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UNIVERSITI PUTRA MALAYSIA

EFFECTS OF LACTOBACILLUS CASEI CONSUMPTION ON INFLAMMATION BIOMARKERS AND HISTOLOGICAL CHANGES IN

SELECTED ORGANS IN NORMAL AND DIABETIC RATS

AIDA ZARFESHANI

FPSK(m) 2011 2

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EFFECTS OF LACTOBACILLUS CASEI

CONSUMPTION ON INFLAMMATION

BIOMARKERS AND HISTOLOGICAL

CHANGES IN SELECTED ORGANS IN

NORMAL AND DIABETIC RATS

AIDA ZARFESHANI

MASTER OF SCIENCES

UNIVERSITI PUTRA MALAYSIA

2010

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EFFECTS OF LACTOBACILLUS CASEI CONSUMPTION ON

INFLAMMATION BIOMARKERS AND HISTOLOGICAL CHANGES IN

SELECTED ORGANS IN NORMAL AND DIABETIC RATS

AIDA ZARFESHANI

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Master of Science

November 2010

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To everyone who believed my abilities,

And supported me in my intention,

To make some of my dreams come true.

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Abstract of thesis presented to the Senate of University Putra Malaysia in fulfilment of the

requirement for the degree of Master of Science

EFFECTS OF LACTOBACILLUS CASEI CONSUMPTION ON INFLAMMATION

BIOMARKERS AND HISTOLOGICAL CHANGES IN SELECTED ORGANS IN

NORMAL AND DIABETIC RATS

By

AIDA ZARFESHANI

November 2010

Chairman: Assoc. Prof. Mohd Sokhini Bin Abd Mutalib, PhD

Faculty: Medicine and Health Sciences

The severity of diabetes mellitus often manifested by a progressive inflammation, indicated by

increased in circulating inflammatory biomarkers. Reducing the rate of the inflammation

progression is one of the many measures to reduce complication of the disease. Many established

evidences have suggested the beneficial effect of probiotic consumption on the progression of

inflammatory bowel syndrome (IBS). In the present study, possible benefit of probiotics on

inflammatory progression of Diabetes mellitus (DM) is investigated. The present study employed

two different approaches to induce hyperglycemia in adult Sprague- Dawley rats. The initial

approach using high fructose diet (HFD), (21% w/v), was unable to induce satisfactorily

hyperglycemia in the animal. Chemical induction using streptozotocin (STZ), (50 mg/ kg body

weight) induced hyperglycemia in all animals injected. Rats in both batches were divided into

four groups. A non-diabetic group (ND), a non-treated group with a standard diet (NT) and two

diabetic groups which were treated with 109 cfu/ml/day (LC1/DLC1) and 10

11 cfu/ml/day

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(LC2/DLC2) of self-cultured Lactobacillus casei strain Shirota (LcS). The probiotic L.cS was

gavaged for three consecutive weeks. Blood was collected through the orbital venous plexus to

measure circulating C-reactive protein (CRP), interleukin-6 (IL-6) and interleukin-4 (IL-4) as the

inflammatory biomarkers. The results have shown no significant difference in blood glucose

level of L.cS fed rats compared with non-treated group (p>0.05). Both doses of L.cS were

observed to induce lower CRP production after three weeks of administration compared to the

diabetic control group. Interleukin-6 was found to be decreased but only at higher dose (1011

cfu/ml) of the L.cS which was comparable with the level that was observed in the non-diabetic

group at the end of the study duration. Interleukin-4 level was found to be significantly decreased

in all treated and the diabetic control group, but was observed to be higher in the normal group.

