Thesis (M. D. S.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available online.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 137-165). Also available in electronic version. Access restricted to campus users.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 74-93) Also available in print.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Ulcerative colitis (UC), a subset of inflammatory bowel disease (IBD), is a chronic uncontrolled inflammatory condition of the intestinal mucosa. As its etiology remains unclear, no specific effective treatment is available. Therefore, development of novel strategies for IBD treatment remains a major medical need. Qing-dai Powder (QDP), an ancient herbal medicinal formula, exerted potent therapeutic effect on intractable UC patients; however, evidence-based support is needed. The aims of this study are: i) to delineate the anti-colitis effect of QDP and its underlying mechanisms in murine colitis; 2) to explore the rationality of QDP formula; 3) to investigate the anti-colitis effects of major component(s) or/and active ingredient(s) of QDP and their underlying mechanisms in murine colitis. In the present study, the therapeutic effect of QDP on UC was investigated on dextran sulfate sodium (DSS)-induced acute murine colitis. Results showed that i) QDP dose-dependently attenuated disease activity index (DAI), colon shortening, histological damage and colonic myeloperoxidase (MPO) activity of DSS-treated mice; ii) QDP significantly decreased the infiltration of immune cells, particularly macrophages and CD4+ T cells, colonic levels of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6, and plasma level of chemokine MCP-1. In RAW 264.7 cells, QDP significantly suppressed lipopolysaccharide (LPS)-induced the production of TNF-α and IL-6, and the expression levels of COX-2 and iNOS via inhibiting IкB-α degradation and p65 nuclear translocation; Also, in primary CD4+ T cells, QDP significantly suppressed the differentiation of Th1 and Th17 cells. These findings indicate that the anti-colitis effects of QDP might be associated with inhibition of inflammatory responses of colonic macrophages and CD4+ T cells. QDP is composed of Qing-dai and Ku-fan. The comparative study of anti-colitis of QDP, Qing-dai and Ku-fan revealed that QDP is a reasonable TCM formula, and Qing-dai is mainly responsible for the anti-colitis effect of QDP and Ku-fan exhibits a weak beneficial effect. Mechanistically, it was found that Qing-dai significantly suppressed Th1 and Th17 responses, characterized as i) suppressing mRNA expression of Th1 cytokine IFN-γ and Th17 cytokine IL-17A, inhibiting the production of Th1 and Th17-related cytokines IFN-γ, IL-17A/F and TNF-α in the colon of DSS-treated mice; ii) restraining the proportions of Th1 and Th17 cells in mesenteric lymph nodes of DSS-treated mice; iii) suppressing the differentiation of Th1 and Th17 cells in vitro. Indirubin is the principle active component of Qing-dai. It was found that indirubin significantly suppressed the generation of Th17 cells in DSS-treated mice, evidenced by i) suppressing the mRNA expression of IFN-γ, IL-17A, and RORγt, and inhibiting the production of IL-17A/F, TNF-α, IL-1β and IL-6 in the colon of DSS-treated mice; ii) reducing Th17 cells in mesenteric lymph nodes of DSS-treated mice through reducing GSK-3β activity and p-STAT3 expression; iii) suppressing the differentiation of Th17 cells through down-regulating the expression of GSK-3β and p-STAT3 in vitro. In summary, the present study provides evidence-based support for the clinical use of QDP in the management of UC, and indicates that indirubin is the main active compound of QDP responsible for its anti-colitis effect.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 81-92) Also available in print.

