心臟病目前仍然是最普遍的威脅人類生命安全的三大病因之一。同西藥相比, 傳統中醫藥具有多靶點，協同作用及小副作用等特性。在中藥歷史中， 丹參和葛根這兩種草藥經常出現在中藥方劑用於治療心血管相關的疾病，已有幾千年的歷史。 我們實驗室發現了一個丹參葛根湯劑具有保護動脈粥樣硬化病人心臟功能的作用，並且可以使收縮的大鼠大動脈舒張的作用。 本研究主要通過舒張豬冠狀動脈，提高大鼠對抗過氧化和離子擾動能力以及提高血管增生四個方面探討丹參葛根複方水提物 (質量比7:3) (DG配方)對血管的作用以提供其治療心血管疾病的藥理基礎。 / 在本研究的第一部， 我們主要探討了DG配方對缺血再灌注損傷的心臟及其心肌細胞的保護作用。我們發現DG配方明顯抑制了心臟損傷相關的肌酸激酶和乳酸脫氫的釋放。同時DG配方顯著促進了再灌注後冠狀動脈內血流量速度和收縮力度的恢復。這些結果說明DG配方可以保護缺血再灌注心臟並且有效促進其功能恢復。我們還觀察了長期給大鼠用DG配方14天後其心臟在缺血再灌注中的表現。類似於再灌注時給藥的結果，DG配方同樣抑制了損傷酶的釋放並且有效促進了冠狀動脈內血流量速度和收縮力度的恢復。 / 同時，在缺氧再灌注離體細胞模型中，我們發現DG配方明顯抑制了缺氧再灌注損傷帶來的細胞死亡。流式細胞儀分析結果表明，藥物處理組中的凋亡類的細胞明顯比對照組中少主要通過抑制促凋亡的caspase3表達明以及促進抗凋亡的Bcl2表達升高。DG配方減少了心肌細胞內細胞色素c從線粒體中釋放明顯以及抑制了線粒體去極化。這說明DG配方也保護了線粒體的膜的完整性，從而確保線粒體功能進而保證細胞的能量系統穩定。最有意思的是DG配方可以直接抑制缺氧再灌注相關的兩條通路, 它不僅抑制活性氧化物質的釋放, 同時也抑制了再灌注後鈣離子的累積。總之，DG配方以抗氧化和抗離子擾動的方式保護了在缺血缺氧再灌注損傷中心臟和心肌細胞的結構和功能。 / 第二部分的研究是關於DG配方對從豬心臟上分離的左冠狀動脈前室間支 (左前降支) 血管的作用及其內在的機制，我們的結果表明對由U46619引起的冠狀動脈血管收縮DG配方表現了濃度依賴的舒血管作用。而該作用並非依賴于內皮細胞及其釋放的舒張血管因數一氧化氮和前列腺素類似物環素和大部分的鉀離子通道。其中只有內向整合鉀離子通道部分參與了舒血管的過程。肌球蛋白輕鏈的磷酸化明顯被DG配方抑制，但是RhoA 的活性並沒有受其影響。鈣離子引發的血管收縮則被DG配方濃度依賴性的受到抑制。這部分的研究證明瞭DG配方主要通過類似鈣離子通道拮抗劑作用抑制鈣離子進入到血管平滑肌細胞減少肌球蛋白磷酸化達到舒張血管的作用。結果說明DG配方可以作為一種安全的藥物用於治療心血管疾病特別是高血壓和心絞痛。 / 本研究的第三部分是關於DG配方的促血管增生的作用。我們發現DG配方可以明顯促進斑馬魚的腸下動脈的出芽並且促進血管增生相關基因的表達，血管內皮細胞生長因數及其受體的mRNA表達。內皮細胞是血管增生的基礎。所以我們利用人源微血管內皮細胞檢測了DG配方在細胞的增生，遷移，分化和形成血管方面的影響以解釋它在斑馬魚中促進血管增生的作用機理。結果發現，DG配方明顯促進了該種內皮細胞的增殖，遷移和形成管狀結構。 / 綜上所述，DG配方可以通過舒張血管，抗氧化，抗離子紊亂和促進血管增生提供心血管保護功能。DG配方通過螯合活性氧化物質和抑制鈣離子的累積保護了因缺血再灌注引起的心臟損傷，說明DG配方可以作為手術的輔助藥物減少心臟病人在缺血再灌注過程中受到的損傷。它以拮抗L型鈣離子通道方式減少鈣離子進入到血管平滑肌細胞來舒張收縮的冠狀動脈血管。說明DG配方可以用於治療高血壓和心絞痛等心臟病。另外DG配方也可以促進血管增生，可用于心肌梗死病人促進其心臟血管系統重建，本研究對於未來臨床實驗具有重要的參考價值。 / Coronary heart diseases (CHD) are one of the most prevalent causes of premature death all over the world. In contrast to western medicine, traditional Chinese medicine (TCM) has shown the benefit of multi-targeting and synergism to treat CHD. Two kinds of Chinese herbs, Danshen (Radix Salviae Miltiorrhiza) (D) and Gegen (Radix Puerariae Lobatae) (G) always present on the TCM formula for treating heart disease. We found a useful formula of Danshen and Gegen decoction with weight ratio of 7:3 (DG) exerting properties of improving the heart function in patient with atheroslcerosis and providing vasodiation and antioxidant protection on the rat cardiovascular system. The present study was designed to evaluate the effects of DG on the vascular activity by its properties on antioxidant and anti-ion stunning to inhibiting the ischemia and reperfusion injury, vasodilation effect on pig coronary artery and angiogenesis effect on zebrafish model. / In the first part of the study, we explored protective effect of DG on rat hearts and cardiomyocytes after ischemia-reperfusion and hypoxia-reoxygenation injury. Comparing to control group, the release of creatine kinase (CK) and lactate dehydrogenase (LDH) significantly decreased in the DG treated groups in a dose-dependent manner. The recovery percentage of coronary flow and contractile force in the DG was higher than that in the control group. These results suggested that DG dose-dependently improved the heart function after ischemia and reperfusion injury in a dose-dependent manner. We also examined chronic effect of DG (14 days pretreatment) on rat heart with ischemia and reperfusion injury. DG induced rat heart with high potential to deal with I/R injury, less damaged enzymes release and high recovery percentage of heart function recovery. / In the cell hypoxia and reoxygenation model, DG significantly inhibited the cell death after H/R treatment with bcl2 expression increase and caspase3 expression decrease. DG also reversed the H/R-induced mitochondrial depolarization and inhibited cytochrome c diffusing out of mitochondria, which confirmed DG anti-apoptosis activity. DG also was found to significantly decrease the intracellular calcium accumulation and reactive oxygen species release within H9c2. / In the second part of present study, results revealed that DG elicited a concentration-dependent relaxation of U46619-preconstricted porcine coronary artery. DG-induced relaxation responses were not altered by the presence of endothelium-related dilator inhibitors, most potassium channel blockers, GMP and AMP pathway inhibitors and endothelium removal. Ba²⁺ (an inward rectifier K⁺ channel blocker) slightly attenuated DG-induced relaxation. The protein expression of phosphorylated myosin light chain (MLC) was inhibited by DG in a concentration-dependent manner whereas the activity of RhoA was not modified. Ca²⁺-induced contraction of coronary artery was inhibited by DG in a concentration-dependent fashion. DG acted as an antagonist of calcium channel inducing the porcine artery dilation. / The third part of the present study is about the pro-angiogenic effect of DG. We found that DG dose-dependently induced zebrafish sub-intestinal vessel sprouting and increased the mRNA expression of vascular endothelial growth factor (VEGF) and its receptors. To explore the underlying mechanism, we also examined the proangiogenic effect of DG on the angiogenesis of endothelial cells. The results showed that DG induced the HMEC-1 proliferation, migration and forming tube. / In conclusion, we found that DG could provide cardiac and cardiovascular protection by its multiple targets. It prevented heart injuries after ischemia or hypoxia and reperfusion through scavenging ROS and inhibiting calcium accumulation. Moreover, it mainly acts as an antagonist of L-type calcium channel to relax the contracted LAD vessel. It also exerted property of inducing angiogenesis in vivo and in vitro. Therefore, DG would be useful for treating coronary artery disease depending on its multiple targets. / 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. / Hu, Fan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 170-215). