Comparative Evaluation of Prednisolone and Infliximab on Intestinal Pathology, Oxidative

Stress, and Clinical Symptoms in TNBS-Induced Crohn's Disease Adult Male Sprague
Dawley Rats

CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGROUND OF STUDY

Inflammatory bowel disease (IBD) is characterized by repetitive episodes of inflammation of the

gastrointestinal tract caused by an abnormal immune response to gut microflora (Hughes et al.,

2019). Inflammatory bowel disease encompasses two types of idiopathic intestinal disease that

are differentiated by their location and depth of involvement in the bowel wall (Halligan et al.,

2019). Ulcerative colitis (UC) involves diffuse inflammation of the colonic mucosa (Reider et

al., 2019). Most often, UC affects the rectum (proctitis), but it may extend into the sigmoid

(proctosigmoiditis), beyond the sigmoid (distal ulcerative colitis), or include the entire colon up

to the cecum (pancolitis) (Taylor et al., 2019). Crohn disease (CD) results in transmural

ulceration of any portion of the gastrointestinal tract (GI), most often affecting the terminal ileum

and colon (Stoker et al., 2019).

TNBS is a chemical compound that has been utilized to induce colitis in experimental animals

(Trigo et al., 2013). This model is particularly valuable for its ability to replicate key aspects of

IBD pathology, making it a relevant tool for studying mucosal immune responses and potential

treatments.2,4,6-Trinitrobenzene sulfonic acid (TNBS) is a hapten, which when binds to tissue

protein turns into an antigen and elicits number of immunologic responses (Cheon et al., 2012).

TNBS-induced colitis is a delayed-type hypersensitivity reaction to haptenized proteins, whereas

DSS colitis is the result of a disruption in epithelial barrier (Wang et al., 2014). The major

inflammatory mediators involved in this TNBS-induced colitis are leukotriene B4 (LTB4) and
the monhydroxyl fatty acids 5-HETE, 12-HETE and 15-HETE (Jaggi et al., 2014). Ethanol is

used in high concentration as a vehicle for the TNBS administration (Kawada et al., 2017).

Ethanol acts as a mucosal barrier breaker so that TNBS can enter the mucosa to induce colitis

where it binds to the amino group of lysine and modify cell surface proteins (Kawada et al.,

2017).

Prednisolone is a synthetic glucocorticoid with potent anti-inflammatory and immunosuppressive

properties. It is widely used in the treatment of inflammatory bowel diseases (IBD), including

ulcerative colitis, due to its ability to reduce inflammation and alleviate symptoms such as

abdominal pain, diarrhea, and rectal bleeding (Toruner et al., 2019). It exerts its therapeutic

effects by binding to glucocorticoid receptors in target cells, leading to the modulation of gene

expression and inhibition of pro-inflammatory pathways. It suppresses the production of various

inflammatory mediators, including cytokines and chemokines, and blocks the recruitment and

activation of immune cells involved in the pathogenesis of ulcerative colitis (Danese & Fiorino,

2024). It is commonly used as a first-line therapy for inducing remission in patients with

moderate to severe ulcerative colitis. It is particularly effective in cases where conventional

treatments such as 5-aminosalicylates or corticosteroids are insufficient to control disease

activity. Prednisolone is often prescribed for short-term use to rapidly suppress inflammation and

alleviate symptoms, followed by tapering to lower doses or discontinuation to minimize long-

term adverse effects (Travis et al., 2018).Despite its efficacy, prednisolone is associated with a

range of adverse effects, especially when used at high doses or for prolonged periods. These

adverse effects include immunosuppression, osteoporosis, hypertension, hyperglycemia, weight

gain, mood changes, and increased susceptibility to infections. Patients receiving prednisolone
therapy require close monitoring and may require additional interventions or alternative

treatments to manage or prevent these adverse effects (Feuerstein et al., 2019).

Monoclonal antibodies, also known as mAbs, are a type of immunoglobulin. These antibodies

are produced by uniform hybrid cells (B cells) originating from the same parent cell. Their

function involves binding to specific proteins on pathogens or abnormal cells, thereby

neutralizing their ability to attach to or invade new cells. Monoclonal antibodies, a form of

biological therapy, have revolutionized IBD treatment by specifically targeting immune-related

pathways implicated in chronic inflammation (Posner et al., 2019).

Studies like the research conducted by Feagan et al., (2016) have explored various monoclonal

antibodies, including anti-tumor necrosis factor (TNF) agents like infliximab and adalimumab,

and other biologics such as vedolizumab and ustekinumab. These antibodies act by neutralizing

key pro-inflammatory molecules, thereby modulating immune responses responsible for

exacerbating IBD symptoms. The use of monoclonal antibodies is particularly prominent in

moderate to severe cases of IBD, offering a more targeted and personalized treatment approach

compared to conventional therapies. These biologics have demonstrated efficacy in inducing and

maintaining remission, reducing the need for surgeries and hospitalizations, and improving

patients' quality of life (Feagan et al., 2016).

1.2 Statement of problem

Inflammatory bowel disease (IBD), encompassing conditions like Crohn's disease and ulcerative

colitis, poses significant challenges in management due to its chronic nature and variable

response to existing therapies. 2,4,6-trinitrobenzene sulfonic acid (TNBS) serves as a reliable

inducer of chronic colitis in animal models, reflecting aspects of human IBD pathology

characterized by chronic inflammation and mucosal damage.

This study seeks to address gaps in current therapeutic strategies for IBD by evaluating and

comparing the therapeutic efficacy of two distinct treatment modalities in TNBS-induced chronic

disease in adult male Sprague Dawley rats. Specifically, the study aims to:

 Assess Histological Changes in the Large Intestine: Investigate the impact of the anti-

inflammatory agent and monoclonal antibodies on large intestine histology in TNBS-

induced chronic disease. This includes evaluating mucosal architecture, epithelial

integrity, inflammatory cell infiltration, and overall tissue damage.

 Evaluate Antioxidant Enzymes Activity: Measure the effect of treatment interventions on

antioxidant enzymes activity within the large intestine. Antioxidant enzymes such as

superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) play

crucial roles in mitigating oxidative stress, which is implicated in the pathogenesis of

IBD.

 Determine Levels of Lipid Peroxidation: Assess the extent of lipid peroxidation in the

large intestine as a marker of oxidative stress-induced tissue damage. Lipid peroxidation

products, such as malondialdehyde (MDA), serve as indicators of oxidative damage and

inflammation severity in colonic tissues.

Despite advancements in IBD therapeutics, there remains a critical need for effective treatments

that can attenuate inflammation, preserve mucosal integrity, and modulate oxidative stress

responses. Understanding how anti-inflammatory agents and monoclonal antibodies impact these

critical aspects of TNBS-induced chronic disease pathology is essential for optimizing treatment

strategies and improving patient outcomes.

1.3 Aim of study

This study aims to evaluate and compare the therapeutic efficacy of anti-inflammatory

agents(Prednisolone) and monoclonal antibodies (infliximab) in the management of TNBS-

Induced Crohn’s disease (CD) model in Adult Male Sprague Dawley Rats. Additionally, the

study aims to investigate the effect of these treatments on large intestine histology, antioxidant

enzymes activity and level of lipid peroxidation, in the CD model.

