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 Bil

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

摘要

學號 : M10218032

研究計劃 : 膽紅素對在葡聚醣硫酸鈉（DSS）小鼠結腸炎模型的影響 總頁數 : 64 頁

學院名稱 : 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

Abstract

Title of thesis : The Effects of Bilirubin in the Dextran Sulphate

Sodium (DSS) Mouse Colitis Model

Name of Institute : National Pingtung University of Science and

Technology

Department of Biological Science and Technology

Graduate Date : June 29th, 2015 Degree Conferred: Master

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 bilirubin-treated 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 8 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 DSS-inducted 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)

Keywords: Bilirubin (BR); dextran sodium sulfate (DSS); Colitis;

Inflammatory bowel disease

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

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

1.1 Background 1

1.2 Aim of the Study 3

1.3 Research Motivation 3

II LITERATURE REVIEW 5

2.1 Inflammatory bowel diseases (IBD) 5

2.1.1 Classification of IBD 5

2.1.2 Pathophysiology 6

2.2 DSS - Animal models of inflammatory bowel diseases 7

2.2.1 Dextran sulfate sodium (DSS) 7

2.2.2 Advantage of DSS colitis mouse model 8

2.2.3 Colitis procedure 9

2.2.4 Dextran sulfate sodium colitis mouse model 9

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

3.7 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

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

List of Figure

Figure 2.1: Conceptual framework for the pathogenesis of IBD 6

Figure 2.2: Molecular structure of Dextran sulfate sodium (DSS) 7

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

I INTRODUCTION

1.1 Background

Inflammatory bowel diseases (IBD), collectively referred to as Crohn's disease (CD) and ulcerative colitis (UC), is a complex multiple disease that

involves inflammation of the intestine (Kumar K.G et al., 2011) These

chronic diseases result in significant morbidity and mortality with

compromised quality of life and life expectancy (Martina Persˇe et al., 2012;

Kanneganti M et al., 2011) Although most of the clinical and pathological

characteristics of CD and UC are same, the exact etiology and pathogenesis of

these disorders remain unclear (Goyal N et al., 2014) However, in recent

years, epidemiologic and genetic studies in man and particular, in IBD-related animal models, have suggested that it appears to involve a complex interaction between genetic susceptibility and environmental triggers including the resident microbial population of the intestinal tract (Abraham C

et al., 2009) These experimental animal models of colitis have also

contributed greatly to our current understanding of the immunological, pathological and physiological features of chronic intestinal inflammation

(Martina Persˇe et al., 2012) Hence, animal models resemble some of the

most important immunological and histopathological aspects of IBD in humans

The Dextran sulfate sodium (DSS) model of experimental colitis is one

of the most popular and widely utilized and characterized animal models of

ulcerative colitis (Chassaing et al., 2015) DSS is a synthetic sulphated

polysaccharide composed of dextran and sulphated anhydroglucose unit

(Ishioka T et al., 1987) The DSS-induced UC model has been well

characterized morphologically and biochemically (Chassaing et al., 2015)

Histologically, DSS produces submucosal erosions, ulceration, inflammatory cell infiltration and crypt abscesses as well as epithelioglandular hyperplasia

cytokines, IL6 and TNFα, which cause colitis (Huang T.C et al., 2010) This

model is particularly useful to study the contribution of the innate immune mechanism towards colitis as well as for the study of epithelial repair

mechanisms (Martina Persˇe et al., 2012)

DSS induces colitis, but the mechanism of action remains unknown

(Chassaing et al., 2015) Chassaing supposed that the result of damage to the

epithelial monolayer lining the large intestine allowing the dissemination of proinflammatory intestinal contents (bacteria and their products) into underlying tissue Colitis onset and severity may vary with many of these factors The varying responses to DSS appear to be dependent on not only DSS but also genetic and microbiological factors of animal Depending on the concentration, molecular weight, the duration, and frequency of DSS administration, animals show differential susceptibilities and responsiveness

to DSS-induced colitis (Martina Persˇe et al., 2012)

The final product of heme catabolism is the bile pigment bilirubin (BR) Bilirubin, a lipophilic linear tetrapyrrole, is generated by the sequential action of heme oxygenase (HO) and bilirubdin reductase (BVR) (Sedlak T.W

et al., 2004; Florczyk U.M et al., 2008) Bilirubin is efficiently metabolized

by the liver and secreted into bile as the water-soluble diglucuronide (Zucker

S.D et al., 2000) In mammalians, bilirubin plays a major role as antioxidant

at physiological concentrations (Qaisiya M., 2014) As little as 10 mM bilirubin protects cell against almost 10,000-fold higher concentrations of