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter 1 --- Intorduction --- p.1 / Chapter 1.1 --- Cardiovascular system and coronary artery diseases --- p.1 / Chapter 1.1.1 --- The cardiovascular system --- p.1 / Chapter 1.1.2 --- Contraction and relaxation of the vascular myocyte in arteries --- p.4 / Chapter 1.1.2.1 --- Ultrastructure of the vascular myocyte --- p.4 / Chapter 1.1.2.2 --- Contraction mechanisms of vascular myocyte --- p.5 / Chapter 1.1.2.3 --- Relaxation mechanisms of vascular myocyte --- p.7 / Chapter 1.1.3 --- Chronic coronary heart disease --- p.9 / Chapter 1.2 --- The way to treat chronic CAD --- p.11 / Chapter 1.2.1 --- Angiogenesis --- p.11 / Chapter 1.2.2 --- Clinical surgery for treating CAD --- p.13 / Chapter 1.2.2.1 --- Three common surgeries for treating CAD --- p.13 / Chapter 1.2.2.2 --- Ischemia and reperfusion (I/R) injury in surgeries --- p.15 / Chapter 1.2.3 --- Drugs for treating CAD --- p.19 / Chapter 1.2.3.1 --- Western medicine therapy in CAD --- p.19 / Chapter 1.2.3.2 --- Traditional Chinese Medicine treatment in CAD --- p.20 / Chapter 1.3 --- Aims of studies --- p.28 / Chapter 2 --- Materials and Methods --- p.29 / Chapter 2.1 --- Solutions and Materials --- p.29 / Chapter 2.1.1 --- Solutions --- p.29 / Chapter 2.1.2 --- Chemicals and enzymes --- p.36 / Chapter 2.2 --- Methods --- p.38 / Chapter 2.2.1 --- Herbal preparation --- p.38 / Chapter 2.2.2 --- Identification and quantification of chemical markers in Danshen and Gegen decoction (DG) --- p.38 / Chapter 2.2.3 --- Assay development for the determination of the DG marker compounds in rat plasma --- p.40 / Chapter 2.2.4 --- Isolation of pig left anterior descending coronary artery --- p.44 / Chapter 2.2.5 --- Isometric tension measurement --- p.45 / Chapter 2.2.6 --- Langendorff related experiment --- p.50 / Chapter 2.2.7 --- Cell culture of H9c2 cells --- p.54 / Chapter 2.2.8 --- Cell viability assay (MTT assay) --- p.56 / Chapter 2.2.9 --- Cell proliferation measurement --- p.57 / Chapter 2.2.10 --- Hypoxia and reperfusion cell model (H9c2) --- p.58 / Chapter 2.2.11 --- Determination of cell apoptosis with Annexin VFITC and PI double staining --- p.59 / Chapter 2.2.12 --- Measurement of mitochondria depolarization --- p.61 / Chapter 2.2.13 --- Measurement of ROS release --- p.63 / Chapter 2.2.14 --- Measurement of calcium localization in H9c2 cells by fluo4 dye and confocal microscopy --- p.64 / Chapter 2.2.15 --- Extraction of proteins from tissue, cell and subcellular fractions --- p.65 / Chapter 2.2.16 --- Western blot assay --- p.67 / Chapter 2.2.17 --- Human microvascular endothelial cells (HMEC1) cell culture --- p.68 / Chapter 2.2.18 --- Cell cycle analysis by PI staining --- p.69 / Chapter 2.2.19 --- Scratch assay for HMEC1cells migration --- p.70 / Chapter 2.2.20 --- Tube formation assay --- p.71 / Chapter 2.2.21 --- Vessel sprouting of Zebrafish --- p.72 / Chapter 2.2.22 --- Real time PCR --- p.74 / Chapter 2.2.23 --- Statistical analysis --- p.76 / Chapter 3 --- Chapter 3 Cardiac protection of Danshen and Gegen decoction in hypoxia/ischemia and reperfusion induced injury --- p.77 / Chapter 3.1 --- Introduction --- p.77 / Chapter 3.2 --- Results --- p.81 / Chapter 3.2.1 --- Cytotoxicity of DG --- p.81 / Chapter 3.2.2 --- The morphology alteration of H9c2 after H/R treatment --- p.83 / Chapter 3.2.3 --- Effect on H H9c2 cell survival after H/R treatment --- p.84 / Chapter 3.2.4 --- Effect on membrane skeleton of H9c2 cells with H/R injury --- p.86 / Chapter 3.2.5 --- Effect on the apoptosis in H9c2 cells induced by H/R injury --- p.88 / Chapter 3.2.6 --- Effect on cytochrome c release from mitochondria of damaged H9c2 cells --- p.92 / Chapter 3.2.7 --- Effect on mitochondria depolarization of H9c2 after H/R treatment --- p.94 / Chapter 3.2.8 --- Effect on reactive oxidant species (ROS) release --- p.96 / Chapter 3.2.9 --- Effect on calcium accumulation within H9c2 in the reperfusion phase --- p.98 / Chapter 3.2.10 --- Effect on heart functions of rat hearts with I/R injury (acute effect) --- p.101 / Chapter 3.2.11 --- Effect on heart function in rats with I/R injury (chronic effect) --- p.107 / Chapter 3.3 --- Discussion --- p.113 / Chapter 4 --- Chapter 4 Vasodilation effects of Danshen and Gegen decoction in porcine coronary artery and its underlying mechanism --- p.118 / Chapter 4.1 --- Introduction --- p.118 / Chapter 4.2 --- Results --- p.121 / Chapter 4.2.1 --- Investigations of endothelium dependent and independent mechanisms --- p.121 / Chapter 4.2.2 --- Effects on cAMP and cGMP pathway --- p.121 / Chapter 4.2.3 --- Effects on potassium channel opening --- p.121 / Chapter 4.2.4 --- Effects on calcium induced contraction and calcium sensitization --- p.122 / Chapter 4.2.5 --- Effects on MLC phosphorylations --- p.123 / Chapter 4.3 --- Discussion --- p.132 / Chapter 5 --- Chapter 5 In vitro and in vivo angiogenic effects of DG --- p.138 / Chapter 5.1 --- Introduction --- p.138 / Chapter 5.2 --- Results --- p.140 / Chapter 5.2.1 --- Effect on subintestinal vessels sprouting in the zebrafish embryo --- p.140 / Chapter 5.2.2 --- Effect on the transcription and expression of VEGFA and VEGF receptors -- Flt1 and KDR/Flk2 --- p.143 / Chapter 5.2.3 --- Effect on HMEC1 proliferation --- p.145 / Chapter 5.2.4 --- Effect on cell cycle of HMEC1 --- p.148 / Chapter 5.2.5 --- Effect on cell migration of HMEC1 --- p.151 / Chapter 5.2.6 --- Effect on tube formation of HMEC1 --- p.154 / Chapter 5.3 --- Discussion --- p.157 / Chapter 6 --- Chapter 6 Conclusions and future work --- p.160 / Chapter 6.1 --- Cardiac protection of DG in the I/R and H/R injury --- p.160 / Chapter 6.2 --- Vasodilation effect of DG on the porcine coronary artery --- p.165 / Chapter 6.3 --- Angiogenic effect of DG in vivo and in vitro --- p.167 / Chapter 6.4 --- Overall conclusion of the study --- p.169 / Chapter 7 --- References --- p.170