1.4 Objectives of the study

The objectives of this study are to;

i. Evaluate the effectiveness of anti-inflammatory agents(Prednisolone) and monoclonal

antibodies (infliximab) in ameliorating pathological changes in the large intestine and

its histology of TNBS- Induced Crohn’s disease (CD) model in Adult Male Sprague

Dawley Rats

ii. Quantify the level of antioxidant enzymes and level of lipid peroxidation such as

Malondialdehyde (MDA), as a marker of oxidative stress in the large intestine of

TNBS- Induced Crohn’s disease (CD) model Sprague- Dawley treated with anti-

inflammatory agents and monoclonal antibodies (infliximab).

iii. Quantify the level of antioxidant enzymes and level of lipid peroxidation such as

Malondialdehyde (MDA), as a marker of oxidative stress in the large intestine of

TNBS- Induced Crohn’s disease (CD) model Sprague- Dawley treated with anti-

inflammatory agents(Prednisolone) and monoclonal antibodies (infliximab)

iv. Evaluate the effectiveness of anti-inflammatory agent (prednisolone) and monoclonal

antibodies (Infliximab) in ameliorating pathological changes in the secretory function

 of the large intestine of TNBS- Induced Crohn’s disease (CD) model Sprague

Dawley rats

v. Assess the therapeutic impact of anti-inflammatory agent (prednisolone) and

monoclonal antibodies (Infliximab) on the changes in body weight in the TNBS-

Induced Crohn’s disease (CD) model in adult male Sprague Dawley rats.

vi. Measure the alterations in total acidity, mucus concentration, diarrhea, loose stools,

gross rectal bloody stools, or any other gross abnormalities following treatment with

anti-inflammatory agent (prednisolone) and monoclonal antibodies (Infliximab) in the

TNBS- Induced Crohn’s disease (CD) model

1.5 Significance of study

The findings from this study can have a significant impact on the management of Crohn's

Disease. By comparing the therapeutic efficacy of Prednisolone and Infliximab, along with

investigating their effects on the large intestine, the study can contribute to:

1. Advancement in IBD Treatment Strategies: The study contributes to the ongoing search

for effective treatment options for inflammatory bowel disease (IBD) by comparing the

therapeutic efficacy of anti-inflammatory agents and monoclonal antibodies. This

comparison can identify which treatment modality provides superior management of

chronic intestinal inflammation, potentially leading to improved therapeutic protocols.

2. Understanding Mechanisms of Action: By evaluating the effects of these treatments on

large intestine histology, antioxidant enzymes activity, and lipid peroxidation levels, the

study provides valuable insights into the mechanisms through which these therapies exert
 their effects. This understanding can help in designing targeted interventions that more

effectively mitigate the pathological features of IBD.

3. Impact on Oxidative Stress and Inflammation: The study's focus on oxidative stress

markers and antioxidant enzyme activities offers a deeper understanding of how oxidative

damage and the body's antioxidant defense mechanisms are modulated by different

treatments. This knowledge is crucial for developing therapies that can reduce oxidative

stress and its associated tissue damage in IBD patients.

4. Preclinical Model Validation: Using the TNBS-induced chronic disease model in Sprague

Dawley rats, the study validates the relevance and reliability of this animal model in

mimicking human IBD. This validation supports its continued use in preclinical research,

facilitating the development and testing of new therapeutic agents.

5. Guidance for Clinical Trials: The findings from this study can inform the design of

clinical trials by highlighting the potential benefits and limitations of anti-inflammatory

agents and monoclonal antibodies. This guidance can lead to more efficient and targeted

clinical studies, accelerating the translation of preclinical findings into clinical practice.

6. Improving Patient Outcomes: Ultimately, the study aims to improve patient outcomes by

identifying more effective treatments for managing chronic inflammatory conditions like

IBD. By providing evidence-based recommendations for the use of anti-inflammatory

agents and monoclonal antibodies, the study contributes to enhancing the quality of life

for IBD patients.

In conclusion, this study has the potential to significantly improve our understanding and

treatment of Crohn's Disease, not only by evaluating treatment efficacy but also by investigating
potential pancreatic involvement. The findings can ultimately contribute to better patient

outcomes and a higher quality of life for those living with Crohn's Disease.

CHAPTER TWO

2.0 Literature review

2.1 Inflammatory bowel disease (IBD)

Inflammatory bowel disease (IBD) represents a group of intestinal disorders that cause prolonged

inflammation of the digestive tract (Masaar et al., 2019). Inflammatory bowel disease (IBD) is a

term that describes disorders involving long-standing (chronic) inflammation of tissues in your

digestive tract (Fiorino et al., 2019).The digestive tract comprises the:mouth, esophagus,

stomach, small intestine, large intestine. It’s responsible for: breaking down food, extracting

nutrients, removing any unusable material and waste products (Lewis et al., 2021).Inflammation

anywhere along the digestive tract interferes with this normal process. IBD can be very painful

and disruptive. In rare cases, it may even be life threatening (Dmochowska et al., 2018).

Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the colon and small

intestine, with Crohn's disease and ulcerative colitis (UC) being the principal types (Talley et al.,

2018).
2.2 Crohn’s disease

Crohn’s disease is a long-term inflammatory condition affecting the gastrointestinal tract,

characterized by symptoms that come and go over time. It's a progressive illness that can lead to

damage and disability in the bowel. While any part of the digestive system can be affected, the

terminal ileum and colon are the most commonly impacted areas. The inflammation tends to be

patchy, and uneven and extends through the entire thickness of the affected area. Initially, most

patients display signs of inflammation, but as time passes, complications like strictures, fistulas,

or abscesses may develop in about half of the cases, often necessitating surgery. Current

treatment approaches focus on achieving sustained and profound remission, aiming to prevent

complications and halt the disease's progressive nature (Torres et al., 2017). Crohn’s disease is

thought to stem from various factors, such as immune system dysfunction, changes in the

microbiota, genetic predisposition, and environmental influences. Despite these associations, the

precise cause of the disease remains elusive (Roda et al., 2020). There are known genetic

tendencies linked to Crohn's disease, and certain environmental factors have been linked to its

onset. Typical symptoms at the outset encompass diarrhea, abdominal pain, rectal bleeding,

fever, weight loss, and fatigue (Veauthier and Hornecker, 2018).

2.2.1 Incidence and prevalence of Crohn’s Diseases

The incidence of CD is 0–20.2 cases per 100,000 person-years in North America and 0.3–12.7

cases per 100,000 person-years in Europe. The highest reported prevalence of CD was in Europe

(322 cases per 100,000 persons in Germany) and North America (319 cases per 100,000 persons

in Canada). CD incidence follows a south-to-north and east-to-west gradient in mainland China .

