6044712633046909955702945 MASTER THESIS In Order to Obtain the RESEARCH MASTER In Immunology Presented and defended by

6044712633046909955702945
MASTER THESIS
In Order to Obtain the
RESEARCH MASTER
In
Immunology
Presented and defended by:
Santia Khoury DiabOn Day 00 September, 2018
Title
Effect of Pitavastatin in carbon tetrachloride – induced liver fibrosis in mice
Supervisor
Dr. Eva HamadeDr. Aida HabibReviewers
Dr.
Dr.
Lebanese University-Faculty of sciences

Acknowledgment
Table of Contents TOC o “1-3” h z u Acknowledgment PAGEREF _Toc522482196 h 2Table of Contents PAGEREF _Toc522482197 h 3List of Abbreviations PAGEREF _Toc522482198 h 5List of Tables PAGEREF _Toc522482199 h 6List of Figures PAGEREF _Toc522482200 h 7Abstract PAGEREF _Toc522482201 h 8Résumé PAGEREF _Toc522482202 h 9Chapter I: Introduction PAGEREF _Toc522482203 h 101. Hepatic fibrosis PAGEREF _Toc522482204 h 102. Architecture of the normal liver PAGEREF _Toc522482205 h 112.1. Anatomy PAGEREF _Toc522482206 h 112.2. Function PAGEREF _Toc522482207 h 122.3. Cells within the Liver PAGEREF _Toc522482208 h 122.4. The liver in health and disease PAGEREF _Toc522482209 h 133. Pathway of liver fibrosis PAGEREF _Toc522482210 h 143.1. Composition and remodeling of ECM PAGEREF _Toc522482211 h 143.2. Immune response PAGEREF _Toc522482212 h 143.3. Profibrotic mediators PAGEREF _Toc522482213 h 144. Regression of fibrosis PAGEREF _Toc522482214 h 145. Effect of Statins on hepatic fibrosis PAGEREF _Toc522482215 h 145.1. Definition PAGEREF _Toc522482216 h 145.2. Pathway PAGEREF _Toc522482217 h 145.3. Role in inflammation and fibrosis PAGEREF _Toc522482218 h 14Aim of the Project PAGEREF _Toc522482219 h 14Chapter II: Materials and methods PAGEREF _Toc522482220 h 151. Animals PAGEREF _Toc522482221 h 152. Experimental Design PAGEREF _Toc522482222 h 152.1 Carbon tetrachloride- (CCL4-) induced liver injury PAGEREF _Toc522482223 h 153. SR PAGEREF _Toc522482224 h 154. Immunohistochemistry staining of hepatic ?SMA PAGEREF _Toc522482225 h 155. ALT and AST detection PAGEREF _Toc522482226 h 156. RNA Extraction PAGEREF _Toc522482227 h 157. Reverse transcription-PCR PAGEREF _Toc522482228 h 158. Real-Time PCR PAGEREF _Toc522482229 h 169. Statistical analysis PAGEREF _Toc522482230 h 16Chapter III: Results PAGEREF _Toc522482231 h 17Chapter IV: Discussion and Conclusion PAGEREF _Toc522482232 h 18Chapter V: Future perspectives PAGEREF _Toc522482233 h 19References PAGEREF _Toc522482234 h 20

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List of Abbreviations
List of Tables
List of Figures TOC h z c “Figure” Figure 1: Structure of the healthy liver. PAGEREF _Toc522498569 h 11Figure 2: Cellular modifications in the sinusoid during liver injury 9. PAGEREF _Toc522498570 h 13

