Exploring Uncaria rhynchophylla and its chemical constituents for the treatment of Alzheimer's disease.

鉤藤是眾多用於治療神經性退行性疾病的傳統中藥複方的組成成份之一。文獻研究發現鉤藤提取物能夠顯著抑制β澱粉樣蛋白纖維的形成和拆卸預製β澱粉樣蛋白纖維。然而鉤藤作用於老年性癡呆模型的實驗研究還未見報道。本課題的研究目的是探討鉤藤提取物對認知功能的改善作用，從而篩選出鉤藤抗老年性癡呆的有效化學成份及探討鉤藤抗老年性癡呆有效化學成份的神經保護作用及其作用機理。首先我們探討了70%乙醇鉤藤提取物對D-半乳糖引起小鼠認知功能障礙的改善作用。水迷宮試驗結果顯示鉤藤提取物(200 和400毫克/千克)能顯著改善D-半乳糖處理小鼠的空間學習和記憶能力。此外，鉤藤提取物(200 和400毫克/千克)還顯著提高D-半乳糖處理小鼠腦組織中乙醯膽鹼和還原型穀胱甘肽的含量，以及超氧化物歧化酶和過氧化氫酶的活性，同時也能降低D-半乳糖處理小鼠腦組織中乙醯膽鹼酯酶的活性和丙二醛的含量。以上研究結果表明鉤藤提取物能改善D-半乳糖處理小鼠認知功能障礙的作用可能是通過抑制腦組織中乙醯膽鹼酯酶的活性和提高腦組織的氧化能力而達成的。其次，我們選用β澱粉樣蛋白引致PC12細胞神經毒性的體外細胞模型來跟蹤篩選出鉤藤提取物中抗老年性癡呆的有效活性成分。結果顯示從鉤藤提取物中分離出六個生物鹼，分別為柯諾辛堿，柯諾辛堿B，去氫鉤藤堿，異鉤藤堿，異去氫鉤藤堿和鉤藤堿。在這六個生物鹼中，只有鉤藤堿和異鉤藤堿具有顯著降低β澱粉樣蛋白導致PC12細胞的死亡，而異鉤藤堿是鉤藤提取物中對β澱粉樣蛋白所致的PC12細胞損傷有最強的保護作用。在明確異鉤藤堿是鉤藤提取物中抗老年性癡呆的主要有效成分的研究基礎上，我們應用β澱粉樣蛋白所致PC12細胞的神經毒性的體外實驗模型來探討異鉤藤堿的神經保護作用及其作用機理。實驗結果顯示異鉤藤堿對β澱粉樣蛋白引起PC12細胞的神經毒性的保護作用呈良好的量效關係。異鉤藤堿對β澱粉樣蛋白引起PC12細胞的神經毒性的保護作用是通過抑制細胞內鈣離子的超載，氧化應激，tau蛋白的過度磷酸化和線粒體細胞凋亡。 此外，異鉤藤堿還顯著抑制3β糖原合成酶激酶的活性，同時啟動磷酸化磷脂醯肌醇3-激酶底物Akt，提示異鉤藤堿對β澱粉樣蛋白所致的PC12細胞的神經毒性的保護作用與PI3K/Akt/GSK3信號通路相關密切相關。最後，我們進一步探討了異鉤藤堿對β澱粉樣蛋白致大鼠認知功能障礙的改善作用及其作用機理。研究結果表明異鉤藤堿(20和40毫克/千克/天)能顯著改善β澱粉樣蛋白所致的大鼠認知功能障礙（用水迷宮試驗來評價）及明顯增加海馬CA1區錐體細胞數目。同時，異鉤藤堿能顯著抑制β澱粉樣蛋白導致大鼠海馬的氧化應激，神經元凋亡以及tau蛋白過度磷酸化。此外，異鉤藤堿能顯著抑制3β糖原合成酶激酶的活性，啟動磷酸化磷脂醯肌醇3-激酶底物Akt，提示異鉤藤堿改善β澱粉樣蛋白導致大鼠認知功能障礙的作用機理與PI3K/Akt/GSK3信號通路相關。綜上所述，鉤藤和異鉤藤堿具有顯著的抗老年癡呆的作用。異鉤藤堿的神經保護作用與其抑制β澱粉樣蛋白導致PC12細胞和大鼠海馬的氧化應激，神經元凋亡以及tau蛋白的過度磷酸化有關。異鉤藤堿神經保護的作用機理與PI3K/Akt/GSK3信號通路密切相關。以上研究結果提示異鉤藤堿具有很好的進一步開發成新的抗老年性癡呆製劑的應用前景。The stem with hooks of Uncaria rhynchophylla (Ramulus Uncariae cum Uncis) is a component herb of many traditional formulae for the treatment of neurodegenerative diseases. Previous studies have demonstrated that the extract of U. rhynchophylla inhibited beta-amyloid (Aβ) fibril formation and disassemble preformed Aβ fibrils. However, scientific evidence concerning the efficacy of U. rhynchophylla in Alzheimer’s disease (AD) experimental models is lacking. The present study aimed at investigating the cognition-improving effect of U. rhynchophylla, identifying the active anti-AD chemical constituents and elucidating the underlying mechanisms of neuroprotective action.Firstly, we investigated whether 70% aqueous ethanol extract of U. rhynchophylla (EUR) could protect against D-galactose (D-gal)-induced cognitive deficits in mice. Mice were given a subcutaneous injection of D-gal (50 mg/kg) and orally administered EUR (100, 200, or 400 mg/kg) daily for 8 weeks. The results showed that EUR (200 or 400 mg/kg) significantly improved spatial learning and memory function in D-gal-treated mice as assessed by the Morris water maze test. In addition, EUR (200 or 400 mg/kg) significantly increased the levels of acetylcholine and glutathione, and the activities of superoxide dismutase and catalase, while it decreased the activity of acetylcholinesterase and the level of malondialdehyde in the brains of D-gal-treated mice. These results indicate that EUR was able to ameliorate cognitive deficits induced by D-gal in mice, and the observed pharmacological action may be mediated, at least in part, by the inhibition of acetylcholinesterase activity and the enhancement of the antioxidant status of the brain tissues.Secondly, we tried to identify the active ingredients of U. rhynchophylla by a bioassay-guided fractionation approach using beta-amyloid (Aβ)-induced neurotoxicity in rat pheochromocytoma (PC12) cells, a well established cellular model of AD. As a result of this work, six