Wang Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 124-135). / Abstracts in English and Chinese. / Acknowledgements / Abstract / 摘要 / Table of contents / List of figures / List of tables / List of abbreviations / CHAPTER / Chapter 1. --- Introduction / Chapter 1.1 --- Background of SARS and epidemiology / Chapter 1.2 --- SARS symptoms and infected regions / Chapter 1.3 --- SARS virus / Chapter 1.4 --- Treatment for SARS at present / Chapter 1.5 --- Vaccine development is a more effective way to fight against SARS / Chapter 1.6 --- Vaccine candidates / Chapter 1.6.1 --- Truncated S protein as a vaccine candidate / Chapter 1.6.2 --- Full-length N protein as a vaccine candidate / Chapter 1.7 --- E.coli expression system / Chapter 1.8 --- Baculovirus expression system / Chapter 1.8.1 --- Characteristics of baculovirus / Chapter 1.8.2 --- Infection cycle of baculovirus / Chapter 1.8.3 --- Control of viral gene expression in virus-infected cells / Chapter 1.8.4 --- Merits of baculovirus expression system / Chapter 1.9 --- Aim of study / Chapter 2. --- "Bacterial expression and purification of rS1-1000(E), rS401-1000(E) and rN(E)" / Chapter 2.1 --- Introduction / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Reagents for bacterial culture / Chapter 2.2.2 --- Reagents for agarose gel electrophoresis / Chapter 2.2.3 --- 2'-deoxyribonucleoside 5'-triphosphate (dNTP) mix for polymerase chain reaction (PCR) / Chapter 2.2.4 --- Sonication buffer / Chapter 2.2.5 --- Reagents for immobilized metal affinity chromatography (IMAC) purification / Chapter 2.2.6 --- Reagents for gel filtration chromatography / Chapter 2.2.7 --- Reagents for sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) / Chapter 2.2.8 --- Reagents for Western blotting / Chapter 2.3 --- Methods / Chapter 2.3.1 --- General techniques in molecular cloning / Chapter 2.3.2 --- "PCR amplification of the S1-400,S401-1000" / Chapter 2.3.3 --- Construction of clone pET-S 1-400 and PET-s401-1000 / Chapter 2.3.4 --- Construction of clone pAC-N / Chapter 2.3.5 --- Expression / Chapter 2.3.6 --- Inclusion bodies preparation / Chapter 2.3.7 --- Inclusion bodies solubilization using urea / Chapter 2.3.8 --- Protein refolding by rapid dilution and dialysis / Chapter 2.3.9 --- Purification of recombinant protein by nickel ion chelating Sepharose fast flow column (IMAC) / Chapter 2.3.10 --- Gel filtration chromatography for further purification / Chapter 2.3.11 --- Bradford assay for the protein concentration analysis / Chapter 2.3.12 --- Protein analysis / Chapter 2.4 --- Results / Chapter 2.4.1 --- SDS-PAGE analysis of the expressed proteins / Chapter 2.4.2 --- Western blot analysis of the bacterial cell lysate / Chapter 2.4.3 --- Protein purification by IMAC / Chapter 2.4.4 --- Purification of rS401-1000(E) by gel filtration / Chapter 2.4.5 --- Determination of production yield of recombinant fusion proteins / Chapter 2.5 --- Discussion / Chapter 2.5.1 --- Expression vector selected for rS1-400(E) and rS401-1000(E) expression / Chapter 2.5.2 --- Protein expression in E.coli / Chapter 2.5.3 --- Purification process / Chapter 3. --- Baculovirus expression and purification of rS401-1000(ACN) and rN(BMN) protein / Chapter 3.1 --- Introduction / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Reagents for insect cell culture and virus work / Chapter 3.3 --- Methods / Chapter 3.3.1 --- "PCR amplification of N and cloning of S401-1000, N genes into the transfer vector pVL1393" / Chapter 3.3.2 --- Cloning of S401-1000 into transfer vector pFastBac HT B / Chapter 3.3.3 --- Virus works / Chapter 3.3.4 --- Identification of recombinant BmNPV or AcMNPV / Chapter 3.3.5 --- Manipulation of silkworm / Chapter 3.3.6 --- Mouse immunization for polyclonal antibody against rN(E) protein / Chapter 3.4 --- Results / Chapter 3.4.1 --- Expression of rN(BMN) in baculovirus / Chapter 3.4.2 --- Expression of rS401-1000(BMN) and rS401-1000(ACN) in baculovirus / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- The expression level of rN(BMN) in both in vitro and invivo / Chapter 3.5.2 --- The rS401-1000(ACN) protein expression level in vitro / Chapter 3.5.3 --- Failure in generating rS401-1000(BMN) / Chapter 3.5.4 --- Purification process of rN(BMN) by IMAC / Chapter 4. --- "Characterization of recombinant rS1-400(E), rN(E), rN(BMN), rS401_1000(E) and rS401-1000(ACN)" / Chapter 4.1 --- Introduction / Chapter 4.2 --- Materials / Chapter 4.2.1 --- Reagents for enzyme-linked immunosorbent assay (ELISA) / Chapter 4.2.2 --- Reagents for purification of human IgG / Chapter 4.2.3 --- Source and identity of Immune sera / Chapter 4.3 --- Methods / Chapter 4.3.1 --- ELISA / Chapter 4.3.2 --- Purification process of human IgG / Chapter 4.4 --- Results / Chapter 4.4.1 --- Validation of Immune sera using SARS viral lysate / Chapter 4.4.2 --- Immunoreactivities of rS1-400(E) and rN(E) against pooled patients sera and normal human serum / Chapter 4.4.3 --- Immunoreactivity comparison of rN(E) and rN(BMN) / Chapter 4.4.4 --- Comparison of the immunoreactivities of rS401-1000(E) and rS401-1000(ACN) / Chapter 4.4.5 --- Immunoreactivity of SARS related proteins against Anti-SARS Antibody (Equine) / Chapter 4.5 --- Discussion / Chapter 4.5.1 --- Comparison of the immunoreactivities of SARS related proteins expressed in the present study / References

Yam Wing Sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 154-160). / Abstracts in English and Chinese. / Abstract English --- p.i / Chinese --- p.iii / Acknowledgments --- p.v / Table of contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Chinese Medicine and Western Medication --- p.1 / Chapter 1.2 --- Chinese Medicine and Compound Formula --- p.2 / Chapter 1.3 --- Cardiovascular disease (CVD) and atherosclerosis --- p.6 / Chapter 1.4 --- General Research Objectives --- p.19 / Chapter Chapter 2 --- Establishment of compound formulation and Extract Preparation --- p.21 / Chapter 2.1 --- Formulation searched from Chinese Pharmacopoeia --- p.21 / Chapter 2.2 --- Aqueous extract preparation --- p.25 / Chapter 2.2.1 --- Materials and Methods --- p.25 / Chapter 2.2.2 --- Discussion --- p.27 / Chapter Chapter 3 --- Vasodilation study --- p.28 / Chapter 3.1 --- Vascular Smooth Muscle Contraction and Relaxation --- p.28 / Chapter 3.2 --- Endothelium and Vasodilation --- p.30 / Chapter 3.3 --- Vasodilation in organ bath --- p.32 / Chapter 3.3.1 --- Materials and Methods --- p.32 / Chapter 3.3.2 --- Results --- p.35 / Chapter 3.3.3 --- Discussion --- p.40 / Chapter 3.4 --- Endothelium