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

Wong Yin-Mei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 119-135). / Abstracts in English and Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iii / Publications --- p.v / Acknowledgements --- p.vi / Table of contents --- p.viii / List of tables --- p.xi / List of figures --- p.xii / Abbreviations --- p.xiv / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Osteoporosis --- p.1 / Chapter 1.1.1 --- Consensus statement --- p.1 / Chapter 1.1.2 --- Epidemiology and outcomes --- p.4 / Chapter 1.1.2.1 --- Hip fractures --- p.4 / Chapter 1.1.2.2 --- Vertebral fractures --- p.5 / Chapter 1.1.2.3 --- Wrist fractures --- p.7 / Chapter 1.1.3 --- Postmenopausal osteoporosis --- p.8 / Chapter 1.1.3.1 --- Pathogenesis --- p.8 / Chapter 1.1.3.1.1 --- Genetics --- p.11 / Chapter 1.1.3.1.2 --- Bone remodeling --- p.14 / Chapter 1.1.3.1.3 --- Calcium homeostasis --- p.21 / Chapter 1.1.3.1.4 --- Life style 一 nutrition and exercise --- p.26 / Chapter 1.1.3.2 --- Current pharmacological treatment --- p.27 / Chapter 1.1.3.2.1 --- Introduction --- p.27 / Chapter 1.1.3.2.2 --- Limitations --- p.31 / Chapter 1.2 --- Traditional Chinese medicine --- p.33 / Chapter 1.2.1 --- The Kidney --- p.33 / Chapter 1.2.2 --- Kidney-tonifying herbs --- p.33 / Chapter 1.3 --- Aim of the studies --- p.36 / Chapter Chapter 2. --- Materials and methods --- p.38 / Chapter 2.1 --- Kidney-tonifying herbs and herbal formula --- p.38 / Chapter 2.1.1 --- Sources --- p.38 / Chapter 2.1.2 --- Herbal extract preparation --- p.38 / Chapter 2.2 --- Animal study --- p.40 / Chapter 2.2.1 --- Reagents --- p.40 / Chapter 2.2.2 --- Animal care --- p.40 / Chapter 2.2.3 --- Herbs and herbal formula preparations for animal studies --- p.41 / Chapter 2.2.4 --- Experimental design --- p.41 / Chapter 2.2.5 --- Gene expression study --- p.44 / Chapter 2.2.5.1 --- Tissue preparation --- p.44 / Chapter 2.2.5.2 --- Isolation of total RNA --- p.45 / Chapter 2.2.5.3 --- Complementary DNA synthesis --- p.47 / Chapter 2.2.5.4 --- Real-time polymerase chain reaction analysis --- p.47 / Chapter 2.3 --- Cell culture study --- p.49 / Chapter 2.3.1 --- Reagents --- p.49 / Chapter 2.3.2 --- Cell lines --- p.49 / Chapter 2.3.2.1 --- "Rat osteosarcoma cell line, UMR-106" --- p.49 / Chapter 2.3.2.2 --- "Human breast cancer cell line, MCF-7" --- p.50 / Chapter 2.3.2.3 --- Cell culture techniques --- p.50 / Chapter 2.3.3 --- Herbs preparations for cell culture --- p.51 / Chapter 2.3.4 --- Cell viability assay --- p.51 / Chapter 2.3.5 --- Cellular alkaline phosphatase activity assay --- p.52 / Chapter 2.3.6 --- Matrix mineralization assay --- p.54 / Chapter 2.3.7 --- Competitive estrogen receptor binding assay --- p.56 / Chapter 2.4 --- Statistical analyses --- p.58 / Chapter Chapter 3. --- Results --- p.59 / Chapter 3.1 --- Extraction yields of Kidney-tonifying herbs and herbal formula --- p.59 / Chapter 3.2 --- Effects of Kidney-tonifying herbs and herbal formula on the gene expressions of calcium absorption and reabsorption related genes --- p.61 / Chapter 3.2.1 --- Gene expression of 25-hydroxyvitamin D3-1 alpha-hydroxylasein the kidney --- p.62 / Chapter 3.2.2 --- Gene expression of vitamin D receptor in the duodenum --- p.65 / Chapter 3.2.3 --- Gene expression of calbindin D9K in the duodenum --- p.67 / Chapter 3.2.4 --- Gene expression of vitamin D receptor in the kidney --- p.69 / Chapter 3.2.5 --- Gene expression of calbindin D28K in the kidney --- p.71 / Chapter 3.3 --- Effects of Kidney-tonifying herbs on osteoblastic UMR-106 cell line --- p.73 / Chapter 3.3.1 --- Effects of Kidney-tonifying herbs on the cell viability of UMR-106 cells --- p.73 / Chapter 3.3.2 --- Effects of Kidney-tonifying herbs on the osteoblastic differentiation of UMR-106 cells --- p.76 / Chapter 3.3.2.1 --- Cellular alkaline phosphatase activity --- p.76 / Chapter 3.3.2.2 --- Degree of matrix mineralization --- p.80 / Chapter 3.4 --- Estrogen receptor binding activities of Kidney-tonifying herbs --- p.85 / Chapter Chapter 4. --- Discussion --- p.89 / Chapter 4.1 --- Safety of Kidney-tonifying herbs and herbal formula --- p.89 / Chapter 4.2 --- Kidney-tonifying herbs and herbal formula preserve bone mineral density --- p.93 / Chapter 4.3 --- Kidney-tonifying herbs and herbal formula modulate calcium homeostasis --- p.97 / Chapter 4.3.1 --- "Roles in renal synthesis of the hormonally active form of vitamin D: 1,25-dihydroxyvitamin D3" --- p.97 / Chapter 4.3.2 --- Roles in calcium absorption in the duodenum --- p.99 / Chapter 4.3.3 --- Roles in calcium reabsorption in the kidney --- p.102 / Chapter 4.3.4 --- Summary --- p.104 / Chapter 4.4 --- Kidney-tonifying herbs modulate bone formation --- p.106 / Chapter 4.4.1 --- Effects on osteoblast proliferation --- p.106 / Chapter 4.4.2 --- Effects on osteoblastic differentiation --- p.107 / Chapter 4.4.3 --- Summary --- p.108 / Chapter 4.5 --- Kidney-tonifying herbs interact with estrogen receptor --- p.110 / Chapter 4.6 --- Active ingredients of Kidney-tonifying herbs --- p.111 / Chapter 4.7 --- Limitations of the present studies --- p.115 / Chapter 4.8 --- Conclusion and future prospect --- p.117 / References --- p.119