Abstract
Résumé
Chapter I: IntroductionLiver disease is a major cause of morbidity and mortality worldwide, and the sequent loss of liver function is a critical clinical challenge. There are many different types of liver disease, which can be broadly grouped into three categories: chronic liver disease caused by metabolic dysfunction, acute liver failure that does not damage normal tissue structure, however is related to direct injury and rapid deterioration of hepatic function. Also, chronic liver failure that is associated with widespread tissue damage and scar-based remodeling, which can eventually lead to end-stage cirrhosis and hepatocellular carcinoma ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”7YU8AqlW”,”properties”:{“formattedCitation”:”1″,”plainCitation”:”1″,”noteIndex”:0},”citationItems”:{“id”:66,”uris”:”http://zotero.org/users/local/dX36zxw6/items/A29872Z6″,”uri”:”http://zotero.org/users/local/dX36zxw6/items/A29872Z6″,”itemData”:{“id”:66,”type”:”article-journal”,”title”:”Cell and Tissue Engineering for Liver Disease”,”container-title”:”Science translational medicine”,”page”:”245sr2″,”volume”:”6″,”issue”:”245″,”source”:”PubMed Central”,”abstract”:”Despite the tremendous hurdles presented by the complexity of the liver’s structure and function, advances in liver physiology, stem cell biology and reprogramming, and the engineering of tissues and devices are accelerating the development of cell-based therapies for treating liver disease and liver failure. This State of the Art Review discusses both the near and long-term prospects for such cell-based therapies and the unique challenges for clinical translation.”,”DOI”:”10.1126/scitranslmed.3005975″,”ISSN”:”1946-6234″,”note”:”PMID: 25031271
PMCID: PMC4374645″,”journalAbbreviation”:”Sci Transl Med”,”author”:{“family”:”Bhatia”,”given”:”Sangeeta N.”},{“family”:”Underhill”,”given”:”Gregory H.”},{“family”:”Zaret”,”given”:”Kenneth S.”},{“family”:”Fox”,”given”:”Ira J.”},”issued”:{“date-parts”:”2014″,7,16}}},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 1.

Hepatic damage can be induced by several factors including viral infection (hepatitis B and C), alcohol abuse, autoimmune hepatitis and chronic cholangiopathies. Also accelerated liver injury due to nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is associated with obesity rates. This situation can cause chronic hepatic inflammation and deregulated wound healing process in the liver, which, if prolonged, can lead to fibrosis ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”nEZrFrAA”,”properties”:{“formattedCitation”:”2″,”plainCitation”:”2″,”noteIndex”:0},”citationItems”:{“id”:61,”uris”:”http://zotero.org/users/local/dX36zxw6/items/2ZDMS72K”,”uri”:”http://zotero.org/users/local/dX36zxw6/items/2ZDMS72K”,”itemData”:{“id”:61,”type”:”article-journal”,”title”:”Management strategies for liver fibrosis”,”container-title”:”Annals of Hepatology”,”page”:”48-56″,”volume”:”16″,”issue”:”1″,”source”:”PubMed”,”abstract”:”Liver fibrosis resulting from chronic liver injury are major causes of morbidity and mortality worldwide. Among causes of hepatic fibrosis, viral infection is most common (hepatitis B and C). In addition, obesity rates worldwide have accelerated the risk of liver injury due to nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Also liver fibrosis is associated with the consumption of alcohol, or autoimmune hepatitis and chronic cholangiophaties. The response of hepatocytes to inflammation plays a decisive role in the physiopathology of hepatic fibrosis, which involves the recruitment of both pro- and anti-inflammatory cells such as monocytes and macrophages. As well as the production of other cytokines and chemokines, which increase the stimulus of hepatic stellate cells by activating proinflammatory cells. The aim of this review is to identify the therapeutic options available for the treatment of the liver fibrosis, enabling the prevention of progression when is detected in time.”,”DOI”:”10.5604/16652681.1226814″,”ISSN”:”1665-2681″,”note”:”PMID: 28051792″,”journalAbbreviation”:”Ann Hepatol”,”language”:”eng”,”author”:{“family”:”Altamirano-Barrera”,”given”:”Alejandra”},{“family”:”Barranco-Fragoso”,”given”:”Beatriz”},{“family”:”Méndez-Sánchez”,”given”:”Nahum”},”issued”:{“date-parts”:”2017″,2}}},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 2.

