UNIVERSITI PUTRA MALAYSIA

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

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 i

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

To everyone who believed my abilities, And supported me in my intention, To make some of my dreams come true. ii

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 10 9 cfu/ml/day (LC1/DLC1) and 10 11 cfu/ml/day iii

(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 (10 11 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. iv

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 Pengerusi Fakulti Oleh AIDA ZARFESHANI November 2010 : Professor Madya Mohd Sokhini Bin Abd Mutalib, PhD : 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 10 9 cfu/ml/hari (DLC1/LC1) dan 10 11 cfu/ml/hari (DLC2/LC2) kultur Lactobacillus casei strain Shirota (LcS). Probiotik diberikan kepada tikus v

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, 10 11 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. vi

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. vii

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. viii

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: ix

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 x

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: xi

Table 4.1 4.2 4.3 4.4 4.5 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 Page 58 61 64 67 69 xii

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 proinflammatory 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 Page 16 22 24 41 43 46 57 60 63 66 71 73 75 77 79 81 xiii

4.11.a Histological structure of rat pancreas sections 83 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 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 84 86 87 89 90 92 93 xiv

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 xv

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 xvi

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 ii iii v vii ix xi xii xiii xv 1 1 4 5 6 6 6 7 7 8 9 11 13 14 15 15 17 21 25 26 27 28 31 32 33 xvii

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 37 37 38 39 42 44 44 47 47 48 48 49 49 49 50 51 52 53 55 55 59 62 65 68 70 70 72 74 76 76 78 80 82 85 88 91 94 5.1 Changes of Body Weight 5.2 Changes of Blood Triglycerides and Glucose 94 96 xviii

5.3 Changes in Inflammatory Biomarkers 5.4 Histological Observations 98 103 6 CONCLUSION AND RECOMMENDATIONS 106 Conclusions Recommendations REFERENCES APPENDICES BIODATA OF STUDENT 106 108 109 126 141 xix