In Korea, studies reported an incidence of 1.68 cases per 100,000 person-years in 2005, after

which it reached a plateau . Moreover, the incidence of CD in Asia has increased more rapidly
than that of UC , although the prevalence of IBD is lower than in Western countries. The

prevalence of CD in Asian populations has also increased (Ng et al, 2017). For example, the

prevalence of CD in Taiwan increased from 0.6 cases per 100,000 persons in 2001 to 3.9 cases

per 100,000 persons in 2015, and the prevalence of CD in Hong Kong in 2014 was 18.6 cases

per 100,000 persons. Few studies have reported CD epidemiology data for Latin America and

Africa. In both regions, CD incidence and prevalence have been reported as low, although a few

studies have reported a high incidence of CD in Brazil. The rapid changes in CD epidemiology

are a global challenge for disease diagnosis, health care delivery and disease prevention. In

newly industrialized countries (such as in Asia), the increasing incidence of CD reflects the

influence of the Western lifestyle, particularly diet, urbanization and industrialization, on risk

(von Haehling et al, 2014). Furthermore, studies of migrants have shown that individuals who

move from low-prevalence to high-prevalence regions are at increased risk of developing CD

and that this risk is even more pronounced in the children of immigrants. The incidence of CD

has surpassed that of UC in many regions in the West. IBD in children younger than 10 years,

and especially those younger than 5 years (very early onset IBD) is becoming more common,

and elderly individuals (older than 65 years) with IBD are a rapidly rising population owing to

new diagnoses in elderly patients and advancing age of those in whom IBD is diagnosed earlier

in adulthood (Lerner et al, 2015). Owing to a lack of population-based prospective studies to

compare CD prevalence and environmental and genetic risk factors for the disease in developing

countries with those in developed countries, it is difficult to predict how this increase in

incidence will affect phenotypic features of CD in developing countries (Vos et al, 2015).

2.2.2 Risk Factor Of Chron Disease

The risk factors for the onset and progression of Crohn's disease (CD) are influenced by

environmental factors in individuals with genetic susceptibility (Kondo et al., 2019; Ng et al.,

2015). In Western countries, smoking has been identified as the only modifiable risk factor for

CD, doubling the risk of developing the disease. The impact of smoking on CD risk is greater

among females and varies with age. Smoking is also associated with early disease onset,

increased need for immunosuppression, higher rates of surgical intervention, and elevated rates

of postoperative disease recurrence (Levine, Sigall Boneh, & Wine, 2018). Meta-analyses have

revealed ethnic differences in the effect of smoking on CD risk, with passive smoking in Japan

also being associated with an increased risk of developing CD (Kondo et al., 2019; Levine et al.,

2018).

Gut dysbiosis, characterized by an imbalance in the gut microbiota, is a feature of CD, and diet is

a significant environmental factor that can influence the intestinal microbiota. Changes in food

composition and the shift from high-fiber, low-fat foods to processed foods containing additives

have altered the host-gut microbiota relationship. Reduced dietary fiber intake and frequent

oscillations between high-fiber and low-fiber foods contribute to decreased gut microbiota

diversity and are associated with the development of CD. The extent to which dietary changes

affect microbial diversity in CD is still a topic of debate. However, studies in Sweden have

shown that greater adherence to a Mediterranean diet is associated with a substantially lower risk

of later-onset CD. The gut microbiota composition can respond to dietary modifications, and

dietary components can induce long-lasting phenotypic changes through epigenetic

modifications. Understanding the complex interaction between diet and gut dysbiosis in CD may

enhance our comprehension of diet's role in CD pathogenesis and facilitate the development of

novel therapeutic approaches (Levine et al., 2018; Khalili et al., 2020).

Childhood antibiotic exposure increases the risk of developing CD, while oral contraceptives,

aspirin, and nonsteroidal anti-inflammatory drugs (NSAIDs) have been reported to elevate CD

risk. Conversely, breastfeeding has been inconsistently associated with reduced CD risk, and

statins have been linked to a decreased risk of CD (Kondo et al., 2019).

2.2.3 Mechanisms/pathophysiology of Chron Diseases

Although the exact etiology of inflammatory bowel disease (IBD) is not known, there is

substantial evidence to suggest that the disease results from an inappropriate immune response in

the bowel to environmental factors such as drugs, toxins, infections, or intestinal microbes in a

genetically susceptible host (Abraham & Cho, 2019; Khor et al., 2019). More than a hundred

genes associated with IBD have been identified, with Crohn disease showing a genetic

association with specific phenotypes, particularly NOD2/CARD15 mutations associated with

early-onset, ileal involvement, and severe disease requiring surgical intervention (Peyrin-Biroulet

et al., 2019). The pathophysiology of Crohn disease is multifactorial, involving genetic

predisposition, infectious, immunological, environmental, and dietary factors (Rieder & Fiocchi,

2019). Characteristic transmural inflammation in Crohn disease can affect the entire GI tract,

commonly involving the terminal ileum and right colon, and may present with classic

cobblestone mucosal appearances and skip lesions (Geltzeiler et al., 2019). Crohn disease

progresses, ongoing inflammation and scarring lead to complications such as bowel obstruction,

stricture formation, and various types of fistulas including enterovesical, enteroenteral,

enterocutaneous, and enterovaginal fistulas (Cuadrado et al, 2018; Rodrigues et al, 2021).

2.2.4 Evaluation of Chron Diseases

Stool tests to rule out infections include culture and sensitivities, ovum and parasites,

Clostridium difficile toxins, leukocyte count. Stools for calprotectin can detect active Crohn

disease and are also used for monitoring disease. Blood tests including complete blood count

and a metabolic panel can highlight the presence of anemia (B12 or iron deficiency) or liver

disease. Special serology such as normal anti-neutrophil cytoplasmic antibodies (ANCA) and

raised anti-saccharomyces cerevisiae antibodies (ASCA) can distinguish Crohn disease from

ulcerative colitis. C-reactive protein (CRP) or erythrocyte sedimentary rate (ESR) can reflect the

severity of the inflammation. A computed tomography scan/magnetic resonance enterography

(MRE) of the abdomen and pelvis can detect abscesses and fistulization. The choice between CT

or MRE is largely directed at minimizing radiation exposure in younger populations. Both give a

higher definition of the diseased intestine. However, MRI can provide more detail when

investigating the fistulizing disease. The use of video capsule endoscopy (VCE) can visualize the

small bowel for CD when regular endoscopy or colonoscopy cannot reach to visualize these

areas. Capsule endoscopy can only detect mucosal changes, whereas MRI can detect transmural

inflammation and also identify other complications. Plain x-rays are ordered when bowel

obstruction is suspected. Small bowel follow-through is often used to assess the involvement of

the terminal ileum and can also detect fistulas. The classic string sign due to stricture formation

or spasm is often seen (Ranasinghe and Hsu, 2023).

2.3 Monoclonal Antibody-Based Therapies

Monoclonal antibodies (mAbs) are a group of antibodies produced by identical clones of B

lymphocytes against a particular antigen (Sadeghalvad and Rezaei, 2021). Monoclonal

antibodies (mAbs) are produce by identical clones of B lymphocytes against a particular

antigen.They are identical in several properties such as protein sequence, antigen-binding site

region, binding affinity for their targets, and identical downstream functional effects

(Sadeghalvad and Rezaei, 2021) .Antibodies or immunoglobulins (Ig) are glycoproteins

produced by differentiated B lymphocytes named “plasma cells” in response to exposure to

antigen (Megha and Mohanan,2021). An antibody molecule has a Y-shaped structure with a total

molecular weight of ~150 kDa, composed of four polypeptide chains including two identical

heavy (H) and two light (L) chains (Hui et al., 2019).Monoclonal antibody-based therapies, also

known as immunotherapy or biologic therapy, are a powerful and targeted approach to treating

various diseases, including Crohn's disease and other diseases like cancer and autoimmune

disorders. In Crohn's disease, monoclonal antibodies is targeted to specific molecules involved in

the inflammatory process. Monoclonal antibody -based therapies work by blocking inflammatory

proteins like TNF-alpha to reduce inflammation in the gut and targeting immune system cells

that contribute to inflammation.The first monoclonal antibody used in IBD were designed to

target the pathway of tumor necrosis factor α (TNFα), which controls cell proliferation and

differentiation and promotes a proinflammatory response (Levin et al., 2016). TNF is secreted by

cells in the inflamed intestinal tract of IBD patients. TNF stimulates macrophages to produce

pro-inflammatory factors, induces the death of epithelial and Paneth cells, regulates the apoptosis

of T cells and promotes the production of degrading protease (Michlewska, S. 2011).