alkaloids, namely corynoxine, corynoxine B, corynoxeine, isorhynchophylline, isocorynoxeine and rhynchophylline were isolated from the extract of U. rhynchophylla. Among them, only rhynchophylline and isorhynchophylline could significantly decrease Aβ-induced cell death in PC12 cells. Moreover, isorhynchophylline (IRN) was found to be the most active ingredient responsible for the protective action of U. rhynchophylla against Aβ₂₅₋₃₅-induced cell death.Thirdly, the neuroprotective effects and its action mechanism of IRN against Aβ₂₅₋₃₅-induced neurotoxicity in PC12 cells, an in vitro experimental model of AD, were examined. The results showed that treatment with IRN dose-dependently protected PC12 cells against Aβ₂₅₋₃₅-induced neurotoxicity. The neuroprotective effect of IRN may be mediated, at least in part, by inhibiting the intracellular calcium overloading, oxidative stress, tau protein hyperphosphorylation and mitochondrial cellular apoptosis induced by Aβ₂₅₋₃₅. Moreover, IRN also inhibited the activity of glycogen synthase kinase (GSK)-3β, an important kinase responsible for tau protein hyperphosphorylation in the development of AD; and activated the phosphorylation of phosphatidylinositol 3-kinase (PI3K) substrate Akt, suggesting that the neuroprotective action of IRN is associated with inhibition of GSK-3β activity and activation of PI3K/Akt signaling pathway.Finally, the ameliorating effect on cognitive deficits of IRN and its underlying mechanism of action in Aβ₂₅₋₃₅-treated rats were investigated. The results showed that oral administration of IRN with two different doses (20 or 40 mg/kg) for 21 days significantly ameliorated cognitive impairments and suppressed the oxidative stress, neuronal apoptosis, and tau protein hyperphosphorylation in the hippocampus of Aβ₂₅₋₃₅-treated rats. In addition, IRN also inhibited the activity of GSK-3β, and activated phosphorylation of phosphatidylinositol 3-kinase (PI3K) substrate Akt, suggesting that the amelioration of cognitive deficits by IRN is associated with inhibition of GSK-3β activity and activation of PI3K/Akt signaling pathway.Taken together, these results confirmed the anti-AD effects of U. rhynchophylla and IRN. The neuroprotective action of IRN may be mediated via inhibition of oxidative stress, neuronal apoptosis and hyperphosphorylation tau protein induced by Aβ₂₅₋₃₅ in vitro and in vivo. The neuroprotective action of IRN is associated with the inhibition of GSK-3β activity and the activation of PI3K/Akt signaling pathway. These experimental findings render IRN a promising candidate worthy of further development into anti-AD pharmaceutical agents.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.Xian, Yanfang.Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.Includes bibliographical references (leaves 242-278).Abstracts also in Chinese.Abstract (English) --- p.I摘要 --- p.IVPublications --- p.VIIAcknowledgements --- p.IXTable of Contents --- p.XList of Figures --- p.XXIList of Tables --- p.XXVIList of Abbreviation --- p.XXVIIChapter Chapter One --- General IntroductionChapter 1.1 --- Alzheimer’s Disease --- p.2Chapter 1.1.1 --- Symptoms --- p.2Chapter 1.1.2 --- Epidemiology --- p.4Chapter 1.1.3 --- Pathology --- p.5Chapter 1.1.4 --- Risk factors --- p.6Chapter 1.2 --- Pathogenesis of AD --- p.10Chapter 1.2.1 --- Neurotransmitter dysfunction --- p.10Chapter 1.2.1.1 --- Cholinergic system dysfunction --- p.10Chapter 1.2.1.2 --- Glutamatergic