폐상피세포에서 Paraquat 에의한아포프토시스에관한연구 단국대학교의과대학내과학교실, 1 건국대학교의과대학내과학교실송탁호, 양주연, 정인국, 박재석, 지영구, 김윤섭, 이계영 1 Paraquat-Induced Apoptotic Cell Death in Lung Epithelial Cells Tak Ho Song, M.D., Joo Yeon Yang, M.D., In Kook Jeong, M.D., Jae Seok Park, M.D., Young Koo Jee, M.D., Youn Seup Kim, M.D., Kye Young Lee, M.D. 1 Department of Internal Medicine, College of Medicine, Dankook University, Cheonan, Korea, 1 Department of Internal Medicine, Konkuk University Hospital School of Medicine, Konkuk University, Seoul Korea Background: Paraquat is extremely toxic chemical material, which generates reactive oxygen species (ROS), causing multiple organ failure. In particular, paraquat leads to irreversible progressive pulmonary fibrosis. Exaggerated cell deaths exceeding the normal repair of type Ⅱ pneumocytes leads to mesenchymal cells proliferation and fibrosis. This study examined the followings; ⅰ) whether or not paraquat induces cell death in lung epithelial cells; ⅱ) whether or not paraquat-induced cell deaths are apoptosis or necrosis; and ⅲ) the effects of N-acetylcysteine, dexamethasone, and bcl-2 on paraquat-induced cell deaths. Methods: A549 and BEAS-2B lung epithelial cell lines were used. The cell viability and apoptosis were evalluated using a MTT assay, Annexin Ⅴ staining was monitored by fluorescence microscopy, The level of bcl-2 inhibition was examined by establishing stable A549 pcdna3-bcl-2 cell lines throung the transfection of pcdna3-bcl-2 with the mock. Results: Paraquat decreased the cell viability in A549 and BEAS-2B cells in a dose and time dependent manner. The Annexin Ⅴ assay showed that apoptosis was the type of paraquat-induced cell death. Paraquat-induced cell deaths was significantly inhibited by N-acetylcysteine, dexamethasone, and bcl-2 overexpression. The cell viability of A549 cells treated with N-acetylcysteine, and dexamethasone on the paraquat-induced cell deaths were increased significantly by ~ %, particularly at high doses. In addition, the cell viability of A549 pcdna3-bcl-2 cells overexpressing bcl-2 was significantly higher than the untransfected A549 cells. Conclusion: Paraquat induces apoptotic cell deaths in lung epithelial cells in a dose and time dependent manner. The paraquat-induced apoptosis of lung epithelial cells might occur through the mitochondrial pathway. (Tuberc Respir Dis 6; 61: 366-373) Key words: Paraquat, Apoptosis, Bcl-2, N-acetylcysteine, Dexamethasone. 