Data from the three inflammatory biomarkers suggested that probiotic L.cS has the potential to

improve inflammatory status among STZ-induced rats. Histological study of the pancreas

showed a huge damage of the Langerhans islets in STZ induced rats. In addition, foamy cells

were found in the kidney cortex of the STZ induced rats. Colon condition was similar among all

groups. Inflammatory invasion was evidenced in the liver where the presence of neutrophils

could be clearly seen in the non-treated group. Treatment with the L.cS observed a significant

reduction in the invasiveness of the neutrophils which highly indicates decreased levels of

inflammation by the probiotics. The results of this study indicate that Lactobacillus casei strain

Shirota, possess an ability to reduce inflammatory biomarkers hence complication caused by

inflammation in DM.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi

keperluan untuk ijazah Master Sains

KESAN PENGAMBILAN LACTOBACILLUS CASEI PERHADAP PENANDA BIOLOGI

KERADANGAN DAN PERUBAHAN HISTOLOGI DALAM ORGAN TERPILIH BAGI

TIKUS NORMAL DAN DIABETES

Oleh

AIDA ZARFESHANI

November 2010

Pengerusi : Professor Madya Mohd Sokhini Bin Abd Mutalib, PhD

Fakulti : Perubatan dan Sains Kesihatan

Tingkat keparahan diabetes mellitus (DM) sering dimanifestasikan oleh keradangan progresif,

ditunjukkan dalam peningkatan edaran penanda biologi radang. Mengurangkan kadar

keradangan adalah salah satu langkah untuk mengurangkan komplikasi penyakit ini. Banyak

bukti menunjukkan kesan pengambilan probiotik adalah bermanfaat untuk sindrom radang usus

(IBS). Kajian ini melaporkan potensi probiotik dalam keradangan progresif DM. Oleh itu, dua

pendekatan berbeza digunakan dalam kajian ini untuk mengaruhkan hiperglisemia pada tikus

dewasa Sprague-Dawley. Pendekatan awal adalah dengan menggunakan diet fruktosa tinggi

yang didapati tidak memuaskan dalam mengaruhkan hiperglisemia tikus. Pengaruhan kimia

menggunakan streptozotocin (STZ), (50 mg/ kg berat badan) dapat mengaruhkan hiperglisemia

pada semua haiwan yang disuntik. Tikus dibahagikan kepada empat kumpulan iaitu satu

kumpulan kawalan normal (ND), kumpulan kawalan diabetes dengan diet piawai (NT) dan dua

kumpulan diabetes yang dirawat dengan 109 cfu/ml/hari (DLC1/LC1) dan 10

11 cfu/ml/hari

(DLC2/LC2) kultur Lactobacillus casei strain Shirota (LcS). Probiotik diberikan kepada tikus

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selama tiga minggu berturut-turut secara oral atau gavage. Darah diambil diawal dan diakhir

kajian melalui pleksus vena orbit dan seterusnya digunakan untuk mengukur aras edaran

Interleukin-4 (IL-4), Interleukin-6 (IL-6) dan protein C-reaktif sebagai penanda biologi

keradangan dan lain-lain parameter biologi. Keputusan kajian menunjukkan tiada perbezaan

signifikan pada aras glukosa darah tikus yang diberi L.cS berbanding kumpulan kawalan diabetes

(p<0.05). Kedua-dua dos L.cS mendorong pengeluaran CRP yang lebih rendah selepas tiga

minggu rawatan berbanding dengan kumpulan kawalan diabetes. IL-6 menunjukkan penurunan

tetapi hanya pada L.cS dos tinggi (DLC2/LC2, 1011

cfu/ml) yang seterusnya dapat dibandingkan

dengan kumpulan ND diakhir kajian. Aras IL-4 dilihat menurun secara signifikan pada semua

tikus yang dirawat dan kumpulan NT tetapi lebih tinggi pada kumpulan ND. Data daripada tiga

penanda biologi keradangan menunjukkan probiotik LcS mempunyai potensi dalam memperbaiki

status keradangan pada tikus aruhan STZ. Kajian histologi pankreas menunjukkan kerosakan

serius pada kelompok Langerhans tikus aruhan STZ. Selain itu, sel berbusa ditemui di korteks

ginjal tikus diabetes aruhan-STZ. Kolon berada pada tahap normal bagi tikus sihat dan lain-lain

kumpulan diabetes. Kehadiran neutrofil dapat dilihat dengan jelas pada kumpulan NT yang

membuktikan keradangan pada organ hati. Rawatan L.cS didapati dapat menurunkan tahap

kemansangan neutrofil secara signifikan yang seterusnya menunjukkan penurunan radang

disebabkan oleh L.cS. Keputusan kajian ini menunjukkan bahawa Lactobacillus casei strain