2.3.1 Physiology of Monoclonal Antibody

Scientists first need to identify a specific antigen on the target cell or molecule. This antigen can

be a protein, carbohydrate, or other molecule that is unique to the target (Wu, et al., 2016).Once

the antigen is identified, monoclonal antibody are design to bind specifically to it. This is done

by isolating B-lymphocytes (immune system cells that produce antibodies) that are specific for
the antigen (Boonyaratanakornkit and Taylor 2019).The B-lymphocytes are then fused with

myeloma cells (cancerous plasma cells) to create hybridoma. Hybridoma can reproduce

indefinitely and produce large quantities of the desired monoclonal antibody (Parray et al.,

2020).Once the monoclonal antibody is produced, it is purified and administered to the patient.

The antibody can be delivered intravenously (through a vein), subcutaneously (under the skin),

or intrathecal (into the spinal canal).Monoclonal antibodies function by binding to the antigen

and block its function (Kumar et al., 2018). For example, some monoclonal antibodies can block

the growth factors that cancer cells need to survive. Monoclonal antibody also trigger the death

of the target cell. This is called antibody-dependent cell-mediated cytotoxicity (ADCC). In

ADCC, immune system cells called macrophages and natural killer cells are attracted to the

antibody-coated target cell and destroy it. The antibody can bind to the antigen and make it more

likely to be engulfed by phagocytes (white blood cells that clear debris) (Zhou et al., 2021)

2.3.2 Mechanism of action monoclonal antibody of crohn’s diseases

Monoclonal antibody, like Infliximab, target TNF-α which is a molecule heavily involved in

inflammation (Jang et al., 2021). The monoclonal antibody is bonded to TNF-α, to prevent it

from interacting with cell receptors, leading to: Reduced activation of immune cells like

macrophages and T cells that contribute to inflammation in the gut and also potentially decreased

cell survival of these immune cells (Griffiths et al., 2020).By preventing TNFα from binding to

its receptors, the monoclonal antibody will disrupt the signaling pathways that would could

trigger any further inflammation. This disruption will leads to a dampened immune response and

reduced inflammatory activity in the gut. The anti-TNF antibodies may indirectly induce the

death (apoptosis) of certain T cells that contribute to inflammation (van Loo and Bertrand 2023).

These monoclonal antibodies might influence the development of macrophages that promote
tissue healing (M2 type) over those that perpetuate inflammation (M1 type) (van Loo and

Bertran 2023).

2.4 Prednisolone

Prednisolone is a glucocorticoid that is prednisone in which the oxo group at position 11 has

been reduced to the corresponding beta-hydroxy group. It is a drug metabolite of prednisone. It

has a role as an adrenergic agent, an anti-inflammatory drug, an antineoplastic agent, an

immunosuppressive agent, a drug metabolite, an environmental contaminant and a xenobiotic. It

is a glucocorticoid, an 11beta-hydroxy steroid, a 21-hydroxy steroid, a 17alpha-hydroxy steroid,

a 20-oxo steroid, a 3-oxo-Delta(1),Delta(4)-steroid, a primary alpha-hydroxy ketone, a tertiary

alpha-hydroxy ketone and a C21-steroid. Prednisolone is a glucocorticoid similar to cortisol used

for its anti-inflammatory, immunosuppressive, anti-neoplastic, and vasoconstrictive effects.

Prednisolone was granted FDA approval on 21 June 1955.Prednisolone is a Corticosteroid. The

mechanism of action of prednisolone is as a Corticosteroid Hormone Receptor Agonist

(Kobayashi, 2017)

Prednisolone can be reversibly metabolized to [prednisone] which is then metabolized to 17α,21-

dihydroxy-pregnan-1,4,6-trien-3,11,30-trione (M-XVII), 20α-dihydro-prednisone (M-V),

6βhydroxy-prednisone (M-XII), 6α-hydroxy-prednisone (M-XIII), or 20β-dihydro-prednisone

(M-IV). 20β-dihydro-prednisone is metabolized to 17α,20ξ,21-trihydroxy-5ξ-pregn-1-en-3,11-

dione(M-XVIII). Prednisolone is metabolized to Δ6-prednisolone (M-XI), 20α-dihydro-

prednisolone (M-III), 20β-dihydro-prednisolone (M-II), 6αhydroxy-prednisolone (M-VII), or

6βhydroxy-prednisolone (M-VI). 6αhydroxy-prednisolone is metabolized to 6α, 11β, 17α, 20β,

21-pentahydroxypregnan-1,4-diene-3-one (M-X). 6βhydroxy-prednisolone is metabolized to

6β,11β,17α,20β,21-pentahydroxypregnan-1,4-diene-3-one (M-VIII), 6β,11β,17α,20α,21-

pentahydroxypregnan-1,4-diene-3-one (M-IX), and 6β,11β,17α,21-tetrahydroxy-5ξ-pregn-1-en-

3,20-dione (M-XIV). MVIII is metabolized to 6β, 11β, 17α, 20β, 21-pentahydroxy-5ξ-pregn-1-

en-3-one (M-XV) and then to MXIV, while MIX is metabolized to 6β, 11β, 17α, 20α, 21-

pentahydroxy-5ξ-pregn-1-en-3-one (M-XVI) and then to MXIV. These metabolites and their

glucuronide conjugates are excreted predominantly in the urine. Reduction of the 4,5 double

bond can occur at both hepatic and extrahepatic sites and yields an inactive substance.

Subsequent reduction of the 3-ketone substituent to a 3-hydroxyl to form tetrahydrocortisol has

been demonstrated only in liver. Most of the ring a - reduced metabolites are enzymatically

coupled through the 3-hydroxyl with sulfate or with glucuronic acid to form water soluble sulfate

esters or glucuronides, and they are excreted as such (Möllmann et al, 2017)

2.4.1 Prednisolone Mechanism of Action of Prednisolone

The short term effects of corticosteroids are decreased vasodilation and permeability of

capillaries, as well as decreased leukocyte migration to sites of inflammation. Corticosteroids

binding to the glucocorticoid receptor mediates changes in gene expression that lead to multiple

downstream effects over hours to days. Glucocorticoids inhibit neutrophil apoptosis and

demargination; they inhibit phospholipase A2, which decreases the formation of arachidonic acid

derivatives; they inhibit NF-Kappa B and other inflammatory transcription factors; they promote

anti-inflammatory genes like interleukin-10. Lower doses of corticosteroids provide an anti-

inflammatory effect, while higher doses are immunosuppressive. High doses of glucocorticoids

for an extended period bind to the mineralocorticoid receptor, raising sodium levels and

decreasing potassium levels.

Although altered homeostatic regulation, including disturbance of 24-h rhythms, is often

observed in the patients undergoing glucocorticoid therapy, the mechanisms underlying the
disturbance remains poorly understood. We report here that chronic treatment with a synthetic

glucocorticoid, prednisolone, can cause alteration of circadian clock function at molecular level.

