Thesis (M.Tech.: Biomedical Technology)-Dept. of Biomedical Technology, Durban University of Technology, 2007 xxiii, 244 leaves / In 2004, it was reported that KwaZulu-Natal had the greatest number of HIV infected people, approximately 1.8 million people, of whom an estimated 450 000 were in need of antiretroviral drug therapy based on their Cluster of Differentiation 4 (CD4) counts and clinical status. Studies on the success of antiretroviral drugs in improving the quality of life in HIV infected individuals have been extensively performed and published. However, there are no published data on the effect that immune boosters have in improving the quality of life in such persons. Considering the side effects, toxicity, multi-drug regimens and drug resistance problems associated with antiretroviral therapy, alternative or supplementary therapies may play an important role in improving the quality of life in HIV infected people. Such therapy might help in situations where some patients who qualify for antiretroviral treatment are unable to access them because of several reasons such as long waiting lists, travelling costs, unwilling to take antiretroviral drugs, etc. Some patients have reservations in taking antiretroviral drugs. The stigma associated with the disease may be a major factor. The aim of this study was to investigate the change in the immune status of HIV infected patients that were on the Inochi New Medicine immune booster, as well as, to assess the safety and efficacy of this immune booster in improving the patients’ quality of life.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: This thesis provides the first scientific evidence on the cancer modulating properties of two unique South African herbal teas, rooibos (Aspalathus Iinearis) and honeybush (Cyclopia intermedia) utilizing in vitro as well as in vivo carcinogenesis assays by: • Demonstrating the in vitro antimutagenic activity of aqueous extracts of the herbal teas against the metabolic activated mutagens, 2-acetylaminofluorene (2- AAF) and the mycotoxin, aflatoxin B1 (AFB,) as well as, to a certain extent, against the direct acting mutagen, hydrogen peroxide, utilizing the Salmonella typhimurium mutagenicity assay. • Increasing the activity of hepatic drug metabolizing enzymes, glutathione Stransferase alpha and UPD-glucuronosyl transferase, and reduced the oxidative stress by stabilizing the level of reduced glutathione (GSH) resulting in an increased hepatic reduced to oxidized glutathione ratio (GSG:GSSG). No toxic effects were noticed in rats consuming the herbal teas for 10 weeks as their sole source of drinking fluid. • Demonstrating the ex vivo modulation of 2-AAF- and AFB1-induced mutagenesis by sub- cellular hepatic fractions of rats consuming the herbal teas in the Salmonella mutagenicity assay. Hepatic cytosolic fractions protected against mutagenesis of both mutagens, while the microsomal fractions exhibited a reduced capacity to metabolize AFB1 to its active mutagenic metabolite. • Providing evidence for the in vivo modulation of tumour promotion using the liver as well as the two-stage skin carcinogenesis animal models. The unprocessed herbal teas arrested proliferation of the placental form of glutathione-Stransferase (GSTP+) altered cells as well as reduced the total number of enzyme altered foci in the liver of rats. Topical application of polyphenolic fractions of the various herbal teas prior to 12-0-tetra-decanoylphorbol-13-acetate (TPA) tumour promotion, reduced tumour formation in mouse skin initiated with 7,12-dimethylbenz[ ajanthracene (DMBA). The protective effect was illustrated by a decreased tumour incidence, a reduction in tumour volume as well as a delayed onset of tumour development. The f1avanol/proanthocyanidin content of the fractions could playa major role in the protection against skin tumour promotion. • Proposing possible mechanisms whereby rooibos and honeybush herbal teas could exert their cancer modulating properties with respect to in vitro and ex vivo antimutagenicity, in vivo oxidative status and reduced tumour promotion. • Providing evidence that the herbal teas mimic the cancer modulating properties of green and black teas although differences exist, presumably due to differences in the polyphenolic constituents. • Suggesting that rooibos and honeybush herbal teas may play an important role as chemopreventive agents in the modulation of cancer. / AFRIKAANSE OPSOMMING: Hierdie tesis bevat die eerste ondersoek na die effek van waterige en polifenoliese ekstrakte van rooibos (Aspalathus Iinearis) en heuningbos (Cyclopia intermedia) op verskeie aspekte van kankerontwikkeling. Die twee kruietees is uniek aan Suid-Afrika en kan 'n belangrike rol speel in die voorkoming van kanker. Verskillende in vitro so wei as in vivo studies het die volgende getoon: • Antimutageniese aktiwiteite teen die metabolies-geaktiveerde mutagene, 2- asetielaminofluoreen (2-AAF) en die mikotoksien, aflatoksien B1 (AFB1) in die Salmonella fyphimurium mutagenisiteitstoets. 'n Beperkte mate van beskerming is ook verleen teen die oksidatiewe mutageen, waterstofperoksied, sonder metaboliese aktivering. • Verhoogde aktiwiteite van die fase II ensieme, glutatioon S-tranferase alfa en UDP-glukuronidase, wat liggaamsvreemde verbindings metaboliseer. Die kruietees verlaag die oksidasietoestand soos weerspieel word deur 'n toename van gereduseerde glutatioon tot die geoksideerde vorm in die lewer van rotte wat 10 weke hierdie kruietees gedrink he!. Die kruietees het geen toksiese uitwerking op die rotte gehad nie. • Antimutageniese aktiwiteite van subselluiE~re fraksies van die lewer teenoor 2- AAF en AFB1 in die Salmonella toets. Die sitosolfraksie van die rotlewer bied beskerming teen die ge"induseerde mutagenese van beide mutagene, terwyl die mikrosomale fraksie ook die metaboliese aktivering van AFB1 na die aktiewe mutageniese metaboliet verminder. • In vivo modulering van kankerpromosie met behulp van bekende rotlewer en muisvel kankerontwikkelingsmodelle. In die lewermodel het die ongeprosesseerde kruietees beide die ontwikkeling en getal van GSTP+ fokusse onderskeidelik vertraag en verminder. In die geval van die velkankermodel het aanwending van polifenoliese fraksies van die kruietees beskerming gebied teen die ontwikkeling van velkankers by muise. Die aantal en grootte van die tumors het afgeneem terwyl die verskyning daarvan ook vertraag is. • Verskeie meganismes waardeur rooibos- en heuningboslee moonllik kanker kan moduleer word voorgeslel. Verskille in die polifenoliese sameslelling asook hul onderskeie konsenlrasies kan 'n belangrike rol speel in die kankerveranderende effekle van die lees. • Oal gereelde inname van rooibos- en/of heuningboslee moonllik 'n belangrike rol kan speel in die voorkoming van dieel- en omgewings-geYnduseerde kankers.

Besides the molecular mechanisms regulating activation of FLS mentioned above, we also investigated anti-inflammatory activities of Chinese herbal medicine sinomenine and Liang Miao San on activated human FLS in RA. Sinomenine, an alkaloid isolated from the root of Sinomenium acutum, has been used to alleviate the symptoms of rheumatic diseases. Liang Miao San (LMS), composed of the herbs Rhizoma Atractylodis (Cangzhu) and Cotex Phellodendri (Huangbai), is another traditional Chinese medicine formula for RA treatment. Since the potential anti-inflammatory activities of sinomenine and LMS have been demonstrated, we investigated the in vitro anti-inflammatory effects of sinomenine and LMS on inflammatory cytokine TNF-alpha activation of human normal and RA-FLS and the underlying intracellular mechanisms. In the present study, sinomenine was found to significantly inhibit TNF-alpha induced cell surface expression of VCAM-1 and release of inflammatory cytokine and chemokine IL-6, CCL2 and CXCL8 from both normal and RA-FLS (all p < 0.05). Our results provide a new insight into the differential anti-inflammatory activities of sinomenine and LMS through the suppression of TNF-alpha activated FLS by modulating distinct intracellular signaling pathways in RA, and help to provide a biochemical basis for the development of a cost-effective human synoviocyte model for the future screening of traditional Chinese medicine (TCM) possessing potential anti-rheumatic activities. (Abstract shortened by UMI.) / IL-27, a novel member of the IL-12 family that is produced early by antigen-presenting cells (APCs), can promote T cell proliferation as well as the production of interferon-gamma by naive T lymphocytes. Recent studies have found that elevated expression of IL-27 has been detected in the synovial membranes and fluid of RA. Herein we investigated the in vitro effects of IL-27, alone or in combination with inflammatory cytokine TNF-alpha or IL-Ibeta on the pro-inflammatory activation of human primary FLS isolated from RA patients and normal control subjects, and the underlying intracellular signaling molecules were also studied. We found that the plasma concentration of IL-27 in RA patients (n=112) was significantly higher than that in control subjects (n=46). Both normal and RA-FLS constitutively express functional IL-27 receptor heterodimer, gp130 and WSX-1, with more potent IL-27-mediated activation of signal transducers and activators of transcription (STAT)1 in RA-FLS. IL-27 was found to induce significantly higher cell surface expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 and release of inflammatory cytokine IL-6, chemokine CCL2, CXCL9, CXCL10 and matrix metalloproteinase (MMP)-1 of RA-FLS than that of normal FLS (all p < 0.05). The above findings therefore provide a new insight into the IL-27-activated immunopathological mechanisms mediated by distinct intracellular signal transductions in joint inflammation of RA and may have important therapeutic implications. / In the present study, we have investigated the mechanisms of the activation of human fibroblast-like synoviocytes (FLS) induced by various stimuli including interleukin (IL)-27, tumor necrosis factor (TNF)-alpha and IL-beta. The activation of human FLS was studied in terms of the release of cytokines and chemokines and the expression of adhesion molecules. / We investigated the in vitro effects of uric acid crystals, alone or in combination with inflammatory cytokine TNF-alpha or IL-beta on the pro-inflammatory activation of human FLS from RA patients and normal control subjects, and the underlying intracellular signaling molecules were also determined. In the present study, uric acid crystals were found to result in a significant increase of inflammatory cytokine IL-6, chemokine CXCL8 and MMP-1 from both normal and RA-FLS (all p < 0.05). Moreover, additive or synergistic effect was observed in the combined treatment of uric acid crystals and TNF-alpha or IL-1beta on the release of IL-6, CXCL8 and MMP-1 from both normal and RA-FLS. Further investigations showed that the release of inflammatory cytokine, chemokine and matrix metalloproteinase stimulated by uric acid crystals was differentially regulated by intracellular activation of extracellular signal-regulated kinase (ERK) and JNK pathways. Our results therefore provide a new insight into the endogenous danger signal uric acid crystals-activated immunopathological mechanisms mediated by distinct intracellular signal transductions in joint inflammation, and also provide biochemical basis for the development of new modality for inflammatory rheumatic diseases. / Chen, Dapeng. / Adviser: Wong Chun Kwok. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 203-240). / 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.

