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Intracellular Pathway and Neuroprotective Effect of Carpinus tschonoskii MAX on 6-Hydroxydopamine-Induced Death of PC12 Cells

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Alternative Title
6-Hydroxydopamine에 의한 PC12 세포 사멸 기전 및 개서어나무의 신경보호 효과
Abstract
파킨슨병에서 도파민성 신경세포의 소실과 관련된 생화학적 기전은 아직도 명확하게 밝혀져 있지 않다. 최근 연구된 바에 의하면, MPTP 모델에서 Calpain과 Cdk5에 의해 neuronal survival에 중요한 transcription factor인 MEF2D의 과인산화가 신경세포의 사멸과 관련되었을 것이라는 보고가 있었다. 그러나 6-hydroxydopamine (6-OHDA)에 의한 PC12 세포의 사멸이 Calpain과 Cdk5에 의해 매개된 MEF2D의 감소와 관련되어 있는지에 대한 연구 보고는 아직까지 없으며, 이를 규명하는 것은 파킨슨병의 도파민성 신경세포의 소실 기전을 이해하는데 큰 기여를 할 것으로 사료된다.
이에 본 연구에서는 PC12 세포에서 6-OHDA이 농도 및 시간 의존적으로 MEF2D를 확연히 감소시키는 것을 확인하였다. 또한, 우리는 6-OHDA에 의해 감소된 MEF2D가 Calpain 억제제인 PD151746과 Cdk5 억제제인 Roscovitine에 의해 각각 증가되는 것을 확인하였다. 이것은 6-OHDA에 의해 감소된 MEF2D는 Calpain과 Cdk5의 활성 모두에 의존적임을 확인할 수 있었다. 한편으로, 세포의 죽음과 생존에서 중요한 역할을 수행하는 MAP kinase와 Akt의 인산화가 6-OHDA에 의해 어떻게 변하는지를 분석하였다. PC12 cell에 6-OHDA를 처리하였을 때, ERK1/2, JNK 및 p38의 인산화가 시간 의존적으로 증가된 반면, Akt의 인산화는 시간 의존적으로 감소되었다. 이러한 결과들로 미루어 보아, MEF2D downregulation, MAP Kinase의 활성화 및 Akt의 비활성화가 6-OHDA에 의한 PC12 세포 죽음에서 중요하게 작용한다고 사료될 수 있었다. 또한 Cdk5와 MAP kinase 사이의 관련성을 확인해 본 결과, Cdk5 억제제인 Roscovitine은 ERK1/2와 JNK의 인산화를 증가시키는 반면, p38의 인산화는 감소시켰다. 이 결과는 Cdk5와 MAP kinase 신호전달 사이가 관련되어 있음을 시사하고, 두 신호전달 사이의 관련성은 신경세포의 생존과 죽음을 결정하는데 중요한 역할을 할 것으로 사료된다. 우리는 또한 6-OHDA에 의한 PC12세포의 죽음에서 항산화효과가 있다고 알려진 개서어나무 (Carpinus tschonoskii MAX)의 신경보호효과를 조사하였다. 개서어나무는 6-OHDA에 의해 유도된 PC12 세포의 죽음을 다음과 같은 메커니즘을 통해 약화시켰다: 1) 증가된 산화적 스트레스 (ROS와 NO)를 억제시켰다; 2) 감소된 MEF2D를 회복시켰다; 3) 활성화된 ERK1/2와 JNK 신호전달을 감소시켰다; 4) 비활성화된 Akt의 인산화를 증가시켰다; 5) Bax의 발현을 감소시키고, Bcl-2, procaspase-3와 PARP의 발현은 증가시켰다. 종합하면, 개서어나무 추출물은 6-OHDA에 의한 PC12세포의 죽음을 예방하고, 이러한 결과는 개서어나무가 파킨슨병의 치료전략으로 이용 가능함을 제시한다.
The biochemical mechanisms involved in dopaminergic neuron loss in Parkinson's disease are not clearly defined. Recent studies reported that neuronal cell death is associated with Calpain/Cdk5-mediated hyperphosphorylation of myocyte enhancer factor 2 (MEF2), a critical transcription factor for neuronal survival, in MPTP model. We therefore investigated the possible involvement of Calpain/Cdk5-mediated MEF2D downregulation for 6-OHDA-induced death of PC12 cells.