Shirota, betpotensi untuk mengurangkan penanda biologi keradangan yang menyebabkan

komplikasi DM.

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ACKNOWLEDGEMENTS

I give my great pleasure in acknowledging the following people whose advice and effort has

made this dissertation possible.

I would like to express my gratitude to my advisor, Assoc. Professor Dr. Mohd Sokhini bin Abd.

Mutalib, for the honor of accepting me as his student, without whom I could not have completed

and written this dissertation. Assoc. Professor Dr. Mohd Sokhini bin Abd Mutalib made it

possible for me to pursue my M.Sc in an intellectually stimulating environment. This dissertation

is part of the research carried out through Assoc. Professor Mohd Sokhini bin Abd. Mutalib’s

vision and interaction with my co-supervisor Prof. Dr. Zaridah binti Hambali.

I really appreciate Dr. Huzwah Khaza’ai for the guidance and advice that made my

interdisciplinary project a success. My thanks also go to Assoc. Professor Dr. Chong Pei Pei and

Dr. Huzwah Khaza’ai for allowing me to use the facilities of their laboratories and departments.

My earnest thanks also extend to Dr. Razana with her knowledge and patience as an excellent

guide.

I am deeply indebted to many people throughout my M.Sc studies. Hence, I extend my thanks to

former and friendly staffs of pathology, hematology and immunology Laboratories.

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My sincere thanks to M.Sc students, Ainun, Aidil and Ibrahim for the comments and assistance

in the some practical parts of my study.

My deepest thanks are due to my beloved mum Fahimeh and brother Kaveh for their unlimited

and overwhelming love that they have shower upon me and also for their constant support which

made my university life a reality.

To all of you, thank you.

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I certify that an Examination Committee has met on 16/Nov/2010 to conduct the final

examination of Aida Zarfeshani on her Master of Sciences thesis entitled “Effects of

Lactobacillus Casei consumption on levels on inflammation biomarkers and histological changes

in selected organs in normal and diabetic rats” in accordance with Universiti Pertanian Malaysia

(Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981.

The Committee recommends that the student be awarded the relevant degree.

Members of Examination Committee were as follows:

Prof. Madya Dr. Zuraini Ahmad

Jabatan Sains Bioperubatan

Fakulti Perubatan dan Sains Kesihatan

Universiti Malaysia

Dr. Roslida Abd. Hamid @ Abd. Razak

Jabatan Sains Bioperubatan

Fakulti Perubatan dan Sains Kesihatan

Universiti Putra Malaysia

Prof. Dr. Asmah Rahmat

Jabatan Pemakanan dan Dietetik

Fakulti Perubatan dan Sains Kesihatan

Universiti Putra Malaysia

Prof. Madya Dr. Siti Balkis Budin

Jabatan Sains Bioperubatan

Fakulti Kesihatan Bersekutu

Universiti Kebangsaan Malaysia

Jalan Raja Muda Abdul Aziz

50300 Kuala Lumpur

BUJANG BIN KIM HUAT, PhD

Professor/Deputy Dean

School of Graduated Studies

Universiti Putra Malaysia

Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as

fulfilment of the requirement for the degree of Master of sciences. The members of the

Supervisory Committee were as follows:

Mohd Sokhini bin Abd. Mutalib, PhD

Assoc. Prof,

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairman)

Zarida binti Hambali, PhD

Assoc. Prof,

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Member)

HASANAH MOHD GHAZALI, PhD

Professor and Dean

School of Graduated Studies

Universiti Putra Malaysia

Date: 18 January 2011

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which have been

duly acknowledged. I also declare that it has not been previously, and is not concurrently,

submitted for any other degree at Universiti Putra Malaysia or at any other institution.

