세포사 경보인자 HMGB1의 펩타이드 억제제를 이용한 뇌졸중 신경보호 효과 연구 = Ischemia intervention by suppression of the danger signal molecule HMGB1 using inhibitory peptide

국문 초록 (Abstract)

Ⅰ. 연구개발의 목적 및 필요성
대표적인내인성세포사경고단백질(endogenousdangersignal)로알려진 HighMobilityGroupBox1(HMGB1)은정상세포에서는주로핵에존재하나,손상된세포에서방출되거나,분비되는방식을통해주변세포손상을가속화시키는한편염증을유도하여세포손상을증폭시킴.HMGB1은뇌졸중후신경세포손상을급격히악화시키고연이은염증반응을유도하는전령단백질로작용할것으로생각됨. 본연구는HMGB1과직접결합하는방식을통해HMGB1의작용을억제하는HMGB1억제펩타이드 (HMGB1InhibitoryPeptide,HIP)인3종류의heptamer펩타이드,HIP-1,-2,-3를대상으로HMGB1작용억제효과및뇌졸중동물모델에서의보호효과를조사하고그작용기전을밝혀HMGB1제어를통한뇌졸중치료방안개발의기반을확보하고자함.
Ⅱ. 연구개발의 내용 및 범위/Ⅲ. 연구개발결과
(1)HMGB1억제펩타이드(HMGB1inhibitorypeptide,HIP)효과검증을통한최종선정:1차선정된세개의HIP의뇌졸중동물모델에서의뇌경색억제효과와이에수반되는운동능력과neurologcaldeficits을조사한 결과HIP-2의 효과가 가장 우수한 것으로 판단하여 HIP-2를 최종선정.(2)HIP-2의작용기전규명:2차선정된 HIP-2이 HMGB1과 직접 결합하는 것을 pull down 실험을 통해 확인하였으며, 그 작용기전을1차배양신경세포와1차배양microglia세포를사용하여조사한 결과, HMGB1이중요한역할을하는것으로알려진염증억제효과와 신경세포apoptosis억제효과를확인하였음. (3)HIP의비침습적(non-invasive)방법인비강내투여(intransasaldelivery):heptamer인HIP의비강내투여한 후 효과를 조사한 결과 MCAO 동물 모델에서 뇌경색 억제 효과와 신경증적 손상 억제 및 운동능력 회복 효과를 확인함 (4)HIP효과 증진을 위한개선 시도:HMGB1억제를포함한복합기능수행을위해 아미노산 변형 길이 변형 등의 시도를 한 결과 약간의 효과 증진이 관찰되었으나, 현저하게 증가되지는 않음. (5)OSEOPONTIN 펩타이드와의 비강내병용 투여 또는 ethyl pyruvate를 정맥투여하면 HIP-2의 보호 효과가 증진되는 것을 확인함.(6)HIP-2 효과최적화를 위한, 용량, 시기, 투여 방법, 병용투여 방안 등을 확인하였음.
Ⅳ. 연구개발결과의 활용계획
(1)HIP의효능및안전성검증을통한안정적이고효과적인뇌졸중치료제개발의기반을제공
(2)뇌졸중후뇌경색을확장시키는2차손상과정의이해및trigger로써의HMGB1의역할이해
(3)뇌졸중에서치료효율을극대화할수있는복합처치방안이확보
(4)뇌졸중이외의중추신경계급성질환(간질,급성저산소성손상,뇌척수외상등)정보제공.
(5)단백질의mis-location에의해새로운기능을수행하는？？dangersignal"에대한이해확대

다국어 초록 (Multilingual Abstract)

High mobility group box 1 (HMGB1) is an endogenous danger signal molecule. In a previous report, we showed that HMGB1 is massively released during NMDA-induced acute damaging process in the postischemic brain and triggers inflammatory processes and induces neuronal apoptosis. We have also reported a robust neuroprotection of intranasally delivered HMGB1 siRNA in the postischemic rat brain (middle cerebral artery occlusion (MCAO), 60 minutes). In the present study, we investigated the therapeutic efficacy of intranasally delivered HMGB1 binding heptamer peptide (HBHP; HMSKPVQ), which was selected using a phage display approach, in the same stroke animal model. A pull-down assay using biotin-labeled HBHP showed that HBHP binds directly to HMGB1, specifically to HMGB1 A box, confirming HMGB1/HBHP interaction. HBHP significantly suppressed HMGB1-mediated neuronal cell death in primary cortical cultures and HMGB1/HBHP binding was detected in NMDA-conditioned culture media. However, a heptamer peptide composed of a scrambled sequence of the seven amino acids in HBHP failed to bind HMGB1 and had no protective effect. Furthermore, HBHP (300 ng) delivered intranasally at 30 minutes before MCAO significantly suppressed infarct volume in the postischemic rat brain (maximal reduction by 41.8± 5.4 %) and ameliorated neurological and behavioral deficits. In contrast, scrambled heptamer peptide had no protective effect at the same dose. Together these results suggest that intranasal HBHP ameliorates neuronal damage in the ischemic brain by binding HMGB1, which might inhibit the function of HMGB1 as an endogenous danger signal molecule.