서 Paraquat (N,N'-dimethyl-4, 4'-bipyridilium; methyl viologen, PQ) 는우리나라를포함하여전세계적으로농업분야에서널리사용되는비선택성제초제이다. 노출시인체내에서용해되어전자수용체로반응성산소유리기 (reactive oxygen species, ROS) 를발생시켜세포막, 단백질, 핵산등과반응하여세포손상을 론 Address for correspondence: Kye Young Lee, M.D. Department of Internal medicine, Konkuk University Hospital School of Medicine, Konkuk University 4-12, Hwayang-Dong, Gwangjin-Gu, Seoul, 143-914, Korea Phone: 82-2--7521 Fax: +82-2--7748 E-mail: kyleemd@kuh.ac.kr Received: Aug. 7. 6 Accepted: Sep. 21. 6 일으킨다 1-3. Paraquat 는폐, 간, 신장, 심장등신체중요장기에손상을초래하나특히폐에심한손상을일으킨다. 폐조직에서는에너지의존과정으로 paraquat가흡수되어혈장과비교해폐에서의 paraquat 농도가 6~배정도높다. 또한폐는대기의산소와가장가깝게접할수있는장기로서반응성산소유리기에의해가장많이손상받는다 4. Paraquat 에의한폐손상은반응성산소유리기에의한직접손상, 염증세포의활성화에의한초기폐손상, 그리고섬유아세포증식및세포기질의변화들이나타나는후기손상등이특징이다. 따라서 paraquat를섭취하면급성폐손상과급성호흡부전증후군및폐섬유화로대부분사망한다 5. 최근의연구보고들에따르면폐상피세포의아포프토시스가급성폐손상및급성호흡부전증후군의발 366
Tuberculosis and Respiratory Diseases Vol. 61. No.4, Oct. 6 생기전에중요한역할을할가능성이크다 6. 폐상피세포에대한세포독성이 paraquat의농도와시간에따라어떠한양상으로나타나는지, 이러한세포독성이아포프토시스인지, 그리고항염증제인 dexamethasone과항산화제인 N-acetylcysteine, 항아포프토시스단백질인 bcl-2가 paraquat 에의한세포독성에어떠한영향을미치는지등을알아보고자하였다. 대상및방법 1. 세포주및시약인체의폐상피세포주로 A549 (type Ⅱ pneumocyte) 세포주와 BEAS-2B (transformed bronchial epithelial cell) 세포주를 DMEM (supplemented with % fetal calf serum, 2 mm L-glutamine, U/mL penicillin and U/mL streptomycin) 배양액을이용하여 37, 5% CO2 incubator에서배양하였다. Paraquat, N-acetylcysteine, 그리고 dexamethasone 은 Sigma사 (St. Louise, USA) 에서구입하였다. 2. Cell viability assay 모세포주를 96 well plate에 well당 5 개의세포가되도록심어배양하였다. Paraquat 에의한세포독성정도를알아보기위해 paraquat의농도는대조군, 1 μ M, μm, μm, 1 mm, 그리고 mm을처치하였으며각각 24시간, 48시간, 그리고 72시간이후에세포의손상정도를알아보았다. Dexamethasone (1 μ M) 은 paraquat 투여 12시간전에전처치하였고, N-acetylcysteine (1 mm) 은 paraquat 투여 1시간전에전처치하였다. 세포의손상정도는 3-(4,5)-2,5- diphenyl-tetrazolium bromide (MTT) assay를이용하였다 7. MTT assay는생존세포에만선택적으로흡수되는황색염료로서생존세포의비율에따라흡광도가증가하는성질을이용한세포독성검사방법으로 Table 1-A. MTT assay for paraquat-induced cell deaths in A549 cells Cell viability(%) 24hr 48hr 72hr Control PQ1μM 96.4±9.1 89.8±8.8 56.6±7.8 PQμM 81.4±7.3 76.3±4.6 47.5±6.1 PQμM 65.9±5.1.3±5. 