KISEP Original Article 대한정신약물학회지 : 제 17 권제 5 호 2006 대뇌피질신경세포일차배양에서리튬의 FeCl 2 에의한 Free Radical Injury 증강기전연구 이승혜 1 이영애 2 변한열 2 곽병주 3 정영기 1 노재성 1 아주대학교의과대학정신과학교실, 1 주식회사뉴로테크중앙연구소, 2 아주대학교의과대학약리학교실 3 ABSTRACT Lithium Potentiates the FeCl 2 Induced Free Radical Injury in Primary Mouse Cortical Cell Culture Seung-Hye Lee, MD 1, Young Ae Lee 2, Han Yeol Byun 2, Byoung Joo Gwag, MD 3, Young Ki Chung, MD 1 and Jai Sung Noh, MD 1 1 Department of Psychiatry, 3 Pharmacology, Ajou University, School of Medicine, 2 Central Laboratory, Neurotech, Suwon, Korea Objectives:For the past half century, lithium has been used for the acute and prophylactic treatment of bipolar disorder and recurrent depression. Recently, new pharmacological effects of Li + have appeared, showing that Li + can influence neuronal injury. We tested the effects of Li + on free radical induced neuronal injury in primary murine cortical cell cultures. Methods:Cortical cells were prepared from fetal mice (embryonic day 15) and exposed to 30 μm Fe 2+ alone or with 5 mm Li + or 5 mm Li + alone for 24 hrs at Days in vitro (DIV) 14. Neuronal death was analyzed by measuring lactate dehydrogenase (LDH) release into media. The fluorescence of 2,7 - dichlorofluorescin (DCF) was measured in as a mean of estimating the formation of reactive oxygen species (ROS). Results:Li + alone does not produce neuronal injury itself but it potentiates Fe 2+ -induced neuronal injury through increasing the production of free radical. Conclusion:This study suggests that the effects of Li + on neuronal survivorship may be injury type dependent and Li + potentiate the free radical injury. Therefore in practice clinician should be cautious in using the lithium in the treatment of brain injured patients. (Korean J Psychopharmacol 2006;17(5):468-474) KEY WORDS:Lithium Free radical injury Necrosis. 서론 리튬은급성조울증상태뿐만아니라반복적인조증과우울삽화의예방에도효과가있고자살예방에도효과가 있는것으로알려져있다. 1) 정신과에도입된최초의약물인리튬은기분안정제 (mood stabilizer) 로서의탁월한효과를설명하기위한다양한연구의대상이었고단일표적 (target) 이라기보다는여러개의표적과작용기전을 접수일자 :2006 년 6 월 5 일 / 심사완료 :2006 년 7 월 1 일교신저자 : 노재성, 442-749 경기도수원시영통구원천동산 5 번지아주대학교의과대학정신과학교실전화 :(031) 219-5180 전송 :(031) 219-5179 E-mail:jsnoh@ajou.ac.kr 468
