吳忠信林榮耀Chung-Hsin WuJung-Yaw Lin鄭瑀萱Yu-Hsuan Cheng2019-09-052020-2-22019-09-052015http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN060143020S%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/104069第十七型小腦脊髓共濟失調症 (Spinocerebellar ataxia type 17; SCA17) 是一種由多麩醯胺(polyglutamine; polyQ)蛋白異常擴增所引起的神經退化性疾病，致病原因是在TATA box binding protein (TBP) 基因上CAG三核苷序列重複出現不正常擴增，因而轉譯出異常的polyQ蛋白表現；polyQ蛋白異常擴增造成蛋白質錯誤折疊及聚集(Aggregation)，另外未折疊及錯誤折疊蛋白若在內質網內堆積會引發內質網壓力 (Endoplasmic reticulum stress; ER stress)，而細胞會啟動未折疊蛋白反應 (Unfolded protein response; UPR) 保護機制以修復內質網功能，一旦壓力持續未解決，反而會誘發由內質網介導的凋亡路徑進而造成細胞死亡。此外內質網功能失調也會增加活性氧 (Reactive oxygen species; ROS) 的生成，誘發氧化壓力而加劇傷害。近年來，越來越多研究認為內質網壓力及氧化壓力可能參與第十七型小腦脊髓共濟失調症致病過程，且許多文獻指出，中草藥的使用具有治療退化性疾病之潛力。因此，本研究設計 (I)人類神經瘤母細胞 (Neuroblastoma SH-SY5Ycells) 以毒蘿蔔素 (Thapsigargin; TG) 及衣黴素 (Tunicamycin; TM) 誘導的內質網壓力、(II) SCA17 nTBP/Q79-EGFP細胞以多西環素 (Doxycycline)誘導出polyQ蛋白表現 (III) SCA17小鼠模式三階段實驗，鑑定金銀花 (Lonicera japonica)之有效成分NH040-1是否具有保護神經細胞的作用。實驗結果顯示NH040-1可增加由TG或TM所造成細胞死亡的生存率、可抑制神經細胞凋亡、可降低內質網壓力路徑相關蛋白的表現並可減少由內質網壓力產生的活性氧生成量，顯示NH040-1對內質網壓力誘導的細胞死亡具有改善效果；並進一步以多西環素誘導出的SCA17 nTBP/Q79-EGFP細胞，我們發現NH040-1可對Q79細胞型態退化有顯著改善效果、減少Q79細胞聚集的現象、且可降低內質網壓力誘導的細胞凋亡表現。實驗進一步轉移至SCA17基因轉殖小鼠上，藉由腹腔注射NH040-1藥物，透過滾輪、步行等實驗觀察其行為，結果顯示NH040-1能夠有效地促進其滾輪上之行為分析以及改善步行印跡，在其運動失調的症狀上發揮療效，並且可以減少基因轉殖小鼠小腦中TBP/polyQ 蛋白的不正常聚集及內質網壓力介導的細胞凋亡現象，以及改善Purkinje cells失序分佈。綜合以上實驗結果，NH040-1具有透過抑制內質網壓力及活性氧生成的表現，進而治療第十七型小腦脊髓共濟失調症的潛在能力。Spinocerebellar ataxia type 17 (SCA17), one of the polyglutamine (polyQ) diseases, is a neurodegenerative disorders caused by the expansion of the polyQ tract (＞43 CAG repeat) in human TATA box binding protein (TBP) gene, leading to an abnormal expansion of a polyQ stretch in the corresponding protein. Previous study indicated that the endoplasmic reticulum (ER) stress or unfolded protein response (UPR) may contribute to the pathogenesis of SCA17, by the accumulation of misfolded proteins, alterations in the calcium homeostasis and reactive oxygen species (ROS) production. This study identified NH040-1 from Lonicera japonica that protecting neurons from Thapsigargin (TG) or Tunicamycin (TM)-induced ER stress and ROS generation in the in vitro SH-SY5Y and SCA17 cell models and the in vivo transgenic mice model. We showed NH040-1 was effective against TG or TM-induced ER stress by increasing the cell viability, supressed ER-mediated apoptotic protein expression and ROS production in SH-SY5Y cell model. Additionally, the results indicated that NH040-1 enhanced the neurite overgrowth and decrease misfolded protein aggregation in SCA 17 cell model. Furthermore, the application of NH040-1 in SCA17 mice model showed that NH040-1: (I) ameliorated motor coordinationin deficits in rotarod analysis and footprint patterns experiments, (II) reduced the polyQ aggregation as well as ER stress-induced apoptotic protein expression, and (III) ameliorated Purkinje neuron degeneration in the cerebella of transgenic mice. Therefore, NH040-1 could be a potentially therapeutic drug to attenuate ER stress and neurodegeneration in SCA17.第十七型小腦脊髓共濟失調症內質網壓力活性氧細胞凋亡金銀花spinocerebellar ataxia type 17endoplasmic reticulum stressreactive oxygen speciesapoptosisLonicera japonica