 hydrogen peroxide 와 singlet oxygen 으로전환된다. 상피세포를포함한일부세포에서활성산소종이 IL-8 유도제로작용하는것으로보고되었으며치주인대세포에서도활성산소종의 IL-8 유도능이보고되었다 15-2). Helicobacter pyroli는위염및십이지장염의원인균으로 H. pyroli 가상피세포에서 IL-8 발현을유도하며활성산소종이신호전달매개체로작용할수있음이제시되었다 21). 이는세균에의한 IL-8 생성에활성산소종이관여할가능성을시사한다. 따라서본연구에서는치주인대세포에서 A. actinomycetemcomitans 의 IL-8 및활성산소종생성유도능을관찰하여치주인대세포의 IL-8 발현시 A. actinomycetemcomitans 의역할을평가하였다. 재료및방법 1. 치주인대세포배양연세대학교치과병원에내원한환자로교정치료를위하여발치한치아의치근으로부터분리배양하였다. 치아의중앙 1/3 부분을나이프로긁어치주인대조직편을분리하였 다. 분리한치주인대조직편을 3 unit/ml penicillin, 3 g/ml streptomycin 및.75 g/ml amphotericin B를함유한 α-mem(gibco BRL, Grand Island, NY, USA) 배지로 3회세척하였다. 세척한조직편을 1 mm 세포배양기에위치시킨후 1unit/ml penicillin, 1g/ml streptomycin,.25 g/ml amphotericin B 및 2% 우태아혈청 (Gibco BRL) 을함유한 α-mem 배지에서배양하였다. 배지는 2일간격으로교체하였으며조직편으로부터세포가자라나기시작한후 1% 우태아혈청을함유한 α-mem 배지에서배양하였으며세포가자라 7% 정도의단층을형성한후.25% trypsin-edta 로처리하여계대배양하였다. 계대배양한세포는질소탱크에보관하여실험에사용하였으며본실험에서는 5-8 계대배양한세포를실험에사용하였다. 이연구는연세대학교치과대학병원임상시험위원회의승인을받았다 (YDDT-28-5). 2. A. actinomycetemcomitans의배양 A. actinomycetemcomitans(atcc43718) 를 1 ml brain heart infusion(bhi)(becton Dickinson, Sparks, MD, USA) 액체배지에접종하여 37, 1% CO 2 항온기에서 24 시간배양하였다. 배양된세균을 1,5 g에서 5분간원심분리하여균체를모았다. 모은균체를 PBS 로 2회세척한후 PBS 에부유시켜 57nm 에서흡광도를측정하였다. A. actinomycetemcomitans 의 colony forming unit(cfu/ml) 와흡광도의표준곡선을기준으로세균수를측정하여실험에사용하였다. 3. 세포독성평가치주인대세포를 24 well 배양기에각 well 당세포수가 1.5 1 5 이되도록분주하였다. 2 시간후세포를 A. actinomycetemcomitans 또는 N-acetylcysteine(NAC) (Sigma, St. Louis, MO, USA) 으로 24 시간처리한후 MTT 방법으로세균또는 NAC 의세포독성능을평가하였다. 배양이끝나는날배양상층액을제거한후각 well 에새배지와 2 μl의 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(mtt) 시약 (5 mg/ml, Sigma) 을첨가하여 MTTformazan 결정체형성을유도하였다. 4시간후배양상층액을제거한후 3 μl의 dimethy sulfoxide(dmso) 를첨가하고실온에서 5분동안 shaking 하여형성된결정체를녹여 57 nm 에서흡광도를측정하였다. 332

4. IL-8 생성평가치주인대세포를 6 mm 배양기에세포수가 1.4 1 6 이되도록분주하였다. 세포가단층을형성한후일정 MOI 가되도록세균을접종하였다. 5시간및 24 시간후배양상층액을모아 ELISA kit(r & D systems, Minneapolis, MN, USA) 를이용하여배양상층액내 IL-8의농도를측정하였다. 5. 활성산소종생성평가 6 mm 배양기에세포수가 1.4 1 6 이되도록분주하였다. 세포가단층을형성한후일정 MOI 가되도록 A. actinomycetemcomitans 로세포를처리하였다. 5시간또는 24 시간후세균으로처리한치주인대세포를 HBSS 완충용액으로일회씻은후 2 μg/ml H 2 DCFDA(Invitrogen, Carlsbad, CA, USA) 로 5분간처리하였다. 세포를 CM-H 2 -DCFDA 로처리하면 CM- H 2-DCFDA 가세포내로들어가세포내 esterase 에의해가수분해되며가수분해된 CM-H 2 -DCF 는세포내형성된활성산소종에의하여산화되어형광을발하게된다. 