이승혜등 통해서기분안정제로서작용할가능성이많다. 2,3) 최근에는리튬이신경세포사에도영향을미쳐신경세포보호효과가있다고알려져있다. 지금까지연구되어온것들을보면리튬이 ceramide, staurosporine, potassium 결핍에의한신경세포의세포자멸사 (apoptosis) 를억제하고, 4-6) 소뇌과립세포 (granular cell) 배양에서리튬을장시간처리할경우 N-methyl-D-aspartate(NM- DA) 수용체활성화에의한괴사 (necrosis) 를억제한다. 7) 리튬의이런신경세포보호효과는 NMDA 독성에국한되지않고항경련제인 phenytoin 과 carbamazepine 에의한세포자멸사를억제하고, 8) 베타아밀로이드에의한신경세포사도억제한다고알려졌다. 9) 그외에도리튬에의한 pro-apoptotic protein인 p53, Bax의발현감소, cytoprotective protein인 Bcl-2의증가, cell survival kinase 인 Akt 의활성화, glycogen synthase kinase 3β (GSK-3β) 의억제등이있다. 3,10) 이러한연구결과들을고려해볼때리튬의신경세포보호는한가지가아닌여러기전과표적을통해이루어진다고생각해볼수있다. 하지만리튬은신경세포보호효과뿐만아니라신경독성 (neurotoxicity) 효과도있다. 리튬의신경학적부작용으로진전 (tremor), 기억력이감소하거나반응시간이느려지는등의인지기능의장애, 드물게는말초신경병증 (peripheral neuropathy) 등이나타날수있으며심하면발작 (seizure) 하거나의식을잃고사망할수도있다. 11) 리튬을장기간사용하였을경우지속적인소뇌의기능장애를나타내거나추체외로증후군 (extrapyramidal syndrome) 이나타나기도하며뇌간 (brainstem) 에기능장애를나타낼수있고치매증상이나타날수있다. 또한비특이적으로안구진탕 (nystagmus) 이나시신경염, 유두부종 (papilledema) 를나타내거나실명을유발할수도있다. 이러한신경독성이혈중치료농도에서도나타난다는것은리튬을임상에서사용함에있어주의깊게생각해보아야할부분이다. 12) 이와같이리튬은신경세포보호와신경독성이라는반대되는효과를모두나타낼수있고각각의작용이어떤작용기전에의해다르게작용하는지에대한연구가분명히밝혀져있지않다. 리튬의신경세포보호효과의긍정적인측면을주로강조하게되면실제임상에서일어날 수있는리튬의신경독성효과에대해간과하게되어여러부작용등을초래할수있다. 리튬이신경세포일차배양에서 calyculin A, cyclosporin A 유도된세포자멸사는효과적으로억제하지만 NMDA 에의한 excitotoxicity 및 FeCl 2 에의한활성산소에의한괴사 (necrosis) 는억제하지못한다. 13) 따라서리튬을신경세포효과로이용하기위해서는리튬의신경손상형태에따라미치는영향을알아보고그기전을규명하는것이필요할것으로생각된다. 이에본연구자들은신경세포일차배양에서산화적손상에의한세포사멸에대한리튬의영향과기전을알아보고자한다. 대상및방법 1. 실험동물및실험기기 1) 실험동물임신 15일된흰쥐 (ICR mouse) 를사용하였다. 2) 실험기기수술용기구는 ROBOZ(Rockvill MA) 를사용했고 CO 2 incubator 는 Forma Scientific(Marietta OH), clean beanch 는삼기 (Korea) 를사용하였다. 위상차현미경과해부현미경은 Olympus(Japan), conforcal scanning laser micorscope(lsm 510) 는 Carl Zeiss(Germany) 를사용하였다. microplate reader 는 molecular device (Menro Park, CA) 를, 원심분리기는 VISION Scientific (Korea) 를사용하였다. minimal essential medium 은 Invitrogen(California, U.S.A), FeCl 2 는 Fluka(Switzerland), Trolox 와 LiCl 는 Sigma 를사용하였다. 2. 배양액및용액조성 1) media stoke(ms) minimal essential medium(earl s salt, L-glutamine free, sodium bicarbonate free) 에 21 mm glucose와 26.5 mm sodium bicarbonate을첨가하였다. 2) growth medium(gm) liquid MEM(L-glutamine free, sodium bicarbonate free) 에 20 mm glucose, 10% horse serum 및 2 mm 469
리튬의 Free Radical Injury 증강기전연구 glutamine 을첨가하였다. 3) plating medium(pm) liquid MEM에 20 mm glucose, 5% fetal bovine serum, 5% horse serum과 2 mm glutamine을첨가하였다. 4) hepes controlled salt solution(hcss) 120 mm NaCl, 5 mm KCl, 1.7mM MgCl 2 2.3 mm CaCl 2 15 mm glucose, 18 mm HEPES, 10 mm NaOH and 0.001% phenol red 로구성하였다. 3. 대뇌피질세포배양대뇌피질세포의배양은이전에보고되었다. 14) 본연구에서사용한방법은임신 15일된흰쥐태아의대뇌피질을분리하여끝을 1/3 크기로줄인파스퇴르피펫을사용하여단세포로분리하였다. PM에희석하여 100 μg/ml poly-d-lysine과 4 μg/ml laminin으로 coating한 24 well plate 에 5 hemispheres/plate(2 105 cell/plate) 의밀도로세포를분주하여 5% CO 2, 37도로유지되는세포배양기에서배양하였다. 