In this study, we found that 6-OHDA significantly decreased MEF2D level in a concentration/time-dependent manner in PC12 cells. We identified that the decrease MEF2D by 6-OHDA in PC12 cells was increased by Calpain inhibitor PD151746 and Cdk5 inhibitor Roscovitine, respectively. We evaluated that 6-OHDA-induced the degradation of MEF2D is dependent on both Calpain and Cdk5 activity. On the other hand, we analyzed the phosphorylation state of several MAP kinase pathway and PI3/Akt pathway, a central role in cell death and survival. After the PC12 cells were treated with 6-OHDA, the phosphoylation of ERK1/2, JNK and p38 was increased, whereas the phosphoylation of Akt was decreased in a time-dependent manner compared to the control. These results suggest that MEF2D downregulation, MAP kinase activation and Akt inactivation plays a central role in PC12 cell death by 6-OHDA. Roscovitine, an inhibitor of Cdk5, increased the phosphorylation of ERK1/2 and JNK, while the phosphorylation of p38 was decreased. These results suggest that a relationship between Cdk5 and MAP kinase signals may play important roles for determining either survival or death of neuronal cells. We also examined the neuroprotective effects of Carpinus tschonoskii MAX on 6-OHDA-induced death of PC12 cells. C. tschonoskii attenuated PC12 cell death induced by 6-OHDA by the following mechanisms: it inhibited the generation of oxidative stress (ROS and NO); it recovered MEF2D levels; it decreased the activities of the ERK 1/2 and JNK pathways; it reduced the expression of Bax and increased those of Bcl-2, procaspase-3 and PARP. Collectively, these results provide evidence that a C. tschonoskii extract prevents PC12 cell death induced by 6-OHDA, and that it may provide the tools as the therapeutic strategies in neurodegenerative disorder including Parkinson's disease.
Author(s)
Kim, Min-Kyoung
Issued Date
2009
Awarded Date
2009. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000004577
Alternative Author(s)
김민경
Affiliation
제주대학교 대학원
Department
대학원 의학과
Advisor
강희경
Table Of Contents
Ⅰ. Introduction = 1
Ⅱ. Materials and Methods = 7
1. Reagents = 7
2. Cell cultures = 7
3. Measurement of cell viability = 8
4. Evaluation of intracellular Reactive Oxygen Species (ROS) = 8
5. Measurement of nitric oxide (NO) production = 9
6. Morphological analyses = 9
7. DNA fragmentation = 9
8. Western blot analyses = 9
9. Statistical analyses = 10
Ⅲ. Results = 13
1. Intracellular pathways involved with 6-hydroxydopamine-induced death of PC12 cells = 13
1.1. 6-OHDA-induced death of PC12 cells = 13
1.2. 6-OHDA-induced apoptosis of PC12 cells = 13
1.3. 6-OHDA generates reactive oxygen species (ROS) and nitric oxide (NO) = 14
1.4. 6-OHDA-induced death of PC12 cells correlates with the degradation of MEF2D = 19
1.5. 6-OHDA-induced MEF2D downregulation is mediated by both Calpain and Cdk5 signaling = 19
1.6. Activation of MAP kinase pathway appears to be involved with PC12 cell death induced by 6-OHDA = 19
1.7. 6-OHDA-induced death of PC12 cells involved in the inactivation of Akt pathway = 20
1.8. Influence of Cdk5 on 6-OHDA-induced alterations of MAP kinase and PI3K/Akt signaling: Cdk5 and MAP kinase 'cross-talk'. = 26
1.8.1. Cdk5 regulates the ERK1/2 pathway = 26
1.8.2. Cdk5 may affects the JNK and p38 MAPK pathways = 26
1.8.3. Cdk5 activity may not affects the PI3/Akt pathway in 6-OHDA-induced death of PC12 cells = 26
2. Neuroprotective effect of Carpinus tschonoskii MAX on 6-hydroxydopamine-induced death of PC12 cells = 31
2.1. Carpinus tschonoskii MAX protects PC12 cells against 6-OHDA-induced death = 31
2.2. Carpinus tschonoskii MAX attenuates 6-OHDA-induced apoptosis in PC12 cells = 31
2.3. Carpinus tschonoskii MAX reduces 6-OHDA-induced generation of intracellular ROS and NO = 32
2.4. Carpinus tschonoskii MAX ameliorates 6-OHDA-induced downregulation of MEF2D = 38
2.5. Carpinus tschonoskii MAX affects 6-OHDA-induced activation of ERK1/2 and JNK = 38
2.6. Carpinus tschonoskii MAX recovers 6-OHDA-induced inactivation of Akt = 38
Ⅳ. Discussion = 43
Ⅴ. Reference = 48
Ⅵ. Abstract in Korean = 57
Degree
Master
Publisher
제주대학교 대학원
Citation
Kim, Min-Kyoung. (2009). Intracellular Pathway and Neuroprotective Effect of Carpinus tschonoskii MAX on 6-Hydroxydopamine-Induced Death of PC12 Cells
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General Graduate School > Veterinary Medicine
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