처리한세포를.25% Trypsin-EDTA 로분리하고 HBSS 완충용액으로씻고 fluorescenceactivated cell sorter(facs, Becton Dickinson) 를이용하여활성산소종을발현하는세포를측정하였다. 6. 통계 ANOVA 및 Tukey's test 로분석하였으며통계적유의성평가를위한유의수준은.5 를기준으로하였다. 결과 1. A. actinomycetemcomitans 에의한 IL-8 생성 치주인대세포를세균대세포수, 즉 MOI 1, 1 및 5 에서 24 시간처리한후 MTT 를시행한결과 5 MOI 이하에서세포독성을보이지않았다 ( 자료제시하지않음 ). IL-8 생성은시간의경과에따라 MOI 에의존적으로증가하여 5 MOI 에서 24 시간처리한경우 12,89 pg/ml 에달하였다 (Fig. 1). 2. A. actinomycetemcomitans 에의한활성산소종생성 활성산소종의생성은 1 MOI 에서증가하기시작하였으며 5 MOI 에서최대생성을보였다 (Fig. 2A). 5 MOI 로치주인대세포를 5시간및 24 시간처리한경우활성산소종의생성은시간이지나면서증가하였다 (Fig. 2B). 3. NAC 의 A. actinomycetemcomitans 에의한 IL-8 생성억제 IL-8 생성및세포독성능에대한결과는 one-way NAC 으로치주인대세포를 3 분전처리한후 5 MOI 가 A B 12 16 1 14 8 12 1 6 8 4 6 2 4 2 1 1 5 1 1 5 A. actinomycetemcomitans (MOI) A. actinomycetemcomitans (MOI) Figure 1. Induction of IL-8 production in periodontal ligament cells treated with A. actinomycetemcomitans. Periodontal ligament cells were treated with A. actinomycetemcomitans at 1, 1, and 5 MOI for 5 h(a) and 24 h (B). The concentration of IL-8 in culture supernatants was estimated by ELISA. * Significantly different from none-treated cells (P <.5). 333

되도록세균으로 5시간및 24 시간처리하여 IL-8 농도를측정하였다 (Fig. 3). 5시간및 24 시간모두에서 NAC 은세균에의한 IL-8 생성을감소시켰다. NAC 은 IL-8 의생성을억제하는농도에서세포독성을보이지않았다 ( 자료제시하지않음 ). 고찰 치주조직의면역형광염색및세균배양을통해유년성치주염환자의치주조직에 A. actinomycetemcomitans 가존재함을확인할수있었고전자현미경상에서도결체조직에서 A. actinomycetemcomitans 의집락을관찰할수있었다 22-23). 이들 결과는 A. actinomycetemcomitans 가치주조직내로침투하여치주조직파괴에관여할수있음을시사한다. 따라서본연구에서는치주인대세포를 A. actinomycetemcomitans 로처리하여 IL-8 생성유도능을평가하였다. 치주인대세포를 A. actinomycetemcomitans 로처리한경우세균의수및세균과의접촉시간이증가함에따라 IL-8 생성이증가하는것을관찰할수있었으며이는 A. actinomycetemcomitans 가치주인대세포의 IL-8 생성을유도할수있음을시사한다. IL-8 발현에 NF-κB 가관여하며 24) 활성산소종의한종류인 H 2O 2 는 NF-B 의활성화를매개한다 24-25). 혈관내피세포를제외한세포, 즉단핵세포, 피부섬유아세포, 상피세포및일부암세포에서 H 2 O 2 가 IL-8 생성을유도하는것으로보고되었 A 1 MOI B 5h 1 1 1 1 2 1 3 1 4 1 1 1 1 2 1 3 1 4 1 MOI 24h 1 1 1 1 2 1 3 1 4 1 1 1 1 2 1 3 1 4 5 MOI none-treated cells Aa-treated cells 1 1 1 1 2 1 3 1 4 Figure 2. Induction of ROS in periodontal ligament cells treated with A. actinomycetemcomitans. Periodontal ligament (PDL) cells were treated with A. actinomycetemcomitans (Aa) at 1, 1, and 5 MOI for 24 h. ROS induction in PDL cells were estimated by FACS (A). PDL cells were treated with A. actinomycetemcomitans at MOI 5 for 5 h and 24 h and ROS induction in PDL cells were estimated by FACS (B). 334