全球過敏性疾病的患病率逐漸增加。大約30 - 40的世界人口患有一個或多個過敏性疾病，它絶對是一個國際性的公共健康問題。在過去三十年，過敏性皮膚炎的發病率增加了2-3 倍，當中患病率最高的是嬰兒和兒童，而且現時並沒有明確的治療方法。然而，對過敏性疾病有效的治療方法仍然缺乏，大多數傳統的治療涉及臨床改善，但不針對促進過敏性炎症發病機制中的主要因素。這些傳統的治療方法都有不良副作用。因此，發展一個更安全和非類固醇的治療方式成為了新的趨勢。 / 從過往的臨床試驗中，患有中度至嚴重過敏性皮膚炎的兒童服用由五種中藥制成的Pentaherbs（PHF）膠囊藥丸後， 顯著地改善他們的生活質數，降低了過敏性皮膚炎指數（SCORAD）及減少使用傳統藥物類固醇的份量，更沒有出現任何不良藥物作用。實驗結果指出PHF 有潛力替代類固醇，成為治療過敏性皮膚炎的取代品。在本研究中，我們使用炎症相關的細胞因子IL-33，激活在有或沒有與皮膚成纖維細胞一起培植下的嗜鹼性細胞系KU812 細胞， 來探討了PHF，牡丹皮（DP，PHF 的五種草藥之一）和沒食子酸（GA，牡丹皮的主要成分之一）的抗炎和抗過敏特性。 / 在過敏性炎症中，嗜鹼性粒細胞是一個重要的效應細胞。我們利用細胞因子IL-33 激活嗜鹼性粒細胞系KU812 細胞，並從研究結果發現出PHF，DP 和GA 能有效及顯著地抑制細胞間粘附分子ICAM-1 的表達，炎症相關趨化因子CCL2，CCL5，CXCL8 和促炎細胞因子IL-6 的釋放。這證實出PHF, DP 和GA有抗炎和抗過敏的特性。在進一步的研究中，我們加入一種常用醫治過敏性炎症藥物的合成類固醇地塞米松, 與PHF, DP 和GA 結合使用。從各種組合的不同濃度地塞米松與PHF，DP 和GA 中，我們發現聯合使用低濃度為0.01 微克/毫升的地塞米松和10 微克/毫升GA 可進一步抑制ICAM-1 在KU812 的表達，趨化因子CCL2 和CCL5 釋放。此外，沒食子酸可顯著抑制細胞內信號分子p38 絲裂原活化蛋白激酶 (MAPK)，IκB-α和JNK 的表達。這表明了黏附分子的表達，趨化因子和細胞因子的釋放的抑製是經由p38 MAPK，IκB-α和JNK 訊息傳遞路徑所調節。實驗證實PHF，DP 和GA 具有抗炎和抗過敏的特性，與過往的臨床試驗結果一致。 / 為了更進一步研究沒食子酸和地塞米松在過敏性炎症的發病機制中扮演的角色， 我們建立了一個體外的模仿患者皮膚皮炎症的模型，共同培養嗜鹼性細系KU812 細胞和皮膚成纖維細胞 。我們發現，單沒食子酸的應用已經可以顯著地抑制在KU812 細胞和成纖維細胞表面粘附分子的表達，並減低釋放過敏性炎症相關的趨化因子CCL2，CCL5，CXCL8 和促炎細胞因子IL-6。此外，地塞米松和沒食子酸的結合使用能增強抑制KU812 細胞面上ICAM-1 的表達，和皮膚成纖維細胞面上ICAM-1 和VCAM-1 的表達，與及趨化因子CCL2 和CXCL8，促炎性細胞因子IL-6 的釋放。 / 上述調查結果表明，天然植物產品PHF，DP 和GA 是具有消炎和抗過敏的作用，抑制嗜鹼性粒細胞趨化遷移至發炎處和隨後釋放的過敏性炎症介質，如炎症相關的趨化因子和促炎細胞因子。結果表明，沒食子酸天然植物產品可能是一個潛在的過敏性皮膚炎的治療劑，而沒食子酸和地塞米松的結合使用，可以有效降低在患者治療皮膚炎症時使用地塞米松的劑量。總結，這項研究結果揭示使用天然植物衍生產品具有更安全,高效力和副作用少的一種新治療方式。 / The worldwide prevalence of allergic diseases has been increasing gradually. Around 30 - 40% of the world population suffers from one or more allergic conditions, it is definitely a national public health issue. The incidence of Atopic Dermatitis (AD) has increased by 2-3 folds in the past 3 decades with no definitive cure, where the case is the highest during the early infancy and childhood. However, effective treatments on allergic diseases are still lacking, with most of the traditional treatment involves clinical improvement but not targeting the primary factors promoting the pathogenesis of allergic inflammation. These traditional treatments have undesirable side effects. Therefore, there has been a rising interest in the development of a safer and nonsteroid immunomodulation formula to cure the disease. / From previous clinical trails, it is revealed that children with moderate-to-severe AD treated with traditional Chinese Medicine, Pentaherbs formula (PHF), have significantly improved their quality of life, lowered the Scoring of Atopic Dermatitis (SCORAD) index and the use of topical steroids without any adverse drug effect, suggesting that PHF can be an alternative potential adjunct therapy for AD. In this present studies, we elucidated the in vitro anti‐inflammatory and anti‐allergic activities of PHF, Cortex Moutan / Danpi (DP, one of the five herbs in PHF) and gallic acid (GA, one of the main ingredients in Danpi) using human basophilic KU812 cells, with or without human dermal fibroblast, upon the activation with alarmin inflammation-related cytokine IL-33. / Human basophilic KU812 cells activated by alarmin cytokine IL-33 were used as basophil cell model for study since basophils are a crucial effector cells in allergic inflammation. Our results showed that PHF, DP and GA exhibited the anti-inflammatory and anti‐allergic activities indicated by significant suppressive effects on the intercellular adhesion molecule (ICAM)‐1 expression, the release of inflammation‐related chemokines CCL2, CCL5, CXCL8 and proinflammatory cytokine IL-6 from IL-33‐activated KU812 basophilic cells. The studies were further investigated with the combined use of synthetic steroid dexamethasone which is a common drug for AD. Among various combinations with different concentrations of dexamethasone with PHF, DP and GA, we demonstrated that the combined use of a concentration as low as 0.01 μg/ml dexamethasone and 10 μg/ml GA could further suppress ICAM‐1 expression, chemokines CCL2 and CCL5 release in IL-33 activated KU812 cells. Furthermore, gallic acid could significantly suppress the intracellular signaling molecules p38 MAPK, IκB-α and JNK in KU812 cells, thereby suggesting the underlying mechanisms for the suppressive effect on adhesion molecules expression, and the chemokines and cytokines release. Both in vivo and in vitro experiments show that PHF, DP and GA exhibit the anti-inflammatory and anti‐allergic activities in concordance to the previous clinical trials using PHF on AD children. / In order to further study the involvement of gallic acid and dexamethasone in the pathogenesis of AD, we then established an in vitro skin inflammatory cell model by co‐culturing human basophilic KU812 cells and human dermal fibroblasts mimicking the skin lesions of the AD patients. We revealed that the application of gallic acid alone could already significantly suppress the adhesion molecules expression on KU812 cell and fibroblasts, and the release of AD-related chemokines CCL2, CCL5, CXCL8 and pro-inflammatory cytokine IL-6 from the co‐culture. In addition, the combined use of dexamethasone and gallic acid showed an enhanced suppressive effect on ICAM-1 on KU812, and ICAM-1 and VCAM-1 on fibroblasts, AD‐releated chemokines CCL2 and CXCL8, and pro-inflammatory cytokine IL-6. / The above findings suggest that PHF, DP and GA are anti-inflammatory and anti-allergic natural plant products by suppressing the transmigration of basophils into the inflamed sites and the subsequent release of allergic inflammation mediators e.g. inflammation-related chemokines and proinflammatory cytokines.The results suggest that natural plant product gallic acid could be a potential therapeutic agent in treating skin inflammation in AD, and the combined use of gallic acid with dexamethasone could lower the dosage of dexamethasone used in AD patients. Together, of the results of this study shed light for a novel therapeutic modality of AD using a safer natural plant derived product with high potency and less side effects to treat AD. / 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. / Liu, Yan Ping Kelly. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 107-122). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.III / 摘要 --- p.VI / PUBLICATIONS --- p.IX / ABBREVIATIONS --- p.XI / TABLES OF CONTENTS --- p.XIII / Chapter CHAPTER 1: --- General Introduction / Chapter 1.1 --- Allergy --- p.1 / Chapter 1.1.1 --- Definition of Allergy --- p.1 / Chapter 1.1.2 --- Allergic diseases and their prevalence --- p.2 / Chapter 1.1.3 --- Allergic inflammation and its characteristics --- p.2 / Chapter 1.1.4 --- Treatment of allergy --- p.4 / Chapter 1.1.5 --- Atopic Dermatitis --- p.7 / Chapter 1.2 --- Biology of basophils --- p.8 / Chapter 1.2.1 --- Development of basophils --- p.8 / Chapter 1.2.2 --- Morphology and phenotype --- p.9 / Chapter 1.2.3 --- Mast cells and basophils --- p.11 / Chapter 1.2.4 --- Basophils and allergic inflammation --- p.12 / Chapter 1.2.5 --- Human basophilic KU812 cell line --- p.13 / Chapter 1.3 --- Adhesion molecules in allergic inflammation --- p.14 / Chapter 1.3.1 --- Selectins --- p.14 / Chapter 1.3.2 --- Integrins --- p.16 / Chapter 1.3.3 --- Immunoglobulin gene super family --- p.16 / Chapter 1.4 --- Chemokines in allergic inflammation --- p.18 / Chapter 1.4.1 --- C chemokines --- p.18 / Chapter 1.4.2 --- CC chemokines --- p.18 / Chapter 1.4.3 --- CXC chemokines --- p.19 / Chapter 1.4.4 --- CX3C chemokines --- p.19 / Chapter 1.5 --- Cytokines in allergic inflammation --- p.20 / Chapter 1.5.1 --- Proinflammatory cytokines --- p.20 / Chapter 1.5.2 --- Anti-inflammatory cytokines --- p.23 / Chapter 1.6 --- Signal Transduction in allergic inflammation --- p.25 / Chapter 1.6.1 --- Intracellular signaling mechanisms --- p.25 / Chapter 1.6.2 --- RAS-RAF-MAPK pathway --- p.27 / Chapter 1.6.3 --- JAK/STAT pathway --- p.28 / Chapter 1.6.4 --- PI3K-Akt pathway --- p.28 / Chapter 1.6.5 --- NF-κB pathway --- p.28 / Chapter 1.7 --- Aim of Study --- p.29 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Cell