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

傳統中醫中藥理論遵從"腎主骨"概念。因此，中醫在治療與骨有關的疾病時一般都處方"補腎"類中藥。 / ELP是一例中藥草本 "補腎" 複方。其包含三種中藥，包括淫羊藿（E）、女貞子（L）和補骨脂（P）。動物體內實驗和臨床研究已證明ELP有效治療絶經後骨質疏鬆症。可是，經口服吸收後的血清中的ELP有效物質對細胞的成骨影響從未進行過相關研究。ELP對預防在缺乏體力活動下所引起的骨質疏鬆症的療效也屬未知。此外，基於其"補腎"的特性，ELP可能潛在著能促進骨折癒合的功能。本研究的目的包括研究血清中ELP的有效物質在細胞和分子水平上的護骨能力，並測試其對預防於失重狀態下引起的骨質疏鬆症（慢性骨紊亂）的效能。本研究還旨在考察 ELP在促進骨癒合 （急性骨紊亂）上的作用。本研究分為三部分。 / 第一部分 -- 骨代謝的體外研究:健康大鼠分別口服草本配方ELP、EL、及單味中草藥提取物E或L、並以蒸餾水作為對照（H2O），口服給藥二小時後收集其血清作體外血清藥理學研究。分別考察含藥血清對各細胞系包括UMR106、RAW264.7、和從大鼠骨中分離出的骨髓間充質幹細胞（MSC）的增殖和分化屬性的影響，並以液質聯用技術（LC-MS）來分析血清內所含中藥的化學成份。 / 第二部分 -- 骨質疏鬆症的體內研究:以尾吊雄性大鼠作為卸荷狀態骨質疏鬆症的動物模型。在不同的給藥組中，大鼠口服高中低三種劑量的ELP（ELP-H、ELP-M和ELP-L），或三個不同抗骨質疏鬆藥物，包括雷洛昔芬（Ral），阿侖膦酸鈉（Aln）和雷奈酸鍶（Strn）作為陽性對照組，並以蒸餾水為安慰劑對照（TS）。另一組大鼠則沒有尾吊，作為正常對照（Non-TS）。本部分分析在吊尾期間大鼠體內生化指標和骨密度（BMD）的變化，及其後各組在骨小梁微結構和骨骼生物力學上的差異。 / 第三部分 -- 骨缺損癒合的體內研究:兩個鑽孔性骨缺損模型分別建立於老年雌性大鼠的左股骨骨幹和右脛骨近端骺端。其後動物分成4組：（1）ELP 口服給藥（ELP）；（2）CDNR外敷治療（CDNR為另一中藥複方，包含紅花（C）、續斷（D）、三七（N）和大黃（R））；（3）ELP口服給藥結合CDNR外敷治療（ELP+CDNR）；（4）和蒸餾水餵養（Control）。通過監測骨缺損癒合的過程、檢測大鼠血液中生化標誌物的變化、骨骼生物力學測試和形態計量學分析，考察ELP及其與CDNR在骨缺損癒合上的協同作用。 / 第一部分的結果顯示，口服給藥二小時後，大鼠血清中淫羊藿的標記化合物淫羊藿苷（icariin）無被檢出。在EL或E的給藥大鼠血清中，檢出淫羊藿苷的其中一個代謝產物icariside I；而其另一個代謝產物icariside II，則在ELP的給藥大鼠血清中檢測到。L和P的常見標記化合物則能從相應餵飼L和P的大鼠血清中檢出。體外血清藥理學研究結果表明含藥（ELP）大鼠血清對細胞無毒性作用，且能促進 UMR106 細胞增殖和上調其Runx2 基因表達。然而，含藥血清無增加UMR106細胞的鹼性磷酸酶活性和鈣沉積。它抑制 RAW264.7細胞的分化及其基質金屬蛋白酶9（MMP-9）和組織蛋白酶 K的基因表達。它亦能促進MSC細胞的增殖，增強其鹼性磷酸酶活性和Runx2與ALP基因的表達。 / 第二部分的結果指出ELP-H能減少吊尾大鼠股骨遠端及腰椎骨密度的百分比損失，抵抗股骨遠端骨小梁微結構惡化和加強股骨骨幹骨缺損部位的生物力學特性。此外，ELP-H還能降低血液骨鈣素和抗酒石酸酸性磷酸酶5b（TRAP5b）的濃度。研究亦發現ELP對骨密度、結構參數和生化指標的影響存在劑量依賴性。整體上而言，ELP在預防卸荷骨質疏鬆症的影響類似於Ral和Aln，而非Strn。 / 第三部分的結果表明，從顯微電腦掃描或形態計量學上分析，所有實驗組跟對照組間均沒有顯著性差異。但值得注意的是，ELP+CDNR大大提高了股骨骨幹骨缺損在癒合過程中的歸一化生物力學屬性。而ELP單獨用藥則減少了TRAP5b的濃度。 / 總之，這項研究結論出血清藥理學研究加上LC-MS的應用能作為找出中藥中有效成分的有效途徑。本研究還展示ELP的含藥血清對骨細胞有護骨作用。ELP可防預在卸荷狀態下形成的骨質疏鬆症，它還有助於提升外敷中藥複方CDNR在骨缺損癒合過程中的療效。從這項研究的三個部分中歸納出的共同點說明，儘管ELP擁有刺激成骨的能力，它的護骨作用主要是透過它的抗骨吸收效果。ELP在慢性（防止骨質疏鬆症）和急性（促進骨癒合）骨紊亂上均有療效。 / Traditional Chinese Medicine (TCM) claims that bone health lies in the functioning of the "Kidneys". When the "Kidney" is strong, our body can stimulate growth and transformation of the bone marrow, which nourishes and strengthens the skeleton. Therefore, "Kindey-tonifying" herbs are usually used to cure bone diseases. / ELP is a "Kidney-tonifying" Chinese herbal formula containing three Chinese herbs including Herba Epimedii (E), Fructus Ligustri Lucidi (L) and Fructus Psoraleae (P). It has been proven effective to treat postmenopausal osteoporosis through in vivo and clinical studies. However, ELP is for oral administration. The osteogenic properties of its post-absorption metabolites have never been studied. The efficacy of ELP on prevention of osteoporosis development due to physical inactivity is also unknown. With its "Kindey-tonifying" property, ELP is also considered as a potential agent to facilitate fracture healing. / The aims of this study included to investigate the osteoprotective effects of ELP metabolites at cellular and molecular levels and to prove the efficacy of ELP on prevention of osteoporosis development in unloading condition - a chronic bone disorder. It also aimed to study the effect of ELP on promotion of bone defect healing - an acute bone disorder. This study was divided into three parts. / Part 1 - in vitro study of bone metabolism: Healthy rats were fed with herbal formula ELP or EL, single herbal extracts of E or L or distilled water as control (H₂O). Sera were then collected for in vitro seropharmacological study. Cell lines including UMR106 and RAW264.7, as well as mesenchymal stem cell (MSC) isolated from rats, were cultured with the sera. Their proliferation and differentiation properties of the cells were analyzed. In addition, the chemical profiles of the herbal extracts within the sera were analyzed using liquid chromatography-mass spectrometry (LC-MS). / Part 2 - in vivo study of osteoporosis: Tail-suspension male rats were used as the unloading osteoporotic animal model. The rats in different groups were fed with three different doses of ELP (ELP-H, ELP-M and ELP-L), or three different anti-osteoporosis drugs including raloxifene (Ral), alendronate (Aln) and strontium ranelate (Strn) as positive controls or distilled water as placebo control (TS). One group of rats was non-tail-suspended as normal control (Non-TS). Changes in bone mineral density (BMD), microarchitecture of trabeculae and biomechanical properties of the bone of the rats were analyzed. Changes in biochemical markers within the tail-suspension period were also