19.1±1.6 PQ1mM.7±3. 12.7±2.1 12.2±1.4 PQmM 36.9±2.8 11.5±.5 9.3±.1 PQ : paraquat Data are the means ± SD.uat Control 1 μm μm μm 1 mm mm 24 hr 48 hr 72 hr Figure 1-A. MTT assay for paraquat-induced cell deaths in A549 cells 367
TH Song et al: Paraquat-induced apoptotic cell deaths 서, 그방법을요약하면, MTT 용액 (5 mg/ml) 을모세포주가있는 96 well plate에 μl씩첨가한후 CO 2 incubator에서 2시간동안배양한후.1 M HCl, isopropanol μl를 96 well plate에첨가하고약 분간 CO 2 배양기에저장하여반응을일으킨뒤 ELISA kit를이용하여 4 nm에서흡광도를측정하여그비율로서세포독성을측정하였다. 3. 아포프토시스 assay 폐상피세포에대한 paraquat 의아포프토시스여부는세포에 Annexin-V를염색한후에형광현미경을이용하여확인하였다. 방법을요약하면세포를 PBS 로세척하고원심분리기로이용해서세포를침전시킨다. 세포침전물을 μl의결합완충제 (binding buffer) 로 resuspension한다. 여기에 Annexin-V 를 5 μl 첨가하고차광하여실온에서 15분간배양한후에분석하였다. 4. Bcl-2 inhibition study Paraquat로인한아포프토시스에 bcl-2 단백질이미치는효과는 A549 세포주에 A549 pcdna3-bcl-2 construct를유전자주입한후 G418을처치하여안정된세포주를얻은뒤에이용하였다. 유전자의 gene construct는강원의대김영명교수로부터공여받았으며유전자전달감염 (gene transfection) 방법은 lipofectamine-plus (Gibco-BRL, Gaithersburg, MD) 를이용하여유전자주입을시행한후 G418 - μ g/ml 농도하에서배양하여내성세포클론을얻었다. 안정된세포주의여부는 monoclonal anti-bcl-2 antibody (Santacruz Biotech, CA) 를이용하여 western blot을시행하여확인하였다. Table 1-B. MTT assay for paraquat-induced cell deaths in BEAS-2B cells Cell viability(%) 24hr 48hr 72hr Control PQ1μM 85.9±7.2 88.5±4.6 78.8±5.1 PQμM 83.9±6.8 76.6±5.4 75.9±5.3 PQμM 75.6±4.5 47.9±3.2 31.9±2.8 PQ1mM 65.6±5.1 27.2±3. 21.7±2.7 PQmM 24.9±1.3 7.1±.8 5.5±.1 PQ : paraquat Data are the means ± SD. Control 1 μm μm μm 1 mm mm 24 hr 48 hr 72 hr Figure 1-B. MTT assay for paraquat-induced cell deaths in BEAS-2B cells 368
Tuberculosis and Respiratory Diseases Vol. 61. No.4, Oct. 6 Table 2. The paraquat-induced between apoptotic cells death and necrotic cells death in A549 cells by Annexin-V assay. Apoptotic cell death (%) Necrotic cell death (%) PQ 1 mm 96.4±4.6 4.4±2.8 PQ mm 82.5±6.8 18.8±3.2 PQ : paraquat Data are the means ± SD. 으며 paraquat 1 mm 이하의농도와다르게 Paraquat 농도 mm에서는아포프토시스와함께세포괴사가일어나는것으로볼때, mm 이상의고농도에서는세포괴사가일어나는것으로생각된다 (Table 2). 