AIDA ZARFESHANI

Date:

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LIST OF TABLES

Body weight in non-diabetic and diabetic rats

during the experimental period

Body weight in non-diabetic and HFD rats

during the experimental period

Blood glucose level in non-diabetic and diabetic

rats during the experimental period

Blood glucose level in non-diabetic and HFD

rats during the experimental period

Blood tiglyceride level in non-diabetic and HFD

rats after 10 weeks HFD induction

Table

4.1

4.2

4.3

4.4

4.5

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64

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LIST OF FIGURES

Figure

2.1

2.2

2.3

3.1

3.2

3.3

4.1

4.2

4.3

4.4

4.5

4.6

4.7

4.8

4.9

4.10

Chronic complications in DM

Schematic diagram of NFκB as an inflammatory regulator

The anti-inflammatory effect of insulin and the pro-

inflammatory effect of glucose

Plating of LcS on MRS agar

Purified PCR product of the 16S rRNA gene fragmentation

spine column of L.cS colonies

Simplified representation of experimental design

Body weight of normal and STZ induced rats during the

experiment

Body weight of normal and HFD induced rats during the

experiment

Blood glucose of normal and STZ induced rats during the

experiment

Blood glucose of normal and HFD induced rats during the

experiment

C-reactive protein of normal and STZ induced rats during the

experiment

Interleukin-6 of normal and STZ induced rats during the

experiment

Interleukin-4 of normal and STZ induced rats during the

experiment

C-reactive protein of normal and HFD induced rats during the

experiment

Interleukin-6 of normal and STZ induced rats during the

experiment

Interleukin-4 of normal and STZ induced rats during the

experiment

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4.11.a

4.11.b

4.12.a

4.12.b

4.13.a

4.13.b

4.14.a

4.14.b

Histological structure of rat pancreas sections

Histological structure of rat pancreas sections

Histological structure of rat liver sections

Histological structure of rat liver sections

Histological structure of rat kidney sections

Histological structure of rat kidney sections

Histological structure of rat colon sections

Histological structure of rat colon sections

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ABBREVIATIONS

ANOVA: Analysis Of Variance

ATP: Adenosine-5’-Tri Phosphate

BG: Blood Glucose

CFU: Colony Forming Unit

CRP: C-reactive Protein

DM: Diabetes Mellitus

DLC1: Diabetic L.cS 1

DLC2: Diabetic L.cS 2

ELISA: Enzyme Linked Immunosorbent Assay

FAO: Food and Agriculture Organization

GDM: Gestational Diabetes Mellitus

GLUT2: Glucose Transporter 2

GLUT4: Glucose Transporter 4

H and E stain: Hematoxylin and Eosin stain

HFD: High Fructose Diet

IBD: Inflammatory Bowel Disease

IBS: Irritable Bowel Syndrome

IDDM: Insulin Dependent Diabetes Mellitus

IFN-γ: Interferon- γ

IκBα: Inhibitor Kappa B Alpha

IκBβ: Inhibitor Kappa B Beta

IL-1,1 b,4,6,10: Interleukin-1,1 b,4,6,10

ILs: Interleukins

LC1: L.cS 1

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LC2: L.cS 2

L.cS: Lactobacillus casei strain Shirota

MANOVA: Multivariate Analysis of Variance

MNT: Medical Nutrition Therapy

MRS: de Man, Rogosa and Sharpe

NAD: Nicotinamide Adenine Dinucleotide