Treatment of cultured hepatic cells (HepG2) with prednisolone induced expression of Period1

(Per1), and the prednisolone treatment also attenuated the serum-induced oscillations in the

expression of Period2 (Per2), Rev-erbalpha, and Bmal1 mRNA in HepG2 cells. Because the

attenuation of clock gene oscillations was blocked by pretreating the cells with a Per1 antisense

phosphothioate oligodeoxynucleotide, the extensive expression of Per1 induced by prednisolone

may have resulted in the reduced amplitude of other clock gene oscillations. Continuous

administration of prednisolone into mice constitutively increased the Per1 mRNA levels in liver

and skeletal muscle, which seems to attenuate the oscillation in the expressions of Per2, Rev-

erbalpha, and Bmal1. However, a single daily administration of prednisolone at the time of day

corresponding to acrophase of endogenous glucocorticoid levels had little effect on the rhythmic

expression of clock genes. These results suggest a possible pharmacological action by

prednisolone on the core circadian oscillation mechanism and indicate the possibility that the

alteration of clock function induced by prednisolone can be avoided by optimizing the dosing

schedule (Ramamoorthy, S, & Cidlowski, 2016).

2.5 Large Intestine

The large intestine, also known as the colon, is a crucial component of the digestive system

responsible for processing and eliminating waste from the body. It plays a vital role in absorbing

water and electrolytes, forming feces, and housing beneficial bacteria. Understanding the

physiology and pathology of the large intestine is essential for comprehending various

gastrointestinal disorders and their management.

2.5.1 Physiology of the Large Intestine

1. Structure: The large intestine extends from the ileocecal valve to the anus and comprises

several distinct regions: the cecum, ascending colon, transverse colon, descending colon,

sigmoid colon, rectum, and anal canal.It is characterized by features such as haustra

(pouches), taeniae coli (three bands of longitudinal muscle), and epiploic appendages

(fat-filled pouches).

2. Motility: Peristalsis, the rhythmic contraction of smooth muscles, facilitates the

movement of fecal matter through the large intestine. Mass movements, powerful

contractions of the colon, occur one to three times daily, propelling feces toward the

rectum.

3. Absorption and Secretion: The large intestine absorbs water, electrolytes, and some

vitamins produced by intestinal bacteria. It secretes mucus, which lubricates feces and

protects the intestinal lining from mechanical damage and bacterial invasion.

4. Microbiota: The large intestine harbors a diverse microbial community, collectively

known as the gut microbiota, which contributes to digestion, metabolism, and immune

function. Beneficial bacteria ferment indigestible carbohydrates, producing short-chain

fatty acids that nourish colonic epithelial cells.

2.5.2 Pathology of the Large Intestine

1. Inflammatory Bowel Diseases (IBD): Crohn's Disease and Ulcerative Colitis are chronic

inflammatory conditions characterized by inflammation of the intestinal mucosa. Crohn's

Disease can affect any part of the gastrointestinal tract and is associated with transmural
 inflammation, while Ulcerative Colitis primarily affects the colon and rectum, leading to

continuous mucosal inflammation.

2. Colorectal Cancer: Colorectal cancer arises from the malignant transformation of

epithelial cells lining the colon or rectum. Risk factors include age, family history,

inflammatory bowel disease, dietary factors, and genetic predisposition. Screening

methods such as colonoscopy, fecal occult blood testing, and stool DNA testing aid in

early detection and prevention.

3. Diverticular Disease: Diverticula are small pouches that protrude through weak points in

the colonic wall, commonly occurring in the sigmoid colon. Diverticulosis refers to the

presence of diverticula without inflammation, while diverticulitis results from

inflammation and infection of diverticula. Symptoms of diverticular disease include

abdominal pain, bloating, changes in bowel habits, and complications such as perforation,

abscess formation, or fistulae.

4. Irritable Bowel Syndrome (IBS): IBS is a functional gastrointestinal disorder

characterized by abdominal pain or discomfort associated with altered bowel habits in the

absence of organic pathology. Subtypes include IBS with predominant diarrhea,

constipation, or mixed bowel habits. Management focuses on symptom relief through

dietary modification, stress management, and pharmacotherapy targeting bowel motility

and visceral hypersensitivity.

2.5 Large Intestine

The large intestine, or colon, is a vital part of the digestive system responsible for processing and

eliminating waste from the body. It plays a crucial role in absorbing water and electrolytes,
forming feces, and housing beneficial bacteria. Understanding its physiology and pathology is

essential for managing various gastrointestinal disorders.

2.5.1 Physiology of the Large Intestine

1. Structure: The large intestine extends from the ileocecal valve to the anus and comprises

several regions: the cecum, ascending colon, transverse colon, descending colon, sigmoid

colon, rectum, and anal canal. It features haustra (pouches), taeniae coli (bands of

longitudinal muscle), and epiploic appendages (fat-filled pouches) (Moore et al., 2019).

2. Motility: Peristalsis, rhythmic contraction of smooth muscles, facilitates fecal movement.

Mass movements propel feces toward the rectum, occurring one to three times daily

(Boron & Boulpaep, 2017).

3. Absorption and Secretion: The large intestine absorbs water, electrolytes, and vitamins

produced by gut bacteria. It secretes mucus for fecal lubrication and protection (Moore et

al., 2019).

4. Microbiota: The gut microbiota in the large intestine aids digestion, metabolism, and

immune function. Fermentation of indigestible carbohydrates produces short-chain fatty

acids, nourishing colonic cells (Sender et al., 2016).

2.5.2 Pathology of the Large Intestine

1. Inflammatory Bowel Diseases (IBD): Crohn's Disease and Ulcerative Colitis are

chronic inflammatory conditions affecting the intestinal mucosa. Crohn's Disease

involves transmural inflammation throughout the gastrointestinal tract, while Ulcerative

 Colitis primarily affects the colon and rectum with continuous mucosal inflammation

(Neurath, 2019).

2. Colorectal Cancer: Malignant transformation of colon or rectum epithelial cells leads to

colorectal cancer. Risk factors include age, family history, diet, and genetic

predisposition. Screening methods like colonoscopy aid in early detection (Kuipers et al.,

2020).

3. Diverticular Disease: Diverticula protruding through weak colonic points characterize

diverticular disease. Diverticulosis is the presence of these pouches without

inflammation, while diverticulitis involves inflammation and infection, potentially

causing complications like abscesses or fistulae (Strate & Morris, 2019).

4. Irritable Bowel Syndrome (IBS): IBS is a functional disorder with abdominal pain and

altered bowel habits. Subtypes include diarrhea-predominant, constipation-predominant,

and mixed patterns. Management includes dietary changes and pharmacotherapy

targeting bowel function (Ford et al., 2020).

CHAPTER THREE

3.0 Material and method

3.1 Material and reagents

3.1.1 Materials

The following were used during the time frame of the project: 40 adult male Sprague-Dawley

rats, electronic compact scale, gloves, plastic cages, gloves, bowls for feed, water bottle, ruler for

measuring , measuring bowl, scraper, syringe, cotton wool, latex foley catheter (size 2-Way

8Fr3-5ml), titrating pipette, oral cannulas, beaker, measuring cylinder, burette, dissecting kit,

conical flask, sodium heparinized capillary tube, light microscope, spectrophotometer, glass

slide, dissecting set, dissecting board, centrifuge, freezer pipette, micro pipettes tips.