3. Paraquat 에의한아포프토시스에서 N-acetylcysteine & dexamethasone의효과 결과 1. Paraquat에의한폐상피세포의생존율 A549 세포에 paraquat를 1 μm, μm, μm, 1 mm, mm 농도로처치하였을때세포생존율 (%) 은농도를증가시킴에따라시간의존적으로감소하였다 (Table 1-A)(Figure 1-A). BEAS-2B 세포에서도 paraquat를 1 μm, μm, μm, 1 mm, mm 농도로처치하였을때 A549 세포와유사하게세포생존율 (%) 의농도의존적이고시간의존적으로감소하였다 (Table 1-B)(Figure 1-B). 2. Paraquat에의한폐상피세포의 apoptosis Annexin Ⅴ 염색을통해서 paraquat 가폐상피세포의아포프토시스를유발한다는것을확인할수있었 항산화제인 N-acetylcysteine 1 mm을 paraquat 투여 1시간전에전처치한군과전처치하지않은군에서세포생존율을비교하였다. Paraquat 를 1 μm, μm, μm, 1 mm, mm 농도로투여하였을때, 시간의경과에따른세포생존율 (%) 은전처치하지않은군에비해서 N-acetylcysteine 전처치후에세포생존율의증가를보였다 (Figure 3). 통계분석은비모수통계법을이용하였고각군간의차이는 Mann- Whitney U test를이용하여비교분석하였으며 P-value <.5를통계적으로유의하다고분석하였다. 그결과 1mmM 이상의농도에서의미있게생존율의증가를보였다. Dexamethasone 1 μm을 paraquat 투여 12시간전에전처치한군과전처치하지않은군에서세포생존율을비교하였다. Paraquat를 1 μm, μm, μm, 1 mm, mm 농도로처치하였을때, N-acetylcysteine과 dexamethasone이 paraquat에의한아포프토시스를억제시킨다는것을확인할수있었으며이 A549 A549 + NAC 1 μm μm μm 1 mm mm 1 μm μm μm 1 mm mm 1 μm μm μm 1 mm mm 24 hr 48 hr 72 hr Figure 3. Inhibition of paraquat-induced cell deaths by NAC (NAC : N-acetylcysteine) 369
TH Song et al: Paraquat-induced apoptotic cell deaths 1 μm μm μm 1 mm mm 1 μm μm μm 1 mm mm 1 μm μm μm 1 mm mm 24 hr 48 hr 72 hr A549 A549 + Dexamethasone Figure 4. Inhibition of paraquat-induced cell deaths by dexamethasone A549 A549 pcdna3- bcl-2 1 μm μm μm 1 mm mm 1 μm μm μm 1 mm mm 1 μm μm μm 1 mm mm 2 4 h r 4 8 h r 7 2 h r Figure 5. The role of bcl-2 in paraquat-induced cell deaths (Inhibition of paraquat-induced cell deaths by bcl-2 overexpression) 러한억제효과는 paraquat 농도가증가할수록, 시간이경과할수록뚜렷하였다. 가지며, paraquat의농도가증가할수록그리고시간이경과할수록억제효과가뚜렷하였다. 4. Paraquat 에의한아포프토시스에서 bcl-2 단백질의효과 고 찰 Bcl-2를 overexpression시킨 A549 pcdna3-bcl-2 군과 A549 군에서세포생존율을비교하였다. Paraquat를 1 μm, μm, μm, 1 mm, mm 농도로처치하였을때, 시간의경과에따른세포생존율은 A549 세포군보다 A549 pcdna3-bcl-2 세포군에서더높게나왔으며농도가증가할수록유의한증가를보였다 (Figure 5). 이러한결과로볼때 paraquat에의한 A549 세포의아포프토시스에 bcl-2 단백질이분명한억제효과를 인체의폐는 가지이상의다른세포들로이루어진다양한기관으로, 이들세포각각은독특한형태학적, 기능적특징을가진다. 호흡기의상피세포들은화학물질, 약물, 그리고무기질과립등에지속적으로노출된다. 폐포의대식세포는폐포공간안에서하나의자유로운세포군으로존재하며변연부폐세포의 5% 를차지한다. 이들은흡입한공기에있는독성과립, 가스들, 그리고병독소들과직접적으로접촉하게되며, 대식세포의식세포작용동안상당량의활동성산소유리기가생산된다. 이들은식세포과정에필요하 3