H2O2 (Baran ̃ ano et al., 2002) Hence, bilirubin elicits substantial antioxidant

effects and is probably the most abundant endogenous antioxidant in mammalian tissue

The bilirubin was able to significantly reduce the release of the inflammatory mediators through inhibits the activation process of the superoxide producing NADPH oxidase by decreasing the potency of the cytosolic fraction and its inhibitory effect seems to be due to the hydrophobic

nature of the tetrapyrrole (Lanone S et al., 2004) However, the effects of

bilirubin on colitis are not fully understood Hence, the objective of this study was to investigate the immunoregulatory activity of bilirubin in the DSS-induced colitis mouse model, including daily clinical assessment of colitis and histopathological analysis

1.2 Aim of the Study

This research indicated that bilirubin has anti-inflammatory activity and may have therapeutic or prophylactic effects on colitis A mouse model of distal colitis induced by DSS that histologically resembles human UC was used in this study to evaluate the anti-colitis activity of bilirubin powder

1.3 Research Motivation

As previous studies, various factors may affect susceptibility to DSS and modify results DSS is sulfated polysaccharide with a highly variable

molecular weight, ranging from 5 kDa to up to 1400 kDa (Chassaing et al.,

2015) It was found that the molecular weight of DSS is very important factor

in the induction of colitis or colitis-induced dysplastic lesions

(carcinogenicity) (Martina Persˇe et al., 2012) Therefore, motivation for this

study is to evaluate the effects of the molecular weight of DSS to induce colitis

In addition, treatment of IBD depends on drugs such as aminosalicylates, corticosteroids, immunosuppressive agents, antibiotics, nutritional support, and the biologic agents However, use of these drugs is

5-sometimes limited by drug-induced toxicity and side effects (Amir A.S et al.,

2010) There is increasing need for alternative agents that may be equally or more effective, but toxicity-free, as well as cheaper Therefore, motivation for this study is to explore the health benefits of natural products particularly the bioactivity of pigment bile, bilirubin The bioactivity compounds that present

in natural products are considered as low toxic substances and safe to

consume Moreover, exploring the molecular mechanism of its immunoregulatory activities in the case of in vivo study With the deeper knowledge about bioactivity of natural product, researchers expect that the using of natural product particularly from bilirubin will be increased in the future, hence increase its value

II LITERATURE REVIEW

2.1 Inflammatory bowel diseases (IBD)

The term 'Inflammatory Bowel Disease' (IBD) refers to conditions characterized by a chronic relapsing inflammatory disorder of the bowel involving mainly the colonic mucosa and submucosa of the colon (Yao J

et al., 2010; Amir A.S et al., 2010) It may result in significant morbidity

and mortality, with compromised quality of life and life expectancy

(Martina Persˇe et al., 2012; Kanneganti M et al., 2011) The annual

incidence of ulcerative colitis was 24.3 per 100,000 person-years in developed countries, 6.3 per 100,000 person-years in developing countries Beside, Crohn’s disease ranges from 20.2 to 5.0 with increasing prevalence

of both IBDs observed over time (Molodecky N.A et al., 2012)

2.1.1 Classification of IBD

IBD includes the two main diseases that are Crohn’s disease (CD) and ulcerative colitis (UC), and both have overlapping and distinct clinical and pathological features (Uhlig, 2013) CD and UC are chronic remittent or progressive inflammatory conditions that may affect the entire gastrointestinal (GI) tract and the colonic mucosa Both of diseases may be associated with an

increased risk for colon cancer (Arthur et al., 2010) Most of the clinical and

pathological characteristics of CD and UC are same, but they also have some markedly different features, for example the location, depth and severity of

inflammation (Goyal N et al., 2014) Complications of UC differ from CD,

with increased risk of perforation, toxic colon and a higher incidence of bowel cancer Histopathological features include the presence of neutrophil infiltrates which form crypt abscesses Inflammation is accompanied by ulceration, edema, and hemorrhage along the length of the colon (Xavier

R.J et al., 2007)

2.1.2 Pathophysiology

Although the exact cause of IBD remains undetermined, it appears to

be related to both genetic and environmental factors Among the pathological findings associated with IBD are dysregulated to response of innate and adaptive immune system, loss of tolerance to commensal bacteria, disrupted mucosal barrier, an increase in inflammatory mediators and oxidative stress

Figure 2.1: Conceptual framework for the pathogenesis of inflammatory

bowel disease (IBD) DC, dendritic cell; IBD, inflammatory

bowel disease; NSAIDs, non-steroidal anti-inflammatory drugs;

TReg cell, regulatory T cell (Neurath M.F et al., 2014)

