咖啡酸和白藜蘆醇對第三型脊髓小腦運動失調症細胞與果蠅模式的神經保護功效及可能機制

Neuroprotective Effects and Possible Mechanisms of Caffeic Acid and Resveratrol in Cell and Drosophila Models of Spinocerebellar Ataxia Type3

巫玉琳(Yu-Ling Wu)

指導教授 ： 劉凱莉

中山醫學大學/健康管理學院/營養學系博士班/博士(2018年)

摘要

第三型脊髓小腦運動失調症 (Spinocerebellar ataxia type 3，SCA3，又稱為Machado–Joseph disease)，為ataxin-3 蛋白質polyglutatmine tract (polyQ)的麩醯胺酸重複個數突變引發的顯性遺傳神經退化疾病，目前無藥物可有效的控制脊髓小腦運動失調症發生。雖然polyQ 突變ataxin-3 蛋白質確切致病的機制仍不清楚，但是研究發現，除了polyQ 突變的ataxin-3 蛋白質會錯誤折疊蛋白質聚集在小腦與脊髓等特定神經細胞核內外， polyQ 突變ataxin-3 蛋白質神經細胞基因轉錄、減少蛋白質分解作用、降低抗氧化物質及抗氧化酵素表現、與增加粒線體損傷和細胞凋亡相關分子表現等作用，致使神經細胞凋亡。食物中所含的植物素 (Phytochemicals)可經由增加神經細胞抗氧化和抗細胞凋亡作用，具有抑制神經退化之功效。咖啡酸 (Caffeic acid, CA) 廣泛存在於水果、蔬菜、酒、橄欖油及咖啡中，屬於hydroxycinnamic acid的酚酸類化合物 (Phenolic acids)，飲食中CA攝取量超過總酚酸類化合物90%。白藜蘆醇 (Resveratrol, Res) 屬於多酚類中stilbene類的化合物，主要食物來源包括紅葡萄、紅酒、莓果、堅果和花生等。雖然研究已證實CA和Res具有良好的抗氧化及抗神經毒殺的功效，但是CA和Res是否具有減緩SCA3 神經退化病程發展的保健功效尚未清楚。本論文以基因轉殖表現polyQ突變ataxin-3蛋白質的SK-N-SH-MJD78神經細胞株與 ELAV-SCA3tr-Q78果蠅模式，評估CA和Res的神經保護角色及可能機制。研究結果證實CA和Res可降低氧化劑tert-butyl hydroperoxide (tBH)誘發SK-N-SH-MJD78神經細胞株細胞凋亡。此外，CA和Res可增加SK-N-SH-MJD78細胞抗氧化及自噬作用相關蛋白質表現，並且降低活性氧化物 (Reactive oxygen species, ROS)、polyQ突變ataxin-3蛋白質和細胞中聚集蛋白質表現量。進一步發現，餵食CA和Res有助於改善ELAV-SCA3tr-Q78果蠅的爬行能力與存活率。CA和Res亦可改善ELAV-SCA3tr-Q78果蠅腦部ROS、polyQ突變ataxin-3蛋白質及與細胞凋亡、抗氧化與自噬作用相關蛋白質表現。在SK-N-SH-MJD78細胞，利用reporter gene assay、暫時性轉殖dominant-negative mutant IκB-α 質體與siNrf2等實驗證實CA和Res對SCA3的神經保護作用與其降低p53與增加NF-κB及Nrf2轉錄活性有關。此研究結果，除可了解CA和Res 對改善polyQ 突變ataxin-3 蛋白質增加腦神經細胞ROS與細胞凋亡的功效與相關機制外，亦可作為日後進行評估補充CA和Res改善SCA3症病臨床症狀保健功效的參考依據。

關鍵字

咖啡酸 ； 白藜蘆醇 ； 第三型脊髓小腦運動失調症

並列摘要

Spinocerebellar ataxia type 3 (SCA3, also called Machado–Joseph disease (MJD), a late-onset and fatally inherited neurodegenerative disease, is caused by an abnormal expansion of the polyglutamine (polyQ) repeat in the protein ataxin-3. Until now, there is no established disease-modifying therapeutic strategy has been available for SCA3. Although the exact mechanism is unknown, the pathogenic effects of polyQ expanded mutant ataxin-3 protein are associated with not only misfolding and aggregation in nuclei of specific neurons but also dysregulation of transcription, protein degradation, mitochondrial function, apoptosis, and antioxidant potency and these thus trigger neuronal death. It is well established that phytochemicals in food through antioxidant and anti-apoptotic effects on neurons exert valuable therapeutic benefits in neurodegenerative diseases. Caffeic acid (CA), widely present in various agricultural products such as fruits, vegetables, wine, olive oil, and coffee, classified as a phenolic compounds of hydroxycinnamic acid and accounts for almost 90% of total phenolic acid intake in the diet. Resveratrol (Res), a polyphenolic stilbene, is present in variety of dietary sources such as red grapes, red wine, berries, nuts and peanuts. Although data have shown the antioxidant and antineurotoxic properties of CA and Res, the health effects of CA and Res against neurodegenerative progress in SCA3 is unknown. Here we investigated the protective role and possible mechanisms of CA and Res in SK-N-SH-MJD78 neuroblastoma cells and ELAV-SCA3tr-Q78 transgenic flies expressing mutant ataxin-3 and mutant ataxin-3 polyQ tract, respectively. Our data showed that CA and Res decreased apoptosis in the pro-oxidant tert-butyl hydroperoxide (tBH)-treated SK-N-SH-MJD78 cells. Moreover, treatments with CA and Res increased the levels of antioxidant and autophagy protein expression as well as diminished ROS, and expression and aggregation of mutant ataxin-3 in SK-N-SH-MJD78 cells. We further discovered that supplementations with CA and Res enhanced survival and motor performance in ELAV-SCA3tr-Q78 transgenic flies. CA and Res also diminished ROS, mutant ataxin-3 polyQ tract, and apoptotic-related molecules, as well as increased antioxidant and autophagy molecules in brain of ELAV-SCA3tr-Q78 transgenic flies. Notably, in SK-N-SH-MJD78 cells, using reporter gene assay, transfection experiments with a dominant-negative mutant IκB-α (DNM IκB-α) plasmid and Nrf2 siRNA demonstrated that the neuroprotective effects of CA and Res on SCA3 are through modulating transcriptional activity of p53, NF-κB and Nrf2. In summary, our findings demonstated the neuroprotective effect and possible mechanisms of CA and Res in improving mutant ataxin-3 induced ROS production and neuronal apoptosis. Moreover, these data could provide information for the preclinical studies of CA and Res in modulating neurodegenerative progression in SCA3.

並列關鍵字

Caffeic Acid ； Resveratrol ； Spinocerebellar Ataxia Type3

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