Tuberculosis and Respiratory Diseases Vol. 61. No.4, Oct. 6 지만, 위험한부작용을유발할수있고폐에손상을주기도한다 8,9. Paraquat는 세기초에산화 환원지시제로영국에서개발되었다. 19년대에제초제로변형개발되어전세계적으로농업분야등에널리이용되지만, 인체에노출시사망할정도로치명적인부작용을가지고있다. Paraquat는인체내에서용해되어전자수용체로작용하며반응성산소유리기 (reactive oxygen species, ROS) 를발생시킨다. 반응성산소유리기는신호전도의두번째전달자로서, 세포신호전달과정에참여한다. 반응성산소유리기는세포막, 단백질, 핵산등과반응함으로써세포손상을일으킨다. Rose 등은 paraquat 가혈중보다폐에 6~배더축적된다고보고하였으며, 산소분압이높고 free radical 의제거효소계가상대적으로적은폐포세포에서가장심한손상이일어난다고하였다. Paraquat에의한반응성산소유리기는폐포상피세포인 type Ⅰ, type Ⅱ 폐포세포에직접작용하여폐포내출혈, 폐부종, 폐포상피세포의파괴를초래하여폐포염을유발하게되고결국폐섬유증을일으킨다. 따라서이러한 paraquat에기인한폐섬유증은간질성폐질환의원인과병리기전을연구하는모델로서이용되고있다 11. 본연구에서인체의폐상피세포주인 A549 세포주와 BEAS-2B 세포주를이용하여 paraquat의세포죽음을알아본결과 paraquat는농도증가및시간경과에비례하여세포죽음이유발됨을알수있었다. 최근의연구보고에의하면급성폐손상및급성호흡부전증후군의발생기전에있어서폐상피세포의아포프토시스가중요한역할을한다고한다 6. 급성호흡부전증후군은과립구의축적, 혈관투과성의증가, 폐탄성의감소, 가스교환의이상, 그리고흉부방사선소견상미만성폐침윤등을특징으로하는염증성폐질환이다 12. 이러한증후군은호흡기질환에의한사망의주요원인이다. Hagimoto 등은동물실험에서 DNA 손상을초래하고, 아포프토시스를유발하는것으로알려진 bleomycin에의해폐섬유증이발생할때폐포상피세포에서 Fas mrna가발현되는것을밝혔다. 또한침윤된임파구에서도 FasL mrna가발현되었으며, 이러한전구아포프토시스분자들의발현은 아포프토시스에의해폐포상피세포손상이일어난다는것을의미한다 11,13-15. 또급성기에활성화된과립구의제거가지연되면기도내에과립구의축적이생기며급성폐손상후반기에폐포상피벽이파괴된다. 이것은아포프토시스분자의적절한발현이폐손상을치유하는데도필수적이라는것을암시한다. 폐포막에서일어나는아포프토시스는급성폐손상과급성호흡곤란증후군의치유와결과에영향을미칠것이다 11,16,17. Paraquat 에의한폐손상은반응성산소유리기의직접작용, 염증세포의활성화에의한초기폐손상, 그리고섬유아세포증식및세포기질들의변화가나타나는후기손상을특징으로한다. 급성폐손상후에폐상피세포가심각하고장기적으로손상되면, 정상적세포들간의관계가장기간파괴된다 5,8. 만약폐상피세포의복구가순조롭게진행되지못하면간엽세포의증식이수반되고결국에는섬유화에이르게된다. 이것은급성폐손상후에발생하는폐상피세포의아포프토시스가폐섬유화의병리적인특징임을보여준다 11,18. 본연구의결과폐상피세포주에서 paraquat 에의해아포프토시스가일어남을확인할수있었다. 다만고농도의 paraquat를투여시에는아포프토시스와세포괴사가같이나타나는데 mm이상의농도에서는세포괴사가주로일어난다. Glucocorticoids 는가장강력한항염증능력과면역억제능력을가지는약제로 NF-κB 의전사활성을억제하는 19, 등다양한기전에의해항염증효과를나타낸다. 또한 glucocorticoid는 phospholipase에의한세포막 phospholipids 의분해를억제함으로써 arachidonic acid cascade에서반응성산소유리기의형성을억제하고, 지속적으로산화물에노출되는폐상피세포에서 glutathione 합성을증가시킴으로써상피세포를보호한다. 최근의연구에서 glucocorticoid 는 caspase 3의발현을억제하고 bcl-2를발현시킴으로써세포의생명력을증가시키고아포프토시스로인한세포손상을억제한다고하였다 21,22. 본연구에서 glucocorticoid 는 paraquat의농도가높을때, 시간이어느정도지났을때 paraquat 에의한폐상피세포의손상을억제하였다. 이는 paraquat에의한세포죽음에서 gluco- 371
