The effects of bilirubin in the dextran sulfate sodium (DSS) mouse colitis model

國立屏東科技大學生物科技系 Department of Biological Science and Technology National Pingtung University of Science and Technology 碩士學位論文 Master Thesis 膽紅素對在葡聚醣硫酸鈉（DSS）小鼠結腸炎模型的影響 The Effects of Bilirubin in the Dextran Sulfate Sodium (DSS) Mouse Colitis Model 指導教授：黃卓治 博士 Adviser：Tzou-Chi Huang, Ph.D 研究生：農氏芳絨 Graduate student：Nong Thi Phuong Nhung 中華民國 104 年 月 29 日 June 29, 2015 摘要 學號 : M10218032 研究計劃 總頁數 學院名稱 : 膽紅素對在葡聚醣硫酸鈉（DSS）小鼠結腸炎模型的影響 : 64 頁 : 國立屏東科技大學 系別:生物科技系 畢業時間及摘要別 : 一百零三學年度第二學期碩士學位論文摘要 研究生 : 農氏芳絨 指導教授: 黃卓治 教授 摘要內容: BALB/c 小鼠分為正常組、模型對照組(500 kDa 的和 40 kDa 的 DSS)和三個不同濃度的膽紅素處理組。口服給藥膽紅素(10，50，100 毫克/公斤體重)一周後，動物給予 3％DSS(40 kDa)的飲用水，除了正 常組外，進一步 個連續天給藥膽紅素有或沒有膽紅素治療。 40 kDa 的 DSS 造成小鼠嚴重的瀰漫性結腸炎，而 500 kDa 的 DSS 處理的小 鼠沒有病變。膽紅素可防止體重減輕和 DSS 誘導的結腸炎增加的疾病 活動指數(DAI)。三種不同濃度的膽紅素中，10mg / kg 的膽紅素組可 取得最好治療的結果。膽紅素抑制 DSS-引導的粘膜水腫、粘膜下糜爛 和結腸及多種組織的損害。本研究發現，膽紅素給藥可改善臨床症狀， 並降低小鼠模型中潰瘍性結腸炎(UC)的損害。 關鍵詞：膽紅素（BR）;葡聚醣硫酸鈉（DSS）;結腸炎;炎性腸病。 I Abstract Student ID : M10218032 Title of thesis : The Effects of Bilirubin in the Dextran Sulphate Sodium (DSS) Mouse Colitis Model Total Page : 64 pages Name of Institute : National Pingtung University of Science and Technology Department of Biological Science and Technology Graduate Date : June 29th, 2015 Name of Student : Nong Thi Phuong Nhung Degree Conferred: Master Adviser: Tzou – Chi Huang, Ph.D The content of abstract in this thesis: BALB/c mice were divided into normal group, colitis control group (500 kDa and 40 kDa DSS), and three different concentrations of bilirubintreated groups Bilirubin (10 or 50 or 100 mg/kg body weight) was administered orally After one week, animals were given 3% DSS (40 kDa) in drinking water, except those of the normal group, and for a further consecutive days with or without bilirubin treatment Mice treated with 40 kDa DSS developed most severe diffuse colitis, while mice treated with DSS of 500 kDa had no lesions Bilirubin prevents body weight loss and an increase in disease activity index (DAI) scores in mice with DSS-induced colitis Among three different concentrations of bilirubin, 10 mg/kg bilirubin group was achieved the best result Bilirubin treatment inhibited DSSinducted mucosal edema, submucosal erosions and colon damage in various tissues Bilirubin administration improves clinical signs and reduces the damage of colonic inflammation in a murine model of ulcerative colitis (UC) II Keywords: Bilirubin (BR); dextran sodium sulfate (DSS); Colitis; Inflammatory bowel disease III Acknowledgements First of all, I have to thank to my principle advisor, Professor Huang Tzou - Chi, for supporting my research with ideas, providing me the opportunity to further my scientific knowledge in such an excellent lab and giving me a push in the right direction in life when I needed the most Also, I wish to extend my gratitude to Mr Ellis Huang for his helps during the experiment in Veterinary Department Secondly, many thanks to all Professors from College of Biological Science and Technology who give me a lot of interesting lectures and good supports when I attended classes in Master program as well as in research My master would have remained a dream if I did not receive a wonderful chance to study at National Pingtung University of Science and Technology I appreciate all your advices and it will prepare me for whatever obstacles I will face in the future I would like to thank all members in BT 204 laboratory who helped me so much when I first come Taiwan in both life and research To my beloved-family in Viet Nam, Mum, and Dad for their support, encouragement and understanding