[보고서]당뇨질환 모델에서 허혈성 뇌손상시 catechin에 의해 변화되는 특정단백질의 탐색 및 뇌세포 방어체계 규명

고필옥

[국가R&D연구보고서] 당뇨질환 모델에서 허혈성 뇌손상시 catechin에 의해 변화되는 특정단백질의 탐색 및 뇌세포 방어체계 규명
Identification of specific proteins and neuronal protective system by catechin in diabetics animal model during ischemic brain injury 원문보기

보고서 정보
주관연구기관	국립경상대학교
연구책임자	고필옥
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2021-06
과제시작연도	2021
주관부처	교육부 Ministry of Education
과제관리전문기관	한국연구재단 National Research Foundation of Korea
등록번호	TRKO202100011069
과제고유번호	1345345869
사업명	개인기초연구(교육부)(R&D)
DB 구축일자	2022-04-23
키워드	퇴행성 뇌질환.당뇨질환.카테킨.뇌세포 방어작용.치료제 개발.neurodegenerative disease.diabetic.catechin.neuroprotection.therapeutic agent development.

초록 ▼

□ 연구개발 목표 및 내용
￭ 최종 목표
당뇨질환과 퇴행성 뇌질환 치료를 위한 catechin의 활용가능성을 확인하고 catechin의 뇌손상시 방어기전에 관여하는 특정 단백질을 탐색하고 추적하여, 뇌질환의 원인을 규명하명하기 위한 구체적인 기초 자료를 제공하고, 이들 질환의 치료법 개발에 기여하고자 한다.
￭ 전체 내용
당뇨질환모델에서 허혈성 뇌손상시 catechin의 뇌세포 보호효과를 밝히며 catechin에 의해 조절되는 다양한 단백질들을 proteomic기법을 통하여 탐색, 추적하고, 탐색된 단백질들의 세포학적, 분자생물학적 방법을 통해 분석한다. 또한, 뇌세포 방어작용에 관여하는 catechin 방어기전에 관여하는 단백질을 규명하고자 한다.

□ 연구개발성과
본 연구과제는 고혈당 상태에서 뇌졸중 동물모델을 확립하였고, 대뇌 피질 세포의 손상시 catechin의 방어효과를 신경기능검사, 대뇌부종 측정, 뇌경색 측정, 조직병리학적 관찰을 통하여 구체적으로 밝혔다. 또한, catechin 투여에 의해 조절되고 변화되는 특정 단백질들을 proteomic 기법을 통하여 탐색하고 추적하였다. 또한, catechin에 의해 조절되는 apoptosis 관련 단백질, oxidative stress 관련 단백질, 칼슘조절단백질, metabolism 관련 단백질 등 특정 단백질들과의 관계 및 역할을 규명하였다. 또한, 해마 신경세포배양에서 glutamate로 유도된 세포 손상시 catechin의 방어기전을 분석하였다. oxidative stress에 의해 유도된 뇌세포 손상시 catechin에 의해 조절되고 변화되는 뇌세포 보호기전을 관찰하고 규명하였다. 또한, catechin은 뇌세포 손상시 증가된 세포내 칼슘농도를 catechin이 조절했다. 세포내 칼슘농도는 hippocalcin과 parvalbumin 단백질의 발현이 관여하고 있었고 catechin은 hippocalcin과 parvalbumin 단백질의 발현을 조절했다.

□ 연구개발성과 활용계획 및 기대 효과
본 연구과제의 수행으로 뇌손상 동물모델에서 catechin의 뇌세포 보호효과와 catechin에 의해 조절되는 특정 단백질을 탐색하고 추적하여 이들 단백질이 작용하는 방어기전을 밝혀 이들 질환의 치료 및 예방을 위한 기초 자료를 제공하였다. 퇴행성 뇌질환의 임상적으로 접근할 수 있는 치료법 개발에 기여하고, 이들 질환의 치료를 위해 catechin을 활용 할 수 있는 구체적인 자료를 제공하는데 중요한 의의가 있다. 또한, catechin을 통한 특정 뇌세포 방어기전을 통하여, catechin을 활용한 치료법 개발 및 조기진단의 정보를 제공하였고, 뇌질환의 치료 또는 완화를 가져오는 기술개발의 초석이 되었다고 판단된다.