신경세포와아교세포 (glia cell) 를포함하는혼합배양세포는 7일째 (DIV 7) 별아교세포 (astrocyte) 가단일층으로자란후아교세포 (glia cell) 가과도하게증식하는것을억제하기위해 10 μm Ara-C 를이틀간처리한후일주일에 2번씩 GM으로갈아주었다. 15) 4. 신경세포사분석 1) LDH 측정독성물질에노출시켰던세포들은 24시간동안위상차현미경으로계속관찰하며세포가죽는정도는신경독성약물에노출이후 24시간이지난다음, 죽은세포로부터배지로분비되는 LDH 의활성을측정하여분석하였다. 16) 세포배양상층액 25 μl 를 96 well plate 에옮기고 100 μl 의 β-nadh 용액 (0.3 mg/ml) 과 25 μl 의 22.7 mm pyruvate 액을넣고 1분간기다린후 microplate reader 로 340 nm에서흡광도변화를 2분간측정하였다. 측정값은거의모든신경세포가죽은조건인 500 μm NMDA 를 24시간동안계속하여노출시킨후그배지로부터얻은 LDH 활성값을 100% 세포 사의기준으로하고 sham wash 를 24시간동안노출하여얻은 LDH 활성값을 0% 세포사의기준으로하여다른 LDH 활성값을보정하였다. 17) 5. Free radicals 측정세포내 free radicals 은 ROS 에의해산화된 dichlorodihydrofluorescein diacetate(dcdhf-da) 의 fluorescent signal인 6-carboxy-2,7 -dichlorofluorescein diacetate(dcf-da) 의변화하는정도를통해측정하였다. 18) Bottom dish 에서배양된대뇌피질세포에 30 μm Fe 2+, 단독혹은표지된농도의리튬과함께처리한후 4시간후에 HCSS 완충액에 DCDHF-DA 의최종농도가 10 μm가되도록신경세포에첨가하여, 30분동안 37 에서배양하였다. 배양한후 conforcal laser scanning microscope(lsh510, Carl Zeiss) 위에 DCDHF-DA 를 490 nm(ex=490 nm, Em=510 nm) 에서흥분시켜그상을 CCD camera 를통하여얻은뒤 LSM 510 expert mode software version 3.2(Carl Zeiss) 를이용하여세포내에서생성된 free radical 의양을정량화하였다. 15) 6. 통계처리자료들은 Systat 사의 Sigmaplot version 9.0을이용하여분석하였다. 각군에서유도된 LDH 크기의평균간의차이를알아보기위해 one-way ANOVA 를시행하였고 post hoc test 로는 Student-Neuman-Keuls test 를시행하였다. 결과 1. 리튬이 Fe 2+ 에의한괴사를강화시킴. 14일간배양된대뇌피질세포에 Li + 5 mm를처리한후 24시간후에 LDH 분석법으로신경세포사를검색하면 Li + 자체만으로는신경세포사에영향을미치지않았다 ( 그림 1D). 역시 14일간배양된대뇌피질세포에 Fe 2+ 30 μm 를단독처리한것과 Fe 2+ 30 μm과 Li + 5 mm를같이처리한것 ( 그림 1C) 을 24시간후에 LDH 분석법으로신경세포사를정량해보면 Fe 2+ 에의한신경세포사가 Li + 첨가로인해증가되었음을알수있다. 또한 Fe 2+ 470 Korean J Psychopharmacol 2006;17(5):468-474
이승혜등 30 μm에항산화제인 Trolox 10 μm를처리하였을때신경세포사가억제됨을할수있다 ( 그림 1E). Fe 2+ 에의한 cell body 의 swelling 되는괴사가진행되고있고 ( 그림 1B), Li + 5 mm를첨가한후에괴사가더진행하고있음을보여준다 ( 그림 1C). 이결과는 Fe 2+ 에의한괴사는 Li + 에의해억제되지않고오히려강화되고 있음을보여주고있다. 2. 리튬이 free radical injury 를증강시킴. Li + 5 mm 단독처리한경우에 Li + 자체만으로 free radical 를형성하지않는다는것을알수있고 ( 그림 2D), Fe 2+ 30 μm 를처리한경우에는 free radical 이형성 A B E 70 60 * C D % Neuronal death 50 40 30 20 10 0 Alone LI Trolox Li alone [Fe 2+ ], 30 μm Figure 1. Li + potentiates necrotic cell death. A-C:Phase contrast photomicroscope of cortical cell cultures (DIV 13-14) exposed to following conditions:sham control (A), 30 μm Fe 2+ (B), 30 μm Fe 2+ plus 5 mm Li + (C), 5 mm Li + alone (D). E:Cortical cell cultures were exposed to 30 μm Fe 2+ alone or with 5 