Genetic and environmental factors induce impaired barrier function in the intestinal mucosa Initiating triggers may involve infections in some patients Altered barrier function subsequently induces the translocation of commensal bacteria and microbial products from the gut lumen into the bowel wall, which leads to immune cell activation and cytokine production If acute mucosal inflammation cannot be resolved by anti-inflammatory mechanisms and the suppression of pro-inflammatory immune responses, chronic intestinal inflammation develops In turn, chronic inflammation may cause complications of the disease and also tissue destruction, which are both driven

by mucosal cytokine responses (Neurath M.F et al., 2014)

2.2 DSS - Animal models of inflammatory bowel diseases

2.2.1 Dextran sulfate sodium (DSS)

Figure 2.2: Molecular structure of Dextran sulfate sodium (DSS)

(http://www.lookfordiagnosis.com)

Dextran sulfate sodium (DSS), (C6H7Na3O14S3)n, is a complex polymer of glucose synthesised by certain bacteria, most commonly

Leuconostoc spp and Streptococcus spp, from sucrose It is a water-soluble,

negatively charged sulfated polysaccharide with a highly variable

molecular weight ranging from 5 to 1400 kDa (Chassaing et al., 2015) DSS

chlorosulphonic acid The sulphur content is approximately 17% which corresponds to approximately two sulphate groups per glucosyl residue of the dextran molecule It is a white powder at room temperature, and highly

soluble in water (100 mg/ml) (Solomon L et al., 2010)

2.2.2 Advantage of DSS colitis mouse model

These mice models can be included: spontaneous colitis models, inducible colitis models, adoptive transfer models and genetically modified

models (Wirtz S et al., 2007a; Wirtz S et al., 2007b) Although these animal

models do not represent the whole complexity disease on human, they are valuable and indispensable tools that provide a wide range of options for investigating involvement of various factors into the pathogenesis of IBD and

evaluate different therapeutic options (Martina Persˇe et al., 2012)

Because chemically induced murine models of intestinal inflammation are simple to induce, the onset, duration, and severity of inflammation is immediate and controllable, they are one of the most commonly used models

in colitis researches Dextran sulphate sodium (DSS) induced colitis is established animal models of mucosal inflammation These models have been used for over two decades in the study of IBD pathogenesis and preclinical

well-studies (Wirtz S et al., 2007a; Wirtz S et al., 2007b)

When compared to other animal models of colitis, the DSS-induced colitis model has some advantages Only changing the concentration of administration of DSS and cycle in rats and other strains of mice, an acute, chronic, or relapsing model can be produced easily Additionally, dysplasia that resembles the clinical course of human UC occurs frequently in the

chronic phase of induced colitis (Martina Persˇe et al., 2012)

DSS-induced model for studying colitis-associated carcinogenesis has been recently reviewed by others (Kanneganti M., 2011) Furthermore, studies that validated DSS model by using different therapeutic agents for IBD show that DSS-induced colitis can be used as a relevant model for the

translation of mice data to human disease (Melgar S et al., 2008a)

2.2.3 Colitis procedure

Colitis is induced by addition of DSS to drinking water (ad libitum) Depending on the susceptibility of the species (such as BALB/c mice are more susceptible) or the molecular weight of DSS, a dose range of 3–10 % DSS is commonly administered for 7 to 10 days to induce an acute inflammation Acute colitis may be extrapolated to chronic colitis by administering in three to five cycles of DSS administration with a one to two

week rest between cycles (Tran et al., 2012), while in some species like adult

female mice of the outbred strain Him: OF1, dose of less than 3 % can also be

used to induce colitis (Mitrovic et al., 2010)

2.2.4 Dextran sulfate sodium colitis mouse model

The animals may easily develop acute or chronic colitis or even colitis- induced dysplastic lesions by changing the concentration, the duration, and frequency of DSS administration Mice show differential susceptibilities and responsiveness to DSS-induced colitis The varying responses to DSS appear

to be dependent on DSS such as concentration, molecular weight, duration of DSS exposure, manufacturer, and batch Moreover, genetic (strain, substrain and gender) and microbiological factors (microbiological state and intestinal flora) of animal also effect significantly in induced colitis in mice (Figure 2.3) Colitis onset and severity may vary with many of these factors (Martina

Persˇe et al., 2012; Goyal N et al., 2014) Additionally, stress can be one of them (Melgar S et al., 2008b)

Figure 2.3: Schematic simplified representation of various factors effect to

DSS-induced colitis (Martina Persˇe et al., 2012)