(출처 : 요약문 2p)

목차 Contents

표지 ... 1
요약문 ... 2
목차 ... 3
1. 연구개발과제의 개요 ... 4
1) 연구개발의 필요성 ... 4
2) 연구의 최종목표 및 연구 범위 ... 6
2. 연구개발과제의 수행 과정 및 수행 내용 ... 6
(1) 연구수행 내용 ... 6
(2) 연구수행 과정 ... 7
3. 연구개발과제의 수행 결과 및 목표 달성 정도 ... 11
1) 연구수행 결과 ... 11
4. 연구개발성과의 관련 분야에 대한 기여 정도 ... 41
5. 연구개발성과의 관리 및 활용 계획 ... 41
6. 참고문헌 ... 41
끝페이지 ... 43

표/그림 (28)

표 Body weight and blood glucose levels in vehicle + sham, catechin + sham, vehicle + middle cerebral artery occlusion (MCAO), catechin + MCAO, diabetes + vehicle + MCAO, and diabetes + catechin + MCAO animals. Data (n=4) are represented as the mean ± S.E.M. * p < 0.05
표 Neurobehavioral scores in vehicle + sham, catechin + sham, vehicle + middle cerebral artery occlusion (MCAO), catechin + MCAO, diabetes + vehicle + MCAO, and diabetes + catechin + MCAO animals. Data (n=4) are represented as the mean ± S.E.M. * p < 0.05
표 Representative photos and infarct volume of brain sections. Brain sections were stained by TTC (A). The ischemic area remained white, while the intact area was stained red. The percentage of ischemic lesion area was calculated as lesion area/whole brain section area
표 Representative photos of Fluoro-Jade B and DAPI staining in the cerebral cortex of vehicle + sham, catechin + sham, vehicle + middle cerebral artery occlusion (MCAO), catechin + MCAO, diabetes + vehicle + MCAO, and diabetes + catechin + MCAO animals. Data (n=4) are represented as the mean ± S.E.M. * p < 0.05
표 Two-dimensional SDS-PAGE analysis of proteins from brain tissue induced MCAO in vehicle + sham, vehicle + MCAO (middle cerebral artery occlusion), diabetic + sham, and diabetic + MCAO groups. Squares indicate the protein spots that were differentially expressed between vehicle + MCAO and diabetic + MCAO groups
표 Neurological deficit score, corner test, and adhesive-removal test in vehicle + middle cerebral artery occlusion (MCAO), epigallocatechin gallate (EGCG) + MCAO, vehicle + sham, and EGCG + sham animals. EGCG improved neurological deficit and biased right turning behavior in ischemic brain damage. Data are graphed as median value (bold black line), upper and lower quartiles (box boundaries), highest and lowest values (whiskers), and black dot (outlier). Mann-Whitney test. Data (n=4) of d and e are shown as the mean ± S.D. # p < 0.001, two-way ANOVA followed by Scheffe’s test
표 Water content, reactive oxygen species, and lipid peroxidation analyses in vehicle + middle cerebral artery occlusion (MCAO), epigallocatechin gallate (EGCG) + MCAO, vehicle + sham, and EGCG + sham animals. EGCG alleviated the increases in DCF and MDA in ischemic-damaged cerebral cortex. Data (n=4) of d and e are shown as the mean ± S.D. # p < 0.001, two-way ANOVA followed by Scheffe’s test
표 Representative photograph of TTC staining (A) and infarct volume (B) in vehicle + sham, epigallocatechin gallate (EGCG) + sham, vehicle + middle cerebral artery occlusion (MCAO), and EGCG + MCAO animals. Intact areas stained red, while ischemic areas remained white in color (A). Infarct volume was calculated as the ratio of infarct area to total brain area (B). EGCG attenuated MCAO-induced infarction. Data (n=4) are represented as mean ± S.E.M. * p < 0.01, ** p < 0.05 vs. vehicle + sham animals, # p < 0.05 vs. vehicle + MCAO animals
표 Representative photograph of hematoxylin and eosin staining and TUNEL staining in vehicle + sham, epigallocatechin gallate (EGCG) + sham, vehicle + middle cerebral artery occlusion (MCAO), and EGCG + MCAO animals. EGCG alleviated histopathological changes caused by MCAO. Filled arrows indicate shrunken and condensed nuclei, and open arrows indicate swelled and vacuolated forms. Scale bar = 100 μm. The number of TUNEL positive cells was markedly increased in vehicle + MCAO animals, while EGCG decreased the number of these positive cells. Arrows indicate cells with positive TUNEL staining. Scale bar: 100 μm. Apoptotic index indicates the percentage of TUNEL-positive cells to total cells. Data (n=4) are shown as mean ± S.E.M. * p < 0.01, ** p < 0.05 vs. vehicle + sham animals, # p < 0.05 vs. vehicle + MCAO animals
