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Title: | 以秀麗隱桿線蟲探討 Tangeretin 及 Didemethyl-tangeretin 經由 DAF-16 降低氧化壓力與類澱粉蛋白造成之毒性 Tangeretin and Didemethyl-tangeretin reduce oxidative stress and amyloid-β toxicity regulated by DAF-16 in Caenorhabditis elegans |
Authors: | KUN-FENG HSIEH 謝昆峰 |
Advisor: | 潘敏雄 |
Co-Advisor: | 廖秀娟 |
Keyword: | 阿茲海默症,類澱粉蛋白,氧化壓力,多甲氧基類黃酮,秀麗隱桿線蟲,DAF-16, Alzheimer’s disease,Amyloid-β,Oxidative stress,Polymethoxy flavone,Caenorhabditis elegans,DAF-16, |
Publication Year : | 2019 |
Degree: | 碩士 |
Abstract: | 阿茲海默症 (Alzheimer’s disease; AD) 為一程序性之神經退化疾病,主要症狀為記憶力下降、情緒失調與運動上之障礙等,造成全世界的健康隱憂。在普遍 AD 之患者腦部會發現類澱粉蛋白 (amyloid-β; Aβ) 堆積之現象,此一蛋白的不正常堆積不僅會造成神經細胞死亡,亦會促使腦部氧化壓力上升,故 Aβ的毒性 被視為造成 AD 主要原因之一。多甲氧基類黃酮 (Polymethoxy flavone; PMF) 為柑橘中含量豐富之機能性成分,在先前研究中顯示其具有許多生理功能,包括抗發炎、抗肥胖與神經保護之功效。然而,PMF 因溶解度差,並會降低其生物利用率。許多文獻指出,PMF 之水解產物 (Hydroxylated PMF; HPMF) 因具有較優良之溶解度,使其較 PMF 更具生理活性。故本篇研究想利用秀麗隱桿線蟲 (Caenorhabditis elegans) 做為評估阿茲海默症之模式生物,評估與比較 Tangeretin (Tan) 與水解型之 5, 4’-didemethyl tangeretin (DTan) 是否具降低氧化壓力與類澱粉蛋白堆積造成之毒性及其調控機制。研究結果顯示,Tan 與 DTan 於 100 μM皆能有效增加線蟲暴露氧化壓力劑 juglone 之存活率,進一步測量線蟲體內之活性氧物質 (ROS) 發現,與控制組相比,暴露 Tan 與 DTan 可顯著降低線蟲體內 ROS 含量約 20.16% 與 46.75%。評估 Tan 及 DTan 之抗 AD 活性,利用能表達人類 Aβ1-42 蛋白之轉機因品系線蟲 CL4176 與 CL2006。抗癱瘓試驗中發現,Tan 與 DTan 皆能有效延後轉基因線蟲 CL4176 與 CL2006 之癱瘓時間,於兩品系中 DTan 之半數致癱瘓時間皆較 Tan 長,推論 DTan 之抗 AD 活性較 Tan 優良。此外,發現暴露 Tan 與 DTan 之組別,CL4176 體內 ROS 含量皆有顯著降低約 18.10% 及 12.88 %。藉由西方墨點法定量 CL4176 中之 Aβ1-42 蛋白,暴露 Tan 及 DTan 皆能抑制 CL4176 中 Aβ1-42 蛋白之表現,然而,Tan 降低 Aβ1-42 蛋白卻優於 DTan;在 CL2006 線蟲 Aβ1-42 蛋白染色結果中發現,暴露 Tan 與 DTan 之組別皆能降低其體內 Aβ1-42 斑塊的堆積,而 DTan 之效果較 Tan 顯著。在暴露 Tan 及 DTan 後發現,皆能誘導轉錄因子 DAF-16/FOXO 轉移進細胞核內;並利用 RNA 干擾技術發現,Tan 與 DTan 延緩轉基因品系 CL4176 之抗 AD 效果與 DAF-16 之有關。由目前的研究結果可推論,Tan 及 DTan 具有降低氧化壓力與 Aβ 蛋白堆積所造成的毒性的能力,並推測為藉由 DAF-16/FOXO 所調控。本篇研究中雖無法進一步比較其優劣,但可推測 Tan 與 DTan 皆具有預防 AD 與氧化壓力相關疾病之潛能。 Alzheimer’s disease (AD) is a progressive neurological disease characterized by deficits in memory, cognition, motor skills, and represents a major healthcare challenge worldwide. The aggregation of amyloid-β (Aβ) is known as the key hallmark in AD, and it causes cell death and oxidative stress in neurons. Polymethoxy flavone (PMF), such as tangeretin and nobiletin, is one of the most abundant bioactive compounds in the peel of citrus fruits. Increasing evidences showed that PMFs have many biological functions including anti-inflammatory, anti-obesity and neuroprotective effects. However, PMFs are difficult to dissolve in water, and hydroxylated PMFs (HPMFs) may solve this problem. This study investigated and compared the tangeretin (Tan) and 5,4’-didemethyl tangeretin (DTan) on reducing oxidative stress and Aβ-induced toxicity in Caenorhabditis elegans AD models, as long as underlying mechanisms. The results showed that both Tan and DTan increased the survival of C. elegans in juglone-induced oxidative stress. In addition, Tan and DTan attenuated endogenous levels of ROS in C. elegans by 20.16% and 46.75%, respectively. Moreover, both Tan and DTan significantly decreased time of the paralysis in transgenic strain CL4176 and CL2006 which express human Aβ1-42 protein in body wall muscle. Comparison of extension time of the paralysis, DTan was better than Tan. On the other hand, Tan and DTan attenuated endogenous levels of ROS in transgenic C. elegans by 18.10% and 12.88%, respectively. Furthermore, by using western blot and Aβ deposit staining assay, the results that Tan and DTan induced a decline in Aβ42 expression and deposits in transgenic strain CL4176 and CL2006. Finally, we showed that Tan and DTan affected the subcellular distribution of the FOXO transcription factor DAF-16. Otherwise, RNA interference by daf-16 showed that Tan and DTan affected on attenuating paralysis were absent in transgenic strain C. elegans CL4176. Current results from this study indicated that Tan and DTan significantly reduced oxidative stress and Aβ42-induced toxicity. Although we can’t compare the neuroprotective effect between Tan and DTan, but suggesting that Tan and DTan have potential for the prevention of AD-associated oxidative stress complications. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74891 |
DOI: | 10.6342/NTU201904172 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 食品科技研究所 |
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