Functional constipation (FC) is a major gastrointestional (GI) disorder which affects about 14% population worldwide. However, due to efficacy and safety concerns, more than 50% FC patients are not completely satisfied with current conventional therapies, thus alternative therapies are needed. A substantial part of FC patients have symptom of slow colonic motility, while therapy targeting a single pathway cannot benefit all of them. In this thesis, we searched for novel FC therapeutics from two distinct sources, both of which can improve colonic motility significantly: (1) MaZiRenWan (MZRW), an herb formula from Traditional Chinese Medicine (TCM); and (2) Spexin (SPX), a newly identified neuropeptide that is deregulated in FC. On the basis of efficacy validation for MZRW by randomized, placebo-controlled clinical studies, we investigated the bioactive compounds and pharmacological actions of MZRW. Firstly, a machine-learning based method, namely MOST, was developed to relate bioactive compounds with their mechanism-of-action targets. MOST demonstrated good performance in 7-fold cross-validation (over 87% accuracy) and temporal validation (over 76% accuracy). In the case laxative effect, MOST predicted that acetylcholinesterase (ACHE) was the mechanism-of-action target of aloe-emodin; in vivo studies validated this prediction. Secondly, we analyzed the bioactive compounds and mechanism-of-actions of MZRW with combination of UPLC-QTOF-MS/MS, clustering analysis, organ bath, and MOST approaches. 97 compounds were identified in MZRW extract, and 35 of them can be found in plasma and feces samples of rats with oral administration of MZRW. Chemical space analysis suggested that these compounds can be classified into component groups, while the corresponding pharmacology can be studied with representative compounds. Emodin, amygdalin, albiflorin, honokiol, and naringin were shown to induce spontaneous contractions of rat colonic smooth muscle in vitro. Biological targets in ACh-, estrogen-, prostaglandin-, cannabinoid-, and purine signaling pathways are able to explain the prokinetic effects of representative compounds and component groups. Pharmacological actions of MZRW are mixture of five classic paradigms. Thirdly, the latest results of three-armed, randomized and controlled clinical study showed that MZRW demonstrated comparable efficacy with the first line drug Senna, the first line drug for constipation in HK, during treatment period, both were better than placebo; and the efficacy was more sustainable in follow-up period when comparing that of Senna and placebo. These data suggested the unique pharmacological profile of MZRW for FC. With pharmacometabolomic analysis, we found that change of oleamide is negatively correlated (pearson r = -0.59, p<0.001) with improvement of Complete Spontaneous Bowel Movement (CSBM) in MZRW group, but not in Senna or placebo group. Oleamide is up-regulated in FC patients compared with healthy controls, and MZRW can significantly reduce oleamide in FC patients (n=30), healthy human volunteers (n=23), and in normal mice (n=12) serum, ileum, and colon. The regulation of oleamide by MZRW is possibly via augmenting FAAH-mediated degradation. Lastly, we investigated the possibility to use SPX, the newly identified, FC-associated neuropeptide to change GI motility. The deregulation of SPX has been found in several disorders including FC, however, the metabolic instability of SPX prevent it to be directly used in clinical practices. Our investigation through combination of molecular dynamics (MD) simulations and NMR analysis suggested a β-turn-helix-β-turn (βαβ) conformation for human spexin (hSPX) adopts in solution. Consistent with this conformation, cyclic analogues of hSPX with a disulfide bond between residue 1 and 13, LH101 (CWTPQAMLYLKGCQ-NH2), activated both GalR2 (EC50=1.19 μM) and GalR3 (EC50=1.56 μM) with potency comparable to wild type, and that the acetylation at the N-terminal, LH101(Ac) raises the potency EC50=0.38 μM on GalR2 and EC50=0.39 μM on GalR3. The serum half lives of LH101 (t1/2=355.7 min) and LH101(Ac) (t1/2=1973.7 min) were significantly longer than the wild type (t1/2=66.5 min), and LH101(Ac) induces the contractions of mice intestinal segment in vitro and attenuates the oleamide-induced slow GI motility in vivo. Collectively, our studies in MZRW suggested that estrogen and oleamide signaling pathways are potential new targets to develop novel therapeutics for FC, while lead compounds targeting these pathways could be found from MZRW. The final study suggested CSAs have potential to be developed as new FC therapy by targeting the galanin receptor associated pathway.