표 Representative images of Fluoro-Jade B (FJB, green) and DAPI (nuclei marker, blue) staining in the cerebral cortex of vehicle + middle cerebral artery occlusion (MCAO), epigallocatechin gallate (EGCG) + MCAO, vehicle + sham, and EGCG + sham animals. FJB-positive cells were increased in the cerebral cortex with ischemic damage. EGCG alleviated these increases (a, b). Arrows indicate FJB-positive cells. Scale bar: 100 μm. Data (n=4) are shown as the mean ± S.D. * p <0.001, two-way ANOVA followed by Scheffe’s test
표 Silver stained gel image of two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis in the cerebral cortex from vehicle + sham, epigallocatechin gallate (EGCG) + sham, vehicle + middle cerebral artery occlusion (MCAO), and EGCG + MCAO animals (n=5 per group). Isoelectric focusing was performed at pH 4-7 IPG strips and electrophoresed on 7.5-17.5% gradient SDS gels. Each square indicates the significantly changed protein spots between vehicle + MCAO and EGCG + MCAO animals
표 List of identified proteins that were significantly differentially expressed in vehicle- and epigallocatechin gallate (EGCG)-treated animals with middle cerebral artery occlusion
표 Magnified protein spots of isocitrate dehydrogenase (ICDH), dynamin-like protein 1 (DLP-1), γ-enolase, pyridoxal-5
표 Western blot analysis of isocitrate dehydrogenase (ICDH), dynamin-like protein 1 (DLP-1), γ-enolase, pyridoxal-5
표 Reverse transcription-PCR (RT-PCR) of isocitrate dehydrogenase (ICDH), dynamin-like protein 1 (DLP-1), γ-enolase, pyridoxal-5
표 Sequence of the primers used for PCR amplification
표 Western blot analysis of poly ADP-ribose polymerase (PARP), caspase-3, bad, and bax proteins in vehicle + sham, epigallocatechin gallate (EGCG) + sham, vehicle + middle cerebral artery occlusion (MCAO), and EGCG + MCAO animals. EGCG prevents MCAO-induced increases of caspase-3, PARP, bad, and bax expressions. Densitometric analysis is represented as a ratio of caspase-3 and PARP staining intensity to actin intensity. Data (n=4) are shown as mean ± S.E.M. * p < 0.01, ** p < 0.05 vs. vehicle + sham animals, # p < 0.05 vs. vehicle + MCAO animals
표 Western blot analysis of parvalbumin and hippocalcinpoly proteins in vehicle + sham, epigallocatechin gallate (EGCG) + sham, vehicle + middle cerebral artery occlusion (MCAO), and EGCG + MCAO animals. EGCG prevents MCAO-induced increases of parvalbumin and hippocalcin expressions. Densitometric analysis is represented as a ratio of parvalbumin and hippocalcin staining intensity to actin intensity. Data (n=4) are shown as mean ± S.E.M. * p < 0.01, ** p < 0.05 vs. vehicle + sham animals, # p < 0.05 vs. vehicle + MCAO animals
표 Western blot analysis of p-Raf, p-MEK1/2, p-PTEN proteins in vehicle + sham, epigallocatechin gallate (EGCG) + sham, vehicle + middle cerebral artery occlusion (MCAO), and EGCG + MCAO animals. Densitometric analysis is represented as a ratio of protein staining intensity to actin intensity. Data (n=4) are shown as mean ± S.E.M. * p < 0.01, ** p < 0.05 vs. vehicle + sham animals, # p < 0.05 vs. vehicle + MCAO animals
표 Peroxiredoxin-2 expression in vehicle + sham, epigallocatechin gallate (EGCG) + sham, vehicle + middle cerebral artery occlusion (MCAO), and EGCG + MCAO animals. Densitometric analysis is represented as a ratio of protein staining intensity to actin intensity. Data (n=4) are shown as mean ± S.E.M. * p < 0.01, ** p < 0.05 vs. vehicle + sham animals, # p < 0.05 vs. vehicle + MCAO animals
