Study of the Neuroprotective Effect of Gastrodia-Uncaria Water Extract against Endoplasmic Reticulum Stress in Cerebral Ischemia in vitro and in vivo

• Other Authors: Choi, Yiu Ting Angus (author.)
• Format: Others
• Language: English; Chinese
• Published: 2016
• Online Access: http://repository.lib.cuhk.edu.hk/en/item/cuhk-1292273

中風是全球第二大死亡原因，缺血性中風佔其中百分之八十七的中風個案。於再灌注時，蛋白質聚集體於受缺血中風影響的神經細胞形成，引致內質網壓力和細胞凋亡。胞漿素原活化劑是現時唯一受美國食品藥品監督管理局批准的急性缺血性中風藥物治療方法。可是，在其短窄治療窗期以及沒有神經保護作用之下，胞漿素原活化劑的應用還是有其限制。所以，針對缺血性中風的藥物研發仍是需求急切。 === 天麻鉤藤飲是一個於二十世紀中所定立的方劑。它現時在中國內地廣泛地用作治療高血壓和中風。天麻和鉤藤是在本方劑的君藥。本研究旨在研究天麻鉤藤水提物在體外和體內實驗中對於缺血性中風的治療作用和機理。 === 體外實驗以大鼠作動物模組，以大腦中動脈閉塞手術來模仿缺血性中風的病發機制。七天的天麻鉤藤水提物治療對中風大鼠的作用以神經功能缺損評分﹑平衡桿行進行評估。TTC法用來評估腦梗死容量。蘇木精-伊紅染色和尼氏染色用評缺血腦組織狀況。免疫組織化學用作檢示素化蛋白質聚集體量和胱天蛋白酶12的表達。 === 在體內實驗的中缺氧缺糖再灌注，天麻鉤藤水提物對神經分化的SK-N-SH神经母细胞瘤细胞的保護作用以MTT法作細胞存活率分析。神經突連接以明視野顯微鏡和免疫細胞化學來檢視。西方墨點法和免疫細胞化學用作評估泛素化蛋白質聚集體量，以用作評價內質網壓力狀態。即時聚合酶鏈鎖反應和西方墨點法用來分析未折疊蛋白反應基因的信使核糖核酸和其蛋白表達。 === 實驗結果顯示天麻鉤藤水提物能夠明顯提升中風大鼠在神經功能缺損評分和平衡桿行的表現。組織實驗顯示天麻鉤藤水提物明顯地減少了腦梗死容量。初級運動皮質﹑初級軀體感覺皮層和海馬體CA3的組織受損和空泡情況皆有改善，亦保護了海馬體CA3錐體神經元及減少高密度尼氏小體神經元數量。免疫組織化學實際顯示天麻鉤藤水提物能明顯減少泛素化蛋白質聚集體量和胱天蛋白酶12的表達。以上結果顯示天麻鉤藤水提物能通過內質網壓力來減低缺血性中風再灌注損傷。降低SK-N-SH細胞於缺氧缺糖再灌注後的存活率和神經突連接。天麻鉤藤水提物能顯著提升葡萄糖調節蛋白78的信使核糖核酸表達。天麻鉤藤水提物在一方面提升蛋白質二硫鍵異構酶﹑葡萄糖調節蛋白78和半胱天蛋白酶12的蛋白表達，另一方面能降低未拼接X-框结合蛋白1蛋白表達。另外，天麻鉤藤水提物能夠明顯地減少26.4%泛素化蛋白質聚集體量，顯示天麻鉤藤水提物能夠提升未折疊蛋白反應機制來減低內質網壓力。 === 從以上結果所見，天麻鉤藤水提物能透過提升未折疊蛋白反應機制來減低內質網壓力來達致神經保護作用，減少缺血性中風再灌注損傷。 === Stroke is the second leading cause of death in worldwide, in which cerebral ischemia accounts for 87% of all stroke cases. Unfolded protein aggregates which forms in the ischemic neurons, leads to endoplasmic reticulum (ER) stress and then neuronal cell death. Currently, recombinant tissue plasminogen activator (rtPA) is the only US Food and Drug Administration approved drug used in treating ischemic stroke. Nonetheless, rtPA is still limited to utilize due to short application period and lack of neuroprotective effect acting on ischemic neurons. Therefore, it is necessary to explore alternative treatment on cerebral ischemia. === Gastrodia-Uncaria Decoction is a traditional Chinese Medicine prescription developed in mid-20th Century. It has been commonly used to treat head related diseases in mainland China, especially hypertension and stroke. Gastrodia elata and Uncaria rhynchophylla are the two main herbs in the decoction. Our study was to investigate the neuroprotective mechanism of the water extract of Gatrodia elata and Uncaria rhynchophylla (GUW) against cerebral ischemia in vitro and in vivo. === For in vivo study, middle cerebral artery occlusion (MCAO) was performed on male Sprague Dawley rats. MCAO rats were administered orally with GUW or water as a control for 7 consecutive days post-operation. Sham group rats were fed with water. TTC staining was used to examine the infarct volume. Hematoxylin and eosin stain and cresyl violet stain were used to examine the tissue integrity at the infarct area. Neurological deficit score and beam walking test were used to evaluate the physiological outcome. Immunohistochemistry on ubiquitinated protein aggregates and caspase-12 was used to evaluate the cell death via ER stress. === For in vitro study, neuronal-differentiated SK-N-SH neuroblastoma cell line was subjected to 7 h of oxygen-glucose deprivation followed by 17 h reoxygenation (OGD/R). MTT assay was used to examine the cyototoxicity profile of GUW and the cell viability after OGD/R. Immunocytochemistry was used to examine the neurite extension and connectivity. Western blot and immunocytochemistry on ubiquitinated protein aggregates were carried out to examine the ER stress status. Proteins from the unfolded protein response were studied by real time PCR and western blot. === Results showed that MCAO rats treated with GUW resulted in a significantly smaller infarct volume and higher tissue integrity at the primary motor cortex, primary somatosensory cortex and hippocampus CA3. Fewer vacuolation and pyramidal neuron shrinkage were observed in GUW rats. Significant improvement in neurological deficit score and beam walking test was observed after 3 days of GUW treatment. A significantly lower caspase-12 activity and amount of ubiquitinated protein aggregates was found in the infarct region of the brain. This suggested GUW protected ischemic rats from ER stress-induced cell death, such that leading to a smaller infarct volume and improving the physiological outcome. From in vitro study, GUW elicited a significant protective effect on SK-N-SH cells after OGD/R. Neurite connectivity of GUW treated cells was preserved more than control cells. GUW led to a significant reduction in unfolded protein aggregates in immunocytochemistry and western blot. GRP78 mRNA expression was found to be significantly upregulated by GUW. Furthermore, GRP78, PDI, and procaspase-12 expression were upregulated by GUW, while unspliced XBP-1 expression was downregulated by GUW, suggesting that UPR was modulated by GUW to counteract ER stress. === To conclude, Gastrodia-Uncaria water extract was potent to protect against endoplasmic reticulum stress-induced reperfusion injury after cerebral ischemia through upregulating unfolded protein response. === Choi, Yiu Ting Angus. === Thesis M.Phil. Chinese University of Hong Kong 2016. === Includes bibliographical references (leaves ). === Abstracts also in Chinese. === Title from PDF title page (viewed on …). === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only.