Cell metabolism disorder is considered as both direct and indirect consequence of oncogenic mutations for tumoigenesis. Autophagy is a metabolic stress response and a mechanism of natural cellular degradation. It is believed that autophagy, as well as metabolism, is a crucial process for the adaptation of cancer cells to changes in nutrient availability. Understanding the relationship between metabolism and autophagy and targeting on the key steps are regarded as a promising strategy to treat cancer. Halofuginone (HF), a semisynthetic quinazolinone alkaloid originally derived from the plant Dichroa febrifuga Lour., has gained attention for its potential therapeutic effects in a variety of cancers. We hypothesize that HF may inhibit cancer cell proliferation by inducing changes in glucose metabolism and modulating related autophagy. A series of studies, from in vitro to in vivo, were designed to approve this concept in colorectal cancer (CRC);Firstly, we found that HF inhibited human CRC cell proliferation and induced the generation of reactive oxygen species (ROS) and apoptosis. As expected, a reduced level of NADPH was also observed, at least in part due to inactivation of glucose-6-phosphate dehydrogenase (G6PD) in pentose phosphate pathway (PPP) upon HF treatment. Given these findings, we further investigated metabolic regulation of HF through Akt/mTORC1-mediated aerobic glycolysis and found that HF downregulated the Akt/mTORC1 signaling pathway. Moreover, metabolomics found slower rates in both glycolytic flux and glucose-derived tricarboxylic acid cycle flux. Meanwhile, both glucose transporter GLUT1 and hexokinase-2 in glycolysis were suppressed in CRC cells by HF. These findings support our notion that HF regulates the Akt/mTORC1 signaling pathway to dampen glucose uptake and glycolysis in CRC cells. Furthermore, HF retarded tumor growth in nude mice inoculated with HCT116 cells and reduced the viability of primary cells from the tissues of CRC patients. This finding further confirmed our hypothesis that HF inhibits CRC cell growth through metabolic regulation of Akt/mTORC1. Because mTORC1 can inhibit autophagy through phosphorylation and inactivation of the initiating kinase ULK1 in cancer cells, we further studied the HF effects on CRC in different nutritional conditions. The results showed that HF in nutrient-rich conditions could reduce SQSTM1/p62 through mTORC1-mediated phosphorylation at Ser757 of ULK1. More interestingly, HF elevated SQSTM1 protein level in low nutrient condition through AMPK-mediated phosphorylation at Ser317/777 of ULK1. It showed that HF could regulate nutrient-sensing mTORC1-ULK1 or AMPK-ULK1 to dually modulate autophagy in CRC cells. Further study by using a variety of methods, including mRFP-GFP-LC3 puncta formation, transmission electron microscope (TEM) analysis and monodansylcadaverine (MDC) staining, found that HF could induce autophagosome formation and inhibit autophagosome membrane elongation, depending on nutrient-sensing pathways. Furthermore, we found HF pronouncedly enhanced expression level of Atg7 under nutrient-rich conditions while it decreased Atg7 in CRC cells under nutrient-poor conditions. These two findings imply that Atg7 is required in dual regulation of autophagic flux depending on nutrient conditions. This conclusion was then validated by comparing with autophagy-related proteins in Atg7 knockout (KO) MEFs and Wild-type (WT) MEFs upon HF treatment. Importantly, through analysis of metabolome and metabolic enzymes, we found that HF inhibited glycolysis under nutrient-rich conditions while it inhibited gluconeogenesis under nutrient-poor conditions in an Atg7-dependent manner. In subsequent studies, we found that caloric restriction (CR) in a xenograft mouse model, which mimics low nutrition in vitro, enhanced the anticancer activity of HF. Further analysis of the expression of TQTSM1 and LC3 in tumor tissues demonstrated that HF is an autophagic inducer in xenograft-bearing nude mice with ad libitum feeding, whereas it is an autophagic inhibitor when using CR.;In summary, this study explains how HF controls CRC cell growth through its influences on glucose metabolism and autophagy regulation. HF not only dually regulates autophagy in vitro and in vivo to inhibit cancer cell growth and proliferation through nutrient-sensing pathways under different conditions, but also modulates glycolysis/gluconeogenesis through an autophagic pathway. These results suggest that HF could turn out to be a potent therapeutic drug for treating CRC.