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Molecular and Biochemical Analyses of Transcriptional Effects of the Stress Hormone Corticosterone on Neuronal Cell Death of Primary Cortical Culture

Title
Molecular and Biochemical Analyses of Transcriptional Effects of the Stress Hormone Corticosterone on Neuronal Cell Death of Primary Cortical Culture
Authors
박선규
Issue Date
2010
Department/Major
대학원 화학·나노과학과
Publisher
이화여자대학교 대학원
Degree
Master
Advisors
한평림
Abstract
Stress hormones such as glucocorticoids are released during stress response from the cortex of the adrenal gland. The major glucocorticoid is cortisol in primates and corticosterone in rodents. Glucocorticoids are beneficial and required for rapid changes of bodily physiology for animals encountering emergency circumstances. However, high levels of glucocorticoids are harmful to the body and increase neurotoxicity caused by seizer, hypoxia, and hypoglycemia. Elevated glucocorticoid levels affect many neuronal diseases including major depression and Alzheimer’s disease. However, the underlying mechanism is not elaborated in detail. Oxidative stress is resulted from excess generation of reactive oxygen species (ROS), such as hydrogen peroxide, nitric oxide, superoxide and hydroxyl radicals. Oxidative stress produces oxidation of lipids, proteins and DNA, causing brain cells to death. Because the brain consumes high oxygen, brain tissue is susceptible to oxidative damages. Oxidative stress is produced by an imbalance in pro-oxidant and antioxidant homeostasis. Several studies demonstrated that glucocorticoid-induced neurotoxicity is accompanied by increased reactive oxygen species (ROS) activities and altered antioxidant enzyme capacity in the brain. However, the molecular basis how glucocorticoids cause ROS stress-mediated cell death is clearly unknown. The present study was undertaken to unravel the mechanism underlying glucocorticoid-induced neuronal cell death. Corticosterone pretreatment for 24 h enhanced neurotoxicity of primary cortical neurons induced by 30 μM Fe²+. Moreover, corticosterone enhanced neurotoxicity induced by other insults, such as zinc, staurosporine, N-methyl-D-aspartate (NMDA), and sodium nitroprusside (SNP). Corticosterone had to be treated for several hours to produce an enhancement of neuronal cell death and pretreatment of corticosterone for 24 h increased the level of neuronal death induced by 30 μM Fe²+ threefold, suggesting that transcriptional changes are an important mechanism. In fact, co-treatment of corticosterone with cyclohexamide blocked corticosterone-induced enhancement of ferrous toxicity. These results suggest that corticosterone-induced enhancement of ferrous toxicity occurs through the mechanism of a new protein synthesis. Corticosterone-enhanced ferrous-toxicity was completely protected by antioxidant, such as trolox, vitamin C, and caffeic acid. Based on these results, this study explored to unravel the mechanism underlying corticosterone-induced changes related to reactive oxygen species. Next, the present study investigated to determine which factors are involved in corticosterone-induced neuronal cell death. Cyclooxygenase-2 (COX-2) expression leads to neuronal cell death via neuro-inflammation. Recently, a number of studies showed that COX-2 may be indirectly or directly involved in ROS generation. The non-selective COX