2.2.5 The molecular weight of DSS

DSS is sulfated polysaccharide with a highly variable molecular weight, ranging from 5 kDa to up to 1400 kDa Based on publish researches, the molecular weight of DSS is assessed as a factor very important in the

induction of colitis (Kitajima S et al., 2000) or colitis-induced dysplastic

lesions (carcinogenicity) The severity of colitis and carcinogenic activity depend on the administration of DSS at different molecular weights (i.e., 5

kDa, 40 kDa, and 500 kDa) (Martina Persˇe et al., 2012)

In 2000, Kitajima and his colleagues compared three commercial preparations of 5% DSS (molecular weight 5, 40 and 500 kDa, respectively) administered for 7 days in BALB/c mice They found that the three preparations led to variations in the disease site, that the severity of colitis observed was greatest with the 40 kDa DSS and that no colitis developed with

the 500 kDa preparation Mice treated with DSS of 5 kDa developed milder

form of colitis (Kitajima S et al., 2000) Given the same as this result, while

carcinogenic activity was not induced by DSS of larger (520 kDa) or smaller (9.5 kDa) molecular weights, only induced by DSS of 54 kDa induced

carcinogenic activity in colon (Martina Persˇe et al., 2012) Molecular

weight of DSS can affect location of colitis as well Mice treated with 5 kDa DSS developed relatively patchy lesions mainly in the cecum and upper colon On the contrary, mice treated with 40 kDa DSS developed most severe

diffuse colitis in the middle and distal third of the large bowel (Kitajima S et

al., 2000)

Table 2.1: Variation of molecular weight and concentration of DSS used in

the induction of colitis in some published studies

MW of DSS

(kDa)

tion (%)

Concentra-Animal model


Duration (days)

mice 2000 54; 520; 9,5 2.5 AC1 rats 480 Hirono I et al.,

of disease will appearance early, such as occult blood and diarrhea are usually the earliest features and may occur as day 2 In the acute model, the inflammation may be fully established within 7–10 days However, animals

tend to recover if the DSS administration is stopped (Solomon L et al., 2010)

2.2.7 Pathological features of DSS colitis

The inflammation is usually limited to the colon with macroscopic

features such as shortened oedematous colon with areas of haemorrhage and ulceration When acute DSS-colitis appearance, histological changes include mucin depletion, epithelial degeneration and necrosis leading to disappearance of epithelial cells which further leads to the formation of cryptitis and crypt abscesses The migration of neutrophils is major cause to lead these cryptitis and crypt abscesses These histological features are the common characteristics of human IBD but rarely reported in DSS-induced colitis The changes in chronic phase appear after few weeks of DSS administration In this period, crypt architectural disarray, increasing the distance (widening of the gap) between crypt bases and muscularis mucosa, deep mucosal lymphocytosis and transmural inflammation are recorded

(Martina Persˇe et al., 2012) In the acute phase of DSS, there is increased

apoptosis and decreased proliferation of epithelium further causing relevant

leaks in the epithelial barrier (Araki et al., 2010)

2.2.8 Pathogenesis of DSS colitis

In fact, the exact mechanism through which DSS initiates colitis is unknown One possible mechanism is that DSS may direct alteration of gut permeability As early as day 1, tight junction proteins such as zona occludens-1 were directly reduced by DSS, leading to increased permeability

by day 3, changes that preceded colonic inflammation (Kitajima et al.,

1999a) There is an uptake of small amounts of DSS by gut macrophages, liver Kupffer cells and mesenteric lymph nodes, detectable as early as day 1

in both acute and chronic DSS-induced colitis (Kitajima et al., 1999b) Taken

together these findings support an early role for a permeability defect in the pathogenesis of DSS colitis

Beside, another mechanism of DSS is also considered A dependent direct cytotoxicity of DSS on the colonic mucosa leads to alteration of integrin-α4 and M290 subunit levels on epithelial cells disrupting

concentration-their interaction with the γδ-intraepithelial T cells (Ni et al., 1996) Although

γδ-intraepithelial T cells have an unknown function, they are thought to be involved in mucosal protection (and healing) against various stimuli including

DSS (Chen et al., 2002) Hence the exact mechanism of induction of colitis

by DSS is clearly undetermined, but the fact that crypt loss and increased permeability usually precede inflammation suggests that the initial insult is most likely at the epithelial cell level, with inflammation developing secondarily

2.3 Bilirubin

Bile pigments are endogenous compounds belonging to the porphyrin

family of molecules They are divided into bilirubin and biliverdin These pigments are intensely coloured and can been seen in the skin during jaundice (bilirubin) and in the green (biliverdin) and yellow (bilirubin) colour of bruises In long time ago, bile pigments and bilirubin were thought of as useless by-products of heme catabolism that can be toxic if they accumulate