studied. / Part 3 - in vivo study of bone defect healing: two drilled-hole bone defects were created in the diaphysis of left femur and proximal metaphysis of right tibia, respectively, of aged female rats. Animals were divided into 4 groups: (1) administered with ELP orally (ELP); (2) treated with another herbal formula CDNR containing Carthami Flos (C), Dipsaci Radix (D), Notoginseng Rhizoma (N) and Rhei Rhizoma (R) topically (CDNR); (3) treated with oral ELP and topical CDNR at the same time (ELP+CDNR); and (4) fed with distilled water (Control). The effects of ELP and the synergistic effects of ELP+CDNR on facilitation of the bone defect healing were monitored in vivo using viva-CT and through measurement of biochemical markers biweekly. After euthanasia of the rats, the bones were harvested for biomechanical test and histomorphometrical analysis. / Results: Part 1 revealed that the common marker compound, icariin, had not been detected in the sera of all the rats. Instead, one of the metabolites of E, icariside I, was found in the sera of the rats fed with EL or E, while another metabolite, icariside II, was detected in the serum of the rats fed with ELP. Common marker compounds of L and P were observed in the sera of the rats fed with the herbal items accordingly. The in vitro studies in this Part showed that there was no cytotoxic effect of the rat sera on the cells. The post-absorbed ELP metabolites in rat serum promoted UMR106 proliferation by 25.7%, (p < 0.05) and upregulated the Runx2 gene expression by 1.18 fold (p < 0.05) after cultured for 2 and 3 days, respectively. However, they could not increase the ALP activity and calcium deposition of UMR106. They also inhibited RAW264.7 differentiation by 29.2 % (p < 0.05) and downregulated the MMP9 and Cathepsin K gene expression of RAW264.7 by 0.46 (p < 0.05) and 0.36 (p < 0.01) fold, respectively. The ELP metabolites promoted the proliferation of MSC by 14.4 % (p < 0.001) and resulted in 42.6 % higher ALP activity than the control serum (p < 0.05). They also upregulated the Runx2 and ALP gene expression at both Day 4 and Day 7 of culture significantly. / Part 2 showed that compared with the tail-suspension control (TS), ELP in high dose (ELP-H) reduced the percentage loss of total and trabecular BMD by 5.46 and 8.52 %, respectively (p < 0.05 both) in distal femur, and by 4.67 % (p < 0.05) in trabecular region of lumbar spine of the tail-suspended rats. Analysis from micro-CT showed that microarchitectural parameters BV/TV, Tb.Th and TV density of the distal femur of ELP-H were 17.62, 11.90 and 8.09 % higher than those of the TS (p < 0.05, for all). 3-point bending test on mid-shaft femur of the rats revealed that the yield load, ultimate load and stiffness of the drill-defect of ELP-H were higher than those of TS significantly. All of the biochemical markers decreased significantly from baseline (Day 0) to Day 28 in ELP-H. In addition, osteocalcin and TRAP5b concentrations of ELP-H were lower than those of TS significantly at Day 28. The effect of ELP on BMD, microarchitectural parameters and biochemical markers were in dose-dependent manner. In general, the osteoprotective effect of ELP-H on unloading bone was similar to Ral and Aln, but not Strn. / Part 3 indicated no significant difference in BV/TV and BMD among all groups at each time point. Histomorphometrical analysis from fluorescent labeling and Goldner’s trichrome staining showed no statistical difference in new bone formation between the Control and other treatment groups. Notably, the normalized yield load, ultimate load and failure of ELP+CDNR were significantly higher than those of Control by 20.38 % (p < 0.05), 23.17 % (p< 0.001) and 25.55 % (p< 0.001), respectively. Analysis on the change of biochemical markers showed that the bone formation marker BALP increased while bone resorption markers Dpd and TRAP5b decreased within the 42-day monitoring period. BALP activity of both Control and ELP increased significantly but only ELP reduced the TRAP5b concentrations starting from Day 14 post-op. There was no statistical difference when the concentrations of the biochemical markers were compared horizontally among the 4 groups at the same time point. / In conclusion, the current study demonstrated that seropharmacological study incorporating with the application of LC-MS can be a potential efficient approach to find out active ingredients of medicine herbs. Post-absorbed metabolites of ELP also showed their osteoprotective effects on bone cells. Aqueous extract of ELP could prevent the development of osteoporosis in unloading condition and such effect was dose-dependent. It also helped elevating the efficacy of a topical applied herbal formula CDNR on improving the bone strength of healing bone defects. A common finding from the 3 parts of this study illustrated that the osteoprotective effect of ELP was mainly achieved by its anti-resorptive efficacy on bone, although it possess an ability to stimulate osteoblastogenesis. ELP was found effective for both chronic (prevent osteoporosis development) and acute (facilitate bone healing) bone disorders. / 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. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Siu, Wing Sum. / "November 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 201-227). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.vi / ACKNOWLEDGEMENTS --- p.ix / TABLE OF CONTENTS --- p.xi / LIST OF FIGURES --- p.xvii / LIST OF TABLES --- p.xxiii / PUBLICATIONS --- p.xxiv / ABBREVIATION --- p.xxv / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter 1.1 --- TRADITIONAL CHINESE MEDICINE (TCM) AND BONE DISEASES --- p.1 / Chapter 1.2 --- CELLULAR AND MOLECULAR MECHANISMS ON BONE METABOLISM --- p.2 / Chapter 1.2.1 --- Bone formation by osteoblast --- p.3 / Chapter 1.2.2 --- Bone resorption by osteoclasts --- p.4 / Chapter 1.3 --- OSTEOPOROSIS --- p.5 / Chapter 1.3.1 --- Postmenopausal osteoporosis --- p.6 / Chapter 1.3.2 --- Disuse osteoporosis --- p.8 / Chapter 1.3.3 --- Basic principle of TCM on osteoporosis --- p.10 / Chapter 1.3.4 --- Common Chinese herbal medicine reported to have anti-osteoporotic effects --- p.11 / Chapter 1.4 --- BONE FRACTURE --- p.11 / Chapter 1.4.1 --- Biology and repair of bone fracture --- p.12 / Chapter 1.4.2 --- TCM on promotion of fracture healing --- p.13 / Chapter 1.4.3 --- Theories of TCM on fracture healing --- p.15 / Chapter CHAPTER 2: --- OSTEOPOROSIS AND HERBS --- p.16 / Chapter 2.1 --- CHINESE HERBAL MEDICINE SELECTED IN THIS PART --- p.16 / Chapter 2.2 --- DESIGN OF STUDY --- p.19 / Chapter 2.3 --- HYPOTHESES AND OBJECTIVES --- p.19 / Chapter 2.4 --- BACKGROUND OF THE STUDY --- p.23 / Chapter 2.4.1 --- In vitro study of ELP on bone cells --- p.23 / Chapter 2.4.2 --- In vivo study of ELP on postmenopausal osteoporosis --- p.23 / Chapter 2.4.3 --- Clinical study of ELP on postmenopausal osteoporosis --- p.24 / Chapter CHAPTER 3: --- PART 1 IN VITRO SEROPHARMACOLOGICAL STUDY ON OSTEOPOROSIS --- p.26 / Chapter 3.1 --- OBJECTIVES --- p.26 / Chapter 3.2 --- SEROPHARMACOLOGICAL APPROACH TO STUDY ELP --- p.26 / Chapter 3.3 --- TYPES OF CELLS INVOLVED IN THE CURRENT STUDY --- p.27 / Chapter 3.3.1 --- UMR106 --- p.28 / Chapter 3.3.2 --- RAW264.7 --- p.28 / Chapter 3.3.3 --- Mesenchymal stem cell (MSC) --- p.28 / Chapter 3.4 --- IN VITRO ASSESSMENTS ON BONE METABOLISM --- p.29 / Chapter 3.4.1 --- Bone formation --- p.29 / Chapter 3.4.1.1 --- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay --- p.29 / Chapter 3.4.1.2 --- Bromodeoxyuridine (BrdU) assay --- p.30 / Chapter 3.4.1.3 --- Total alkaline phosphatase (ALP) activity measurement --- p.30 / Chapter 3.4.1.4 --- Calcium deposition analysis --- p.30 / Chapter 3.4.2 --- Bone degradation --- p.31 / Chapter 3.4.2.1 --- Tartrate-resistant acid phosphatase (TRAP) staining --- p.31 / Chapter 3.4.3 --- Phenotypic markers of cells involved in bone remodeling using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) --- p.31 / Chapter 3.5 --- MATERIAL AND METHODS --- p.37 / Chapter 3.5.1 --- Preparation of herbal extracts --- p.37 / Chapter 3.5.2 --- Serum preparation for seropharmacological study --- p.38 / Chapter 3.5.2.1 --- Administration of herbal extracts and blood collection --- p.38 / Chapter 3.5.2.2 --- Serum preparation --- p.38 / Chapter 3.5.3 --- Analysis of marker compounds in serum using liquid chromatographymass spectrometry (LC-MS) --- p.39 / Chapter 3.5.3.1 --- Serum preparation --- p.39 / Chapter 3.5.3.2 --- Operation of LC-MS --- p.39 / Chapter 3.5.4 --- Isolation and characterization of MSC from bone marrow --- p.40 / Chapter 3.5.5 --- Cell culture --- p.42 / Chapter 3.5.5.1 --- General materials --- p.42 / Chapter 3.5.5.2 --- UMR106 --- p.43 / Chapter 3.5.5.3 --- RAW264.7 --- p.44 / Chapter 3.5.5.4 --- Bone Marrow MSC --- p.45 / Chapter 3.5.6 --- Assays analyzing the responses of cells on the effect of metabolites of herbs in serum --- p.46 / Chapter 3.5.6.1 --- General materials --- p.46 / Chapter 3.5.6.2 --- Assays for bone formation --- p.50 / Chapter 3.5.6.3 --- Assays for bone degradation --- p.55 / Chapter 3.5.7 --- Statistical analysis --- p.56 / Chapter 3.6 --- RESULTS --- p.57 / Chapter 3.6.1 --- Chemical characterization of ELP extract --- p.57 / Chapter 3.6.2 --- Marker compounds found in rat serum using LC-MS --- p.58 / Chapter 3.6.3 --- Effects of herbal metabolites on UMR106 --- p.61 / Chapter 3.6.3.1 --- Effect on cell viability --- p.61 / Chapter 3.6.3.2 --- Effects on cell proliferation and differentiation --- p.61 / Chapter 3.6.3.3 --- Regulation on osteogenesis through gene expression --- p.63 / Chapter 3.6.4 --- Effects of herbal metabolites on RAW264.7 --- p.67 / Chapter 3.6.4.1 --- Effect on cell viability --- p.67 / Chapter 3.6.4.2 --- Inhibitory effect on RAW264.7 --- p.67 / Chapter 3.6.4.3 --- Regulation on osteoclastogenesis through gene expression --- p.67 / Chapter 3.6.5 --- Effects of herbal metabolites on bone marrow mesenchyma stem cell (MSC) --- p.70 / Chapter 3.6.5.1 --- Confirmation of MSC isolated from bone marrow of rat using flow cytometry --- p.70 / Chapter 3.6.5.2 --- Effect on cell viability --- p.70 / Chapter 3.6.5.3 --- Effects on cell proliferation and differentiation --- p.71 / Chapter 3.6.5.4 --- Regulation on osteogenesis through gene expression --- p.71 / Chapter 3.7 --- DISCUSSION --- p.75 / Chapter CHAPTER 4: --- PART 2 IN VIVO STUDY ON DISUSE OSTEOPOROSIS . --- p.83 / Chapter 4.1 --- OBJECTIVES --- p.83 / Chapter 4.2 --- POTENTIAL EFFECT OF ELP ON DISUSE OSTEOPOROSIS --- p.83 / Chapter 4.3 --- ANIMAL MODELS FOR OSTEOPOROSIS STUDY --- p.84 / Chapter 4.3.1 --- Conventional ovariectomized animal model for the studies of osteoporosis --- p.85 / Chapter 4.3.2 --- Animal models for study of disuse osteoporosis --- p.85 / Chapter 4.3.2.1 --- Bandaging or casting --- p.86 / Chapter 4.3.2.2 --- Tail-suspension (TS) --- p.86 / Chapter 4.4 --- ASSESSMENTS ON DISUSE OSTEOPOROSIS DEVELOPMENT --- p.87 / Chapter 4.4.1 --- Bone mineral density (BMD) measurement --- p.87 / Chapter 4.4.2 --- Micro-architecture analysis --- p.87 / Chapter 4.4.3 --- Bone strength assessment --- p.88 / Chapter 4.4.4 --- Bone turnover monitoring by measuring biochemical markers --- p.89 / Chapter 4.4.4.1 --- Bone formation markers --- p.89 / Chapter 4.4.4.2 --- Bone resorption markers --- p.91 / Chapter 4.5 --- MATERIAL AND METHODS --- p.95 / Chapter 4.5.1 --- Preparation of herbal extracts --- p.95 / Chapter 4.5.2 --- Tail-suspension rat model --- p.95 / Chapter 4.5.3 --- Animal arrangement and grouping --- p.97 / Chapter 4.5.4 --- Administration of herbal extracts and drugs --- p.97 / Chapter 4.5.5 --- Assessments on disuse osteoporosis development --- p.98 / Chapter 4.5.5.1 --- Bone mineral density measurement using Peripheral Quantitative Computed Tomography (pQCT) --- p.98 / Chapter 4.5.5.2 --- Bone micro-architecture analysis using Micro-computed Tomography (μCT) --- p.99 / Chapter 4.5.5.3 --- Bone strength assessment through biomechanical bending test --- p.100 / Chapter 4.5.5.4 --- Bone turnover monitoring by measuring biochemical markers --- p.100 / Chapter 4.5.5.4.1 --- Serum collection --- p.100 / Chapter 4.5.5.4.2 --- Measurements of biochemical markers --- p.101 / Chapter 4.5.6 --- Statistical analysis --- p.105 / Chapter 4.6 --- RESULTS --- p.106 / Chapter 4.6.1 --- Effects of ELP on bone mineral density (BMD) --- p.106 / Chapter 4.6.2 --- Effects of ELP on bone micro-architecture --- p.118 / Chapter 4.6.3 --- Effects of ELP on biomechanics of bone --- p.122 / Chapter 4.6.4 --- Effects of ELP on bone turnover --- p.125 / Chapter 4.7 --- DISCUSSION --- p.132 / Chapter CHAPTER 5: --- PART 3 IN VIVO STUDY ON BONE DEFECT HEALING --- p.140 / Chapter 5.1 --- HERBAL ITEMS SELECTED IN THIS PART --- p.140 / Chapter 5.2 --- DESIGN OF STUDY --- p.143 / Chapter 5.3 --- HYPOTHESES AND OBJECTIVES --- p.144 / Chapter 5.4 --- SPECIFIC STRATEGY ON PROMOTION OF FRACTURE HEALING OF TCM --- p.144 / Chapter 5.5 --- POTENTIAL EFFECT OF ELP ON BONE HEALING --- p.144 / Chapter 5.6 --- ANIMAL MODELS --- p.146 / Chapter 5.6.1 --- Bone fracture model --- p.147 / Chapter 5.6.2 --- Drill-hole bone defect model --- p.147 / Chapter 5.7 --- ASSESSMENTS ON BONE HEALING --- p.149 / Chapter 5.7.1 --- Micro-architecture analysis --- p.149 / Chapter 5.7.2 --- Bone strength assessment --- p.150 / Chapter 5.7.3 --- Bone turnover monitoring by measuring biochemical markers --- p.151 / Chapter 5.7.4 --- Histomorphometry --- p.151 / Chapter 5.8 --- MATERIALS AND METHODS --- p.153 / Chapter 5.8.1 --- Preparation of herbal extracts --- p.153 / Chapter 5.8.1.1 --- ELP --- p.153 / Chapter 5.8.1.2 --- CDNR --- p.153 / Chapter 5.8.2 --- Production of drill-hole bone defect --- p.154 / Chapter 5.8.2.1 --- Femur --- p.155 / Chapter 5.8.2.2 --- Tibia --- p.155 / Chapter 5.8.2.3 --- Animal arrangement and grouping --- p.157 / Chapter 5.8.3 --- Herbal formulae administration and application --- p.157 / Chapter 5.8.3.1 --- Oral administration --- p.157 / Chapter 5.8.3.2 --- Topical application --- p.157 / Chapter 5.8.4 --- Assessments on bone healing --- p.158 / Chapter 5.8.4.1 --- Bone micro-architecture and bone density measurement using in vivo micro-computed tomography (vivaCT) --- p.158 / Chapter 5.8.4.2 --- Bone strength assessment through biomechanical bending test --- p.159 / Chapter 5.8.4.3 --- Bone turnover monitoring by measuring biochemical markers --- p.160 / Chapter 5.8.4.4 --- Histomorphometry --- p.160 / Chapter 5.8.4.4.1 --- Fluorochrome double labeling --- p.160 / Chapter 5.8.4.4.2 --- Tissue processing and sectioning --- p.161 / Chapter 5.8.4.4.3 --- Staining of sections --- p.162 / Chapter 5.8.4.4.4 --- Image analysis --- p.164 / Chapter 5.8.5 --- Statistical analysis --- p.165 / Chapter 5.9 --- RESULTS --- p.166 / Chapter 5.9.1 --- Effect of ELP and CDNR on bone micro-architecture --- p.and / Chapter bone --- density at the bone defect site --- p.166 / Chapter 5.9.2 --- Histomorphometrical findings in treatment of bone healing --- p.172 / Chapter 5.9.3 --- Effect of ELP and CDNR on biomechanics of bone --- p.175 / Chapter 5.9.4 --- Effect of ELP and CDNR on bone turnover --- p.178 / Chapter 5.10 --- DISCUSSION --- p.184 / Chapter CHAPTER 6: --- GENERAL DISCUSSION AND CONCLUSION --- p.193 / Chapter 6.1 --- UNKNOWN AREAS FOR THE STUDY OF ELP --- p.193 / Chapter 6.2 --- SUMMARY OF CRUCIAL FINDINGS OF THE OSTEOGENIC EFFECTS OF ELP IN EACH PART OF THIS STUDY --- p.194 / Chapter 6.2.1 --- Part 1: in vitro seropharmacological study on osteoporosis --- p.194 / Chapter 6.2.2 --- Part 2: in vivo study on disuse osteoporosis --- p.195 / Chapter 6.2.3 --- Part 3: in vivo study on bone healing --- p.196 / Chapter 6.3 --- COMMON OSTEOGENIC EFFECT OF ELP IN THE THREE PARTS OF THE WHOLE STUDY --- p.197 / Chapter 6.4 --- LIMITATIONS OF THE PRESENT STUDY --- p.197 / Chapter 6.5 --- SIGNIFICANCES OF THIS STUDY --- p.199 / Chapter 6.6 --- FUTURE STUDIES --- p.199 / BIBLIOGRAPHY --- p.201

In order to gain insights into the underlying mechanisms, several methods including, flow cytometric, western blot and quantitative real-time PCR analyses were employed. AITC-induced cell growth inhibition in SW620 cells was mainly caused by G2/M arrest, which was accompanied by regulatory proteins modifications. Results of western blot and quantitative real-time PCR analysis showed clear downregulation of pivotal phosphatases Cdc25B and Cdc25C at both transcriptional and post-translational levels in AITC-treated cells. Subsequently, accumulation of inhibitory phosphorylation of Cdc2 on Thr14 and Tyr15 were resulted. Furthermore, an AITC induced apoptosis after prolonged exposure was observed. It was a caspase-mediated apoptosis as evidenced by the activation of initiator caspases (-8 and -9), effector caspases (-3 and -7) and cleavage of Poly (ADP-ribose) polymerase (PARP). Besides in vitro studies, the antitumor activity of AITC was further illustrated by a nude mice xenografts experiment. Treatment with 10 micromol AITC could effectively suppress the growth of SW620 xenografts in vivo. Taken together, our results suggest that AITC is an attractive candidate for future research in chemotherapy and chemoprevention. / Many epidemiological studies indicate that a high intake of cruciferous vegetables, such as cabbage, broccoli and Brussels sprouts, may reduce the risk of certain types of cancer. Glucosinolates in cruciferous vegetables and their digested products are suggested to play an important role in such chemoprevention. When plant tissue is physically damaged, glucosidic bonds are cleaved by endogenous myrosinase to produce various products. Among these products, isothiocyanates (ITCs) draw most of the attention because of their potent antitumor activities. But the molecular mechanism leading to such effects has not yet been defined. / The objective of this study was to investigate the chemotherapeutic potential of allyl isothiocyanate (AITC) towards human colorectal adenocarcinoma cells. Another commonly founded ITC, phenylethyl isothiocyanate (PEITC) was employed as a reference sample. The growth inhibitory effects of ITCs on different colorectal adenocarcinoma cells were investigated using in vitro cell models. Both AITC and PEITC were found to inhibit the growth and proliferation of Caco-2, COLO 201 and SW620 cells in a time- and dose-dependent manner. Based on sensitivity, the most vulnerable SW620 cells were chosen for further studies. In the following BrdU assay, IC50 values for 24-h AITC and PEITC treatments were determined to be 30.2 and 9.21 microM, respectively. At the same time, the effects of ITCs on human normal skin fibroblast Hs68 cells were also investigated. It was found that the survival of Hs68 cells was not affected by the treatments of AITC. However, the survival of Hs68 cells was greatly affected by PEITC-treatments in a dose- and time-dependent manner. / Lau, Wing Sze. / Adviser: Wong Yum Shing. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 115-128). / 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.