ND: Non-diabetic

NT: Non-treated

NFκB: Nuclear Factor Kappa B

NIDDM: Non Insulin Dependent Diabetes Mellitus

NK: Natural Killer

NO: Nitric Oxide

PCR: Polymeras Chain Reaction

ROS: Reactive Oxygen Species

RPM: Revolutions Per Minutes

STZ: Streptozotocin

TG: Triglyceride

TNF-α: Tumor Necrosis Factor Alpha

WHO: World Health Organization

Weight/Volume: w/v

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TABLE OF CONTENTS

DEDICATION

ABSTRACT

ABSTRAK

ACKNOWLEDGEMENTS

APPROVAL

DECLARATION

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

CHAPTERS

1 INTRODUCTION

1.1 Over View 1.2 Rationale of the Study

1.3 Problem Statement

1.4 Objective 1.4.1 Specific Objectives

1.5 Hypothesis

2 LITERATURE REVIEW

2.1 Diabetes Mellitus

2.2 Types of Diabetes Mellitus

2.2.1 Diabetes Mellitus Type 1

2.2.2 Diabetes Mellitus Types 2

2.2.3 Gestational Diabetes Mellitus

2.3 Role of Insulin in Diabetes Mellitus

2.4 Chronic Complication of Diabetes Mellitus

2.4.1 Non-Vascular Complications of Diabetes Mellitus

2.4.2 Vascular Complications of Diabetes Mellitus

2.5 Inflammations and Diabetes Mellitus

2.6 Treatment of Diabetes Mellitus

2.6.1 Pharmacological Treatments of Diabetes Mellitus

2.6.2 Non- Pharmacological Treatments of Diabetes Mellitus

2.7 Probiotic’s Strains

2.8 Probiotics and Inflammation

2.9 Probiotics and Diabetes Mellitus

2.10 Experimental Model Used for Diabetes

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3 METHODOLOGY

3.1 Chemicals

3.2 Major Equipments

3.3 Preparation of Lactobacillus casei strain Shirota

3.4 Identification of Lactobacillus casei strain Shirota

3.5 Experimental Animals

3.6 Study Design

3.7 Induction of Diabetes Mellitus by High Fructose Diet

3.8 Induction of Diabetes Mellitus in STZ group

3.9 Blood Collection

3.10 Blood Glucose Measurement

3.11 Blood Triglyceride Measurement

3.12 Measurement of Inflammatory Biomarkers

3.12.1 Measurement of CRP Level

3.12.2 Measurement of IL-6 Level

3.12.3 Measurement of IL-4 Level

3.13 Organ Collection

3.14 Statistical Analysis

4 RESULTS

4.1 Changes of Body Weight in STZ Induced Rats

4.2 Changes of Body Weight in the HFD Fed Rats

4.3 Changes of Blood Glucose in STZ Induced Rats

4.4 Changes of Blood Glucose in HFD Rats

4.5 Changes of Triglycerides in HFD rats

4.6 Effect of Probiotics on Inflammation 4.6.1 Changes of CRP in STZ Induced Rats

4.6.2 Changes of IL-6 in STZ Induced Rats

4.6.3 Changes of IL-4 in STZ Induced Rats

4.7 Changes in Inflammatory Biomarkers in HFD Fed Rats

4.7.1 Changes of CRP in HFD Induced Rats

4.7.2 Changes of IL-6 in HFD Induced Rats

4.7.3 Changes of IL-4 in HFD Induced Rats

4.8 Histological Observations of the Pancreas

4.9 Histological Observations of the Liver

4.10 Histological Observations of the Kidney

4.11 Histological Observations of the Colon

5 DISSCUSION

5.1 Changes of Body Weight

5.2 Changes of Blood Triglycerides and Glucose

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5.3 Changes in Inflammatory Biomarkers

5.4 Histological Observations

6 CONCLUSION AND RECOMMENDATIONS

Conclusions

Recommendations

REFERENCES

APPENDICES

BIODATA OF STUDENT

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106

106

108

109

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141