For the countless times that I fell and stumbled, their unconditional love got my chin off the floor, helped me to overcome the obstacles and carry on with my long educational journey to get to where I am now Nong Thi Phuong Nhung 2015.06.29 IV Table of Content 摘要 I Abstract II Acknowledgements IV Table of Content V List of Table VIII List of Figure IX I INTRODUCTION 1.1 Background 1.2 Aim of the Study 1.3 Research Motivation II LITERATURE REVIEW 2.1 Inflammatory bowel diseases (IBD) 2.1.1 Classification of IBD 2.1.2 Pathophysiology 2.2 DSS - Animal models of inflammatory bowel diseases 2.2.1 Dextran sulfate sodium (DSS) 2.2.2 Advantage of DSS colitis mouse model 2.2.3 Colitis procedure V 2.2.4 Dextran sulfate sodium colitis mouse model 2.2.5 The molecular weight of DSS 10 2.2.6 Clinical features 12 2.2.7 Pathological features of DSS colitis 12 2.2.8 Pathogenesis of DSS colitis 13 2.3 Bilirubin 14 2.3.1 Bilirubin: chemical structure and formation 14 2.3.2 Bilirubin metabolism 15 2.3.3 Toxicity of bilirubin 17 2.3.4 Bilirubin as an antioxidant 19 III MATERIALS AND METHODS 22 3.1 Materials 22 3.1.1 Preparation of bilirubin 22 3.1.2 Preparation DSS solution 22 3.2 Experimental Design 22 3.3 Animals 24 3.4 Induction of colitis 24 3.5 Assessment of DSS colitis 25 3.6 Histopathological analysis 25 VI 3.7 Statistical Analysis 27 IV RESULTS AND DISCUSSIONS 27 4.1 The effects of DSS molecular weight to induce colitis in mice 27 4.2 Effects of bilirubin (BR) in the dextran sulphate sodium (DSS) mouse model – Induced experimental colitis 33 4.2.1 Oral administration of bilirubin prevents body weight loss in the DSS-induced colitis model 33 4.2.2 Assessment of disease activity index in mice 34 4.2.3 Bilirubin prevented the colonic shortening induced and prevented the reducing liver weight induced by DSS 36 4.2.4 Histologic findings in DSS-induced colitis 38 V CONCLUSION 47 REFERENCE 48 APPENDIX 58 Information of Author 64 VII List of Table Table 2.1: Variation of molecular weight and concentration of DSS used in the induction of colitis in some published studies 11 Table 2.2: Effects of Bilirubin in animal model in some published studies 20 Table 3.1: Disease activity index (DAI) scoring system 25 VIII List of Figure Figure 2.1: Conceptual framework for the pathogenesis of IBD Figure 2.2: Molecular structure of Dextran sulfate sodium (DSS) Figure 2.4: Chemical structure of the naturally occurring unconjugated BR 15 Figure 2.5:Oxidation-reduction cycles for bilirubin and GSH 16 Figure 3.1: The flowchart of experimental design 24 Figure 4.1: Changes in the body weight of mice with DSS-induced colitis 28 Figure 4.2: The disease activity index in mice 29 Figure 4.3: Histological analysis of mice organs 30 Figure 4.4: Oral administration of bilirubin prevents body weight loss in the DSS-induced colitis model 33 Figure 4.5: The disease activity index in mice 34 Figure 4.6: Bilirubin prevented the colonic shortening induced and prevented the reducing liver weight induced by DSS 36 Figure 4.7: Bilirubin reduces disease manifestation during DSS model in large intestine 39 Figure 4.8: Histological analysis of large intestine in DSS mice group 39 Figure 4.9: Histological analysis of spleen 42 Figure 4.10: Histological analysis of liver 43 Figure 4.11: Histological analysis of kidney 44 IX Neurons Against Oxidative Stress Injury Proceedings of the National Academy of Science of the United States of America 96: 2445–2450 Endler, G 2003 Is Low Serum Bilirubin an Independent Risk Factor for Coronary Artery Disease in Men but not in Women? 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