system dysfunction --- p.11Chapter 1.2.2 --- Hippocampus atrophy --- p.15Chapter 1.2.3 --- “Amyloid Cascade hypothesis --- p.18Chapter 1.2.4 --- Increased oxidative stress --- p.21Chapter 1.2.5 --- Increased neuronal apoptosis --- p.23Chapter 1.2.6 --- Mitochondrial dysfunction --- p.27Chapter 1.2.7 --- Calcium dysregulation --- p.31Chapter 1.2.8 --- Increased tau protein hyperphosphorylation --- p.34Chapter 1.2.9 --- GSK3 hypothesis of AD --- p.37Chapter 1.3 --- Animal Models of AD --- p.41Chapter 1.3.1 --- Non-transgenic animal models of AD --- p.42Chapter 1.3.1.1 --- Spontaneous models --- p.42Chapter 1.3.1.2 --- Scopolamine-induced rodent models --- p.43Chapter 1.3.1.3 --- Aluminum-induced rodent models --- p.44Chapter 1.3.1.4 --- D-galactose-induced rodent models --- p.45Chapter 1.3.1.5 --- Aβ infusion rodent models --- p.46Chapter 1.3.2 --- Transgenic animal models of AD --- p.48Chapter 1.3.2.1 --- Transgenic rodent models for AD --- p.49Chapter 1.3.2.2 --- AD models in D. rerio --- p.53Chapter 1.3.2.3 --- AD models in D. melanogaster --- p.54Chapter 1.3.2.4 --- AD models in C. elegans --- p.54Chapter 1.4 --- Treatments for AD --- p.55Chapter 1.4.1 --- Current symptomatic treatments --- p.56Chapter 1.4.1.1 --- AChEIs --- p.56Chapter 1.4.1.2 --- NMDA antagonist --- p.57Chapter 1.4.2 --- Disease-modifying approaches --- p.61Chapter 1.4.2.1 --- Amyloid-directed therapies --- p.61Chapter 1.4.2.2 --- Tau-directed therapies --- p.61Chapter 1.4.2.3 --- Anti-oxidant agents --- p.62Chapter 1.4.2.4 --- NSAIDs --- p.63Chapter 1.4.2.5 --- Estrogen replacement therapy (ERT) --- p.64Chapter 1.4.3 --- Herbal medicines --- p.67Chapter 1.5 --- Uncaria rhynchophylla --- p.69Chapter 1.5.1 --- Chemical constituents --- p.69Chapter 1.5.2 --- Alkaloids --- p.72Chapter 1.6 --- Pharmacological Activities of Uncaria rhynchophylla and Its Alkaloids --- p.75Chapter 1.6.1 --- Effects on cardiovascular system --- p.75Chapter 1.6.2 --- Effects on central nervous system --- p.77Chapter 1.6.3 --- Antioxidant activities --- p.79Chapter 1.6.4 --- Anti-inflammatory and analgesic effects --- p.80Chapter 1.6.5 --- Effects on platelet aggregation and thrombosis --- p.81Chapter 1.6.6 --- Other pharmacological effects --- p.81Chapter 1.7 --- Hypothesis and Objectives of the Present Study --- p.83Chapter Chapter Two --- Uncaria rhynchophylla Ameliorates Cognitive Deficits Induced by D-galactose in MiceChapter 2.1 --- Introduction --- p.86Chapter 2.2 --- Materials and Methods --- p.88Chapter 2.2.1 --- Drugs and chemical reagents --- p.88Chapter 2.2.2 --- Plant materials and extraction --- p.89Chapter 2.2.3 --- Animals --- p.90Chapter 2.2.4 --- Experimental design and drugs treatment --- p.90Chapter 2.2.5 --- Morris water maze test --- p.91Chapter 2.2.6 --- Preparation of brain tissue samples --- p.92Chapter 2.2.7 --- Measurement of intracellular ROS level --- p.92Chapter 2.2.8 --- Assay of MDA level --- p.92Chapter 2.2.9 --- Assay of GSH level --- p.93Chapter 2.2.10 --- Measurement of SOD activity --- p.93Chapter 2.2.11 --- Measurement of CAT activity --- p.94Chapter 2.2.12 --- Assay of Ach level --- p.94Chapter 2.2.13 --- Measurement of AChE activity --- p.95Chapter 2.2.14 --- Statistical analysis --- p.95Chapter 2.3 --- Results --- p.95Chapter 2.3.1 --- Quality determination of EUR --- p.95Chapter 2.3.2 --- Effects of EUR on Morris water maze in D-gal-treated mice --- p.97Chapter 2.3.3 --- Effects of EUR on the level of intracellular ROS