dependent vasodilation --- p.40 / Chapter 3.4.1 --- Materials and Methods --- p.43 / Chapter 3.4.2 --- Results --- p.45 / Chapter 3.4.3 --- Discussion --- p.54 / Chapter 3.5 --- Adrenoceptor and vasodilation --- p.55 / Chapter 3.5.1 --- Materials and Methods --- p.57 / Chapter 3.5.2 --- Results --- p.58 / Chapter 3.5.3 --- Discussion --- p.62 / Chapter 3.6 --- Potassium Channels and Vasodilation --- p.63 / Chapter 3.6.1 --- Materials and Methods --- p.65 / Chapter 3.6.2 --- Results --- p.67 / Chapter 3.6.3 --- Discussion and Summary --- p.77 / Chapter 3.7 --- Potential active components from Fenge and Danshen --- p.82 / Chapter 3.7.1 --- Materials and Methods --- p.82 / Chapter 3.7.2 --- Results --- p.83 / Chapter 3.7.3 --- Discussion --- p.87 / Chapter Chapter 4 --- Comparison of Fenge and Yege --- p.88 / Chapter 4.1 --- Vasodilative effects of Fenge and Yege --- p.89 / Chapter 4.1.1 --- Materials and Methods --- p.89 / Chapter 4.1.2 --- Results --- p.89 / Chapter 4.1.3 --- Discussion --- p.101 / Chapter 4.2 --- The comparison of antioxidative effect between Yege and Fenge --- p.104 / Chapter 4.2.1 --- Red blood cell hemolysis model --- p.106 / Chapter 4.2.1.1 --- Materials and Methods --- p.106 / Chapter 4.2.1.2 --- Results --- p.108 / Chapter 4.2.1.3 --- Discussion --- p.110 / Chapter 4.2.2 --- Ischemia-reperfusion on Langendroff --- p.112 / Chapter 4.2.2.1 --- Materials and Methods --- p.114 / Chapter 4.2.2.2 --- Results --- p.117 / Chapter 4.2.2.3 --- Discussion --- p.125 / Chapter Chapter 5 --- Comparison of Chemical Profiles of Fenge and Yege --- p.127 / Chapter 5.1 --- The application of HPLC --- p.127 / Chapter 5.2 --- HPLC standardization --- p.129 / Chapter 5.2.1 --- Materials and Methods --- p.132 / Chapter 5.2.2 --- Results --- p.133 / Chapter 5.2.3 --- Discussion --- p.144 / Chapter Chapter 6 --- "Summaries, Discussion and prospects" --- p.146 / Chapter 6.1 --- Summaries and Discussion --- p.146 / Chapter 6.2 --- Prospects --- p.148 / Chapter 6.2.1 --- "Cardiovascular tonic effect of pure compounds, extracts with difference solvents and their vasodilative mechanism." --- p.148 / Chapter 6.2.2 --- Macrophage Foam Cell and Atherosclerosis --- p.149 / Chapter 6.2.3 --- The D:F (7:3) and D:Y (7:3) compound formulae capsule with GMP --- p.152 / References --- p.154

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.IV / Publications --- p.VII / Acknowledgements --- p.IX / Table of Contents --- p.X / List of Figures --- p.XXI / List of Tables --- p.XXVI / List of Abbreviation --- p.XXVII / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Alzheimer’s Disease --- p.2 / Chapter 1.1.1 --- Symptoms --- p.2 / Chapter 1.1.2 --- Epidemiology --- p.4 / Chapter 1.1.3 --- Pathology --- p.5 / Chapter 1.1.4 --- Risk factors --- p.6 / Chapter 1.2 --- Pathogenesis of AD --- p.10 / Chapter 1.2.1 --- Neurotransmitter dysfunction --- p.10 / Chapter 1.2.1.1 --- Cholinergic system dysfunction --- p.10 / Chapter 1.2.1.2 --- Glutamatergic system dysfunction --- p.11 / Chapter 1.2.2 --- Hippocampus atrophy --- p.15 / Chapter 1.2.3 --- “Amyloid Cascade hypothesis --- p.18 / Chapter 1.2.4 --- Increased oxidative stress --- p.21 / Chapter 1.2.5 --- Increased neuronal apoptosis --- p.23 / Chapter 1.2.6 --- Mitochondrial dysfunction --- p.27 / Chapter 1.2.7 --- Calcium dysregulation --- p.31 / Chapter 1.2.8 --- Increased tau protein hyperphosphorylation --- p.34 / Chapter 1.2.9 --- GSK3 hypothesis of AD --- p.37 / Chapter 1.3 --- Animal Models of AD --- p.41 / Chapter 1.3.1 --- Non-transgenic animal models of AD --- p.42 / Chapter 1.3.1.1 --- Spontaneous models --- p.42 / Chapter 1.3.1.2 --- Scopolamine-induced rodent models --- p.43 / Chapter 1.3.1.3 --- Aluminum-induced rodent models --- p.44 / Chapter 1.3.1.4 --- D-galactose-induced rodent models --- p.45 / Chapter 1.3.1.5 --- Aβ infusion rodent models --- p.46 / Chapter 1.3.2 --- Transgenic animal models of AD --- p.48 / Chapter 1.3.2.1 --- Transgenic rodent models for AD --- p.49 / Chapter 1.3.2.2 --- AD models in D. rerio --- p.53 / Chapter 1.3.2.3 --- AD models in D. melanogaster --- p.54 / Chapter 1.3.2.4 --- AD models in C. elegans --- p.54 / Chapter 1.4 --- Treatments for AD --- p.55 / Chapter 1.4.1 --- Current symptomatic treatments --- p.56 / Chapter 1.4.1.1 --- AChEIs --- p.56 / Chapter 1.4.1.2 --- NMDA antagonist --- p.57 / Chapter 1.4.2 --- Disease-modifying approaches --- p.61 / Chapter 1.4.2.1 --- Amyloid-directed therapies --- p.61 / Chapter 1.4.2.2 --- Tau-directed therapies --- p.61 / Chapter 1.4.2.3 --- Anti-oxidant agents --- p.62 / Chapter 1.4.2.4 --- NSAIDs --- p.63 / Chapter 1.4.2.5 --- Estrogen replacement therapy (ERT) --- p.64 / Chapter 1.4.3 --- Herbal medicines --- p.67 / Chapter 1.5 --- Uncaria rhynchophylla --- p.69 / Chapter 1.5.1 --- Chemical constituents --- p.69 / Chapter 1.5.2 --- Alkaloids --- p.72 / Chapter 1.6 --- Pharmacological Activities of Uncaria rhynchophylla and Its Alkaloids --- p.75 / Chapter 1.6.1 --- Effects on cardiovascular system --- p.75 / Chapter 1.6.2 --- Effects on central nervous system --- p.77 / Chapter 1.6.3 --- Antioxidant activities --- p.79 / Chapter 1.6.4 --- Anti-inflammatory and analgesic effects --- p.80 / Chapter 1.6.5 --- Effects on platelet aggregation and thrombosis --- p.81 / Chapter 1.6.6 --- Other pharmacological effects --- p.81 / Chapter 1.7 --- Hypothesis and Objectives of the Present Study --- p.83 / Chapter Chapter Two --- Uncaria rhynchophylla Ameliorates Cognitive Deficits Induced by D-galactose in Mice / Chapter 2.1 --- Introduction --- p.86 / Chapter 2.2 --- Materials and Methods --- p.88 / Chapter 2.2.1 --- Drugs and chemical reagents --- p.88 / Chapter 2.2.2 --- Plant materials and extraction --- p.89 / Chapter 2.2.3 --- Animals --- p.90 / Chapter 2.2.4 --- Experimental design and drugs treatment --- p.90 / Chapter 2.2.5 --- Morris water maze test --- p.91 / Chapter 2.2.6 --- Preparation of brain tissue samples --- p.92 / Chapter 2.2.7 --- Measurement of intracellular ROS level --- p.92 / Chapter 2.2.8 --- Assay of MDA level --- p.92 / Chapter 2.2.9 --- Assay of