Culture --- p.32 / Chapter 2.1.2 --- Serum Supplements --- p.33 / Chapter 2.1.3 --- Recombinant human cytokine --- p.33 / Chapter 2.1.4 --- Dexamethasone --- p.33 / Chapter 2.1.5 --- Phosphate-buffered saline --- p.34 / Chapter 2.1.6 --- Dimethyl sulfoxide --- p.34 / Chapter 2.1.7 --- Nucleotide-binding oligomerization domain ligands --- p.34 / Chapter 2.1.8 --- BAY 117082 --- p.34 / Chapter 2.1.9 --- Cell surface and intracellular immunofluorescence staining --- p.35 / Chapter 2.1.10 --- In vitro XTT based toxicology assay kit --- p.38 / Chapter 2.1.11 --- Quantitative analysis of inflammatory mediators release --- p.38 / Chapter 2.1.12 --- Natural Products --- p.39 / Chapter 2.1.13 --- Animal Experiment --- p.40 / Chapter 2.2 --- Methods --- p.41 / Chapter 2.2.1 --- Cell Culture --- p.41 / Chapter 2.2.2 --- Preparation of plant extracts --- p.42 / Chapter 2.2.3 --- Cell toxicity of the natural products --- p.42 / Chapter 2.2.4 --- Flow cytometric analysis of cell surface expression of molecules --- p.43 / Chapter 2.2.5 --- CBA assay --- p.43 / Chapter 2.2.6 --- Flow cytometric analysis of activated intracellular molecules --- p.44 / Chapter 2.2.7 --- Allergic asthmatic mice model --- p.45 / Chapter 2.2.8 --- Statistical analysis --- p.45 / Chapter Chapter 3: --- Anti-inflammatory and anti-allergic properties of Pentaherbs formula, Danpi and Gallic acid / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.1.1 --- Basophils in inflammation --- p.46 / Chapter 3.1.2 --- IL-33 --- p.47 / Chapter 3.1.3 --- Natural plant products --- p.48 / Chapter 3.1.4 --- Dexamethasone --- p.51 / Chapter 3.1.5 --- Hypothesis and aim of study --- p.52 / Chapter 3.2 --- Results --- p.54 / Chapter 3.2.1 --- Cell cytotoxicity of PHF, DP and GA on human basophilic KU812 cells --- p.54 / Chapter 3.2.2 --- Effect of adhesion molecules expression on IL-33-activated KU812 cells treated with PHF, DP and GA --- p.56 / Chapter 3.2.3 --- Effect of PHF, DP and GA on inflammation-related chemokines CCL2,CCL5, CXCL-8 production from IL-33-activated KU812 cells --- p.59 / Chapter 3.2.4 --- Effect of PHF, DP and GA on pro-inflammatory cytokine IL-6 production from IL-33-activated KU812 cells --- p.64 / Chapter 3.2.5 --- Intracellular signaling pathways involved in GA treatment on IL33-activated KU812 cells --- p.67 / Chapter 3.2.6 --- Effect on the adhesion molecules expression, chemokines and cytokines release of IL-33-activated human basophilic KU812 cells upon the combined treatment of PHF/DP/GA with dexamethasone --- p.73 / Chapter 3.2.7 --- In vivo effect of PHF and DP on Th2 and inflammatory cytokines concentration in serum or BALF in allergic inflammatory mice models --- p.76 / Chapter 3.3 --- Discussion --- p.79 / Chapter Chapter 4: --- Gallic acid and Dexamethasone in Atopic Dermatitis / Chapter 4.1 --- Introductions --- p.84 / Chapter 4.1.1 --- Atopic Dermatitis --- p.84 / Chapter 4.1.2 --- Basophils in AD --- p.86 / Chapter 4.1.3 --- Dermal fibroblasts in AD --- p.86 / Chapter 4.1.4 --- Hypothesis --- p.87 / Chapter 4.2 --- Results --- p.88 / Chapter 4.2.1 --- Effect of the combined use of GA and dexamethasone on ICAM-1 expression on KU812 cells co-cultured with fibroblasts --- p.88 / Chapter 4.2.2 --- Effect of the combined use of GA and dexamethasone on ICAM-1 and VCAM-1 expression on fibroblasts co-cultured with KU812 cells --- p.90 / Chapter 4.2.3 --- Effect on chemokines release from the co-culture upon the treatment with GA and dexamethasone --- p.93 / Chapter 4.2.4 --- Effect on cytokine release from the co-culture treated with GA and dexamethasone --- p.96 / Chapter 4.3 --- Discussions --- p.98 / Chapter Chapter 5: --- Concluding Remarks and Future Prospective / Chapter 5.1 --- Concluding remarks --- p.100 / Chapter 5.2 --- Future prospective --- p.101 / References --- p.107

糖尿病是一種以不正常的高血糖為主要特徵的長期性的糖代謝紊亂疾病。二型糖尿病是常見的糖尿病類型，多於九成的糖尿病病人患有此種類型。各種引起糖尿病的病因最終都會導致血糖過高，並且最終會引起有關眼睛，腎臟，神經和血管系統的併發癥。迄今，糖尿病正影響著大約世界6%的人口，而現在患病率依然在逐年增加。在香港，由於高能量的食和缺乏運動，越來越多的老年人和青年人正在遭受著糖尿病的困擾。糖尿病不是一種致命性的疾病，但是如果沒有採取好的治療控制措施，糖尿病最終會引起一些併發癥，這些併發癥最終會使糖尿病患者走向死亡。高血糖癥不僅是糖尿病的主要特徵，而且也是引起各種糖尿病併發癥的重要因素，在二型糖尿病的治療當中，根據各種病理因素，市場上已經研製出了很多西藥來治療糖尿病。然而，它們都有一些副作用的限制。因此，我們需要通過綜合治療和通過新的途徑研製新的製劑來控制血糖水平，保護病人遠離長期併發癥的困擾。如今，腎臟在血糖平衡中的重要角色已經被很好的認知。 在過去的二十年裡， 通過減少血糖在腎臟的重吸收來增加尿液中血糖的排出，從而達到降低體內血糖水平的方法已經被提出并認為是治療糖尿病的一直新的途徑。 在腎臟中，鈉葡萄糖共轉運體2（SGLT 2）主要負責葡萄糖的重吸收，因此，鈉葡萄糖共轉運體2（SGLT 2）抑製劑被認為是一種有潛質的新型的治療糖尿病的製劑。然而，市場上至今沒有成功研製這種製劑。达格列嗪（dapagliflozin），作為一種最有潛質的鈉葡萄糖共轉運體2（SGLT 2）抑製劑，依然處於臨床三期實驗。至今，對具有鈉葡萄糖共轉運體2（SGLT 2）抑製作用的天然產物和傳統中醫藥的信息報導非常少。中醫中藥的治療理念強調整體治療，從此點看來，爲了使糖尿病患者遠離長期的糖尿病併發癥的困擾，中醫中藥可能比西藥更有優勢。 / 因此，本研究的目的是尋找那些具有體外能專門抑制鈉葡萄糖共轉運體2（SGLT 2）並且體內能通過增加尿糖排出來降低血糖水平的抗糖尿天然產物或傳統中藥。從文獻分析中找到了經常用於治療糖尿病的11種中藥和兩種天然產物。 / 試管實驗確立了五味子醇提物和丹皮酚對表達了人的鈉葡萄糖共轉運體2（SGLT 2）基因的COS 7細胞鏈中鈉葡萄糖共轉運體2對¹⁴C-α-甲基- D-葡萄糖苷的吸收作用具有很強的抑制作用。 / 生物活性引導的片段分析確立了五味子醇提物中的活性片段--乙酸乙酯：甲醇（4:6）（F8）片段具有明顯的專門抑制鈉葡萄糖共轉運體2的作用。本實驗也對F8進行了高效液相色譜和液質聯用色譜分析。五味子中三種常見的化合物：五味子甲素，五味子乙素和五味子醇甲存在于F8中，但濃度都很低。試管實驗顯示，這三種常見化合物均無抑制鈉葡萄糖共轉運體2的作用。因此得出結論，這三種常見的五味子化合物不是F8中有效的抑制鈉葡萄糖共轉運體2的活性成份。 / 本實驗也利用動物實驗調查了丹皮酚的抗糖尿作用。糖尿病大鼠被餵食了三個星期的丹皮酚，基礎血糖實驗和尿糖排出實驗均無陽性結果。 / Diabetes Mellitus (DM) is a chronic disorder of glucose metabolism characterized by abnormally high blood glucose level. Type 2 DM is the common form of diabetes which accounts for more than 90% of all DM cases. All causes of diabetes ultimately lead to hyperglycemia, and it can cause the late complications involving the eyes, kidneys, nerves and blood vessels, which are harmful to health. DM is now affecting about 6% population of the world, and the prevalence is still increasing quickly year by year. In Hong Kong, more and more elderly and youth are suffering from diabetes because of lacking of exercise and high energy diet. DM is not a fatal disease, but if no good action is taken, it can finally cause some kinds of complications, which can lead the patients to the end of their lives. Hyperglycemia is the major characteristics of diabetes, and it is also an important factor which induces all kinds of diabetic complications. In the therapy of type 2 diabetes, a lot of western medicine have been developed in the market according to various pathological causes. However, they have limitations such as existence of side effects. Therefore, combination therapy and development of new agents with novel mechanisms should be required to control the glycemic level and protect the patients from the long-term complications. Nowadays, the significance of the kidney's role in glucose homeostasis is well recognized. Glucose excretion with urine by reducing the renal glucose reabsorption to attenuate the glycemic level has been considered as a new mechanism to treat diabetes since the past two decades. Inhibitors on sodium glucose co-transporters 2 (SGLT 2) which are responsible for the glucose reabsorption in kidney are considered as a kind of new agents that have a potential on the treatment of diabetes. However, there is still no such kind of drug developed in the market, since the most potential one, dapagliflozin, is still on Phase III clinical trial. So far, only few information is found on natural products/traditional Chinese medicines (TCMs) that possess SGLT inhibitory action. Regarding the protection of patients from long-term complications, Chinese medicine which consider the body as a whole, may have advantages over western drugs. / Therefore, the aim of this study is to search for anti-diabetic TCM/natural products which specifically inhibit the activity of SGLT2 in vitro and attenuate plasma glucose level in vivo via increasing glucose excretion through urination. From literature review, 11 TCMs and 2 natural products frequently used in treating DM were selected for screening. / Using hSGLT 1 and hSGLT 2-expressed COS-7 cell lines as a model, in vitro study demonstrated that Fructus Schisandrae chinensis (ethanolic extract) and paeonol posses the most potent inhibitory effect on SGLT 2 in the in vitro ¹⁴C-α-methyl-D-glucopyranoside (¹⁴C-AMG) uptake assay. / The purification of active fraction(s) in ethanolic extract of Schisandrae chinensis fructus was carried out using the bioassay-guided fractionation assay. The ethyl acetate-methanol (4:6) fraction (F8) was selected with significant specific inhibitory effect on SGLT 2. UPLC and LC/MS-MS profiles of F8 were also given in this study. The concentrations of three common compounds of Fructus Shisansrae chinensis: deoxyschisandrin, schisandrin B (γ-schisandrin) and schisandrin were shown very low concentration in F8, the results of uptake assay showed none of these three compounds have inhibitory effects on SGLT 2. It is concluded that these three common compounds in Schisandrae chinensis fructus are not the effective ingredients in F8 which can specifically inhibit SGLT 2. / The anti-diabetic effects of paeonol in treating type 2 DM was investigated in animal study. Paeonol (200 and 300 mg/mL) was given to the type 2 diabetic rat model - Zucker Diabetic Fatty (ZDF) rats for three weeks, the results showed no positive effects on the basal glycaemia test and urinary glucose excretion test. / 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. / Qu, Yue. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 141-153). / Abstracts also in Chinese. / TABLE OF CONTENTS / ABSTRACT --- p.iv / 摘要 --- p.vii / ACKNOWLEDGEMENT --- p.ix / LIST OF ABBREVIATIONS --- p.x / LIST OF TABLES --- p.xiii / LIST OF FIGURES --- p.xiv / TABLE OF CONTENTS --- p.1 / Chapter CHAPTER 1 --- INTRODUCTION --- p.8 / Chapter 1.1 --- Definition, diagnosis, classification and epidemiology of Diabetes Mellitus --- p.8 / Chapter 1.1.1 --- Definition of Diabetes Mellitus --- p.8 / Chapter 1.1.2 --- Diagnosis of Diabetes Mellitus --- p.8 / Chapter 1.1.3 --- Classification of Diabetes Mellitus --- p.9 / Chapter 1.1.4 --- Prevalence of Diabetes Mellitus --- p.11 / Chapter 1.2 --- Glucose Homeostasis and Diabetes Mellitus --- p.12 / Chapter 1.2.1 --- General Description --- p.12 / Chapter 1.2.2 --- Kidney's role in Glucose Homeostasis --- p.14 / Chapter 1.2.2.1 --- Gluconeogenesis in the Kidney --- p.15 / Chapter 1.2.2.2 --- Glucose Reabsorption in the Kidney --- p.15 / Chapter 1.2.2.3 --- Renal glucose transporters --- p.17 / Chapter 1.2.2.4 --- Disorders with abnormal renal glucose transport --- p.19 / Chapter 1.3 --- Etiology of Diabetes Mellitus --- p.20 / Chapter 1.3.1 --- Pancreatic β cell dysfunction --- p.21 / Chapter 1.3.2 --- Insulin resistance --- p.21 / Chapter 1.4 --- Diabetic complications --- p.23 / Chapter 1.5 --- Treatment of type 2 Diabetes Mellitus --- p.25 / Chapter 1.5.1 --- Conventional therapy of type 2 Diabetes Mellitus --- p.25 / Chapter 1.5.2 --- New mechanism for the treatment of type 2 Diabetes Mellitus - Inhibition of glucose reabsorption by glucose transporters in Kidney --- p.29 / Chapter 1.6 --- Traditional Chinese Medicine for Diabetes Mellitus --- p.30 / Chapter 1.7 --- Project objective --- p.33 / Chapter CHAPTER 2 --- TRADITIONAL CHINESE HERBAL MATERIALS AND NATURAL PRODUCTS --- p.36 / Chapter 2.1 --- Materials --- p.36 / Chapter 2.2 --- General description and anti-diabetic effects of selected herbs/natural products --- p.38 / Chapter 2.3 --- Extraction Method --- p.45 / Chapter CHAPTER 3 --- IN VITRO STUDIES OF THE INHIBITORY EFFECT OF SELECTED TRADITIONAL CHINESE HERBS AND NATURAL PRODUCTS ON SODIUM GLUCOSE COTRANSPORTERS (SGLT) --- p.48 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.2 --- Materials --- p.49 / Chapter 3.3 --- Methods and Methods --- p.52 / Chapter 3.3.1 --- In vitro model for screening of SGLT inhibitor --- p.52 / Chapter 3.3.1.1 --- Preparation of hSGLT1 and hSGLT2 Plasmid --- p.52 / Chapter 3.3.1.2 --- Transient Transfection of SGLT1 or SGLT2 clone --- p.53 / Chapter 3.3.1.3 --- Detection of mRNA expression level by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.54 / Chapter 3.3.1.4 --- Development of SGLT1 or SGLT2 stable cell lines --- p.56 / Chapter 3.3.1.5 --- Results --- p.56 / Chapter 3.3.2 --- Cell proliferation assay (MTT assay) --- p.57 / Chapter 3.3.2.1 --- Methods --- p.57 / Chapter 3.3.2.2 --- Results --- p.58 / Chapter 3.3.3 --- Uptake Assay of ¹⁴C-α-methyl-D-glucopyranoside (¹⁴C-AMG) in cultured COS-7 cells expressing SGLT1 or SGLT2 --- p.63 / Chapter 3.3.3.1 --- Methods --- p.63 / Chapter 3.3.3.2 --- Screening Results of Effective Chinese Herbs/Natural Products --- p.64 / Chapter 3.4 --- Discussion --- p.83 / Chapter CHAPTER 4 --- FRACTIONATION OF SCHISANDRAE CHINENSIS FRUCTUS --- p.86 / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Organic Extraction of Schisandrae Chinensis Fructus --- p.86 / Chapter 4.2.1 --- Material and Methods --- p.86 / Chapter 4.2.2 --- Result --- p.86 / Chapter 4.3 --- Bioassay-guided Fractionation of Ethanolic Extract of Schisandrae Chinensis Fructus --- p.87 / Chapter 4.3.1 --- Materials --- p.87 / Chapter 4.3.2 --- Methods --- p.87 / Chapter 4.3.2 --- Results --- p.89 / Chapter 4.4 --- ¹⁴C-α-methyl-D-glucopyranoside (¹⁴C-AMG) Uptake Assay of fractions in cultured COS-7 cells expressing SGLT1 or SGLT2 --- p.92 / Chapter 4.4.1 --- Methods --- p.92 / Chapter 4.4.2 --- Results --- p.93 / Chapter 4.5 --- Characterization of F8 of Schisandrae chinensis fructus using Ultra Performance Liquid Chromatography (UPLC) --- p.98 / Chapter 4.5.1 --- Introduction --- p.98 / Chapter 4.5.2 --- Materials and Methods --- p.98 / Chapter 4.5.3 --- UPLC chromatograms --- p.99 / Chapter 4.6 --- Characterization of F8 using Liquid Chromatography/Mass Spectrometry-Mass Spectrometry (LC/MS-MS) --- p.101 / Chapter 4.6.1. --- Materials --- p.101 / Chapter 4.6.2 --- Methods --- p.102 / Chapter 4.6.3 --- Results --- p.103 / Chapter 4.7 --- ¹⁴C-α-methyl-D-glucopyranoside (¹⁴C-AMG) Uptake Assay of three chemical standards in cultured COS-7 cells expressing SGLT1 or SLGT2 --- p.108 / Chapter 4.7.1 --- Methods --- p.108 / Chapter 4.7.2 --- Results --- p.108 / Chapter 4.8 --- Discussion --- p.111 / Chapter CHAPTER 5 --- IN VIVO STUDIES OF THE ANTI-DIABETIC EFFECT OF SELECTED TRADITIONAL CHINESE HERBS AND NATURAL PRODUCTS IN TYPE 2 DIABETIC RAT MODEL --- p.114 / Chapter 5.1 --- Introduction --- p.114 / Chapter 5.1.1 --- Diabetic Animal Models --- p.114 / Chapter 5.2 --- In vivo Study Tests --- p.117 / Chapter 5.2.1 --- Introduction --- p.117 / Chapter 5.2.2 --- Animals --- p.117 / Chapter 5.2.3 --- Methods --- p.118 / Chapter 5.2.4 --- Results --- p.120 / Chapter 5.3 --- Discussion --- p.125 / Chapter CHAPTER 6 --- GENERAL DISCUSSION --- p.128 / Chapter 6.1 --- Importance of SGLT --- p.128 / Chapter 6.2 --- Current developed SGLT 2 Inhibitors --- p.130 / Chapter 6.3 --- Importance and Treatment of DM by TCMs --- p.132 / Chapter 6.4 --- Screening and Developing drugs from Traditional Chinese medicinal plants --- p.134 / Chapter 6.5 --- Limitations and Improvements --- p.136 / Chapter 6.6 --- Future Works --- p.137 / Chapter 6.7 --- Conclusions --- p.139 / REFERENCES --- p.141

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