in the brains of D-gal-treated mice --- p.101Chapter 2.3.4 --- Effects of EUR on the levels of GSH and MDA in the brains of D-gal-treated mice --- p.103Chapter 2.3.5 --- Effects of EUR on the activities of SOD and CAT in the brains of D-gal-treated mice --- p.105Chapter 2.3.6 --- Effects of EUR on the level of ACh and the activity of AChE in the brains of D-gal-treated mice --- p.107Chapter 2.4 --- Discussion --- p.109Chapter Chapter Three --- Bioassay-Guided Isolation of Neuroprotective Compounds from Uncaria rhynchophylla Against Beta-Amyloid-Induced NeurotoxicityChapter 3.1 --- Introduction --- p.113Chapter 3.2 --- Materials and Methods --- p.114Chapter 3.2.2 --- Drugs and chemical reagents --- p.114Chapter 3.2.2 --- Preparation of aggregated Aβ₂₅₋₃₅ --- p.115Chapter 3.2.3 --- Extraction, fractionation, isolation and identification processes --- p.115Chapter 3.2.4 --- Cell culture and drug treatment --- p.119Chapter 3.2.5 --- Cell viability assay --- p.119Chapter 3.2.6 --- Statistical analysis --- p.120Chapter 3.3 --- Results --- p.120Chapter 3.3.1 --- Isolation and structural determination of the isolated compounds --- p.120Chapter 3.3.2 --- Effects of different fractions and isolated compounds on Aβ₂₅₋₃₅-induced cells death in PC12 cells --- p.122Chapter 3.4 --- Discussion --- p.126Chapter Chapter Four --- Neuroprotective Effects of Isorhynchophylline Against Beta-Amyloid-Induced Neurotoxicity in PC12 Cells and Its Possible MechanismsChapter 4.1 --- Introduction --- p.130Chapter 4.2 --- Materials and Methods --- p.131Chapter 4.2.1 --- Drugs and chemical reagents --- p.131Chapter 4.2.2 --- Cell culture and drugs treatment --- p.134Chapter 4.2.3 --- Cell viability assay --- p.134Chapter 4.2.4 --- Lactate dehydrogenase (LDH) activity assay --- p.135Chapter 4.2.5 --- Measurement of intracellular ROS production --- p.135Chapter 4.2.6 --- Malondialdehyde (MDA) and glutathione (GSH) assay --- p.136Chapter 4.2.7 --- Measurement of SOD activity --- p.137Chapter 4.2.8 --- Measurement of CAT activity --- p.137Chapter 4.2.9 --- Measurement of intracellular calcium concentration --- p.138Chapter 4.2.10 --- Measurement of mitochondrial membrane potential --- p.139Chapter 4.2.11 --- Quantification of DNA fragmentation --- p.139Chapter 4.2.12 --- Cytochrome c assay --- p.140Chapter 4.2.13 --- Western blotting analysis --- p.140Chapter 4.2.14 --- Real time-polymerase chain reaction (RT-PCR) analysis --- p.141Chapter 4.2.15 --- Statistical analysis --- p.142Chapter 4.3 --- Results --- p.143Chapter 4.3.1 --- Effects of IRN on Aβ₂₅₋₃₅-induced cytotoxicity in PC12 cells --- p.143Chapter 4.3.2 --- Effects of IRN on the level of intracellular ROS in Aβ₂₅₋₃₅-treated PC12 cells --- p.145Chapter 4.3.3 --- Effects of IRN on the levels of GSH and MDA in Aβ₂₅₋₃₅-treated PC12 cells --- p.147Chapter 4.3.4 --- Effects of IRN on the activities of SOD and CAT in Aβ₂₅₋₃₅-treated PC12 cells --- p.149Chapter 4.3.5 --- Effects of IRN on intracellular calcium level in Aβ₂₅₋₃₅-treated PC12 Cells --- p.151Chapter 4.3.6 --- Effects of IRN on MMP in Aβ₂₅₋₃₅-treated PC12 cells --- p.153Chapter 4.3.7 --- Effects of IRN on DNA fragmentation in Aβ₂₅₋₃₅-treated PC12 cells --- p.155Chapter 4.3.8 --- Effects of IRN on the release of cytochrome c in Aβ₂₅₋₃₅-treated PC12 cells --- p.157Chapter 4.3.9 --- Effects of IRN on the protein and mRNA levels of the ratio of Bcl-2/Bax in Aβ₂₅₋₃₅-treated PC12 cells --- p.159Chapter 4.3.10 --- Effects of IRN on the