白藜蘆醇對MPTP誘導人類神經瘤SK-N-SH細胞死亡的保護效果

Abstract

巴金森氏症是常見的漸進性神經退化性疾病，許多相關研究認為氧化壓力在造成巴金森氏症上，扮演極重要的角色。MPTP（1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine）是一種選擇性破壞多巴胺神經細胞的神經毒素，目前被廣泛應用於建立巴金森氏症實驗動物模式。Resveratrol是一種存在於紅葡萄酒的植物性動情素，具有神經保護的效果。本實驗是採用MPTP細胞模式來探討MPTP對細胞傷害及處理resveratrol的保護效果。實驗材料是以retinoic acid誘導SK-N-SH神經瘤細胞分化之SK-N-SH神經細胞。實驗結果發現在10 mM MPTP 處理SK-N-SH細胞24小時只有70%的存活率；MPTP對SK-N-SH細胞的氧化性傷害並非呈現在提高細胞內的ROS量，細胞亦不會有提高HSP70量的反應。以40~50 nM resveratrol處理細胞3小時會顯著的降低細胞的存活率，低於30 nM resveratrol處理對細胞是沒有毒性的。如以10~30 nM resveratrol先處理細胞3小時再共同處理10 mM MPTP 24小時，則有使細胞的存活率提高約為對照組的1.08倍的效果。resveratrol 處理細胞27小時組比其對應的處理3小時組，細胞內ROS量都有下降的趨勢，以10~40 nM resveratrol先處理細胞3小時再共同處理10 mM MPTP 24小時，則細胞的ROS量均顯著的下降。以20nM resveratrol處理SK-N-SH細胞3小時或27小時，均會讓細胞HSP70量上升，經AG490（JAK抑制劑）處理證實細胞是藉由JAK/STAT pathway 的訊息傳遞路徑誘導HSP70產生。根據以上結果推測resveratrol先處理對細胞是一種壓力，低劑量時細胞HSP70增加，可以用以抵抗的MPTP的傷害，而細胞內HSP70的產生並不一定藉由細胞內ROS量升高引起的。高濃度40~50 nM resveratrol的處理，對細胞造成的壓力大，所以會顯著的傷害細胞存活、上升ROS量；如又同時與MPTP共同處理細胞就會加成的傷害細胞及增加ROS的產生量。本研究探討resveratrol對神經細胞保護效果的劑量及機制，祈能在人類巴金森氏症的預防及治療運用上能有所助益。Large numbers of experimental evidences supported that oxidative stress played an important role in the mediation of nerve cell death in Parkinson’s disease (PD), a common progressively neurodegenerative disease. 1-Methyl-4-phenyl-1,2,3,6-tetra- hydropyridine (MPTP)-induced neurotoxicity is the most common model to study the pathogenesis of PD. Resveratrol, a phytoestrogen in red wine extracts, had been shown to exhibit neuroprotective effects in several experimental models. In this study, the protective effects of resveratrol on MPTP-induced cell death in differentiated SK-N-SH neuronal cell were investigated. The data indicated that cells treated with 10 mM MPTP for 24 h, the viability was near 70%, MPTP damage on SK-N-SH cells was not by the process of higher ROS leveling and the MPTP-treated cells did not exhibited the response to rise their cellular HSP70 proteins. SK-N-SH cells treated with 40~50 nM resveratrol for 3 h, their cell viability were significantly decreased. Concentrations below 30 nM resveratrol for 24h treatment were not exhibited cytotoxicity. Cells pretreated with resveratrol at 10~30 nM for 3 h and then cotreated with 10 mM MPTP cells for 24 h, the viabilities rise up to near 1.08 fold. The ROS quantities in resveratrol/27 h-treated cells were lower than that in resveratrol/3 h-treated cells respectively. In cells pretreated with 10~40 nM resveratrol for 3 h and then cotreated with 10 mM MPTP for 3 h, the ROS quantities were decreased significantly. The quantities of HSP 70 proteins were increased in SK-N-SH cells treated with 20 nM resveratrol for 3 or 27 h. The induction of HSP 70 proteins through signal transduction of JAK/STAT pathway were proofed by AG490 inhibition. Based on above data, we speculated that resveratrol pretreatment was a kind of cell stress. In cells treated at lower concentrations, resveratrol enhanced HSP 70 production and increased in resistance toward MPTP damage and it might indicate that cellular HSP 70 production was not only mediated through rising ROS quantities. SK-N-SH cells pretreated in 40~50 nM resveratrol resulted in sever cell damage and higher ROS production, and additional effect of MPTP co-treatment to enhance cell damage and ROS production. This study may provide some information of dosage effect and protection mechanism of resveratrol for prevention and curing of PD.