3.1.2Drugs and Chemicals

Ciprofloxacin Hydrochloride (Ratnamani Healthcare PVT. LTD., India), Ketamine

hydrochloride injection (Psychotropics LTD., India), Infliximab (Cliag AG, Switzerland),

Xylaxine (Bioveta,a.s.,Czech Republic), Acetic acid glacial 99.5% (Loba chemical LTD.,India),

Phosphate buffer(pH7.4, 0.1M) (Oxford laboratories Mumbai India), Bluing solution,

Hydrochloric acid(HCL), Haematoxylin and eosin staining, Ellman reagent, Gluthathion acid,

Thiobarbituric acid, Carbonate buffer, Analytical glucose(Oxford laboratories ,Mumbai india),

Distilled water, Methylated spirit, H2SO4, Ammonia solution, Olive oil, Aqueous hydrochloric

acid, Ferric chloride, Potassium ferricyanide, Phosphate buffer, Griess reagent, Sodium

nitroprusside, Thiobarbituric acid, HCL, Trichloroacetic acid(TCA), FeCl2, Ascorbic acid,

Ammonium molybdate, Sodium Phosphate, Sulphuric acid, Potassium acetate, Aluminium

chloride , Methanol, Sodium carbonate solution (7.5% w/v), Folin- Ciocalteu reagent, FeCl3,
Potassium ferrocyanide, Dragendroff’s reagent, Mayer’s reagent, Sulphuric acid, all other

chemicals and reagent used for this study were of analytical grade.

3.2 Ethical statement

All the animal experiments and protocol were maintained according to the rules and regulations

of the National Research Council, for laboratory animal care and use. Ethical clearance was

obtained from the Olabisi Onabanjo University Teaching Hospital Human Research Ethics

Committee (OOUTH-HREC). All the animal carcasses was buried deep in the ground covered

with lime and disinfectant at least two feet beneath the natural surface and covered with soil.

3.3 Animals

Mature male rats (seven weeks old) of the Sprague-Dawley strain were used for this study. The

rats were housed in plastic cages, kept under standard laboratory conditions and were allowed

free access to feed and water throughout the experimental period. They were acclimatized for 2

weeks. All guidelines with the use and care of laboratory animals was strictly adhered to. Animal

identification was done before the beginning of the research work. At the end of the experiment,

the carcasses was buried deep in the ground, covered with lime and disinfectant.

3.4 Induction of anesthesia

Anesthesia was induced using, For a 10mL vial usng ketamine 100 mg/mL and xylazine 100

mg/mL add: 1.75mL ketamine (1000 mg/mL) and 0.25 mL xylazine (1000 mg/mL, anesthesia

agent will be administerd via inter- peritoneal route

3.5 Induction of Chron diseases

This was done according to the method of Loeuillard et al, (2014). The Rats was food-deprived

for 24 h prior to induction of chron disease and was allowed free access to water throughout the

study. Chron diseases was induced by weekly administration of increasing concentrations of

TNBS (15, 30, 45, 60, 60 and 60 mg) over 6 wk. After instillation of TNBS, the rats was then

maintained in a head-down position for a few minutes to prevent leakage of the intracolonic

instillate. Control groups received the same volume of the vehicle

3.6 Experimental procedure

After the induction of Chron diseases, treatment was done for a period of 42 days. After fourteen

days acclimatization the animals were randomized into five different groups consisting of ten

animals in each, as shown in the Table 3.1 below

Table 3.1 Animal grouping

Group Treatment Number

of rats

per

group

A: normal control physiological saline (0.9% w/v NaCl, p.o) 10

B: Chron diseases TNBS solution transrectally for six weeks 10

groups

C: Anti- TNBS solution transrectally for six weeks and 10

inflammatory group 5mg/Kg of prednisolone (P.O)

D: MCA B group TNBS solution transrectally for six weeks and 10

5 mg/kg of infliximab (i.p)

P.O: oral route of administration

I.P: Inter Peritoneal route of administration:

3.7 Administration of treatment

3.7.1 Administration of anti-inflammatory

The choice anti-inflammatory agent to be used in this study is prednisolone (Witaicenis et al,

2012), was adminsiterd every 72 hours.

3.7.2 Administration of MCAB

The choice MCAB, used in this study was infliximab, the dose was picked according to the result

of Triantafillidis et al., (2005), 5 mg/kg, i.p. was administed bi weekly for 6 weeks Dadsetaney

al., 2016

3.8 Clinical course assessment

The animals were observed on a daily basis and checked for diarrhea, loose stools, gross rectal

bloody stools, or any other gross abnormalities. The weight of each animal was obtained on a

weekly basis to check for weight loss after induction of colitis. These observations were reported

as a numerical score (Table 3.2).

Table 3.2 Animal grouping

Feature 0 1 2 3

Stool Normal Loose stool Diarrhea Diarrhea with

blood

Hyperemia None Focal Focal and Extensive

thickening of thickening of

bowel wall bowel wall

Adhesions None Mild Moderate Extensive

Megacolon None Mild Moderate Toxic megacolon

Ulcerations None Mild ulceration Moderate Severe damage

on one side < 1 ulceration > 1 cm extending > 2 cm

cm
3.9 Histological studies:

After careful removal of organs, they were trimmed of fat. They were weighed and immediately

fixed in 10% formal saline. After fixing the tissues, they were put into ascending grades of

alcohol and then cleared in xylene. They were embedded in paraffin, and serial sections of 5μm

was obtained. Sections were stained with hematoxylin and eosin.. The microscopic alterations

were assessed according to the criteria shown in Table 3.3, and a numerical score of the colonic

abnormalities was obtained. The histologic grades ranged from 0 (normal) to 18 (intense

inflammatory reaction).
Table 3.3 Animal grouping

Feature 0 1 2 3

Abnormalities None (Normal) Mild or focal, Moderate, not Severe &

of mucosal not exceeding exceeding the diffuse,

architecture lamina propria submucosa exceeding the

submucosa

Crypt None Mild atrophy Moderate Severe atrophy,

abnormalities atrophy branched crypts,

Branched crypts cryptitis, crypt

abscess

Inflammatory Normal Scattered cells Moderate or Massive

cell infiltration confluent cells infiltration of

cells

Vascular Normal blood Mild dilatation Moderate , Severe

dilatation vessels (localized) dilatation of generalized

several blood dilatation of

vessels blood vessels

Edema None Low level In the All over the

limited to villi submucosa section

Mast cells Normal Three cells Clusters of > 3 Clusters in

scattered cells clustered in cells in the submucosa and

submucosa submucosa serosa

3.10 Determination of pH, volume free and total acidity of the gastric content

The abdomen of each rat was opened under a mild anesthesia without damaging any blood

supply and measurements of pH, total and free acidity of gastric juice were performed as

described by Hisam et al. Gastric pH was measured by pH meter and recorded. The gastro-

esophageal and gastro-duodenal junctions were secured before the stomach was isolated. One

milliliter (1 ml) of distilled water was introduced into the stomach and the organ was carefully

shaken. Gastric juice was collected and centrifuged at 3000 rpm for 10 minutes. The supernatant

was taken and diluted 10 times. Two to three drops of phenolphthalein were added to the

solution. Titration was done using 0.01 M solutions of NaOH until the color of the test solution

changed to light pink, indicating a pH of 7. The volume of NaOH was used to determine the

hydrogen ion concentration. Acidity was determined using the equation described by Blood, as

follows:

volume of NaOH × normality of NaOH × 100

mEq /ltr
0.1

3. 11 Determination of gastric mucus concentration

22
Gastric mucus concentration was determined using the method described by Corne et al. The

excised stomach was soaked for 2hours in 0.1% Alcian blue dissolved in a buffer solution

containing 0.1M sucrose and 0.05M Sodium acetate (pH adjusted to 5.8 with hydrochloric acid).