GSH level --- p.93 / Chapter 2.2.10 --- Measurement of SOD activity --- p.93 / Chapter 2.2.11 --- Measurement of CAT activity --- p.94 / Chapter 2.2.12 --- Assay of Ach level --- p.94 / Chapter 2.2.13 --- Measurement of AChE activity --- p.95 / Chapter 2.2.14 --- Statistical analysis --- p.95 / Chapter 2.3 --- Results --- p.95 / Chapter 2.3.1 --- Quality determination of EUR --- p.95 / Chapter 2.3.2 --- Effects of EUR on Morris water maze in D-gal-treated mice --- p.97 / Chapter 2.3.3 --- Effects of EUR on the level of intracellular ROS in the brains of D-gal-treated mice --- p.101 / Chapter 2.3.4 --- Effects of EUR on the levels of GSH and MDA in the brains of D-gal-treated mice --- p.103 / Chapter 2.3.5 --- Effects of EUR on the activities of SOD and CAT in the brains of D-gal-treated mice --- p.105 / Chapter 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.107 / Chapter 2.4 --- Discussion --- p.109 / Chapter Chapter Three --- Bioassay-Guided Isolation of Neuroprotective Compounds from Uncaria rhynchophylla Against Beta-Amyloid-Induced Neurotoxicity / Chapter 3.1 --- Introduction --- p.113 / Chapter 3.2 --- Materials and Methods --- p.114 / Chapter 3.2.2 --- Drugs and chemical reagents --- p.114 / Chapter 3.2.2 --- Preparation of aggregated Aβ₂₅₋₃₅ --- p.115 / Chapter 3.2.3 --- Extraction, fractionation, isolation and identification processes --- p.115 / Chapter 3.2.4 --- Cell culture and drug treatment --- p.119 / Chapter 3.2.5 --- Cell viability assay --- p.119 / Chapter 3.2.6 --- Statistical analysis --- p.120 / Chapter 3.3 --- Results --- p.120 / Chapter 3.3.1 --- Isolation and structural determination of the isolated compounds --- p.120 / Chapter 3.3.2 --- Effects of different fractions and isolated compounds on Aβ₂₅₋₃₅-induced cells death in PC12 cells --- p.122 / Chapter 3.4 --- Discussion --- p.126 / Chapter Chapter Four --- Neuroprotective Effects of Isorhynchophylline Against Beta-Amyloid-Induced Neurotoxicity in PC12 Cells and Its Possible Mechanisms / Chapter 4.1 --- Introduction --- p.130 / Chapter 4.2 --- Materials and Methods --- p.131 / Chapter 4.2.1 --- Drugs and chemical reagents --- p.131 / Chapter 4.2.2 --- Cell culture and drugs treatment --- p.134 / Chapter 4.2.3 --- Cell viability assay --- p.134 / Chapter 4.2.4 --- Lactate dehydrogenase (LDH) activity assay --- p.135 / Chapter 4.2.5 --- Measurement of intracellular ROS production --- p.135 / Chapter 4.2.6 --- Malondialdehyde (MDA) and glutathione (GSH) assay --- p.136 / Chapter 4.2.7 --- Measurement of SOD activity --- p.137 / Chapter 4.2.8 --- Measurement of CAT activity --- p.137 / Chapter 4.2.9 --- Measurement of intracellular calcium concentration --- p.138 / Chapter 4.2.10 --- Measurement of mitochondrial membrane potential --- p.139 / Chapter 4.2.11 --- Quantification of DNA fragmentation --- p.139 / Chapter 4.2.12 --- Cytochrome c assay --- p.140 / Chapter 4.2.13 --- Western blotting analysis --- p.140 / Chapter 4.2.14 --- Real time-polymerase chain reaction (RT-PCR) analysis --- p.141 / Chapter 4.2.15 --- Statistical analysis --- p.142 / Chapter 4.3 --- Results --- p.143 / Chapter 4.3.1 --- Effects of IRN on Aβ₂₅₋₃₅-induced cytotoxicity in PC12 cells --- p.143 / Chapter 4.3.2 --- Effects of IRN on the level of intracellular ROS in Aβ₂₅₋₃₅-treated PC12 cells --- p.145 / Chapter 4.3.3 --- Effects of IRN on the levels of GSH and MDA in Aβ₂₅₋₃₅-treated PC12 cells --- p.147 / Chapter 4.3.4 --- Effects of IRN on the activities of SOD and CAT in Aβ₂₅₋₃₅-treated PC12 cells --- p.149 / Chapter 4.3.5 --- Effects of IRN on intracellular calcium level in Aβ₂₅₋₃₅-treated PC12 Cells --- p.151 / Chapter 4.3.6 --- Effects of IRN on MMP in Aβ₂₅₋₃₅-treated PC12 cells --- p.153 / Chapter 4.3.7 --- Effects of IRN on DNA fragmentation in Aβ₂₅₋₃₅-treated PC12 cells --- p.155 / Chapter 4.3.8 --- Effects of IRN on the release of cytochrome c in Aβ₂₅₋₃₅-treated PC12 cells --- p.157 / Chapter 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.159 / Chapter 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.162 / Chapter 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.165 / Chapter 4.3.12 --- Effects of IRN on tau protein hyperphosphorylation in Aβ₂₅₋₃₅-treated PC12 Cells --- p.168 / Chapter 4.3.13 --- Effects of IRN on Aβ₂₅₋₃₅-induced activation of GSK-3β in PC12 cells --- p.170 / Chapter 4.3.14 --- Effects of IRN on Aβ₂₅₋₃₅-induced inactivation of PI3K/Akt pathway --- p.173 / Chapter 4.4 --- Discussion --- p.177 / Chapter Chapter Five --- Isorhynchophylline Treatment Improves Cognitive Deficits Induced by Beta-Amyloid in Rats: Involvement of PI3K/Akt Signaling Pathway / Chapter 5.1 --- Introduction --- p.186 / Chapter 5.2 --- Materials and Methods --- p.187 / Chapter 5.2.1 --- Drugs and chemical reagents --- p.187 / Chapter 5.2.2 --- Animals --- p.188 / Chapter 5.2.3 --- Aβ₂₅₋₃₅ injections --- p.188 / Chapter 5.2.4 --- Experimental design and drugs treatment --- p.189 / Chapter 5.2.5 --- Morris water maze test --- p.190 / Chapter 5.2.6 --- Nissl’s staining for neurons --- p.193 / Chapter 5.2.7 --- Preparation of brain tissue samples --- p.193 / Chapter 5.2.8 --- Measurement of intracellular ROS level --- p.194 / Chapter 5.2.9 --- Assay of MDA level --- p.194 / Chapter 5.2.10 --- Assay of GSH level --- p.195 / Chapter 5.2.11 --- Measurement of SOD activity --- p.195 / Chapter 5.2.12 --- Measurement of CAT activity --- p.195 / Chapter 5.2.13 --- Cytochrome c assay --- p.196 / Chapter 5.2.14 --- Western blotting analysis --- p.196 / Chapter 5.2.15 --- RT-PCR analysis --- p.197 / Chapter 5.2.16 --- Statistical analysis --- p.198 / Chapter 5.3 --- Results --- p.199 / Chapter 5.3.1 --- IRN treatment rescued behavioral impairment in the Morris water maze test --- p.199 / Chapter 5.3.2 --- Effects of IRN on the number of pyramidal neuronal cells in the hippocampal CA1 region of Aβ₂₅₋₃₅-treated rats --- p.203 / Chapter 5.3.3 --- Effects of IRN on the intracellular ROS level in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.205 / Chapter 5.3.4 --- Effects of IRN on the levels of GSH and MDA