Finally, the neuroprotective effects of TGP against corticosterone-induced neurotoxicity in rat pheochromocytoma (PC12) cells, an in vitro experimental model of depression were studied. The results showed TGP treatment dose-dependently protected the cells against corticosterone-induced toxicity. The cytoprotection afforded by TGP treatment was shown to be associated with an enhanced antioxidant activity, and increased expressions of neurotrophins including brain-derived neurotrophic factor, nerve growth factor and neurotrophin-3. / Secondly, the antidepressant-like effect of TGP was evaluated by a rat model of depression induced by chronic unpredictable mild stress (CUMS). The results showed that a 5-week CUMS caused depression-like behavior in rats, as indicated by a significant decreases in sucrose consumption (assessed by sucrose preference test) and locomotor activity (assessed by open-field test), and an increase in immobility time (assessed by forced swim test). Intragastric administration of TGP during the five weeks of CUMS procedure significantly suppressed these behavioral changes induced by CUMS. / Taken together, the results confirmed the antidepressant-like effect of TGP. The antidepressive action of TGP may be mediated by the modulation of the hypothalamic-pituitary-adrenal axis function, the inhibition of oxidative stress, and the up-regulation of neurotrophins, thereby leading to the neuroprotective effects. / The antidepressant-like effect of TGP was firstly evaluated by the behavioral despair test, forced swim test and tail suspension test. The results showed that intragastric administration of TGP caused a significant reduction of immobility time in both forced swim and tail suspension tests in mice. TGP treatment also significantly reduced the duration of immobility time in the forced swim test in rats. / The root of Paeonia lactiflora Pall. (Family: Ranunculaceae), commonly known as peony, is a component herb of many traditional formulae for the treatment of depression-like disorders. Previous studies have demonstrated the antidepressive effect of peony extract in mouse models of depression. Total glycosides of peony (TGP) is regarded as the major active ingredients of peony. The present study aims to confirm the antidepressive potential of TGP and evaluate its action mechanisms. / Thirdly, the neuroprotective effects of TGP on CUMS-treated rats and its possible mechanisms were investigated. The results showed that treatment with TGP for 5 weeks produced neuroprotective effects on the hippocampus of CUMS-treated rats. This effect was associated with the attenuation of hypothalamic-pituitary-adrenal axis hyperactivation (characterized by a decreased serum corticosterone level and an increased hippocampal glucocorticoid receptor expression), an inhibition of oxidative stress, and up-regulation of neurotrophins such as brain-derived neurotrophic factor and neurotrophin-3 in the hippocampus. / Mao, Qingqiu. / Adviser: Che Chun-Tao. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 158-186). / 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.

Asthma has long been considered as one of the most common health problems in the world. In Hong Kong, the prevalence of childhood asthma has increased from 4.8% in 1989, to 10.2% in 2002. In spite of the popularity of using Chinese herbs to treat asthma in Hong Kong, evidence on the effectiveness of herbal treatments is lacking. The Chinese herbal formula CUF2 is an innovative formula developed in the Institute of Chinese Medicine, The Chinese University of Hong Kong and it is composed of 5 commonly used Chinese herbs: Radix astragali, Cordyceps sinesis, Radix Scutellaria, Bulbus fritillariae cirrhosae and Radix stemonae. These herbs are chosen because of their well-known effects on either reducing coughing and sputum production, or anti-inflammatory and immunomodulatory activities. Based on the theoretical benefits of CUF2, we conducted a series of animal, in vitro and clinical studies to explore the efficacy, safety and mechanism of action of CUF2. / Following the establishment of the animal model, we have investigated the effect of CUF2 using this model of asthma. We found that 28 days pretreatment with CUF2 could reduce total cell number and eosinophilia in bronchoalveolar lavage fluid (BALF), prevent the eosinophil infiltration of airways, decrease pulmonary inflammatory cells, and reduce mucus and goblet cell hyperplasia. Especially in the reduction of goblet cell hyperplasia, we demonstrated that there was no significant difference between the effects of high dose CUF2 and dexamethasone (DEX). The eosinophilic immune-inflammatory responses in the airways in OA-sensitized/challenged rats were completely blocked by DEX returning to almost the same as those in normal rats, but the loss of thymus index and body weight were also observed. In contrast to the overall immunosuppressive effects of DEX, decreased production of inflammatory cytokines and chemokines [interleukin (IL)-4, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-2 and monocyte chemotactic protein-1 (MCP-1)], increased production of IL-10 and interferon-gamma (IFN-gamma) in BALF and no suppression of body weight and thymus index were demonstrated in the CUF2-treated groups. There was a dose-response relationship with more prominent effects seen with higher doses of CUF2. These findings indicate that the CUF2 has anti-airway inflammatory activity and exhibits immunomodulatory effect on Th1/Th2 responses in ovalbumin sensitized rats after allergen challenge, and this may imply its potential application to patients with allergic asthma. / In order to evaluate the clinical efficacy and safety, we conducted a multicenter, randomized, double blind, parallel and placebo controlled clinical trial. The same Chinese herbal formula was used in this clinical trial in 85 children aged 7-15 years with mild to moderate perennial asthma as an adjuvant therapy for 6 months. The primary outcome measure was the steroid dosage reduction. Other outcome measures included changes in disease severity score (DSS), lung function, serum concentrations of total IgE and the levels of some key allergy and inflammatory markers in peripheral blood, fractional exhaled nitric oxide (FENO), frequency of asthma attacks and quality of life (QOL). To assess safety, we did urinalysis, complete blood count, liver function and renal function at baseline and the end of the study. Drug compliance and adverse effects were also checked at each monthly visit. All patients were maintained on inhaled corticosteroid at their usual dose and dosing interval, and continued to receive short-acting, inhaled beta2-agonists as needed. There were no serious adverse events reported in the 6-month study period by any of the subjects. Hematological (except eosinophils count) and biochemical profiles (including renal function and liver function) remained within normal limits in the CUF2 group and placebo group at the end of the study. CUF2 was well tolerated in asthmatic children. Both CUF2 group and placebo group showed an improvement in most of clinical parameters. The dosage of inhaled corticosteroid was successfully reduced in both groups. Both groups had similar decrease in DSS, improved QOL and improved lung function parameter PEFR (L/min). Although these parameters showed no statistically significant difference between two groups, the percentage of eosinophils and lymphocytes were significantly decreased in CUF2 group as compared with the placebo group. The CUF2 group also showed improved diary symptom score, reduced expression of TNF-alpha and slight increase in anti-inflammation cytokine IL-18 in the blood. A trend of greater improvement in frequency of upper respiratory infection (URI) in CUF2 group was noted, but no statistical significance was attained. The changes in lung function parameter FEV1%, FENO, frequency of asthma attacks and serum concentrations of total IgE, IgE HDM, IgE cat, cockroach, TARC, LTB4 and LTC4D4E4 showed no statistically significant difference CUF2 group and placebo group. Overall, our data demonstrated that CUF2 treatment had some immunomodulatory effect in childhood asthma. Our findings should support further investigations of Chinese herbal medicine in the area of asthma without steroid therapy. / In the animal study, firstly we attempted to establish a novel murine model of asthma. We adopted a modified sensitization procedure using 10-point subcutaneous and intraperitoneal injections of Ovalbumin (OA) with freshly prepared Al(OH)3 and successfully induced severe airway allergic reactions in young Sprague Dawley (SD) rats. In this SD rat model, allergen exposure triggered accumulation of inflammation cells and eosinophils in the airway submucosa and goblet cells hyperplasia in mucosa, lung function test revealed obstructive lung function changes that included increase of lung resistance (RL) and decrease of dynamic lung compliance (Cdyn). Cytokine and chemokine assays showed that there was a change of the TH1/Th2 balance as illustrated by the high Th2 (interleukin-4)/Th1 (interferon-gamma) ratio. These results demonstrated the feasibility and