The dye formed complexes with mucus after washing two times in 0.25M sucrose at 15 and 45

minutes. The mucus was eluted by immersion in 10mL aliquots of 0.5M MgCl 2 for 2 hours. The
resulting blue solution was shaken with equal volumes of diethyl ether and centrifuged at 3000

rpm for 10 minutes. The optical density of the aqueous phase was measured at 580 nm using a

spectrophotometer. Values of the absorbance measured in the different treatment groups was

used to determine the corresponding concentration of alcian blue which formed complexes with

mucin on the wall of the glandular portion of the stomach. Finally, the amount of mucin per

gram of the net glandular tissue was calculated with a formula as shown below;

Alcian blue(µg )
Mucin content (µg Alcianblue /g wet tissue)=
Weight of glandular tissue ( g)

3.12 Determination of malondialehyde activity (lipid peroxidation)

The malondialehyde activity of the testis and prostrate was estimated using the method of Stocks

and Dormandy (1971). Malondialdehyde, formed from the breakdown of of polyunsaturaated

fatty acids, serves as a convenient index for determining the extent of lipid peroxidaton reaction.

Malondialdehyde has been identified as a product of lipid peroxidation that reacts with

thiobarbituric acid to give a red specie absorbing at 535 nm.

Procedure: 1 ml of serum or tissue homogenate was combined with 2 ml of TCA-TBA-HCL

and mixed thoroughly. The solution was heated for 15 minutes in a boiling water bath. After

cooling, the flourescent precipitate was removed by centrifugation at 1000g for 10 mins. The

absorbance of the sample was determined at 535 nm against a blank that contains all the reagents

minus the sample. The malondialdehyde concentration of the sample was calculated using an

extinction coefficeient of 1.56 × 10-5 M-1 Cm-1. Calculation of lipid peroxidation

V
MDA (nmol /ml )=OD ∑ ×
v
OD = Absorbance (optical density) of sample

∑ = Molar extinction coefficient

V = Total volume of the reacting sample

v = Volume of the sample

3. 13 Statistical Analysis

All the values are expressed as mean ± standard error of mean (SEM). Analysis of data was done

using GraphPad Prism version 5 for Windows. Differences between groups were analyzed by

one-way ANOVA followed by Bonferroni post-hoc test. Differences were considered significant

when P < 0.05.

CHAPTER FOUR
4.0 Result and Analysis
4.1 Observational changes on the stool types in TNBS induced Crohn's Disease model in

adult male Sprague Dawley rats after treatment with anti-inflammatory (Prednisolane)

agents and monoclonal antibodies (Infliximab)

Table 4.1, which presents observational changes in stool types in a TNBS-induced Crohn's

Disease model in adult male Sprague Dawley rats after treatment with anti-inflammatory agents

(Prednisolone) and monoclonal antibodies (Infliximab), the Pre-Induction colum shows Stool

consistency before inducing Crohn's disease. The Induction Weeks 1-6 showed Weekly

observations after inducing Crohn's disease, at week three of Induction there was an increase in

stool type 1 and 2 in test group B to D, as at week 6 induction there was an increase in stool type

3 across all test group. In all groups, the average stool score increases (indicates looser stools)

after inducing Crohn's disease (Induction Weeks 1-6).

Treatment Weeks 1-6: showed weekly observations after starting treatment. Both Prednisolone

(Group C) and Infliximab (Group D) treatments appear to improve stool consistency compared

to the control group (Group B) during treatment weeks. Week 3 of treatment seems to be a

turning point where treatment groups show the most significant difference from the control

group in terms of stool consistency as there was a continuous shift in the stool type from type 3

and 2, to type 1 and 0.

 Table 4.1 Observational changes on the stool types in TNBS induced Crohn's Disease

model in adult male Sprague Dawley rats after treatment with anti-inflammatory

(Prednisolane) agents and monoclonal antibodies (Infliximab)

A B C D
0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3
20.2
20.1 2.40 0.00 0.00 7.60 5.47 0.00 22.5 6.00 6.12 0.00 21.5 4.50 7.21 0.00
3
Pre 4± ± ± ± ± ± ± 1± ± ± ± 1± ± ± ±
±
0.71 0.29 0.00 0.00 1.07 1.43A 0.00 1.03 0.45 0.69A 0.00 0.58 2.28 0.73A 0.00
1.28
Indu
20.03 7.76 12.95 0.00 15.46 6.69 9.32 7.79 7.63±
ction 0.00± 0.00± 9.01± 5.93± 8.65± 0.00± 0.00±
± ± ± ± ± ± ± ± 1.72A,
week 0.00 0.00 0.81A 1.02 1.06A 0.00 0.00
2.92 1.43 2.45A 0.00 1.10 0.47 0.65A 1.93 B
1
Indu
13.72 0.00 13.07 6.13 6.90± 14.95 7.34
ction 17.37 7.11± 0.00± 0.00± 6.48± 5.71± 0.00± 6.2±1 0.00±
± ± ± ± 0.39A, ± ±
week ±1.08 1.73 0.00 0.00 2.87A 0.87 0.00 .35A,B 0.00
2.45A 0.00 2.50 0.56 B
1.94B 1.67
2
Indu
14.72 6.92± 6.35±
ction 20.60 6.78± 0.00± 0.00± 1.08± 5.51± 0.00± 7.20± 5.41± 0.00± 4.25± 6.47± 0.00±
±2.45 0.22 1.38A,
week ±2.35 1.78 0.00 0.00 1.08A 0.83 A 0.00 1.84A 0.68 0.00 0.91A 1.09 0.00
A,B B
3
Indu
15.72 5.77± 5.50±
ction 16.00 8.95± 0.00± 0.00± 6.77± 7.94± 0.00± 6.23± 8.13± 0.00± 5.43± 9.11± 0.00±
±2.45 1.35A, 0.69A,
week ±1.00 1.73 0.00 0.00 1.97 2.09 A 0.00 0.94A 1.07 B 0.00 0.85A 1.45 B 0.00
4
Indu
16.72 15.00 5.00± 11.74
ction 19.20 7.27± 0.00± 0.00± 0.10± 1.870 1.37± 7.42± 0.00± 2.57± 3.77± 0.00±
±2.45 ±0.71 2.22A, ±1.94
week ±2.83 1.27 0.00 0.00 0.10A .54 A A 0.64A 0.45 B 0.00B 0.69A B 0.55B 0.00B
5
Indu
3.95 17.72 15.60
ction 14.40 5.98± 0.00± 1.47± 3.57± 2.32± 7.50± 0.00± 0.00± 5.40± 8.88± 2.30± 0.00±
± ±2.45 ±0.68
week ±1.59 1.11 0.00 1.23A 0.47 A A 1.04A 0.90 0.00B 0.00B 2.60A 2.43 0.54B 0.00B
0.85
6
Treat
18.72 16.20 13.25
ment 9.25± 4.67± 0.00± 0.00± 3.80± 4.51± 11.30 0.00± 0.00± 9.10± 6.48± 0.00± 0.00±
±2.45 ±0.72 ±3.63
week 1.17 0.80 0.00 0.00 3.80 1.90 A A ±5.21 A,B 0.00B 0.00B 5.21 0.73C 0.00B 0.00B
1
Treat
19.72 16.80 12.80 10.82
ment 12.83 6.87± 0.00± 0.00± 4.46± 4.25± 8.73± 0.00± 0.00± 12.68 0.00± 0.00±
±2.45 ±0.86 ± ±1.35
week ±2.72 1.12 0.00 0.00 1.93 2.20 A 0.76 0.00B 0.00B ±0.69 B 0.00B 0.00B
A 1.28
2
Treat
20.72 17.40
ment 8.42± 9.18± 0.00± 0.00± 4.83± 9.45± 10.18 8.65± 0.00± 0.00± 11.44 11.40 0.00± 0.00±
±2.45 ±1.03
week 1.38 1.56 0.00 0.00 2.71 4.70 A ±2.03 1.02 0.00B 0.00B ±2.21 ±0.89 0.00B 0.00B
A
3
Treat
21.72 17.80 11.73 14.51 10.66
ment 8.71± 11.82 0.00± 0.00± 6.00± 4.18± 10.64 0.00± 0.00± 0.00± 0.00±
±2.45 ±1.20 ±2.46 ±0.88 ±1.63
week 1.59 ±2.31 0.00 0.00 3.70 2.56A A ±1.54 B 0.00B 0.00B B B 0.00B 0.00B
A
4
Treat
22.72 18.20
ment 11.97 10.61 0.00± 0.00± 3.95± 2.96± 8.03± 6.43± 0.00± 0.00± 10.74 7.53± 0.00± 0.00±
±2.45 ±1.39
week ±1.33 ±0,92 0.00 0.00 2.65 1.91A A 0.77 1.05 0.00B 0.00B ±1.31 1.69 0.00B 0.00B
A
5
Treat 14.72 3.54± 0.00± 0.00± 4.85± 0.99± 22.72 18.60 9.87± 0.67± 0.00± 0.00± 13.21 0.60± 0.00± 0.00±
ment ±1.63 0.31 0.00 0.00 3.68 0.64 ±2.45 ±1.60 0.95 0.42 0.00B 0.00B ±1.61 0.60 0.00B 0.00B
week A A
6
0: Normal stool;1: Loose stool; 2: Diarrhea; 3:Diarrhea with blood. Each value is an expression of mean ± SEM. (P <0.05)
A - Values were significant when compared to group A, B-Values were significant when compared to group B, C- Values
were significant when compared to group C.
4.2 Observational changes on the body weight in of TNBS-induced Crohn's Disease (CD)