in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.207 / Chapter 5.3.5 --- Effects of IRN on the activities of SOD and CAT in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.209 / Chapter 5.3.6 --- Effects of IRN on cytochrome c in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.211 / Chapter 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.213 / Chapter 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.216 / Chapter 5.3.9 --- Effects of IRN on the protein and mRNA levels of caspase-8 in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.219 / Chapter 5.3.10 --- Effects of IRN on the tau protein hyperphosphorylation in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.222 / Chapter 5.3.11 --- Effects of IRN on the activation of GSK-3β in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.224 / Chapter 5.3.12 --- Effects of IRN on the PI3K/Akt pathway in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.226 / Chapter 5.4 --- Discussion --- p.228 / Chapter Chapter Six --- General Discussion and Future Perspectives / Chapter 6.1 --- General Discussion and Conclusions --- p.237 / Chapter 6.2 --- Future Perspectives --- p.243 / References by Alphabetical Order --- p.246

Alzhemier's disease (AD) is a common form of dementia which is characterized by the deposition of amyloids in affected neurons and a cholinergic neurotransmission deficit in the brain. Current therapeutic intervention for AD is primarily based on inhibition of brain acetylcholinesterase (AChE) to restore the brain acetylcholine level. Cryptotanshinone (CT) is a diterprene which is extracted from the root of Salvia miltiorrhiza, an herb that is commonly prescribed in Chinese medicine to treat cardiovascular disease. The present study is aimed at verifying CT's property as an AChE inhibitor using different models. By AChE activity assay, CT was found to be a dual inhibitor which inhibits both human acetylcholinesterase (AChE) and butylcholinesterase (BuChE) with similar IC50. CT inhibited human AChE in a reversible manner, and the inhibition showed the characteristics of mixed-type. To human BuChE, CT is an uncompetitive inhibitor. CT can also inhibit AChE from rat cortical neurons. Apart from AChE inhibition, CT was demonstrated to have ameliorating effect on glutamate excitotoxicity, which is a cause of neuron death in AD. Further study showing that CT treatment can reduce cellular tau phosphorylation, which is the downstream effector of glutamate-induced excitotoxicity. In animal model, the effect of CT on learning impairment in scopolamine-treated rats was also evaluated by the acquisition protocol of Morris water maze. The task learning ability of scopolamine-treated rats was significantly reversed by CT, and the CT-fed rats were able to develop spatial searching strategy comparable to the control animals. Chronic administration of CT at effective doses did not cause significant hepatotoxicity. Cholinergic side effect of muscle weakness was not observed in CT treated rats. On the contrary CT was found to increase the locomotor activity of NIH mice in forced swimming test through reducing the lactic acid in the circulation. Data in this study gives further support on CT's potential as a therapeutic drug for treating AD. / by Wong, Kin Kwan Kelvin. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 144-167). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

3-Hydroxy-3-Methylglutaryl Coenzyme A (HMG CoA) reductase is a 97 kDa glycoprotein located in the endoplasmic reticulum responsible for cholesterol biosynthesis in mammalian liver and intestine. HMG CoA reductase inhibitors (statins) (e.g. simvastatin, mevastatin and parvastatin) are used clinically to treat and prevent coronary artery diseases by reducing plasma LDL-cholesterol level. Recent studies have demonstrated that statins can provide beneficial effects (pleiotropic effects) beyond its lipid-lowering activity. However, the modulatory effects of statins on ion channels activities have not been fully explored. Hence, this study is designed to demonstrate the existence of the HMG CoA reductase in various human isolate cardiovascular preparations and the modulatory effect(s) of simvastatin on both large-conductance calcium-activated (BKCa) and ATP-sensitive (IKATP) potassium channels of porcine isolated coronary vascular smooth muscle cells. / In conclusion, our results demonstrated the biochemical existence of HMG CoA reductase in various human isolated cardiovascular preparations and porcine isolated coronary artery. Simvastatin modulates the BKCa and IKATP channels of the porcine isolated coronary artery via different and multiple cellular mechanisms. / In this study, we demonstrated the biochemical existence of the HMG CoA reductase in various human isolated cardiovascular preparations and porcine isolated coronary artery. In addition, we demonstrated that simvastatin modulates both the BKCa channels and IKATP channels of porcine isolated coronary artery via different mechanisms. Acute application of simvastatin (100 nM) slightly enhanced whereas simvastatin (≥ 1 muM) inhibited the BKCa amplitude of porcine coronary artery smooth muscle cells. The classical HMG CoA reductase-mevalonate cascade is important in mediating the inhibitory effect of simvastatin observed at low concentrations (1 and 3 muM), whereas an increased PKC-delta protein expression and activation is important in simvastatin (10 muM)-mediated inhibition of BKCa channels. In contrast, the basal activity of the IKATP channels was not affected by simvastatin (1, 3 and 10 muM). However, acute application of simvastatin (1, 3 and 10 muM) inhibited the opening of the IKATP channels by cromakalim and pinacidil in a PP2A-dependent manner (sensitive to okadaic acid, a PP2A inhibitor). The okadaic acid-sensitive, simvastatin-mediated inhibitory effect on IKATP channel is mediated by an activation of AMPK in a Ca2+-dependent manner. Activation of AMPK probably increased the activity of the Na+/K+ ATPase and subsequently caused an influx of glucose via the SGLT1 down the Na + concentration gradient for the ouabain-sensitive, glucose-dependent activation of PP2A. / Seto, Sai Wang. / Adviser: Yiu-Wa Kwan. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3456. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 221-254). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

