Naloxone 對於H2O2 在小鼠運動神經細胞株 NSC-34 所引起的細胞毒性之保護作用

Protective effects of naloxone on H2O2-induced cytotoxicity in mouse motor neuronal NSC-34 cells

林于婷(Yu-Ting Lin)

指導教授 ： 羅怡卿

高雄醫學大學/醫學院/藥理學科研究所/碩士(2010年)

摘要

許多證據和研究指出由氧化壓力、粒線體的破壞、蛋白質不正常堆積和發炎所造成的神經細胞死亡、運動終板 (endplate) 的傳遞失敗以及軸突傳導 (axonal transport) 退化等的情況會啟動運動神經相關疾病病程。先前的研究指出naloxone 可藉由抑制 microglia 上NADPH oxidase的活化，而有抗發炎和神經保護的作用。本研究的目的為探討 naloxone 是否可以減少H2O2 對於小鼠運動神經細胞株 NSC-34 的傷害。結果顯示 H2O2 的確會減少細胞的存活率、增加氧化壓力的相關因子(NOX2 和胞內活性氧化物質，intracellular ROS) 、減少NSC-34細胞內運動神經細胞存活蛋白質 (protein of survival motor neuron gene, Smn) 表現、粒線體膜電位的減少以及增加細胞的耗氧率。然而，naloxone 的前處理可以藉由增加NSC-34 細胞存活率和運動神經細胞存活蛋白質 (Smn) 表現、減少氧化壓力相關因子的表現、維持粒線體膜電位以及 cytochrome c 的釋放來減少H2O2 對細胞的傷害。除此之外，在給予 H2O2 刺激的神經細胞內也發現，naloxone 的前處理可以減少細胞內caspase 3 的活化、增加 Bcl-2/ Bax 的比例和減少細胞的耗氧率。整體來說，naloxone 不僅具有減少細胞內氧化壓力的效果，同時也可以增加有益於運動神經細胞存活的Smn表現，並且具有調控細胞凋亡路徑中，蛋白質的表現和活化能力。因此，naloxone 的神經保護作用具有極大潛力應用在運動神經退化性疾病的臨床治療上。

關鍵字

NSC-34 ； naloxone ； 氧化壓力 ； 細胞凋亡

並列摘要

Increasing evidence indicates that motor neuron diseases characterized by loss of motor endplate, axonal degeneration, and cell death of motor neurons are caused by oxidative stress, mitochondrial dysfunction, protein aggregation, axonal transport defects, and inflammation. Naloxone has been reported to possess anti-inflammatory and neuroprotective effects, at least in part, by its inhibition on NADPH oxidase. The aim of this study is to investigate the protective effect of naloxone on H2O2-induced cytotoxicity in a motor neuronal cell line, NSC-34. Results indicated H2O2 decreased cell viability, down-regulated protein of survival motor neuron gene (Smn) expression, decreased mitochondria membrane potential and increased oxidative stress factors (NOX2 and intracellular reactive oxygen species, intracellular ROS). In addition, H2O2 also increased oxygen consumption rate in NSC-34. However, naloxone pre-treatment increased cell viability and Smn protein expression and attenuated oxidative stress factors, oxygen consumption and release of cytochrome c from mitochodria in H2O2-treated cells. Moreover, naloxone also attenuated H2O2-induced overexpression of cleaved-caspase-3. Naloxone increased Bcl-2/Bax ratio in H2O2-treated cells as well. Taken together, naloxone not only attenuated oxidative stress, but also up-regulated Smn protein, and modulated apoptotic and anti-apoptotic protein expression. The characterization in neuroprotective effect has potential benefits on motor neuronal degenerative diseases.

並列關鍵字

NSC-34 ； naloxone ； oxidative stress ； apoptosis

參考文獻

Ano Y, Sakudo A, Kimata T, Uraki R, Sugiura K, Onodera T (2010) Oxidative damage to neurons caused by the induction of microglial NADPH oxidase in encephalomyocarditis virus infection. Neurosci Lett 469:39-43.

Barber SC, Mead RJ, Shaw PJ (2006) Oxidative stress in ALS: a mechanism of neurodegeneration and a therapeutic target. Biochim Biophys Acta 1762:1051-1067.

Barber SC, Higginbottom A, Mead RJ, Barber S, Shaw PJ (2009) An in vitro screening cascade to identify neuroprotective antioxidants in ALS. Free Radic Biol Med 46:1127-1138.

Baumer D, Lee S, Nicholson G, Davies JL, Parkinson NJ, Murray LM, Gillingwater TH, Ansorge O, Davies KE, Talbot K (2009) Alternative splicing events are a late feature of pathology in a mouse model of spinal muscular atrophy. PLoS Genet 5:e1000773.

Bedard K, Krause KH (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87:245-313.