(Bulmer A.C et al., 2008) However, in the past 20 years, a growing number

of research groups have investigated the possible beneficial effect of bile pigments in the human body Very important early findings showed bile pigments were part of a group of compounds called antioxidants, thereby protecting from free radicals that are produced constantly in the human body

(Stocker R et al., 2004)

2.3.1 Bilirubin: chemical structure and formation

Although bilirubin appears to be a simple molecule, the unconjugated bilirubin (UCB) IXa 4Z, 15Z molecule, the major compound in mammals, has

a peculiar stereo-chemical structure (Figure 2.4) In fact, all hydrophilic groups are involved in strong hydrogen bonds to lead turning the molecule

into a closed molecule with a ridge-tile conformation (Bonnett R et al.,

1976) Because these hydrogen bonds render UCB hydrophobic and they also shield the central –CH2–, the diazo-reagent cannot accessible with them UCB base on the pH of the plasma, bile or urine, they can be present flexible such

as uncharged diacid, as a mono anion or as a dianion The uncharged diacid is

by far the dominant species at low and physiological pH but the ionized

fractions become more important in an alkaline milieu (Ostrow J.D et al.,

1994)

Figure 2.4: Chemical structure of the naturally occurring unconjugated

bilirubin IXa (Fevery J et al., 2008)

2.3.2 Bilirubin metabolism

Bilirubin is an oxidative end product of heme catabolism It is a lipophilic linear tetrapyrrole, abundant in blood plasma, which occurs uniquely in mammals Heme oxygenase (HO) catalyzes the cleavage of the heme ring to form ferrous iron (Fe2+), carbon monoxide (CO) and biliverdin (BV) Biliverdin is rapidly reduced by biliverdin reductase (BVR) to bilirubin

(BR), the major physiological antioxidant (Baran ̃ ano et al., 2002; Florczyk U.M et al., 2008) Bilirubin is a highly lipophilic molecule despite containing

hydrophilic carboxyl groups They are unavailable for interaction with water because intramolecular hydrogen bonds to the pyrrole nitrogen atoms Bilirubin is found in blood bound to plasma albumin, which transports it to the liver, where it is conjugated to hydrophilic acceptors Because bilirubin is toxic and insoluble, it must be glucuronidated before being excreted in the

bile (Kapitulnik J et al., 2004)

Figure 2.5: Oxidation-reduction cycles for bilirubin and GSH

(Sedlak T.W et al., 2004)

Hemoglobin, released from senescent red blood cells and heme containing enzymes, are the major source of heme for bile pigment synthesis

(Schmid R et al., 1975) The catabolism of heme occurs in the cells of the

reticulo-endothelial system both liver and spleen From the body each day, bile pigments are excreted of approximately 300 mg (Stocker R., 2004) The metabolism of bile pigments in the human body is illustrated in Figure 2.5 Heme is released from a series of hemeproteins, including hemoglobin and cytochrome P450, and metabolized by heme oxygenase to form carbon

monoxide, biliverdin, and free iron (Schmid R et al., 1975) Lipophilic

reactive oxygen species act directly on bilirubin, leading to its oxidation to biliverdin Biliverdin is being reduced by biliverdin reductase to bilirubin,

permitting bilirubin to detoxify a 10 000-fold excess of oxidants Soluble oxidants are detoxified by glutathione (GSH), a cycle that requires 2 enzymes,

glutathione peroxidase and glutathione reductase (Sedlak T.W et al., 2004)

Interestingly, the metabolism of heme is colorfully displayed in the time course of bruising where the blue-green color of biliverdin is followed

by the yellow coloration of bilirubin After bilirubin is formed, this hydrophobic compound is bound to serum albumin, for which it has a strong affinity (Brodersen R., 1979) The circulation delivers bilirubin to the liver where it is actively and passively absorbed into the hepatocyte Intercellular glutathione-S-transferase then transports bilirubin to the endoplasmic reticulum where glucuronic acid conjugates are formed by UDP glucuronosyl

transferase (UGT1A1) (Kamisako T et al., 2000) Conjugation renders

bilirubin soluble water It is then actively transported into the bile caniculi by multidrug resistance protein 2 (MRP2) and the bilirubin conjugates are then directed into the duodenum via the bile duct As bilirubin glucuronides enter the gastrointestinal tract, bacterial enzymes including b-glucuronidase, hydrolyse the bilirubin esters forming unconjugated pigment Some of the

unconjugated bilirubin is reabsorbed (Alonso E.M et al., 1991) and

re-excreted The remaining pigments are reduced by the intestinal bacterial flora

to urobilins and stercobilins, which provide the distinctive coloration of faeces