I established the phage antibody library platform for the identification of specific antibodies. In the first part of my study, I tried to identify antibody against SARS-CoV. Two fragments on the spike protein, which is responsible for inducing viral entry, was chosen as target for the selection of antibody. An antibody was identified which can selectively recognize the SARS-CoV infected cells, but not non-infected cells. Although this antibody was found to retain no neutralizing ability, this specific antibody may have potential to develop for diagnostic purpose. / I utilized the phage system-based cloning method as an attractive approach to screen and identify virus-specific antibodies that can be encoded by the human genome. Once a useful phage clone is identified, unlimited amounts of human monoclonal virus-specific antibodies can be manufactured, and potentially applied clinically for prophylactic and therapeutic uses. The study focuses on two of these new infections, both of which cause severe respiratory disease: SARS and avian influenza. / Identification of specific antibodies, either for diagnostic or therapeutic use, was successfully demonstrated in the two infectious disease models. The phage antibody platform offers a fast and cost-effective method to identify phage antibodies, which can easily be converted to human viral specific monoclonal antibodies for clinical use. / In the 21st century, a number of novel infectious diseases emerged suddenly and spread rapidly, endangering the lives and well-being of people around the world. Severe acute respiratory syndrome (SARS) is a life threatening form of atypical pneumonia that ravaged Hong Kong, Taiwan, China, Canada and many cities in 2003. In the same year, novel avian influenza viruses infected human beings on two continents. Both of these diseases originated in animals and crossed over into the human population. These emerging diseases pose significant public health threats while providing a chilling reminder that another influenza pandemic could occur at any time. Thus, the development of effective therapeutics to control the disease is of paramount importance. Although several vaccines against SARS and avian influenza are available nowadays, the poor clinical performance and frequent mutation of viral strains may limit the practical use and value of the vaccines. Moreover, there are no promising antiviral drugs available for the treatment. Therefore, I aimed to develop an immunotherapy as an alternative treatment option against these diseases. / In the second part of my study, the extracellular domain of matrix protein of avian influenza virus was chosen as target for the selection of antibody. I successfully identified an antibody which can neutralize the avian influenza virus infection. This promising result indicated this antibody has potential to develop for therapeutic use and these antibodies can be easily manufactured in unlimited amounts for clinical application. / Leung, Ka Man. / Adviser: Kwok Pui Fung. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0212. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 112-123). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

EGb761 consists of two major groups of substances, flavonoids and terpenoids. Using human neuroblastoma SH-SY5Y cells, the present study demonstrated that, EGb761 could block Abeta-42 (a 42-amino acid cytoxic form of beta amyloid protein)-induced cell apoptosis, reactive oxygen species (ROS) accumulation, mitochondrial dysfunction and activation of c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt signaling pathways, possibly via its antioxidant and platelet activating factor (PAF) antagonizing activities. Two active constituents of EGb761, quercetin (a flavonoid) and ginkgolide B (a terpenoid) might contribute to the protective effects of EGb761. Quercetin but not ginkgolide B might be responsible for the antioxidant action of EGb761. Both compounds might be involved in the PAF antagonist activity of EGb761. / EGb761, a Ginkgo biloba extract, is a medicinal product for the treatment and prevention of cardiovascular and neuronal diseases, including Alzheimer's disease (AD). While considerable researches have documented its neuroprotective effects, its clinical effect is inconclusive and the precise neuroprotective mechanisms are not clearly known. / In conclusion, EGb761 may have beneficial effects in treatment and prevention of neurodegenerative diseases like AD. Its neuroprotective effects may be associated with constituent multiplicity, the dosage and BBB permeability. / The ability of EGb761 to cross the blood brain barrier (BBB) is unclear. In this study, the ability of EGb761 to cross the BBB was speculated through comparison of the effects of EGb761 on mitochondrial function between platelets and central nervous system in two animal models, the senescence accelerated prone 8 (SAMP8) mouse strain and ovariectomized rats. Mitochondrial function was evaluated as cytochrome c oxidase (COX) activity, mitochondrial ATP content and mitochondrial glutathione (GSH) content. SAMP8 mice have been widely used as a model of age-related cognitive decline with relevance to biochemical and genetic alterations in AD. Using two age groups (3-week-old and 40-week-old) of SAMP8 mice, this study found that, EGb761 protected against mitochondrial dysfunction in both platelets and hippocampi of old mice, but only showed protective effects on platelet mitochondria of young mice. Estrogen withdrawal was suggested to play a primary role in the onset of post-menopausal AD. Using ovariectomized middle-aged rats to mimic the post-menopausal pathophysiological changes, this study also demonstrated that, EGb761 protected against mitochondrial dysfunction in both platelets and hippocampi of ovariectomized rats. In contrast, in sham-operated rats, EGb761 increased mitochondrial GSH content in platelets but failed to show similar effect on hippocampi. These results suggested that the effects of EGb761 on the brain might be interfered by the BBB permeability. / The effective dosage of EGb761 in the brain remains undetermined. Using SH-SY5Y cells, this study demonstrated that low doses of EGb761 (50--100 mug/ml) inhibited hydrogen peroxide (H2O2)-induced cell apoptosis via inactivation of Alet, JNK and caspase 3 while high doses of EGb761 (250--500 flg/ml) enhanced H2O2 toxicities via inactivation of Akt and enhancement of activation of JNK and caspase 3. Additional experiments suggested that the dosage effect of EGb761 on apoptotic signaling proteins might be correlated with regulation of the cell redox state. / Shi, Chun. / Adviser: Lee Ka Ho Kenneth. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 81-99). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Waterborne diseases continue to plague the poorest people in low-income countries and are estimated to cause 4,600,000 acute incidents of diarrhea resulting in over 2,000 deaths daily. A major challenge is performing microbiology tests to monitor drinking water quality. Friends of the Old (FOTO) implemented a novel strategy using evidence-based microbiology to educate communities about the relationship between contaminated water and disease. Two commercially available tests for E.coli, adapted for fieldwork, provided easily interpreted results of contamination that correlate with WHO's disease risk categories. Simple and effective household water treatment options 'solar pasteurization and/or chlorination' were provided to all 14,400 families and 42 schools in Lower Nyakach, Kenya. From February to May, 2015, adjacent districts had serious cholera outbreaks, but in Lower Nyakach, where education and the use of chlorine were nearly universal, there were no cases of cholera and steadily decreasing rates of diarrhea. A cross-sectional study was conducted to verify self-reported water treatment practices with evidence-based microbiological testing. A random sample of 377 households revealed that 95% treat their water each and every time they collect. Microbiological verification found 96% of household safe water storage vessels were low risk compared to their very high risk source water. A strong association (p < 0.001) existed between the observed decrease in diarrhea trends from health facilities in Lower Nyakach and exposure to the novel training. The strategy used by FOTO could be replicated to empower communities worldwide to identify contaminated drinking water sources and to reduce the incidence of waterborne disease.

[Truncated abstract] The main objective of this study was to develop and validate a demographic/epidemiologic Markov model for population modelling/forecasting of CARPs as well as CHD deaths and incidence in Western Australia using population, linked hospital morbidity and mortality data for WA over the period 1980 to 2000. A key feature of the model was the ability to count events as individuals moved from one state to another and an important aspect of model development and implementation was the method for estimation of model transition probabilities from available population data. The model was validated through comparison of model predictions with actual event numbers and through demonstration of its use in producing forecasts under standard extrapolation methods for transition probabilities as well as improving the forecasts by taking into account various possible changes to the management of CHD via surgical treatment changes. The final major objective was to demonstrate the use of model for performing sensitivity analysis of some scenarios. In particular, to explore the possible impact on future numbers of CARPs due to improvements in surgical procedures, particularly the introduction of drug eluting stents, and to explore the possible impact of change in trend of CHD incidence as might be caused by the obesity epidemic. ... When the effectiveness of PCI due to introduction of DES was increased by reducing Pr(CABG given PCI) and Pr(a repeat PCI), there was a small decline in the requirements for PCIs and the effect seemed to have a lag. Finally, in addition to these changes when other changes were incorporated which captured that a PCI was used more than a CABG due to a change in health policy after the introduction of DES, there was a small increase in the requirements for PCIs with a lag in the effect. Four incidence scenarios were developed for assessing the effect of change in secular trends of CHD incidence as might be caused by the obesity epidemic in such a way that they gradually represented an increasing effect of obesity epidemic (assuming that other risk factors changed favourably) on CHD incidence. The strategy adopted for developing the scenarios was that based on past trends the most dominant component of CHD incidence was first gradually altered and finally the remaining components were altered. iv The results showed that if the most dominant component of CHD incidence, eg, Pr(CHD - no history of CHD) levelled off and the trends in all other transition probabilities continued into future, then the projected numbers of CABGs and PCIs for 2001-2005 were insensitive to these changes. Even increasing this probability by as much as 20 percent did not alter the results much. These results implied that the short-term effect on projected numbers of CARPs caused by an increase in the most dominant component of CHD incidence, possibly due to an ?obesity epidemic, is small. In the final incidence scenario, two of the remaining CHD incidence components-Pr(CABG - no history of CHD) and Pr(CHD death - no CHD and no history of CHD) were projected to level off over 2001-2005 because these probabilities were declining over the baseline period of 1998-2000. The projected numbers of CABGs were more sensitive (compared to the previous scenarios) to these changes but PCIs were not.