UNIVERSITI TEKNOLOGI MARA
ATHEROPROTECTIVE POTENTIAL OF LACTIC ACID BACTERIA AND
THE UNDERLYING
CHOLESTEROL-LOWERING MECHANISM(S)
NUR SYAKILA BINTI ROHAWI
Thesis submitted in fulfillment of the requirements for the degree of
Doctor of Philosophy (Pharmacology)
Faculty of Pharmacy
December 2021
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ABSTRACT
Current treatments and management against atherosclerosis rely predominantly on lipid lowering and diet modification, however it appears to be rather modest, often compromised by the lack of response in high-risk patients and adverse effects. This raises the possibility of using hypocholesterolaemic probiotics for atheroprotection.
Lactic acid bacteria (LAB), the most common, non-pathogenic and safe probiotics, were reported to possess cholesterol-lowering effects. Nevertheless, the beneficial effects of LAB are believed to be strain-dependent and their mechanisms are not completely understood. As such, the present study aimed to identify a superior strain of LAB with excellent cholesterol lowering and atheroprotective properties as well as to elucidate the underlying mechanisms. The present study identified Pediococcus pentosaceus LAB6 and Lactobacillus plantarum LAB12 with promising cholesterol- lowering effects from a preliminary screening of 12 locally sourced lactic acid bacteria (LAB; 5 lactobacilli and 7 pediococci) using the new method of cholesterol quantification through high-performance thin layer chromatography (HPTLC). The LAB yielded strain-dependent cholesterol reducing activity in two different growth media (MRSC: broth containing cholesterol and MRSBC:broth containing cholesterol and bile). LAB6 (MRSC, 51.95% and MRSBC, 57.49%) and LAB12 (MRSC, 58.89%; MRSBC, 68.50%) emerged as the best cholesterol-reducing LAB. Both LAB assimilated fluorescently-tagged cholesterol at the outermost layer of cell membranes.
The LAB also facilitated the deconjugation activity (LAB6:100% vs >38%;
LAB12:100% vs >75% deconjugation) against glyco- and tauro-conjugated bile salts, respectively. Capitalising on these beneficial properties, this study went on to investigate the anti-atherosclerotic and atheroprotective potentials of LAB in vitro and in vivo, respectively. For the in vitro study, the sub-toxic concentration of 24h LAB- fermented cell-free supernatant against RAW264.7 and HUVEC were determined using the sulforhodamine B assay. The LAB-derived CFS was also assessed against the oxLDL-induced foam cell formation assays [i.e., lipid staining by Oil Red O stain, mitochondrial dysfunction using the mitochondrial membrane potential (MMP) assay and antioxidant assays (i.e., glutathione and superoxide dismutase). The effects of LAB6- and LAB12-derived CFS against oxLDL-induced endothelial dysfunction was explored in HUVEC on monocyte-endothelial adhesion and MMP assay. The LAB6- and LAB12-derived CFS not only significantly (p<0.05) reduced oxLDL uptake, reduced MMP depolarisation and increased antioxidant activity in oxLDL-induced RAW264.7 cells, but also reduced monocytes adhesion and prevented MMP depolarisation on oxLDL-induced endothelial dysfunction. For the in vivo study, the atheroprotective properties of freeze-dried LAB12 (LAB12-FD) was explored using C57BL/6J mice fed with high-fat diet for 20 weeks. Daily consumption of LAB12-FD significantly reduced total cholesterol content in the livers, reduced fatty streak formation and infiltration of macrophages in the aortas, as well as improved intestinal barrier (i.e., reduced serum lipopolysaccharide content and urine lactulose mannitol ratio) of C57BL/6J mice. Altogether, the present findings strongly support the possibility of using LAB12 as a dietary supplement for prevention of hypercholesterolaemia and atherosclerosis. The current data also provides important insights into the complementary use of LAB in management of metabolic diseases.
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ACKNOWLEDGEMENT
In the name of Allah, the Most Gracious and the Most Merciful. Alhamdullillah, all praises to Allah for His blessings, strength and patience that had enabled me to complete this thesis. First and foremost, I would like to express my sincere gratitude to my main supervisor, Prof. Dr. Kalavathy Ramasamy, for the precious opportunity to be involved in this important project. I am grateful for her trust, supervision, motivation, encouragement and sharing of knowledge throughout the course of this study. My sincere appreciation also goes to my co-supervisor, Assoc. Prof. Dr. Lim Siong Meng, for his constant help, constructive comments and careful reading of my write-up. The present work would not have been possible without both my supervisors’ guidance and support. Special thanks from me to Prof. Dr. Snenaza Agatonovic Kustrin (Sechenov First Moscow State Medical University, Russia), Dr.
Lim Fei Tieng (Hi-Tech Instrument Sdn Bhd), Dr. Hasseri Halim (Faculty of Pharmacy, UiTM), Assoc. Prof. Dato' Dr. Vellayan Subramaniam (Faculty of Pharmacy, UiTM), and staffs at LAFAM, Dr. Zolkapli Bin Eshak and Madam Nor Hayati Binti Monzai (Imaging Centre, UiTM) for their timely advice, sharing information and generous help at different stages of my PhD project.