2.3.3 Toxicity of bilirubin

Bilirubin is widely known as an end product of heme metabolism The great interest in understanding the regulation of expression and enzymatic activity of heme oxygenase results from the wide array of biological effects displayed by the products of heme degradation Carbon monoxide is a putative neural messenger and a major cardiovascular regulator but can also compete with O2 for binding to hemoglobin The iron released from heme has been shown to be involved in cellular toxicity as a result of its capacity to

induce the formation of reactive oxygen species (ROS) Biliverdin and bilirubin are potent antioxidants and protect cells from oxidative stress on the one hand, whereas bilirubin displays neurotoxicity on the other hand This review provides a molecular insight into the complex cytotoxic and cytoprotective effects of bilirubin under both physiological and pathological

conditions (Kapitulnik J et al., 2004) Very high levels of serum bilirubin

lead to its accumulation in the brain, causing kernicterus Almost all newborns display some level of jaundice, and some display high enough serum bilirubin levels that phototherapy or exchange transfusion is considered

(Sedlak T.W et al., 2014)

Free unconjugated bilirubin exhibits a wide range of toxicity to many cell types, particularly neuronal cells When the plasma levels of UCB are excessively elevated and surpass the capacity of albumin for high-affinity binding of UCB, the unbound (free) fraction of the pigment increases Free UCB can easily enter the cells by passive diffusion and cause toxicity The most vulnerable site is the central nervous system UCB binds to discrete brain areas, such as the basal ganglia (kernicterus), and produces a wide array

of neurological deficits collectively known as bilirubin encephalopathy These include irreversible abnormalities in motor, sensory (auditory and ocular), and

cognitive functions (Kapitulnik J et al., 2004) All known toxic effects of

bilirubin are abrogated by binding to albumin Cerebral toxicity (kernicterus) from bilirubin occurs when the molar ratio between bilirubin and albumin exceeds 1.0 Severe and chronically elevated bilirubin production/re-absorption, which exceeds the liver’s capacity to excrete bilirubin, i.e in neonatal hyperbilirubinemia, can result in bilirubin toxicity and acute or chronic neurological dysfunction The toxic mechanism of bilirubin has been extensively investigated Bilirubin toxicity is usually seen during exaggerated neonatal hyperbilirubinaemia and in patients with Crigler–Najjar syndrome at

all ages (Wang X et al., 2006)

In contrast to the great interest in elucidating the molecular

mechanisms of UCB-induced neurotoxicity, fewer studies addressed the

possibility that UCB is also toxic for non-neural cells (Kapitulnik J et al.,

2004) Blood erythrocytes are the immediate target for binding of free (unbound) UCB This binding is greatly increased at UCB/albumin molar ratios higher than 1.0, leading to morphologic changes, cell lysis, and loss of membrane lipids Alterations of membrane dynamic properties of erythrocytes were accompanied by the release of phospholipids and cholesterol The loss of inner-located phospholipids induced the externalization of phosphatidylserine This membrane perturbation was increased by acidosis, indicating that the UCB species interacting with the membrane is the uncharged diacid

2.3.4 Bilirubin as an antioxidant

Early, indirect evidence for an antioxidant activity of bilirubin and biliverdin comes from studies showing that small quantities of the pigment stabilize vitamin A and β-carotene during intestinal uptake, and that animals with low plasma bilirubin show early symptoms of vitamin E deficiency In particular, UCB has a major capacity to protect phospholipids against oxidative damage from free radicals generated from the peroxisome (Stocker

et al., 1987) UCB effectively inhibited oxidation of LDL near physiologic

serum levels This effect was nearly twenty times more potent than that of

Vitamin E, a known scavenger of free radicals (Wu et al., 1994) There have

also been early reports on the reaction of bilirubin with reactive oxygen species Unconjugated bilirubin efficiently scavenges singlet oxygen and serves as a reducing agent for certain peroxidases, including horseradish peroxidase and prostaglandin H synthase in the presence of hydrogen

peroxide or organic hydroperoxides (Brodersen R et al., 1969) Similarly,

bilirubin glucuronides are oxidized by rat liver microsomes in the presence of

lipid peroxides (Cuypers H.T et al., 1983)

Bilirubin in its free, albumin-bound, and conjugated forms contains an

extended system of conjugated double bonds and a pair of reactive hydrogen atoms (depicted in Figure 2.5), the latter of which are most likely involved in antioxidant activity via H-donation to an incipient radical, such as lipid peroxyl radical (LOO·), to form lipid hydroperoxide (LOOH) and bilirubin radical (Bilirubin·):