validity of the SD rat model for studying allergic asthma. This SD model is much cheaper and readily available than the Brown Norway rat model and may facilitate further drug trial in asthma. / In the in vitro study, we investigated the effect of CUF2 on the release of cytokines and/or gene expression using human mast cell line HMC-1, human bronchial epithelial cell line BEAS-2B, peripheral blood mononuclear cells (PBMCs) from healthy subjects and airway cells present in induced sputum from asthmatic patients. We have shown (1) the CUF2 had no cytotoxic effects in final working concentration; (2) CUF2 had inhibitory effects on IL-6, TNF-alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion from HMC-1 in a dose-dependent manner. However, no reduction of IL-8 production in HMC-1 was demonstrated. (3) In addition, study of the effect of CUF2 on the expression of cytokine gene from HMC-1 showed that IL-4, IL-6 and GM-CSF mRNA expressions were down regulated at 24 hours, 24 hours, 16 hours and 24 hours of time points, respectively. No effects on IL-8 and TNF-alpha mRNA expression was observed. (4) Furthermore, CUF2 also significantly inhibited in vitro IL-6 and GM-CSF secretion in TNF-alpha stimulated BEAS-2B cell and reduced GM-CSF production in airway cells present in induced sputum from asthmatic children. (5) We observed that CUF2 enhanced TNF-alpha and IL-6 production but did not alter the levels of GM-CSF and IL-8 in mitogen-stimulated PBMCs from health subject. These findings suggest that pharmacological activities of the CUF2 may be mediated by regulating the production of cytokines in human mast cell, bronchial epithelial cell, airway cell and PBMCs. / In this study, a novel animal asthma model has been established. This model has extensively characterized and exhibited several inflammatory, immunological features that resemble those of human asthma and may facilitate further drug trial in asthma. CUF2 showed its efficacy treating the animal model of allergic asthma. In vitro study also provided evidence of its beneficial dichotomous effects on cytokine and chemokine production in HMC-1, PBMCs and airway cells. A multi-center, randomized, double blind, placebo controlled clinical trial showed that CUF2 had a certain degree of clinical efficacy. Furthermore, the use of CUF2, with the study dose and treatment period, was safe. The efficacy of individual ingredient and the mechanism of CUF2 have not been clarified and further investigations are warranted. In conclusion, our results provided evidence of the potential beneficial effect of CUF2 on immune system functions and supported the potential use of TCM as therapeutic drugs for allergic inflammatory diseases. / by Wong Yeuk Oi. / "September 2005." / Advisers: Yn Tz Sung; Kowk Pui Fung. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6296. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 330-349). / 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.

For the development of a G6PD-deficient mouse model, we introduced the mutant Gpdxa-m1Neu allele (a severe ENU-induced mutation that results in 13-15% G6PD activities of wild type littermates) into the C57L/J background (a strain that constitutively exhibits low G6PD activity) through a breeding program. Of significance is that 78% of the F2 generation had G6PD activities &lt;2 U/g Hb, levels similar to those of severe G6PD deficiency in human. The efficacy of this model was preliminary verified by the known haemolytic agent, naphthalene, as demonstrated by the decrease of GSH/GSSG ratio by 24.6% (P=0.032) and increase of methaemoglobin by 4.5 fold (P=0.8) when compared with the respective control without treatment. / Genetic analysis of 14 mutation hotpots was performed on 98 hemi-/homozygous and 17 heterozygous G6PD-deficient human subjects. We developed a novel Multiplex Primer Extension Reaction (MPER) assay and detected seven specific mutations in 97 subjects: c.1376G>T (33.7%), c.1388G>A (29.6%), c.871G>A + c.1311C>T (12.3%), c.95A>G (9.2%), c.392G>T (7.1%), c.1024C>T (6.2%) and c.1360C>T (1.0%). For the genotyping of 15 heterozygous female, all mutations were identified as follows: c.1376G>T/Normal (33.3%), c.1388G>A/Normal (26.7%), c.871G>A/Normal + c.1311C>T/Normal (20.0%), c.95A>G/Normal (13.3%) and c.392G>T./Normal (6.7%). The c.871G>A and 'silent' mutation c.1311 C>T was newly found to coexist in a high proportion of genotype in our population. / Glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects are vulnerable to chemical-induced haemolysis if exposed to oxidative agents. Little is known, however, of the haemolytic effects of Chinese herbal medicine on G6PD-deficient subjects. Only one case study has reported that a G6PD-deficient newborn developed severe haemolysis after ingestion of Rhizoma Coptidis. Besides, recent studies reported that green tea and its constituents exerted pro-oxidative effects on cellular systems in culture. / Glucose-6-phosphate dehydrogenase deficiency is a genetic disorder inherited in the X-linked manner. The condition is prevalent in the Mediterranean region, Africa and Southeast Asia. In Hong Kong, the frequency of G6PD deficiency is around 4.5% in males and 0.3% in females. Over 140 specific mutations of the X-linked gene for G6PD have been characterized in various geographic regions. However, the local mutation pattern has not been clearly determined. / In conclusion, some Chinese herbal medicine, tea and tea polyphenols significantly altered the oxidative status of G6PD-deficient erythrocytes in vitro. Their in vivo effects on G6PD-deficient individuals would be further investigated by the novel G6PD-dificient mouse model. / In this study, we aim (1) to investigate effects of (a) a panel of Chinese Herbal Medicine (CHM), (b) tea and its constituents, on the oxidative status of human G6PD-deficient erythrocytes in vitro ; (2) to characterize the genotype of G6PD-deficiency in the Chinese population and their specific response to oxidative stress; (3) to develop a novel strain of mice as a model for study of chemicals agents on G6PD-deficient red cell in vivo. / Our results showed that six of eighteen CHM significantly reduced GSH levels in the G6PD-deficient erythrocytes (p&lt;0.05, n=10). After exposure to 1 mg/mL of Rhizoma Coptidis, GSH levels in G6PD-deficient erythrocytes was decreased by 48.9 +/- 5.4% (P&lt;0.001, n=10). At 5 mg/mL of Cortex Moutan, Radix Rehmanniae, Radix Bupleuri, Rhizoma Polygoni Cuspidati and Flos Chimonanthi, GSH levels were decreased significantly (P=0.001 to 0.004) by 51.8 +/- 7.6%, 25.9 +/- 6.7%, 21.0 +/- 6.9%, 17.5 $ 6.7% and 8.7 +/- 6.8% respectively. There were noticeable increases in levels of methaemoglobin by 2.8 fold (5 mg/mL, P=0.012) and 3.4 fold (10 mg/mL, P=0.016) in the presence of Rhizoma Coptidis and Cortex Moutan, respectively, in G6PD-deficient erythrocytes. / We also investigated the pro-oxidative effect of tea and its polyphenolic components on G6PD erythrocytes from G6PD-deficient (n=8) and normal adult (n=8) subjects. The tea extracts significantly reduced GSH and increased GSSG levels in G6PD-deficient erythrocytes in a dose-dependent manner (0.5-10 mg/mL), but not in normal erythrocytes. Similar dose-dependent responses to (-)-Epigallocatechin (EGC) and (-)-Epigallocatechin-3gallate (EGCG), but not to the other polyphenols, were observed. In G6PD-deficient cells, GSH was reduced by 43.3% (EGC at 0.05 mg/mL) and 33.3% (EGCG at 0.5 mg/mL), compared with pre-challenged levels. The concentration of methaemoglobin was increased significantly when these cells were challenged with tea extracts, and EGC. Plasma haemoglobin levels were higher in G6PD-deficient samples after exposure to tea extracts, EGCG, EGC and gallic acid, compared with those in normal blood. / Ko Chun Kay. / "August 2006." / Advisers: Tai Fai Fok; Kwai Har Karen Li. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1577. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. xxii-xliii). / 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.