I am really thankful for the financial support by the Ministry of Higher Education Malaysia and the Universiti Teknologi MARA (UiTM). I acknowledge receipt of funding under the Long Term Research Grant Scheme [600-RMI/LRGS 5/3 (2/2012)], Geran Insentif Penyeliaan (GIP) [600-IRMI/MyRA 5/3/GIP (046/2017)], 600-IRMI 5/3/GIP (089/2018), Fundamental Research Grant Scheme [600-IRMI/FRGS 5/3 (317/2019)] and UiTM Postgraduate Assistance Scheme (UPTA). I wish to give a shout-out to all members of the Collaborative Drug Discovery Research (CDDR) Laboratory, in particular Dr. Muhamad Fareez, Dr. Muhammad Zaki, Muhammad Zaki, Siti Hajar, Fatin Umirah, Dr. Syamimi, Dr. Yuganthini, Umi Khalsom, Naemah, Faezah, and Muhammad Syukri for all their assistance, kindness, cooperation and moral support. I cherish our friendship and all wonderful memories we had together. I am also thankful for the support and assistance from staffs and members of the Pharmacology and Toxicology Laboratory, the BRAIN Research Laboratory as well as all post-graduate students of the Faculty of Pharmacy, UiTM. I would also like to thank the dean, Assoc. Prof. Dr. Shariza Sahudin, lecturers, technical and supporting staffs of the Faculty of Pharmacy, UiTM for their direct and indirect contributions over the course of my postgraduate candidature.
Last but not least, I wish to express my deepest gratitude to my beloved husband (Mr Muhamad Azim Bin Azmi), parents (Mr Rohawi Bin Hassim and Madam Makhani Binti Ahmad), sisters (Ezatul Farita and Intan Nur Farahin) and brother (Muhammad Nur Ezzat), for their endless love, unceasing prayers and constant encouragement. My family members have always stood by me through good and bad times. To those whose names are not mentioned here, but has contributed to this research in one way or the other, your kindness meant a lot to me. Thank you very much.
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TABLE OF CONTENTS
Page
CONFIRMATION BY PANEL OF EXAMINERS ii
AUTHOR’S DECLARATION iii
ABSTRACT iv
ACKNOWLEDGEMENT i
TABLE OF CONTENTS ii
LIST OF TABLES vii
LIST OF FIGURES x
LIST OF ABBREVIATIONS xiv
LIST OF UNITS xxii
CHAPTER ONE INTRODUCTION 1
1.1 Research Background 1
1.2 Problem Statement 3
1.3 Objectives 5
1.4 Significance of the study 6
CHAPTER TWO LITERATURE REVIEW 7
2.1 Atherosclerosis 7
2.1.1 Overview 7
2.1.2 Epidemiology 7
2.1.3 Risk Factors 10
2.1.4 Pathophysiology 13
2.1.5 Therapeutic and Lifestyle Intervention 22
2.1.6 Diet-Induced Dysbiosis 28
2.2 Probiotics Improved Dysbiosis: Potential Atheroprotection 32
2.2.1 Overview 32
2.2.2 Probiotics Reverse Dysbiosis in Atherosclerosis 36
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2.2.3 Cholesterol-lowering Properties of Probiotics In Vitro 39 2.2.4 Hypocholesterolaemic Properties of Probiotics In Vivo 44 2.2.5 Hypocholesterolaemic Properties of Probiotics: Clinical Studies 51 2.2.6 The Effects of Probiotics against Atherosclerotic Parameters 57
CHAPTER THREE RESEARCH METHODOLOGY 69
3.1 Introduction 69
3.2 Materials 72
3.2.1 In Vitro Assessment of Pediococci- and Lactobacilli-Induced Cholesterol-Lowering Effects Using Digitally Enhanced HPTLC and
Confocal Microscopy 72
3.2.2 A New HPTLC Method for Determination of Pediococci- and Lactobacilli-Induced Bile Salt Deconjugation 73 3.2.3 Anti-Atherosclerotic Effects of CFS Fermented by P. pentosaceus
LAB6 and L. plantarum LAB12 In Vitro 74
3.2.4 Hypocholesterolaemic and Atheroprotective Effects of L. plantarum
LAB12 in HFD-Induced C57BL/6J Mice 76
3.3 Methodology 81
3.3.1 In Vitro Assessment of Pediococci- and Lactobacilli-Induced Cholesterol-Lowering Effects Using Digitally Enhanced HPTLC and
Confocal Microscopy 81
3.3.2 A New HPTLC Method for Determination of Pediococci- and Lactobacilli-Induced Bile Salt Deconjugation 89 3.3.3 Anti-Atherosclerotic Effects of CFS Produced by P. pentosaceus
LAB6 and L. plantarum LAB12 In Vitro 94
3.3.4 Hypocholesterolaemic and Atheroprotective Effects of L. plantarum
LAB12 in HFD-Induced C57BL/6J Mice 104
3.3.5 In Vivo Study of the Atheroprotective potential of LAB12 105
CHAPTER FOUR RESULTS 115
4.1 Introduction 115