표 PP2A subunit B expression in vehicle + sham, epigallocatechin gallate (EGCG) + sham, vehicle + middle cerebral artery occlusion (MCAO), and EGCG + MCAO animals. Densitometric analysis is represented as a ratio of protein staining intensity to actin intensity. Data (n=4) are shown as mean ± S.E.M. * p < 0.01, ** p < 0.05 vs. vehicle + sham animals, # p < 0.05 vs. vehicle + MCAO animals
표 Image of thioredoxin (TRX) protein spots using proteomics (A, D), Western blot (B, E), and reverse transcription-PCR analyses (C, F) in vehicle + middle cerebral artery occlusion (MCAO), epigallocatechin gallate (EGCG) + MCAO, vehicle + sham, and EGCG + sham animals. Squares of A photos indicate TRX protein spot. TRX protein level is expressed as a ratio of spot intensity in each group relative to that in the vehicle + sham group. TRX protein and mRNA levels are expressed as the intensity of TRX to that of β-actin. Data (n=4) are shown as the mean ± S.D. * p < 0.01 and ** p < 0.001, two-way ANOVA followed by Scheffe’s test
표 Images of immunofluorescence staining with thioredoxin (TRX, green) and DAPI (nuclei marker, blue) in vehicle + middle cerebral artery occlusion (MCAO), epigallocatechin gallate (EGCG) + MCAO, vehicle + sham, and EGCG + sham animals. Quantified number of TRX-positive cells in the cerebral cortex region. Arrows indicate TRX-positive cells. Data (n=4) are shown as the mean ± S.D.* p < 0.001, two-way ANOVA followed by Scheffe’s test. Scale bars = 100 μm
표 Immunoprecipitation result of thioredoxin (TRX) and apoptosis signal-regulating kinase 1 (ASK1) interaction in vehicle + middle cerebral artery occlusion (MCAO), epigallocatechin gallate (EGCG) + MCAO, vehicle + sham, and EGCG + sham animals. Densitometric results are represented as intensity ratio of identified protein to IgG intensity (b). Data (n=4) are represented as the mean ± S.D. * p < 0.01, two-way ANOVA followed by Scheffe’s test
표 Cell viability in glutamate exposured HT22 cells. Epigallocatechin gallate (EGCG, 10, 20, 40 μM) was applied for 1 h and glutamate (Glu, 5 mM) was subsequently treated. Cell images were obtained by phase-contrast microscopy to confirm the morphological changes. MTT assay was performed to access cell viability. Data (n=5) are shown as the mean ± S.D. *p < 0.01 and **p < 0.001, two-way ANOVA followed by Dunnett’s multiple comparison. Scale bar=100 μm
표 Western blot analysis of thioredoxin (TRX) in HT22 cells. Epigallocatechin gallate (EGCG, 10, 20, 40 μM) was applied for 1 h and glutamate (Glu, 5 mM) was subsequently treated. Densitometric results are expressed as the intensity of thioredoxin to that of β-actin. Data (n=5) are shown as the mean ± S.D. *p < 0.01 and **p < 0.001, two-way ANOVA followed by Dunnett’s multiple comparison. Scale bar=100 μm
표 Western blot analysis of thioredoxin (TRX, A, B), caspase-3 and cleaved caspase-3 (C, D) in siRNA transfected cells. Epigallocatechin gallate (EGCG, 10, 20, 40 μM) was applied for 1 h and followed with glutamate (Glu, 5 mM). Densitometric results are expressed as the ratio of TRX or caspase-3 intensity to that of β-actin. Data (n=5) are shown as the mean ± S.D. * p <0.001 in un-transfected group, # p <0.05, ## p <0.001 in transfected group, two-way ANOVA followed by Scheffe’s test
표 Calcium concentration in glutamate-treated HT22 cells. Glutamate, vehicle, catechin was exposed to HT22 cells for 24 h. ata (n=4) are represented as the mean ± S.E.M. * p < 0.05

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