胰腺癌是一種致死率極高的惡性疾病,在全世界所有的癌症中死亡率排列第八, 在美國排列第四。 很多因素造成了胰腺癌較差的預後,其中包括: 早期檢出率極低; 較少胰腺癌患者的腫瘤適宜手術切除;高轉移率;以及對傳統放療和化療具有較高抗性等。 因此,發展新的治療藥物迫在眉睫。 / 近年來, 植物藥以及從這些植物藥裡分離出的天然化合物, 單獨使用或者與傳統化療藥物合併使用時, 都顯示出對不同類型的癌症具有較好療效。植物藥毛萼香茶菜(唇形科)含有豐富的具有抗癌活性的二萜類化合物。其中毛萼乙素(EriB) 是一個擁有最好抗癌活性的對映-貝殼杉烷型二萜化合物。 基於此背景, 本研究的目標為:利用胰腺癌體外體內模型, 研究EriB的抗胰腺癌活性以及誘導胰腺癌細胞凋亡的機理。 / 體外實驗中, EriB對四種胰腺癌細胞株都顯示了顯著的細胞毒活性,其活性與化療藥物喜樹堿類似。其中, EriB對胰腺癌細胞株CAPAN-2活性最強, 半數致死濃度IC₅₀為0.73 μM。細胞凋亡特徵:細胞核凝聚, 磷脂醯絲氨酸外翻, DNA梯狀條帶以及片斷化,在EriB誘導的胰腺癌細胞株CAPAN-2中出現。此外, EriB還造成癌細胞在細胞週期G2/M期的阻滯。機理研究發現, EriB是通過啟動絲裂原活化蛋白激酶(MAPK), caspase及 p53信號通路來誘導細胞凋亡和細胞週期阻滯的。抗凋亡蛋白與促凋亡蛋白比率(bcl-2/bak)的減少也可能對啟動細胞凋亡內途徑發揮一定作用。除此以外, EriB對癌細胞的細胞毒活性及致凋亡作用依賴于活性氧分子(ROS)的產生。在對細胞進行抗氧化劑預處理的實驗中發現, 只有含巰基基團的抗氧化劑能夠有效的阻斷EriB對癌細胞的活性。進一步實驗證明, EriB對細胞內兩個抗氧化系統: 谷胱甘肽系統及硫氧還蛋白系統的抑制作用導致了ROS在癌細胞中的積聚。同時,ROS的產生啟動了MAPK,熱休克蛋白70以及caspase信號通路,卻抑制了NFκB通路。 / 動物體內實驗證實, 每天對胰腺癌細胞移植瘤裸鼠進行腹腔注射EriB(2.5 毫克/千克),能有效的抑制腫瘤生長, 並且對心臟,肝臟和腎臟沒有引起顯著毒性。 對腫瘤組織的分析表明, 給藥組(EriB)比溶劑對照組出現更多的細胞凋亡, 並產生較多的ROS積聚。 / 綜上所述, 本項研究首次闡述了EriB具有顯著的體內外抗胰腺癌活性。機理研究證明, EriB抑制胰腺癌細胞內兩個含巰基基團的抗氧化系統, 從而導致ROS在細胞中積聚, 並啟動(或抑制)了包括MAPK, p53, caspase和NFB在內的信號通路, 最終導致癌細胞死亡。 此外, 動物體內研究證明EriB的抗腫瘤生長活性和低毒性, 令該化合物具有潛力進一步研究發展成為抗胰腺癌的新藥物。 / Pancreatic cancer is the fourth and eighth leading cause of cancer-related deaths in the U.S. and worldwide, respectively. Its poor prognosis is attributed to its late diagnosis, limitation to surgical resection, aggressive local invasion, and early metastases, as well as high resistance to chemotherapy and radiotherapy. Therefore, a search for an alternative to therapeutic agents is in desperate need. / In recent years, herbal medicines or natural compounds isolated from herbs either used alone or in combination with conventional anti-cancer agents have been shown to have beneficial effects on various cancers. In this context, the Chinese herb Isodon eriocalyx (Dunn.) Hara (family Lamiaceae) is a well-known source of anti-cancer diterpenoids, the most potent one being Eriocalyxin B (EriB, an ent-kauranoid). Therefore, the aims of the present study are to investigate the anti-tumor activities of EriB in human pancreatic adenocarcinoma cells and tumor-bearing mouse model, as well as the underlying mechanisms. / Our results showed that EriB exhibited significant cytotoxic effects on four pancreatic adenocarcinoma cell lines, with potencies being comparable to that of chemotherapeutic agent camptothecin. EriB had the most potent cytotoxicity in CAPAN-2 cells with IC₅₀ = 0.73 μM. The hallmark features of apoptosis, such as nuclear condensation, translocation of phosphatidylserine, DNA laddering, and DNA fragmentation were observed in EriB-treated CAPAN-2 cells. On the other hand, EriB also induced G2/M phase cell cycle arrest. Mechanistic studies revealed that EriB induced apoptosis and cell cycle arrest through the activation of MAPKs (p38, ERK1/2), caspase cascade, and p53/p21/cdk1-cyclinB1 signaling pathways. A decrease in the ratio of anti-apoptotic to pro-apoptotic proteins (bcl-2/bak) also contributed to the activation of intrinsic apoptotic pathway. Further investigation showed that EriB-induced cytotoxic and apoptotic effects were dependent on reactive oxygen species (ROS) production. Such demonstrated effects could be inhibited by pre-treatment with thiol-containing antioxidants. Furthermore, EriB induced ROS was mediated via the inhibition of two main antioxidant systems, namely glutathione and thioredoxin systems. EriB-mediated ROS activated multiple targets or signal pathways, including MAPK, heat shock protein (Hsp) 70, and caspase cascade, while inhibiting the NFκB pathway. / On the other hand, in vivo study demonstrated that daily intraperitoneal administration of EriB (2.5mg/kg/day) in human pancreatic tumor xenografts BALB/c nude mice significantly inhibited tumor growth, but without having toxicity in the heart, liver and kidney. In addition, EriB treatments induced in vivo cell apoptosis and superoxide production as observed in tumor tissues. / In conclusion, the present study reports for the first time that EriB has possessed anti-proliferative activities in pancreatic cancer cells. The anti-proliferative effects of EriB on CAPAN-2 cells could be attributable to the regulation of cellular apoptosis and cell cycle arrest. The inhibitory effects of EriB on two antioxidant systems result in the accumulation of ROS, which in turn activate MAPK, p53, Hsp70 and caspase cascade, while inhibiting NFB pathway and finally leading to pancreatic cancer cell death. Meanwhile, in vivo study further confirms the anti-tumor properties of EriB, suggesting that EriB could be considered as a potential chemotherapeutic agent for patients with pancreatic cancer. / 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. / Li, Lin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 207-230). / Abstracts also in Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Publications --- p.vi / Acknowledgements --- p.vii / Table of contents --- p.ix / List of figures --- p.xv / List of tables --- p.xix / List of abbreviations --- p.xx / Chapter Chapter1 --- General Introduction --- p.1 / Chapter 1.1 --- The pancreas --- p.2 / Chapter 1.1.1 --- Anatomy of the pancreas --- p.2 / Chapter 1.1.2 --- Histology of the pancreas --- p.4 / Chapter 1.1.3 --- Exocrine pancreas --- p.5 / Chapter 1.1.3.1 --- Structure of secretory acini, ducts and stroma in pancreas --- p.5 / Chapter 1.1.3.2 --- Functions of exocrine pancreas --- p.6 / Chapter 1.1.4 --- Endocrine pancreas --- p.9 / Chapter 1.1.4.1 --- Structure of islets cells --- p.10 / Chapter 1.1.4.2 --- Functions of endocrine pancreas --- p.10 / Chapter 1.1.5 --- Disorders of the pancreas --- p.11 / Chapter 1.2 --- Pancreatic cancer --- p.14 / Chapter 1.2.1 --- Epidemiology --- p.14 / Chapter 1.2.2 --- The risks and causes of pancreatic cancer --- p.15 / Chapter 1.2.3 --- Signs and symptoms of pancreatic cancer --- p.18 / Chapter 1.2.4 --- Types of pancreatic cancer --- p.19 / Chapter 1.2.5 --- Diagnosis of pancreatic cancer --- p.21 / Chapter 1.2.6 --- Staging of pancreatic cancer --- p.27 / Chapter 1.3 --- Treatments for pancreatic cancer --- p.29 / Chapter 1.3.1 --- Surgery --- p.29 / Chapter 1.3.2 --- Chemotherapy --- p.30 / Chapter 1.3.2.1 --- 5-fluorouracil (5-FU) --- p.32 / Chapter 1.3.2.2 --- Gemcitabine (Gem) --- p.33 / Chapter 1.3.2.3 --- Other cytotoxic agents --- p.34 / Chapter 1.3.3 --- Radiotherapy --- p.35 / Chapter 1.3.4 --- Target therapies --- p.37 / Chapter 1.3.4.1 --- Antiangiogenic therapy --- p.37 / Chapter 1.3.4.2 --- Epidermal growth factor receptor (EGFR) signaling inhibitors --- p.39 / Chapter 1.3.4.3 --- Hedgehog and Notch signaling pathways inhibitors --- p.41 / Chapter 1.3.5 --- Gene therapy --- p.42 / Chapter 1.3.6 --- Immunotherapy --- p.45 / Chapter 1.3.7 --- Combination therapies --- p.46 / Chapter 1.4 --- Molecular targets for pancreatic cancer chemotherapy --- p.49 / Chapter 1.4.1 --- Therapies-induced apoptosis --- p.49 / Chapter 1.4.1.1 --- Caspase cascade and bcl-2 Family --- p.49 / Chapter 1.4.1.2 --- Role of mitogen-activated protein kinases (MAPKs) in apoptosis --- p.50 / Chapter 1.4.2 --- Nuclear factor-κB activation in pancreatic cancer --- p.50 / Chapter 1.4.3 --- The PI3K and AKT pathway --- p.51 / Chapter 1.4.4 --- JAK/STAT pathway --- p.51 / Chapter 1.4.5 --- Other molecular targets --- p.52 / Chapter 1.5 --- Herbal medicine as an alternative treatment for cancer treatment --- p.53 / Chapter 1.5.1 --- Herbal medicines for different types of cancer treatment --- p.53 / Chapter 1.5.2 --- Herbal medicines for pancreatic cancer treatment --- p.59 / Chapter 1.6 --- Introduction of Isodon eriocalyx (Dunn.) Hara --- p.61 / Chapter 1.6.1 --- Background of Isodon genus and Isodon eriocalyx (Dunn.) Hara --- p.61 / Chapter 1.6.2 --- Diterpenoids from Isodon species and their activities --- p.62 / Chapter 1.6.3 --- The potential anti-cancer activity of Eriocalyxin B, a diterpenoid isolated from Isodon eriocalyx (Dunn.) Hara --- p.62 / Chapter 1.7 --- Aims and objectives of this study --- p.66 / Chapter Chapter 2 --- Eriocalyxin B induces apoptosis and cell cycle arrest in pancreatic adenocarcinoma cells through caspase- and p53-dependent pathways --- p.67 / Chapter 2.1 --- Introduction --- p.68 / Chapter 2.2 --- Materials and methods --- p.71 / Chapter 2.2.1 --- Preparation and quality control of Eriocalyxin B --- p.71 / Chapter 2.2.2 --- Materials --- p.72 / Chapter 2.2.3 --- Cell culture --- p.72 / Chapter 2.2.4 --- Preparation of human peripheral blood mononuclear cells (PBMC) --- p.73 / Chapter 2.2.5 --- Cytotoxicity assay --- p.75 / Chapter 2.2.6 --- Hoechst 33258 staining for morphological evaluation --- p.76 / Chapter 2.2.7 --- DNA fragmentation detection by DNA ladder --- p.76 / Chapter 2.2.8 --- Cell death detection ELISA --- p.77 / Chapter 2.2.9 --- Apoptosis detection by flow cytometry --- p.78 / Chapter 2.2.10 --- Cell cycle analysis by flow cytometry --- p.78 / Chapter 2.2.11 --- Western blot analysis --- p.79 / Chapter 2.2.12 --- Statistical analysis --- p.80 / Chapter 2.3 --- Results --- p.81 / Chapter 2.3.1 --- EriB induces cytotoxic effect in human pancreatic cancer cells --- p.81 / Chapter 2.3.2 --- EriB induces apoptosis in CAPAN-2 cells --- p.85 / Chapter 2.3.3 --- Activation of pro-apoptotic caspases in EriB-treated CAPAN-2 cells --- p.89 / Chapter 2.3.4 --- Modulation of bcl-2/bak ratio in EriB-treated CAPAN-2 cells --- p.92 / Chapter 2.3.5 --- EriB causes G2/M cell cycle arrest --- p.94 / Chapter 2.3.6 --- EriB modulates expression of G2/M cell cycle regulatory proteins through activation of the p53 pathway --- p.96 / Chapter 2.4 --- Discussion --- p.99 / Chapter Chapter 3 --- Eriocalyxin B induces apoptosis in pancreatic cancer CAPAN-2 cells via mediation of reactive oxygen species --- p.107 / Chapter 3.1 --- Introduction --- p.108 / Chapter 3.2 --- Materials and methods --- p.113 / Chapter 3.2.1 --- Materials --- p.113 / Chapter 3.2.2 --- Cell culture and MTT assay --- p.113 / Chapter 3.2.3 --- Apoptosis detection by flow cytometry --- p.114 / Chapter 3.2.4 --- Reactive oxygen species (ROS) detection by flow cytometry --- p.114 / Chapter 3.2.5 --- Glutathione assessment --- p.115 / Chapter 3.2.6 --- Glutathione peroxidase (GPx) activity detection --- p.116 / Chapter 3.2.7 --- Thioredoxin reductase (TrxR) activity detection --- p.116 / Chapter 3.2.8 --- Nuclear and cytosolic fractionation --- p.117 / Chapter 3.2.9 --- Western blot analysis --- p.117 / Chapter 3.2.10 --- Electrophoretic mobility shift assay --- p.119 / Chapter 3.2.11 --- Statistical analysis --- p.119 / Chapter 3.3 --- Results --- p.120 / Chapter 3.3.1 --- Thiol-containing antioxidants inhibits EriB-induced cytotoxic effects --- p.120 / Chapter 3.3.2 --- Thiol-containing antioxidants inhibits EriB-induced apoptotic effects --- p.122 / Chapter 3.3.3 --- Effects of EriB on hydrogen peroxide production --- p.125 / Chapter 3.3.4 --- EriB depletes glutathione level and suppresses GPx activity --- p.128 / Chapter 3.3.5 --- EriB inhibits thioredoxin system and activates ASK1 --- p.130 / Chapter 3.3.6 --- EriB increases Hsp70 and cleaved-PARP expression through ROS --- p.134 / Chapter 3.3.7 --- EriB inhibits NFkB pathway in CAPAN-2 cells --- p.137 / Chapter 3.4 --- Discussion --- p.142 / Chapter Chapter 4 --- In vivo study of the anti-tumor efficacy of Eriocalyxin B in human pancreatic tumor xenograft model --- p.149 / Chapter 4.1 --- Introduction --- p.150 / Chapter 4.2 --- Materials and methods --- p.154 / Chapter 4.2.1 --- Establishment of a subcutaneous pancreatic cancer xenograft model --- p.154 / Chapter 4.2.2 --- Evaluation of the effects of EriB on tumor growth --- p.155 / Chapter 4.2.2.1 --- Pilot study for EriB and camptothecin treatment --- p.155 / Chapter 4.2.2.2 --- Confirmation study of effective dose of EriB --- p.156 / Chapter 4.2.2.3 --- Dose-comparison study of CPT-11 --- p.156 / Chapter 4.2.2.4 --- Comparison study of EriB and CPT-11 treatments --- p..157 / Chapter 4.2.3 --- Measurement of plasma-specific enzyme levels --- p.157 / Chapter 4.2.4 --- Assays of terminal deoxytransferase-catalyzed DNA nick-end labeling (TUNEL) --- p..158 / Chapter 4.2.5 --- Histological evaluation --- p.159 / Chapter 4.2.6 --- Detection of superoxide by DHE staining --- p.159 / Chapter 4.2.7 --- Establishment of an orthotopic model (SW1990) of pancreatic cancer and detection of the plasma biomarker CA19-9 --- p.160 / Chapter 4.2.7.1 --- Detection of CA19-9 expression by immunofluorescent staining and western blot --- p.161 / Chapter 4.2.7.2 --- Establishment of an orthotopic pancreatic cancer xenograft model by SW1990 cells --- p.162 / Chapter 4.2.8 --- Statistical analysis --- p.164 / Chapter 4.3 --- Results --- p.165 / Chapter 4.3.1 --- EriB inhibits the growth of CAPAN-2 human pancreatic tumor xenografts --- p.165 / Chapter 4.3.2 --- EriB treatments induce cell apoptosis in tumor tissues --- p.173 / Chapter 4.3.3 --- Toxicity tests for EriB --- p.175 / Chapter 4.3.3.1 --- Plasma enzyme levels after EriB treatments --- p.175 / Chapter 4.3.3.2 --- No apparent alterations in histology of the heart, liver and kidney tissues --- p..176 / Chapter 4.3.4tEriB induces superoxide production in the tumor tissues --- p.178 / Chapter 4.3.5 --- Successful establishment of an orthotopic xenograft model --- p.180 / Chapter 4.4 --- Discussion --- p.184 / Chapter Chapter 5 --- General Discussion --- p.188 / Chapter 5.1 --- Discussion --- p.189 / Chapter 5.2 --- Conclusion --- p.204 / Chapter 5.3 --- Limitations of the study --- p.205 / Chapter 5.4 --- Future work --- p.206 / Chapter Chapter 6 --- References --- p.207