model in adult male Sprague Dawley rats rats after treatment with anti-inflammatory

agents(Prednisolone) agents and monoclonal antibodies (Infliximab)

Table 4.2 Observational changes on the body weight in of TNBS-induced Crohn's Disease

(CD) model in adult male Sprague Dawley rats rats after treatment with anti-inflammatory

agents(Prednisolone) agents and monoclonal antibodies (Infliximab)

A B C D

PRE 165.20 ± 16.07

INDUCTION 165.20 ± 16.07

WK1

WK2 160.52 ± 13.50

WK3 184.90 ± 11.10

WK4 206.02 ± 9.26

WK5 228.40 ± 7.36

WK6 230.42 ± 10.93

TRT WK1 243.77 ± 6.69

TRT WK2 249.97 ± 9.93

TRT WK3 248.17 ± 25.87

TRT WK4 249.17 ± 25.87

TRT WK5 250.17 ± 25.87

TRT WK6 251.17 ± 25.87

Each value is an expression of mean ± SEM. (P <0.05)

A - Values were significant when compared to group A, B-Values were significant when compared to group B, C- Values
were significant when compared to group C.
4.4 Clinical Observational of the effect of TNBS-induced Crohn's Disease (CD) model in

adult male Sprague Dawley rats after treatment with of TNBS-induced Crohn's Disease

(CD) model in adult male Sprague Dawley rats

Table 4.4 Clinical Observational of the effect of TNBS-induced Crohn's Disease (CD)

model in adult male Sprague Dawley rats after treatment with anti-inflammatory

agents(Prednisolone) agents and monoclonal antibodies (Infliximab)

A B C D

0.00 ± 0.00
ULCERATION

0.00 ± 0.00
HYPERMIA

ADHESION 0.00 ± 0.00

1.00 ± 0.63
MEGACOLON

Each value is an expression of mean ± SEM. (P <0.05)

A - Values were significant when compared to group A, B-Values were significant when compared to group B, C- Values
were significant when compared to group C, D- Values were significant when compared to group D.
4.5 Comparison of the Therapeutic effect of anti-inflammatory anti-inflammatory

agents(Prednisolone) agents and monoclonal antibodies (Infliximab) on the mucin content

on in the stomach, small intestine and large intestine of TNBS-induced Crohn's Disease

(CD) model in adult male Sprague Dawley rats

Table 4.5 Comparison of the Therapeutic effect of anti-inflammatory agents(Prednisolone)

agents and monoclonal antibodies (Infliximab) on the mucin content on in the stomach,

small intestine and large intestine of TNBS-induced Crohn's Disease (CD) model in adult

male Sprague Dawley rats

A B D E

Large Intestine 160.40 ± 2.94

(µg Alcian blue/g

wet tissue)

Each value is an expression of mean ± SEM. (P <0.05)

A - Values were significant when compared to group A, B-Values were significant when compared to group B, C- Values
were significant when compared to group C, D- Values were significant when compared to group D.
4.6 Comparison of the Therapeutic effect of anti-inflammatory agents(Prednisolone) agents

and monoclonal antibodies (Infliximab) on the PH content in the stomach, small intestine

and large intestine of TNBS-induced Crohn's Disease (CD) model in adult male Sprague

Dawley rats
Table 4.6 Comparison of the Therapeutic effect of anti-inflammatory agents(Prednisolone)

agents and monoclonal antibodies (Infliximab) on the PH content in the stomach, small

intestine and large intestine of TNBS-induced Crohn's Disease (CD) model in adult male

Sprague Dawley rats

A B D E

Large 4.25 ± 0.48

Intestine

Each value is an expression of mean ± SEM. (P <0.05)

A - Values were significant when compared to group A, B-Values were significant when compared to group B, C- Values
were significant when compared to group C.
4.7 Comparison of the Therapeutic effect of anti-inflammatory agents(Prednisolone) agents

and monoclonal antibodies (Infliximab) on the total acidity in the small intestine and large

intestine of of TNBS-induced Crohn's Disease (CD) model in adult male Sprague Dawley

rats
Table 4.7 Comparison of the Therapeutic effect of anti-inflammatory agents(Prednisolone)

agents and monoclonal antibodies (Infliximab) on the total acidity in the small intestine and

large intestine of of TNBS-induced Crohn's Disease (CD) model in adult male Sprague

Dawley rats

A B D E

Large Intestine 142.55 ± 6.20

(mEq/ltr)

Each value is an expression of mean ± SEM. (P <0.05)

A - Values were significant when compared to group A, B-Values were significant when compared to group B, C- Values
were significant when compared to group C, D- Values were significant when compared to group D.
 CHAPTER FIVE

5.0 Discussion, Conclusion and recommendation

5.1 Discussion