白藜蘆醇衍生物SRT1720在百草枯誘發帕金森氏症實驗模式的神經保護機制：針對粒線體功能之研究

Abstract

帕金森氏症 (Parkinson’s disease，PD) 為目前最普遍的神經退化性疾病之一，該病因主要是由於中腦黑質區的多巴胺細胞的死亡造成運動系統的失能。環境常用農藥百草枯 (Paraquat，PQ) 目前已知是導致帕金森氏症的環境因子之一。它主要作用在粒線體上，阻斷粒線體的功能、造成大量氧化自由基生成、並誘導細胞凋亡的發生。沉默調節因子蛋白Sirtuin家族 (Sirtuin Family，Sirt1-Sirt7) 是一群 Nicotinamide adenine dinucleotide (NAD+) 依賴性去乙醯化酶，具有抗老化、以及預防神經退化性疾病等能力。SRT1720是根據天然植物酚類白藜蘆醇製造出來的化學衍生物，具有活化Sirtuin的能力。先前研究也顯示SRT1720具有增加糖尿病小鼠的存活率、抗腫瘤、抗發炎等功能，但SRT1720對於神經退化性疾病的保護性並不清楚。為了解SRT1720是否具有對抗PQ的細胞毒性，用以評估SRT1720是否具有治療帕金森氏症的潛力，本研究使用人類神經瘤母細胞株 (SH-SY5Y) 作為帕金森氏症的離體外實驗模式，來探討SRT1720及PQ對於細胞的作用及影響。實驗結果顯示，PQ造成細胞存活率呈劑量反應地下降，而SRT1720可以回復因PQ所造成細胞存活率的下降、細胞凋亡的產生、粒線體的型態變化，以及降低氧化自由基的生成等。這證明SRT1720對細胞具有神經保護的效果。本研究也利用西方點墨法證實了當細胞暴露在PQ下，SRT1720會回復因PQ所導致Sirt1-Sirt7蛋白含量的下降。其中，大量表現Sirt1可以對抗PQ造成細胞的死亡。本研究也發現SRT1720可回復PQ自噬小體在細胞中的堆積情形，利用西方點墨法觀察SRT1720可以回復LC3-I/II的蛋白質再細胞間的堆積。此外，在對於20週大的C57BL/6小鼠注射PQ (10 mg/kg) 及SRT1720 (0.1mg/kg或1 mg/kg)，並利用滾輪及獨木橋試驗觀察其運動行為。結果顯示，SRT1720可以回復PQ所造成運動能力上的下降，並且減緩PQ所造成中腦區多巴胺神經元的傷害。綜觀以上結果，在細胞暴露在PQ時，SRT1720或許可以經由保護粒線體功能，使ROS生成量達到回復及降低細胞凋亡的發生。同時SRT1720也能保持自噬作用的平衡，降低自噬小體在細胞中的堆積。這些機轉也許與SRT1720可以保護多巴胺神經元有關。另一方面，由於Peroxisomal proliferator-activated receptor-coactivator 1α (PGC-1α) 與粒線體的生合成與神經保護有關，本實驗也發現SRT1720可改變PGC-1α去乙醯化的程度，但SRT1720對於Sirtuin蛋白以及Sirt1下游PGC-1α的活化與否還需做進一步的調查及研究。此研究顯示SRT1720對於保護細胞免於受到PQ所引發氧化壓力以及粒線體損傷之神經退化模型提供了一個具有潛力的治療方法。

Parkinson’s disease (PD) is one of the most common neurodegenerative disorder and mainly affecting the motor system because of the dopamine neuronal death in the substantia nigra. The exposure to environmental neurotoxin paraquat (PQ) is a widely used herbicide. It induces the increase of ROS stress, leads to mitochondrial dysfunction, and results in apoptotic cell death. Epidemiologically, it could be the risk for PD incidence. Mammalian silent information regulator 2 Sirtuin Family (Sirt1-Sirt7) is a NAD+ dependent deacetylase enzyme and it protects against such as anti-aging and neurodegenerative disease. SRT1720 which derives from resveratrol is able to activate Sirt1. SRT1720 has been reported to improve survival in obese mice, anti-tumor, and anti-inflammatory, but the effect in the neurodegenerative disease it still unknown. We thus proposed if SRT1720 could have neuron- protective effect in PQ-induced toxicity. We used SH-SY5Y cell to evaluate the effect of SRT1720 and PQ. First, we confirmed that PQ could dose- and time-dependently decrease SH-SY5Y cell viability, increase ROS formation, and induce mitochondrial dysfunction. However, SRT1720 pretreatment improved cell viability, decreased apoptosis and ROS formation, and prevented mitochondrial dysfunction in PQ-treated SH-SY5Y cells. By Western blot analysis, SRT1720 pretreatment could preserve Sirt1-Sirt7 protein contents during PQ intoxication. In autophagy studies, we also found that SRT1720 could reduce PQ-induce autophagic vacuoles accumulation. Furthermore, we also found that intraperitoneally injection of 10 mg/kg PQ once a week in mice can decrease the level of motor activity after 6 weeks treatment. However, SRT1720 (0.1 mg/kg or 1mg/kg) treatment, reversed PQ- induced motor defect. Taken together, SRT1720 could protect mitochondrial function and improve cell survival during PQ intoxication. This work provided a promising therapeutic way for treating aging-related neurodegenerations, such as PD.