다 15-19,26). 또한본연구팀의이전연구를통해치주인대세포에서도 H 2 O 2 가 IL-8 생성을유도하며 mitogen activated protein kinases(mapks) 가이러한과정에관여함을확인할수있었다 2). 본연구에서 A. actinomycetemcomitans 로치주인대세포를처리한경우세포내활성산소종의생성은세균수및세균과의접촉시간이증가함에따라증가하는것을관찰할수있었다. 또한항산화제인 NAC 으로세포를전처리한경우 A. actinomycetemcomitans 에의한 IL-8 생성이감소하는것을확인하였다. 이는치주인대세포에서본세균에의한 IL-8 생성에활성산소종이관여할수있음을시사한다. 위상피세포및치은섬유아세포에서 IL-8 발현시세포내신호전달매개체로서활성산소종의역할이일부연구자들에의해보고되었다. O'hara 등 27) 은위상피세포에서활성산소종이 TNF 에의한 IL-8 발현을매개하는것으로보고하였다. Seo 등 21) 은위염및십이지장염의주요한원인균인 H. pyroli 에의한 IL-8 생성에있어서활성산소종의역할을보고하였다. 위상피세포에서 H. pyroli 는 IL-8 및활성산소종의생성을유도하였으며 NAC 은 H. pyroli에의한 IL-8 생성을억제하였다. Kim 등 28) 은치은섬유아세포에서치주염의원인균인 P. gingivalis 및 A. actinomycetemco mitans 의 lipopolysaccharide(lps) 가 IL-8 와활성산소종의생성을유도하며 NAC 이이들세균의 LPS 에의한 IL-8 생성억제함을보고하여활성산소종과 IL-8 발현과의관련가능성을제시하였다. 치주염동물모델을이용한연구에서도활 성산소종제거제인 tempol 또는 NAC 이치주염의염증소견인호중구의침윤, 혈관의투과도, 치조골흡수를감소시킴을관찰하여항산화제요법이치주염치료의보조법으로유익할수있음이제시되었다 29-3). 이전연구자들의이와같은실험결과및본연구의실험결과는활성산소종이 IL-8 발현의매개체로작용할수있음을시사한다. 치주인대세포에서보고된 IL-8 발현유도인자로는염증유도 cytokine 및일부치주염원인균이있다. 염증유도 cytokine 으로는 IL-1 및 tumor necrosis factor(tnf) 가치주염의원인균으로는 P. gingivalis와 P. intermedia 가또한 T. maltophilum 과 T. lecithinolyticum 의주외막단백질이 IL-8 발현을유도하는것으로보고되었다 11-12,31-32). 이들유도물질중 T. maltophilum 과 T. lecithinolyticum 의주외막단백질에의한 IL-8 생성에 NF-κB 가관여하는것으로보고되었을뿐활성산소종의역할은연구되지않았다 12). 본연구를통해 A. actinomycetemcomitans 가치주인대세포에서 IL-8 및활성산소종의생성을유도하며항산화제인 NAC 은 A. actinomycetemcomitans 에의한 IL-8 생성을감소시킴을관찰하였으며이들결과는 A. actinomycetemcomitans 도다른치주염관련세균들과마찬가지로치주인대세포의 IL-8 생성을유도할수있으며활성산소종이 A. actinomycetemcomitans 에의한 IL-8 생성의세포내신호전달매개체로작용할수있음을시사한다. IL-8 은호중구의이동 / 활성화및파골세포의분화를매개하므로 A. actino- A 16 14 12 1 8 6 4 2 NAC(15 mm) Aa(5 MOI) B 14 12 1 8 6 4 2 NAC(15 mm) Aa(5 MOI) Figure 3. Effect of N-acetylcysteine on A. actinomycetemcomitans induced IL-8 production in periodontal ligament cells. Periodontal ligament cells were pretreated with N-acetylcysteine (NAC, 15 mm) for 3 min and then treated with A. actinomycetemcomitans (Aa) at 5 MOI for 5 h (A) and 24 h (B). The concentration of IL-8 in culture supernatants was determined by ELISA. * Significantly different from none-treated cells (P <.5). ** Significantly different from A. actinomycetemcomitans-treated cells (P <.5). 335

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