LOO· + Bilirubin → LOOH + Bilirubin·

The powerful antioxidant effect of UCB translate clinically: asymptomatic males with low total bilirubin levels have more severe coronary artery disease on angiography In fact, low total serum bilirubin is a strong, independent risk factor for coronary disease, with a significance approaching that of established risk factors such as smoking and hypertension (Schwertner

et al., 1994) The inverse relationship between total bilirubin and coronary

artery disease has been consistently confirmed (Hopkins et al., 1996; Kronenberg et al., 2002; Endler et al., 2003) Overall, a 40–50% reduction in

coronary artery disease prevalence is noted in males with total bilirubin

higher than 8 mg/L independent of other risk factors (Endler et al., 2003)

However, what matters most is what goes on inside cells Remarkably, as little as 10 nanomolar bilirubin can protect cultures from the oxidant stress of

10 000 times higher concentrations of hydrogen peroxide (Dore S et al.,

1999) During the oxidant stress associated with myocardial and cerebral infarcts, infection, inflammation, and various causes of ischemia, the intracellular environment is exposed to high concentrations of reactive oxygen species It has long been assumed that the principal cellular antioxidant is the peptide glutathione (GSH), whose tissue concentrations are millimolar, presumably sufficient to cope with most instances of oxidative stress By contrast, levels of bilirubin in rodent tissues are only 10 to 50 nanomolar, at least 10 000 times lower than concentrations of GSH (Sedlak

T.W et al., 2004)

Table 2.2: Effects of Bilirubin in animal model in some published studies

Animal Model Treatment Effects of BR Reference

injection, hind paw injection

Inhibits iNOS expression and

Reduce ischemia injury on rat small intestine

Kadl A et al.,

2007 Rats CsA-

induced

tubular

injury

Intravenously inject

Protected against CsA-induced tubular injury

Ameliorates the extent of

intestinal IR injury

Protect Against Oxidative Stress-Induced Retinal Degeneration

Oveson et al.,

2011

III MATERIALS AND METHODS

3.1 Materials

3.1.1 Preparation of bilirubin

Bilirubin (MW: 584.66; Sigma, Switzerland) was used in these experiments Because bilirubin are sensitive to light, all preparations and reactions were carried out in dim light Bilirubin powder was solubilized in DMSO and adjusted with oil (1volume DMSO: 9 volume oil)

3.1.2 Preparation DSS solution

DSS (500 kDa; Sigma, Sweden) and DSS (40 kDa; Sigma, Sweden)

were used in these experiments DSS powder was solubilized in water ad

libitum with 3 % concentration

3.2 Experimental Design

The present study was carried out at Biological Science and Technology, National Pingtung of Science and Technology, Taiwan from September 2014 to June 2015 This study was performed with a total 36 mice, including 6 different treatments The mice were pathogen-free male The experiment divided into 6 parts based on the difference of DSS molecular weigh and the difference of bilirubin level treatments All experiments in this research were performed following the below diagram:

Figure 3.1: The flowchart of experimental design

Male BALB/c mice

12 weeks, 24-28 g

Effects of BR in DSS mouse model

Effects of molecular weight DSS to induce

colitis

Control group

Only water (n=6)

3% DSS (500 kDa) (n=6)

3% DSS (40 kDa) (n=6)

3% DSS (40 kDa)

10mg/kg BR (n=6)

Assessment of DSS colitis daily

Body weight Blood

in stool

Stool consistency

Histopathalogical anaalysis Collecting organ Sacrifice

50mg/kg BR (n=6)

100mg/kg BR (n=6)

8 days

8 days

7 days

3.3 Animals

Pathogen-free male BALB/c mice, 12 weeks of age were studied Mice were maintained for one week on normal laboratory before initiating the study All mice weighted about 24–28 g at the beginning of the study The animals were bred under standard conditions and maintained in a 12-h light/12-h dark cycle at 22°C plus or minus 1°C and given food and tap water

ad libitum in accordance with Taiwan Office Regulations throughout the

experiments

3.4 Induction of colitis

Colitis was induced by modification of the method of DSS-induced colonic inflammation as previously described Colitis was induced by administering 3% (wt/vol) DSS dissolved in distilled water to mice for 8 days

Control animals received distilled water ad libitum The mice were checked

daily for body weight and assessed for signs of rectal bleeding and diarrhea

The animals were divided into 6 groups including: normal group, two colitis groups, and three bilirubin-treated groups In the first group, designated

as the normal control group (n = 6), colitis was not induced and no therapeutic intervention was performed The second group and the third group, designated

as the DSS group (n = 6), received 3% DSS (500 kDa) and 3% DSS (40 kDa) for 8 days in the second week of experiment In the bilirubin-treated groups, bilirubin (10 or 50 or 100 mg/kg body weight) (n=6/group) was administered daily per oral route for 7 days After one week of treatment with bilirubin, animals were given 3% DSS (40 kDa) in drinking water for a further 8 consecutive days At the end of the treatment period, the animals were sacrificed Organs were removed and analysed histologically

