雷公根和酸棗仁之有效成分對 Aβ 造成神經損傷之保護作用

Abstract

阿茲海默症 (Alzheimer’s disease, AD) 是好發於 65 歲以上老年人之神經退化性疾病，使患者在記憶、語言與行為等多種認知功能出現退化，嚴重影響病人的生活品質與人際關係。其致病原因是聚合之類澱粉樣蛋白質 β (amyloid β, Aβ) 於腦部大量沉積，Aβ 會造成神經細胞氧化壓力與細胞凋亡，被視為造成阿茲海默症的主因。目前許多新開發中的藥物或保健食品，皆以 Aβ 作為目標，包含降低含量，減緩其造成的氧化壓力等。傳統的中草藥被應用在疾病預防已有數千年歷史，近年來也開發用於改善阿茲海默症。其中以銀杏萃取物，發展最為成功，已經被廣泛使用在歐美的保健食品中。 根據本研究室的研究結果，雷公根與酸棗仁在動物模式與細胞模式中，皆有保護大鼠與神經細胞的能力。本論文希望找出其有效成分並針對抗氧化壓力與細胞凋亡進行研究。 本研究首先先以外觀與顯微鑑定法鑑定雷公根酸棗仁是否為正品。確認為正品後，以 Aβ25-35 誘導分化的 PC12 細胞造成傷害作為細胞模式，討論雷公根與酸棗仁的有效成分之保護作用，並深入探討抗氧化壓力與細胞凋亡的方式。研究結果發現，雷公根內含量較多的兩個成分，madecoside (MS) 和asiaticoside (AS) 以及酸棗仁的 jujuboside A (JA)，皆可降低 Aβ25-35 造成的細胞毒性。此外將此三種物質再和 Aβ25-35 共培養 6 小時後進行抗氧化能力測試，發現 reactive oxygen species (ROS) 與 glutathione/glutathione disulfide (GSH/GSSG) 兩種氧化壓力指標皆會改善，三種物質皆能顯著提高 catalase (CAT) 的活性，但只有 MS 能顯著提高 glutathione peroxidase (GPx) 的活性，而 AS 與 JA 則較能顯著提高superoxide dismutase (SOD) 的活性，顯示三種成分雖藉由不同方式但皆具有抗氧化的能力。 細胞凋亡可以分為兩個途徑，死亡受器途徑 (death receptor pathway) 與粒線體途徑。死亡受器途徑中，物質活化 caspase-8，進而活化 caspase-3，造成細胞凋亡。粒線體途徑中，壓力藉由活化 caspase-9，進而活化 caspase-3，造成細胞凋亡。我們研究發現，MS 和 JA 抗細胞凋亡的效果較好，且可透過調節死亡受器與粒線體兩個途徑。但 AS 效果較差，僅能調節死亡受器途徑。本研究顯示 MS 與 JA 可能為雷公根與酸棗仁能改善阿茲海默症的主要有效成分，而其機制則為增強抗氧化酵素活性與降低細胞凋亡。

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder mostly affecting the population of above 65 years of age. AD patients suffer from many cognitive abilities losses such as memory loss, language disability and behavior dysfunction, etc, and seriously affected their quality of life and social relationship. The most important pathology of AD is Amyloid β (Aβ) deposit, and Aβ causes oxidative stress and apoptosis in neuron, which is considered as the major reason for AD. Now lots of new drugs or health food in development begin with Aβ as the target, including decreasing levels and oxidative stress. Traditional Chinese herbal medicine has been used for thousands of years in the disease, in recent years, it developed to improve Alzheimer's disease. Among them, Ginkgo biloba extract, the most successful development has been widely used as health foods in Europe and U.S. According to the experimental results of our laboratory, Centella asiatica and Ziziphi spinosae semen have the abilities to protect rat and neuron in animal model and cell model. This study tries to find the effective compounds and research for the anti-oxidative ability and anti-aoptosis anbility. At first, we identify Centella asiatica and Ziziphi spinosae semen by appearance and microscope. Then our study established the Aβ25-35-induced damage in differentiated PC12 cells as a cell model to discuss the protective effect of effective compounds in Centella asiatica and Ziziphi spinosae semen, and dive into the anti-oxidative stress and apoptosis. The experimental results show that the abundant component, madecoside (MS) and asiaticoside (AS) in Centella asiatica and the jujuboside A (JA) in Ziziphi spinosae semen, can reduce Aβ25-35 induced cytotoxicity. Co-culture the three substances with Aβ25-35 in 6 hr, and test the antioxidant capacity, we found they can improve both reactive oxygen species (ROS) and glutathione/glutathione disulfide (GSH/GSSG), enhance the activity of catalase (CAT), but only MS can enhance the activity of glutathione peroxidase (GPx) , and AS and JA can enhance the activity of superoxide dismutase (SOD). Those results show that they have antioxidant capacity by different ways. Apoptosis can be divided into two pathways, death receptor pathway and mitochondrial pathway. In death receptor pathway, ligands activate caspase-8, and then activate caspase-3, in the end they induce apoptosis. In mitochondrial pathway, stress activates caspase-9, and then activates caspase-3, in the end they induce apoptosis. In our study, we find MS and JA have better anti-apoptosis abilities, and they can regulate death receptor pathway and mitochondrial pathway. But AS has poor effect, it can only regulate death receptor pathway. This study shows that MS and JA may be the effective compound in Centella asiatica and Ziziphi spinosae semen, and can improve Alzheimer's disease by enhancing the activities of antioxidant enzymes and reducing apoptosis.