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a common clinical problem. Tanshinone IIA is a compound purified from the Chinese herb Danshen ( Radix Salviae Miltiorrhiza Bge). Thrombopoietin (TPO) and Erythropoietin (Epo) are hematopoietic growth factors. The effects of tanshinone IIA, EPO and TPO on hypoxia-ischemia brain injury were investigated in this study, using in vitro model of neural cell culture and an in vivo model of hypoxic-ischemic brain damage. / Our observation provided the first evidence showing the expression of functional TPO receptor c-mpl in central nervous system. It revealed that novel agents TPO, EPO and tanshinone IIA have neuroprotection effects against brain injury induced by hypoxia-ischemia in neonatal rats, and these agents could be developed for clinical applications. / To investigate the effect of TPO, EPO and tanshinone IIA on in-vivo neural protection, a neonatal rat model of hypoxic-ischemic brain damage was established. Our results demonstrated significant and sustained brain injury in the hypoxic-ischemic and vehicle-treated group, measured by the reduction in relative weights of the ipsilateral (right) to the contralateral (left) brain at 1 and 3 weeks post-surgery, compared with those of sham-operated animals. At 3 weeks post-surgery, the hypoxic-ischemic animals had decreased cortical neuron density quantified by neuron-specific enolase (NSE) staining, and compromised sensorimotor functions in response to the postural reflex test. Treatment with TPO, EPO and tanshinone IIA significantly reduced the severity of brain injury, as indicated by the significantly increased ipsilateral brain weight and neuron density. Recoveries of sensorimotor functions (p &lt; 0.05) and histopathology were also observed in animals that received TPO, EPO and tanshinone IIA. The plasma of tanshinone IIA-treated animals exhibited higher antioxidant activities (oxygen radical absorbance capacity assay) than those from vehicle-treated rats. / TPO and TPO receptor (c-mpl) mRNA was identified in human cerebral hemispheres, cerebellum, mouse neural progenitor cell line C17.2 and four neuroblastoma cell lines (SK-N-MC, MHH-NB-11, SK-N-AS and SH-SY-5Y) using RT-PCR methods. TPO proteins were detected in human cerebrospinal fluid (CSF) and plasma by ELISA. Furthermore, TPO receptor c-mpl was confirmed in human cerebral hemispheres, hippocampus, cerebellum, brainstem and spinal cord using immunohistostaining. TPO had a stimulating effect on the growth of neural progenitor cell C17.2 in culture via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway as demonstrated by Western blot. The anti-apoptotic effects of TPO, EPO on C17.2 cells were demonstrated by staining with Annexin-V and PI. EPO exerted a protective effect against SHSY-5Y cell damage induced by NMDA (N-methyl-d-aspartate), as demonstrated by the MTT and LDH assay. The anti-oxidative property of tanshinone IIA was studied in the C17.2 cell line. Tanshinone IIA increased the viability of these cells subjected to 2,2'-azobis (2-amidino propane hydrochloride) (AAPH)-induced oxidative stress. / by Xia Wen-Jie. / "May 2005." / Advisers: Kwok-Pui Fung, Tai-Fai Fok. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0126. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 126-146). / 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.

Absorption study showed that gossypetin was methoxylated and conjugated to form glucuronide during the first-pass metabolism after oral administration. Glucuronide conjugate was the major circulating form in the plasma. As determined by HPLC analysis, the total gossypetin concentration in the plasma was higher than the unchanged gossypetin indicating that most of gossypetin underwent first-pass metabolism. Moreover, urinary excretion was not a main elimination route. / Uses of foods and dietary supplements present a safe chemopreventive strategy. The application of phytochemicals for cancer prevention currently receives a great deal of attention. Flavonoids are known to be antiproliferative and may play an important role in the prevention of carcinogenesis. In addition to epidemiologic studies, basic science research to elucidate mechanisms and evaluate chemopreventive potential of phytochemicals is also necessary. In this study, gossypetin was found to have stronger antiproliferative activity when compared with quercetin, a well studied flavonoid, in human hepatocellular carcinoma (HepG2) cells and human breast carcinoma (MCF-7) cells. The results demonstrated that gossypetin induced growth inhibition in MCF-7 cell line by arresting cell cycle at G0/G1 phase. The inhibition of cell cycle progression was associated with the decrement of cyclin D1 expression, cdk6 kinase activity and phosphorylation of retinoblastoma protein (pRb). Although the cdk inhibitor p21 could not be detected, its upstream protein, p53 tumor suppressor protein, was activated by gossypetin in the MCF-7 cell line. Also, the proliferation of MCF-7 cells was suppressed through down-regulating the Erk1/2 pathway. / Ngai Lei-ka. / "August 2005." / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6156. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 222-250). / 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.

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.