mm Li + or with 10 μm Trolox. Neuronal death was analyzed 24h later by measuring LDH efflux into the bathing medium, mean±sem (n=8 culture wells/ condition). *:p<0.05, significant difference from relevant control (alone) by analysis of variance and Student- Neuman-Keuls test. A B E 800 * 600 C D DCF intensity (%) 400 200 0 BL Li alone Alone LI [Fe 2+ ], 30 μm Figure 2. Li + potentiates free radical injury induced by Fe 2+. A-D:Fluorescence photomicrographs showing 6- carboxy-2,7 -dichlorofluorescin diacetate, the oxidation product of DCDHF-DA with hydroxyl radical, in cortical cell cultures after 4h exposure to sham control (BL, A), 30 μm Fe 2+ (B), plus 5mM Li + (C), 5 mm Li + alone (D), E:Levels of hydroxyl radical were analyzed by measuring fluorescence intensity of 6-carboxy-2,7 -dichlorofluorescin diacetate, mean±sem (n=50-60 neurons randomly chosen from two culture dishes for each condition). *:p<0.05, significant difference from relevant control (alone) by analysis of variance and Student-Neuman-Keuls test. 471
리튬의 Free Radical Injury 증강기전연구 되며 ( 그림 2B), Fe 2+ 30 μm에 Li 5 mm를첨가한경우에는 free radical 생성이더증강됨을알수있다 ( 그림 2C). 따라서 Li + 자체만으로 free radical 을형성하지않으나 Fe 2+ 에의한유발된 free radical 의생산을증강시켜 free radical injury 에의한괴사를강화시킨다 ( 그림 2E). 고찰 본연구를통해쥐의대뇌신경세포일차배양에서리튬단독처리로는신경세포사에유의미한영향을미치지않으나 FeCl 2 를처리하여괴사를유발한후에는신경세포사를더강화시키는것을알수있다. 또한그기전으로리튬이 FeCl 2 에의한 free radical injury를증강시킴으로써괴사에의한신경세포사를더증강시키는것으로생각된다. 본연구에서는신경세포사의형태에따른리튬의영향에대해알아보고자하는것으로신경세포사는세포자멸사와괴사로나누어볼수있다. 첫번째로세포자멸사는정상적발달과정중일어나며형태학적으로세포가죽을때세포질이수축하여핵염색질의응축이일어나고결국은핵막과세포질이여러개로분리되어주변의식균세포에의해제거되는세포사의한형태이다. 19,20) 신경성장단백질 (neurotrophic factors) 제거, 베타아밀로이드처리, protein kinase 활성억제, oxidative stress 등을겪는신경세포들에서세포자멸사가일어난다. 21-24) 두번째로괴사는형태학적으로세포질 (plasma) 과핵막 (nuclear membrane) 의파괴, 마이토콘드리아의팽창으로인한세포용적이특징적으로증가하여결국세포가파괴되는병적인현상으로정상적인발달과정에서는발생하지않으며주로 Ca 2+, Na 2+, Cl - 등이온들의급격한불균형을유도하는손상에의해일어난다. 이때신경세포에서는 ionotropic glutamate receptor 가활성화되어 Ca 2+ 과 Na 2+ 이세포안으로들어가고이에따라 Cl - 와 H 2O가신경세포안으로따라들어가서신경세포는수분후에팽창하여, 수시간이내에세포막이완전히유리된다. 25-29) 리튬의신경독성은이에대한작용기전이명확히밝 혀져있지않지만생검상 (biopsy) 말초신경계에광범위한말이집탈락 (demyelination) 이발견되었고특히소뇌를포함한중추신경계의여러부위에광범위한말이집탈락되어조롱박세포 (Purkinje cell) 의손실, 소뇌피질의신경아교증 (gliosis), 위축등에영향을미친다고보고된다. 리튬의신경세포보호와신경독성효과의두가지다른형태를가지게되는원인중의하나로장기간리튬에노출되면 PKC(Protein kinase C) 가활성화되고 GCK-3(glycogen synthase kinase-3β) 를억제되는신호전달과정이 Lithium-response gene network 에작용하여 Lithium-response transcription factors 나 promoter elements에의해유전자발현을변화시킴으로해서신경세포보호효과가있다고알려졌다. 