inhibitor, aspirin (1-3 mM) and the COX-2 selective inhibitor, NS-398 (30-100 μM), attenuated the corticosterone-enhanced Fe²+ toxicity. Whereas the COX-1 selective inhibitor, SC-560 (30-100 μM), did not block the corticosterone-enhanced cell death. These results suggest that COX-2 plays critical role in corticosterone induced increase of Fe²+ toxicity. The nitric oxide toxicity is accentuated in the presence of oxidative stress. The non specific nitric oxide synthase (NOS) inhibitor, L-NAME (1-5 mM), and the neuronal NOS (nNOS) selective inhibitor, 7-NI (30-100 μM), blocked the corticosterone enhanced Fe²+ toxicity. Reactive oxygen species accumulated during the corticosterone-induced enhanced Fe²+ toxicity were visualized using 2’, 7’-dichlorodihydro-fluoresceindeacetate (CM-H₂DCFDA; DCF), an indicator of ROS accumulation. Cortical neurons pretreated with corticosterone for 24 h, followed by Fe²+, showed increased DCF fluorescence compared with neurons treated with corticosterone (400 ng/ml) or Fe²+ 30 μM alone. In contrast, the COX-2 specific inhibitor, NS-398 (100 μM), or the nNOS specific inhibitor, 7-NI (100 μM), suppressed the DCF fluorescence levels. Enhanced ROS accumulation in neurons treated with corticosterone followed by was confirmed by MDA assay. High lipid peroxidation level was produced in neurons undergoing corticosterone-induced enhanced Fe²+ toxicity, while NS-398 (100 μM) or 7-NI (100 μM) completely blocked ROS stress. The results of the present study suggest that chronic treatment of corticosterone potentiated ferrous-toxicity in primary cortical culture by the mechanism involving COX-2- and nNOS- related to ROS generation.;스트레스를 받을 시에 인체 내 부신 피질에서 스트레스 호르몬인 glucocorticoid가 방출된다. Glucocorticoid의 주 종류는 영장류에서는 cortisol, 설치류에서는 corticosterone이다. 스트레스 호르몬은 신체 내 대사 활동을 증가시켜 긴급하게 필요로 하는 에너지 공급을 가능하게 하고, 면역억제작용이나 항염증작용에 중요한 역할을 하지만 이 호르몬이 높은 농도로 오랫동안 지속적으로 존재하면 대부분의 장기에 유해하다. 지속적인 스트레스 호르몬의 방출은 뇌신경계에 있어서 신경세포독성을 증가시키고, 우울증, 알츠하이머 병과 같은 뇌질환에도 영향을 미친다. 뇌는 산소를 많이 소비하기 때문에 다른 기관보다 산화적 스트레스에 취약하다. 활성산소종(reactive oxygen species, ROS)의 과도한 생성은 지질, 단백질, DNA를 산화시켜 세포가 죽게 만든다. 최근 연구에서 glucocortioid에 의한 신경세포독성은 산화적 스트레스를 수반하고 뇌 안의 항산화 효소에 양적인 변화를 가져온다고 밝혔다. 하지만 glucocorticoid와 활성산소종 간의 분자적 관련성에 대해서는 자세히 밝혀지지 않았다. 본 연구는 1차 배양 신경세포를 통해 glucocorticoid에 의한 신경세포사에 대한 메커니즘을 밝히고자 하였다. 설치류의 스트레스 호르몬인 corticosterone의 24 시간 전처리는 철 이온에 의한 대뇌 피질의 신경세포사를 3배 가량 증가시켰고, 이러한 신경세포사의 증가는 cyclohexamide에 의해 보호되었다. 이것은 corticosterone에 의한 전사적 변화, 즉 새로운 단백질의 합성으로 신경세포사에 영향을 미친다는 것을 시사한다. Corticosterone에 의해 증가된 철이온의 독성은 항산화제인 trolox나 vitamin C, caffeic acid에 의해 감소하였다. 이러한 결과들을 통해 신경세포가 corticosterone에 장기적으로 노출되어 독성에 민감하게 되는 것은 활성산소종과 깊은 관련이 있다. Corticosterone에 의해 유도된 철 이온의 신경세포독성 증가는 cyclooxygenase-2 (COX-2)와 neuronal nitric oxide synthase(nNOS)의 억제제에 의해 감소하였다. Western blot 실험, 면역세포염색법 등의 실험을 통해 corticosterone의 농도가 증가함에 따라 COX-2 및 nNOS의 단백질 발현이 증가하는 것을 관찰했고, 세포 형광 염색을 통해서 1차 배양 신경세포 내의 COX-2 및 nNOS 발현 증가를 확인했다. 이외에, caspase, PARP, PKC 억제제는 corticosterone에 의한 철 이온 독성 증폭을 부분적으로 억제하는 것을 확인하였다. 하지만 NADPH oxidase나 xanthine oxidase의 억제제들은 corticosterone에 의해 증가된 신경세포사를 보호하지 못했다. Corticosterone이 ROS와 연관이 있는 것을 확인하기 위하여 ROS를 탐지하는 염색법을 사용하여 corticosterone에 의한 철 이온 독성을 가진 신경세포에서 ROS의 증가를 관찰했고 지질산화 또한 증가하는 것을 확인하였다. 이상의 연구 결과들은 COX-2 및 nNOS와 관련되어 있는 활성산소종의 발생에 의해 corticosterone의 장기적인 노출이 산화적 스트레스에 의한 신경세포 독성을 민감하게 만든다는 것을 시사한다.
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