After adjusted for the effect of baseline MCE risk and reduction in SBP, the multivariate meta-regression showed baseline SBP was not significantly related to the RD for all the relevant outcomes examined (p>0.22) except MCE (p=0.0226). However, the baseline MCE risk remained significantly related to the RD for all the outcomes (p&lt;0.01) except CHD (p=0.1011). The reduction in SBP remained significantly related to the RD for deaths due to CVD, MCE, CHF and stroke (p&lt;0.01) but not to the RD for all-cause death (p=0.3788) and CHD (p=0.8755). / Conclusions. This study showed that baseline CVD risk and reduction in blood pressure were strongly and consistently related to the absolute effect of treatment and surprisingly the baseline blood pressure was not. The findings lend direct support to the overall risk approach to primary prevention and suggest that contrary to conventional wisdom and current practice, the overall CVD risk rather than blood pressure alone should be used to identify and treat people to prevent major CVD events through anti-hypertensive drugs. These findings suggest that anti-hypertensive drugs should be given to those who have a high future CVD risk rather than high blood pressure alone so as to achieve better cost-effectiveness of anti-hypertensive drugs for primary prevention. / Data extraction and analyses: Two reviewers independently abstracted data on baseline variables, variables that determine methodological quality, and outcomes. The following main outcomes were assessed: all-cause deaths, deaths due to cardiovascular disease, death due to causes other than CVD, major cardiovascular events (MCE), congestive heart failure (CHF), stroke, and coronary heart disease (CHD). / Key words. hypertension, antihypertensive drugs, cardiovascular disease, meta-analysis, systematic review, randomized controlled trial, primary prevention, baseline risk, evidence based medicine / Meta-analysis was used to obtain the overall odds ratio (OR) and risk difference (RD). Forest plots, bubble plots and funnel plots were used to show the results visually or to check biases. Meta-regression was used to identify factors that may independently determine the effect of antihypertensive drugs. The control CVD risk, initial mean blood pressure and reduction in blood pressure were examined. / Method. Identification of studies: The databases searched included ACP Journal Club, Cochrane Database of Systematic Reviews, Database of Abstracts of Reviews of Effects, Cochrane Central Register of Controlled Trials, MEDLINE, EMBASE, Cumulative Index to Nursing & Allied Health Literature, Chinese Medical Current Contents to identify relevant studies between 1966 and 2005. We also examined references from relevant trials, reviews and meta-analyses. For trials to be included in this review, they have to have the following characteristics: (1) essential hypertension in patients of any age, sex and race; (2) treatment intervention is antihypertensive drugs; (3) control intervention is a placebo or no treatment; (4) endpoint outcomes are all-cause death and major cardiovascular events; and (5) randomized controlled trials. / Objective. Although the overall risk approach to cardiovascular disease (CVD) primary prevention has been widely adopted, direct evidence that supports this policy is however weak and in some aspects lacking. Importantly, there is no direct evidence to show, between blood pressure and CVD risk, which is a better predictor of the absolute benefit from anti-hypertensive drugs. The evidence that the absolute benefit increases as the future CVD risk increases does not necessarily mean that treating high risk people will be more cost-effective than treating hypertensive people as blood pressure may also be positively related to treatment benefit. The high risk approach would be preferable only when we can show with strong evidence that blood pressure is not related to the absolute benefit of treatment or the CVD risk is much more strongly related to the benefit than blood pressure. We thus conducted this systematic review to examine the evidence from randomized controlled trials to directly show how blood pressure and CVD risk are related to the absolute benefit from anti-hypertensive drugs and compare the capability of the two factors in predicting the benefit. The stronger predictor should be a better indicator for identifying those who should be treated with anti-hypertensive drugs. / Results. Twenty-two eligible randomized controlled trials with a total of 55,448 participants were identified from 1967 to 2004. The average follow-up was 45.6 months ranging from 13 to 84 months. The combined RD and OR for all-cause deaths, deaths due to CVD, MCE, CHF, Stroke and CHD were all statistically significant, showing a consistent and considerable reduction in the risk of these outcomes due to the treatment of anti-hypertensive drugs (p&lt;0.01). / Jiang, Yu. / "September 2007." / Adviser: Jin Ling Tang. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4657. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 107-115). / 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. / Abstract in English and Chinese. / School code: 1307.

Tang Chi-wing, Wendy = 飲品味道對炎熱中運動的華籍男女小童之體液平衡及體温調節之影響 / 鄧稚穎. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 74-86). / Text in English; abstracts in English and Chinese. / Tang Chi-wing, Wendy = Yin pin wei dao dui yan re zhong yun dong de hua ji nan nü xiao tong zhi ti ye ping heng ji ti wen diao jie zhi ying xiang / Deng Zhiying. / Dedication --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / Table of Contents --- p.vi / List of Tables --- p.viii / List of Figure --- p.ix / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Background --- p.1 / Chapter 1.2 --- Purpose of the Study --- p.4 / Chapter 1.3 --- Hypotheses --- p.5 / Chapter 1.4 --- Definition of Terms --- p.6 / Chapter 1.5 --- Assumptions --- p.7 / Chapter 1.6 --- Delimitations --- p.7 / Chapter 1.7 --- Limitations --- p.8 / Chapter 1.8 --- Significance of Study --- p.8 / Chapter CHAPTER TWO --- REVIEW OF LITERATURE --- p.9 / Chapter 2.1 --- Water Balance during Exercise --- p.9 / Chapter 2.2 --- Effect of Dehydration on Human Body during Exercise --- p.11 / Chapter 2.3 --- Physiological Responses of Dehydration for Children when Exercising in the Heat --- p.17 / Chapter 2.4 --- Effect of Fluid Ingestion during Exercise --- p.20 / Chapter 2.5 --- Drivers of Fluid Replacement --- p.23 / Chapter CHAPTER THREE --- METHODOLOGY --- p.30 / Chapter 3.1 --- Participants --- p.30 / Chapter 3.2 --- Equipment and Instrumentation --- p.31 / Chapter 3.3 --- Preliminary Measurement --- p.31 / Chapter 3.4 --- Study Design and Procedures --- p.35 / Chapter 3.5 --- Collection and Analysis of Blood Samples --- p.44 / Chapter 3.6 --- Statistical Analysis --- p.44 / Chapter CHAPTER FOUR --- RESULTS --- p.46 / Chapter 4.1 --- Exercise Intensity --- p.46 / Chapter 4.2 --- Body Fluid Balance --- p.50 / Chapter 4.3 --- Thermoregulatory Responses --- p.61 / Chapter 4.4 --- Taste Perception Analysis --- p.62 / Chapter 4.5 --- Summary of the Results --- p.66 / Chapter CHAPTER FIVE --- DISCUSSION --- p.67 / Recommendations and Applications --- p.73 / REFERENCES --- p.74 / APPENDIXES --- p.87