12) 또한 60세이상에서치매가발병한군과대조군을비교해보았을때리튬을사용하였을경우치매발병을예방하지못하였다. 30) Alzheimer s disease 에서리튬이 GSK-3β 을억제하여 β-amyloid peptide 에의한 tau hyperphosphorylation과세포사를억제함으로써신경보호효과가있다는보고도있지만 31) 한편에서는리튬이 GSK-3β 의 translocation 은억제하지만 tau hyperphosphorylation과 DNA의산화성손상 (oxidative DNA damage) 의억제와는연관이없는것으로알려졌다. 따라서리튬을단독으로사용함으로해서 β-amyloid peptide 에의한여러가지부작용들을예방한다고할수없으므로 oxidative DNA damage 나 tau hyperphosphorylation 를막을수있는다른약물의사용을고려해보아야한다. 32) 본연구는리튬이세포사멸형태에따라다르게작용한다는것과그작용기전에대해알아보고자하였고, 리튬이그자체로는괴사를유발하지않으나신경세포에 FeCl 2 로인한 free radical injury 를유발하였을때 free radical 의생산을증가시킴으로써괴사를더욱강화시킴을알수있었다. 리튬이오랫동안광범위하게쓰여온약물이고뇌혈관장벽 (Brain-blood barrier, BBB) 를잘통과하고, 단백질에결합하지않는등그대사과정등이간단하고잘알려진약물이다. 따라서리튬의신경보호효과와신경독성효과가다르게나타나는것에대한작용기전을연구하여임상실제에서적응증을분명 472 Korean J Psychopharmacol 2006;17(5):468-474
이승혜등 히하여사용하는것이필요할것으로생각된다. 이를위해서 FeCl 2 외에다른 free radical injury에대한리튬의신경독성효과와작용기전에대해연구가되어야할것이며본연구결과를바탕으로해서리튬을임상에서적용시에항산화제를같이투여하는것에대해서도고려해보아야할것이다. 요약 목적 : 리튬은조울증및반복적우울증의급성기치료및유지기의재발방지를위해가장많이쓰이는약물이다. 이러한리튬의기분안정에대한연구들뿐만아니라신경세포보호에대한연구들이늘어나고있다. 하지만리튬이신경세포사의형태에따라특이적으로작용하여세포자멸사 (apoptosis) 는억제하지만괴사 (necrosis) 를더증강시킨다는연구가보고되고있어본연구에서는리튬이괴사 (necrosis) 를더증강시킨다는가설을세웠고그기전에대해알아보고자하였다. 방법 : 임신 15일된횐쥐를사용하여 14일간배양된대뇌피질세포에 24시간동안 Fe 2+ 30 μm, Li + 5 mm을각각단독처리하고 Fe 2+ 30 μm에 Li + 5 mm를처리하였다. 신경세포사는죽은세포로부터배지로분비되는 lactate dehydrogenase(ldh) 의활성을측정하여분석하였다. 세포내 free radicals 은 reactive oxygen species(ros) 에의해산화된 dichlorodihydrofluorescein diacetate(dcdhf-da) 의 fluorescent signal 를통해측정하였다. 결과 : 리튬자체로 free radical 을형성하여신경세포사를유발하지는않지만 Fe 2+ 에의해유도된 free radical 의생산을증가시킴으로써괴사를더욱증강시킴을알수있었다. 결론 : 본연구를통해리튬이그자체로는괴사를유발하지않으나신경세포에서 FeCl 2 로인한 free radical injury 를유발하였을때 free radical 의생산을증가시킴으로써괴사를더욱강화시킴을알수있었다. 이를통해리튬의다양한다른 free radical injury 에대한증강효과를연구하여임상실제에서리튬과함께항산화제를병용함으로써신경세포를보호할뿐만아니라세포괴사를예방할수있을것이다. 중심단어 : 신경세포사 리튬 free radical injury 세포자멸사 (apoptosis) 괴사 (necrosis). 참고문헌 1) Goodwin FK, Jamison KR. Manic-depressive illness. New york: Oxford university press;1999. 2) Jope RS. Anti-bipolar therapy: mechanism of action of lithium. Mol Psychiatry 1999;4:117-128. 3) Manji HK, Moore GJ, Chen G. Lithium at 50: have the neuroprotective effects of this unique cation been overlooked? Biol Psychiatry 1999;46:929-940. 4) Bijur GN, De SP, Jope RS. Glycogen synthase kinase-3beta facilitates staurosporine and heat shock-induced apoptosis. Protection by lithium. J Biol Chem 2000;275:7583-7590. 