TH Song et al: Paraquat-induced apoptotic cell deaths corticoid의효과가항염증효과가아니고폐포상피세포를보호하는효과라는것을의미한다. 폐의산화손상에있어서 N-acetylcysteine (NAC) 의보호역할은이미알려져있다. 본연구에서 N-acetylcysteine은시간과 paraquat 농도에따라서약간의차이를보이나전반적으로 ~% 정도의방어효과가있었다. 이러한효과는현재까지몇가지의기전을통해서설명이되는데 alveolar type Ⅱ 세포를보호하거나 glutathione 을증가시킴으로써산화손상에대한세포의저항을증가시킨다고한다 23-25. Bcl-2는마이토콘드리아의외부막에위치하는항아포프토시스단백질로잘알려져있다. 아포프토시스와관련된세포의손상은마이토콘드리아기능의손상과반응성산소유리기의과다한생산때문인데 bcl-2는산화성자극에의한세포손상을보호한다. Bcl-2는반응성산소유리기의생산혹은항산화경로에영향을미칠것으로생각된다. 본연구에서 paraquat에의한폐상피세포의손상에 bcl-2가어떠한영향을주는지알아보았으며, bcl-2를과발현시켰을때세포독성이약 ~% 정도감소하였다. 이러한결과는이전의연구결과와일치한다 26-28. 결론적으로, paraquat 는저농도에서아포프토시스를일으킨다. 또한 bcl-2 단백질과 N-acetylcysteine 의항아포프토시스역할을고려할때, paraquat에의해유발되는아포프토시스는마이토콘드리아경로를거칠것으로생각된다. 요약연구배경 : Paraquat는 P-4 reductase 에의해반응성산소유리기 (ROS) 를발생시켜세포막, 단백질, 핵산등과반응함으로써세포손상을유도하며급성폐손상을일으킨다. 최근급성폐손상및급성호흡곤란증후군에있어서폐상피세포의아포프토시스가중요한역할을한다고알려지기시작하였다. 이에반응성산소유리기에의한폐손상의대표적물질인 paraquat로인한폐상피세포의세포죽음이아포프토시스인지확인하고 dexamethasone, N-acetylcysteine, 그리고 bcl-2가 paraquat로인한폐상피세포죽 음에어떠한영향을미치는지등을연구하였다. 방법 : 폐상피세포주인 A549와 BEAS-2B 세포주, 그리고 bcl-2 construct를유전자주입한 A549 pcdna3-bcl-2 세포주를이용하였다. 아포프토시스는 Annexin Ⅴ assay를이용하여서판정하였으며세포독성검사는 MTT assay를이용하였다. Paraquat 는, 1 μm, μm, μm, 1 mm, mm의농도로사용하였다. Dexamethasone 은 1 μm의농도로 paraquat 투여 12시간전에전처치하였고, N-acetylcysteine은 1 mm의농도로 paraquat 투여 1시간전에전처치하였다. 결과 : 양세포주모두에서 paraquat는농도와시간경과에따라서세포죽음을증가시켰고, 이러한세포죽음은아포프토시스였다. N-acetylcysteine 과 dexamethasone 은시간과농도에따라약간의차이가있으나전반적으로 ~% 의방어효과가있었다. Bcl-2를과발현시킨 A549-bcl-2 세포주에서 A549-neo 세포주에비해 paraquat 에의한세포독성이약 ~% 정도차단되었다. 결론 : Paraquat는폐상피세포에서아포프토시스를유도하며, paraquat에의한아포프토시스는마이토콘드리아경로에의해일어날것으로추정된다. 참고문헌 1. Aldrich TK, Fisher AB, Cadenas E, Chance B. Evidence for lipid peroxidation by paraquat in the perfused rat lung. J Lab Clin Med 1983;1:66-73. 2. Skillrud DM, Martin WJ 2nd. Paraquat-induced injury of type II alveolar cells. Am Rev Respir Dis 1984;129:995-9. 3. TH Lee, SW Yang, TG LEE, SY Choi, YW Lee, KY Lee, Therapeutic effect of hemoperfusion in paraquat poisoning patients. Kor J Int Med 1997;52:114-9. 4. SI Lee, KW Ahn, CH Chung, The effect of aminotriazole on pulmonaty toxicity of paraquat poisoning. Tuber Respir Dis 1994;41:222-. 5. Uhal BD, Joshi L, True AL, Mundle S, Raza A, Pardo A, et al. Fibroblasts isolated after fibrotic lung injury induce apoptosis of alveolar epithelial cells in vitro. Am J Physiol 1995;269:L819-28. 6. Kitamura Y, Hashimoto S, Mizuta N, Kobayashi A, Kooguchi K, Fujiwara I,et al. Fas/FasL-dependent 372
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