3.5 Assessment of DSS colitis

A disease activity index (DAI) was determined by scoring changes in body weight, stool hemoccult positivity or gross bleeding, and stool

consistency in accordance with the method previous described (Huang T.C

et al., 2010) At indicated time points, animals were assessed for body

weight Additionally, rectal bleeding and stool consistency were monitored daily Based on the obvious differences in appearance between the treatment groups, grading was performed by independent researchers Stool consistency was assessed using the following 4-point scale: 0, normal; 2, Loose stools; and 4, Watery diarrhea The intensity of the guaiac paper test was scored by the following scale: 0, negative; 2, Slight bleeding; and 4, Gross bleeding A validated clinical disease activity index ranging from 0 to 4 was calculated using the following parameters: stool consistency, presence of fecal blood, and changes in body weight (Table 3.1) The mice were sacrificed at day 15, and the length and weight of the colons were measured

Table 3.1: Disease activity index (DAI) scoring system

2 Slight bleeding Loose stools

3.6 Histopathological analysis

Histological analysis of animal tissues following an experimental procedure or intervention generally encompasses the following steps performed: tissue fixation, processing, embedding and sectioning, slide preparation, staining, and microscopic analysis (Tulis D.A., 2007) Processing

is performed as following steps:

- Tissue fixation: Organs (colon, kidney, spleen, lung, liver and heart) were fixed in 10% formalin solution (pH=7) for at least 24 hours

- Tissue processing: The tissue must be dehydrated after fixed Then, they were cleared in order for adequate paraffin infiltration, embedding, and sectioning This step involves tissues incubated in a series of graded alcohols

to remove water, usually 70% through 100% alcohols All incubation steps are performed under vacuum, pressure and at room temperature except the liquid paraffin steps, which are performed under elevated temperature Tissue processing was set up overnight hours with the sample incubating in liquid paraffin and ready for embedding the next morning

- Embedding and sectioning: The embedder should be pre-heated (at least 3 degrees above the melting point of the wax) when using a paraffin-embedder All the paraffin pellets are in liquified form, and that the cold plate

is pre-chilled In short time the block will start to harden Once the paraffin is completely solidified, move the block from the cold plate, release the stainless steel base mold After that, the block was stored at room temperature until ready for sectioning

- Slide preparation: Sections of tissue were cut at 5 μm After that, mounted on clean glass slide Then, dried overnight at 37oC Once dried, store the slides in an appropriate dry location until ready for further histological assays

- Staining: The sections were cleared, hydrated, stained with hematoxylin and eosin (H&E) Immune organs were being stained were colon, kidney, spleen, lung, liver and heart

- Analysis: Sections were evaluated using light microscopy

3.7 Statistical Analysis

All data were presented as mean ± standard deviation (SD) The statistical significances between each treatment groups were compared and analyzed by one way ANOVA and Duncan’s multiple range tests using SPSS software

(version 20.0) Data with p < 0.05 were considered statistically significant

IV RESULTS AND DISCUSSIONS

4.1 The effects of DSS molecular weight to induce colitis in mice

Spontaneous and drug-induced forms of Ulcerative colitis (UC) in

animals have been used as models for human UC (Okayasu et al., 1990)

These animal models have been used to study the mechanism of inflammatory bowel disease (IBD) and the therapeutic effects of anti-inflammatory agents Dextran sulphate sodium (DSS)-induced colitis is well-established animal models of mucosal inflammation that has been used for over 2 decades in the study of IBD pathogenesis and preclinical studies

(Martina Persˇe et al., 2012)

In recent year, researchers published about the effects of the molecular weight of DSS on the induction of colitis or carcinogenicity in the rodent model Axelsson suggested that a high sulfate content per molecular of DSS is important for the induction of colitis They observed a significant reduction in colitis-induction when the sulphur content is below 16.6% and augmentation

of inflammatory activity with increasing molecular weights (Axelsson et al.,

1996) In 1983, Hirono reported a relationship between the molecular weight

of DSS and carcinogenicity in rats They found that DSS of large or smaller molecular weights had no carcinogenic activity when DSS at three different molecular weights, 9.5 kDa, 54 kDa, and 520 kDa, was administered to rats at

2.5 % for 480 days (Hirono et al., 1983) These studies indicate that the

molecular weight of DSS is an important factor in the induction of colitis and colonic tumors in rodents