5) Centeno F, Mora A, Fuentes JM, Soler G, Claro E. Partial lithium-associated protection against apoptosis induced by C2- ceramide in cerebellar granule neurons. Neuroreport 1998;9: 4199-4203. 6) D Mello SR, Anelli R, Calissano P. Lithium induces apoptosis in immature cerebellar granule cells but promotes survival of mature neurons. Exp Cell Res 1994;211:332-338. 7) Nonaka S, Hough CJ, Chuang DM. Chronic lithium treatment robustly protects neurons in the central nervous system against excitotoxicity by inhibiting N-methyl-D-aspartate receptor-mediated calcium influx. Proc Natl Acad Sci USA 1998;95:2642-2647. 8) Nonaka S, Katsube N, Chuang DM. Lithium protects rat cerebellar granule cells against apoptosis induced by anticonvulsants, phenytoin and carbamazepine. J Pharmacol Exp Ther 1998; 286:539-547. 9) Wei H, Leeds PR, Qian Y, Wei W, Chen R, Chuang D. Betaamyloid peptide-induced death of PC 12 cells and cerebellar granule cell neurons is inhibited by long-term lithium treatment. Eur J Pharmacol 2000;392:117-123. 10) Chuang DM, Chen RW, Chalecka-Franaszek E, Ren M, Hashimoto R, Senatorove V, et al. Neuroprotective effects of lithium in cultured cells and animal models of diseases. Bipolar Disord 2002;4:129-136. 11) Benjamin JS, Virginia AS. Biological therapies. In: Edmondson J, Gabbard GO, Grebb JA, Manley M, Pataki CS, Sussman N. editors. Synopsis of psychiatry. behavioral sciences/clinical psychiatry. Lippincott Williams & Wilkins;2003. p.1069-1072. 12) Adityanjee, Munshi KR, Thampy A. The syndrome of irreversible lithium-effectuated neurotoxicity. Clin Neuropharmacol 2005; 28:38-49. 13) Kang HJ, Noh JS, Bae YS, Gwag BJ. Calcium-dependent prevention of neuronal apoptosis by lithium ion: essential role of phosphoinositide 3-kinase and phospholipase C gamma. Mol Pharmacol 2003;64:228-234. 14) Noh JS, Gwag BJ. Attenuation of oxidative neuronal necrosis by a dopamine D1 agonist in mouse cortical cell cultures. Exp Neurol 1997;146:604-608. 15) Noh JS, Kang HJ, Kim EY, Sohn S, Chung YK, Kim SU, et al. Haloperidol-induced neuronal apoptosis: role of p38 and c-jun- NH(2)-terminal protein kinase. J Neurochem 2000;75:2327-2334. 16) Koh JY, Choi DW. Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J Neurosci Methods 1987;20:83-90. 17) Jung JH. 6-hydroxydopamine induces neuronal apoptosis in murine primary cortical culture[master s thesis]. Gyeonggi: Ajou Univ.;1999. 18) Seo SY, Kim EY, Kim H, Gwag BJ. Neuroprotective effect of 473
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