protein and mRNA levels of cleaved caspase-3 and caspase-9 in Aβ₂₅₋₃₅-treated PC12 cells --- p.162Chapter 4.3.11 --- Effects of IRN on the protein of pro-caspase-8 and mRNA levels of the full length of caspase-8 in Aβ₂₅₋₃₅-treated PC12 cells --- p.165Chapter 4.3.12 --- Effects of IRN on tau protein hyperphosphorylation in Aβ₂₅₋₃₅-treated PC12 Cells --- p.168Chapter 4.3.13 --- Effects of IRN on Aβ₂₅₋₃₅-induced activation of GSK-3β in PC12 cells --- p.170Chapter 4.3.14 --- Effects of IRN on Aβ₂₅₋₃₅-induced inactivation of PI3K/Akt pathway --- p.173Chapter 4.4 --- Discussion --- p.177Chapter Chapter Five --- Isorhynchophylline Treatment Improves Cognitive Deficits Induced by Beta-Amyloid in Rats: Involvement of PI3K/Akt Signaling PathwayChapter 5.1 --- Introduction --- p.186Chapter 5.2 --- Materials and Methods --- p.187Chapter 5.2.1 --- Drugs and chemical reagents --- p.187Chapter 5.2.2 --- Animals --- p.188Chapter 5.2.3 --- Aβ₂₅₋₃₅ injections --- p.188Chapter 5.2.4 --- Experimental design and drugs treatment --- p.189Chapter 5.2.5 --- Morris water maze test --- p.190Chapter 5.2.6 --- Nissl’s staining for neurons --- p.193Chapter 5.2.7 --- Preparation of brain tissue samples --- p.193Chapter 5.2.8 --- Measurement of intracellular ROS level --- p.194Chapter 5.2.9 --- Assay of MDA level --- p.194Chapter 5.2.10 --- Assay of GSH level --- p.195Chapter 5.2.11 --- Measurement of SOD activity --- p.195Chapter 5.2.12 --- Measurement of CAT activity --- p.195Chapter 5.2.13 --- Cytochrome c assay --- p.196Chapter 5.2.14 --- Western blotting analysis --- p.196Chapter 5.2.15 --- RT-PCR analysis --- p.197Chapter 5.2.16 --- Statistical analysis --- p.198Chapter 5.3 --- Results --- p.199Chapter 5.3.1 --- IRN treatment rescued behavioral impairment in the Morris water maze test --- p.199Chapter 5.3.2 --- Effects of IRN on the number of pyramidal neuronal cells in the hippocampal CA1 region of Aβ₂₅₋₃₅-treated rats --- p.203Chapter 5.3.3 --- Effects of IRN on the intracellular ROS level in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.205Chapter 5.3.4 --- Effects of IRN on the levels of GSH and MDA in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.207Chapter 5.3.5 --- Effects of IRN on the activities of SOD and CAT in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.209Chapter 5.3.6 --- Effects of IRN on cytochrome c in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.211Chapter 5.3.7 --- Effects of IRN on the protein and mRNA level of the ratio of Bcl-2/Bax in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.213Chapter 5.3.8 --- Effects of IRN on the protein and mRNA levels of cleaved caspase-3 and caspase-9 in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.216Chapter 5.3.9 --- Effects of IRN on the protein and mRNA levels of caspase-8 in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.219Chapter 5.3.10 --- Effects of IRN on the tau protein hyperphosphorylation in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.222Chapter 5.3.11 --- Effects of IRN on the activation of GSK-3β in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.224Chapter 5.3.12 --- Effects of IRN on the PI3K/Akt pathway in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.226Chapter 5.4 --- Discussion --- p.228Chapter Chapter Six --- General Discussion and Future PerspectivesChapter 6.1 --- General Discussion and Conclusions --- p.237Chapter 6.2 --- Future Perspectives --- p.243References by Alphabetical Order --- p.24