1. Hepatic fibrosisHepatic fibrosis is the main complication of chronic liver failure and characterized by the excessive accumulation of an altered extracellular matrix, that is extremely rich in type I and III collagens. Deposition of scar tissue results from a wound healing response that occurs to maintain liver integrity after several insults from various biochemical metabolites ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”PR8G59F5″,”properties”:{“formattedCitation”:”3″,”plainCitation”:”3″,”noteIndex”:0},”citationItems”:{“id”:78,”uris”:”http://zotero.org/users/local/dX36zxw6/items/6PR3LU25″,”uri”:”http://zotero.org/users/local/dX36zxw6/items/6PR3LU25″,”itemData”:{“id”:78,”type”:”article-journal”,”title”:”Cellular mechanisms of tissue fibrosis. 5. Novel insights into liver fibrosis”,”container-title”:”American Journal of Physiology. Cell Physiology”,”page”:”C789-799″,”volume”:”305″,”issue”:”8″,”source”:”PubMed”,”abstract”:”Liver fibrosis is the common scarring reaction associated with chronic liver injury that results from prolonged parenchymal cell injury and/or inflammation. The fibrogenic response is characterized by progressive accumulation of extracellular matrix components enriched in fibrillar collagens and a failure of matrix turnover. This process is driven by a heterogeneous population of hepatic myofibroblasts, which mainly derive from hepatic stellate cells and portal fibroblasts. Regression of fibrosis can be achieved by the successful control of chronic liver injury, owing to termination of the fibrogenic reaction following clearance of hepatic myofibroblasts and restoration of fibrolytic pathways. Understanding of the complex network underlying liver fibrogenesis has allowed the identification of a large number of antifibrotic targets, but no antifibrotic drug has as yet been approved. This review will highlight recent advances regarding the mechanisms that regulate liver fibrogenesis and fibrosis regression, with special focus on novel signaling pathways and the role of inflammatory cells. Translation of these findings to therapies will require continued efforts to develop multitarget therapeutic approaches that will improve the grim prognosis of liver cirrhosis.”,”DOI”:”10.1152/ajpcell.00230.2013″,”ISSN”:”1522-1563″,”note”:”PMID: 23903700″,”journalAbbreviation”:”Am. J. Physiol., Cell Physiol.”,”language”:”eng”,”author”:{“family”:”Mallat”,”given”:”Ariane”},{“family”:”Lotersztajn”,”given”:”Sophie”},”issued”:{“date-parts”:”2013″,10,15}}},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 3. However, the continuous unbalanced synthesis of matrix protein and degradation leads to an incomplete matrix remodeling and irreversible cirrhosis ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”PSctsZ2D”,”properties”:{“formattedCitation”:”4″,”plainCitation”:”4″,”noteIndex”:0},”citationItems”:{“id”:76,”uris”:”http://zotero.org/users/local/dX36zxw6/items/EINLTY6N”,”uri”:”http://zotero.org/users/local/dX36zxw6/items/EINLTY6N”,”itemData”:{“id”:76,”type”:”article-journal”,”title”:”Remodeling and homeostasis of the extracellular matrix: implications for fibrotic diseases and cancer”,”container-title”:”Disease Models ; Mechanisms”,”page”:”165-178″,”volume”:”4″,”issue”:”2″,”source”:”PubMed Central”,”abstract”:”Dynamic remodeling of the extracellular matrix (ECM) is essential for development, wound healing and normal organ homeostasis. Life-threatening pathological conditions arise when ECM remodeling becomes excessive or uncontrolled. In this Perspective, we focus on how ECM remodeling contributes to fibrotic diseases and cancer, which both present challenging obstacles with respect to clinical treatment, to illustrate the importance and complexity of cell-ECM interactions in the pathogenesis of these conditions. Fibrotic diseases, which include pulmonary fibrosis, systemic sclerosis, liver cirrhosis and cardiovascular disease, account for over 45% of deaths in the developed world. ECM remodeling is also crucial for tumor malignancy and metastatic progression, which ultimately cause over 90% of deaths from cancer. Here, we discuss current methodologies and models for understanding and quantifying the impact of environmental cues provided by the ECM on disease progression, and how improving our understanding of ECM remodeling in these pathological conditions is crucial for uncovering novel therapeutic targets and treatment strategies. This can only be achieved through the use of appropriate in vitro and in vivo models to mimic disease, and with technologies that enable accurate monitoring, imaging and quantification of the ECM.”,”DOI”:”10.1242/dmm.004077″,”ISSN”:”1754-8403″,”note”:”PMID: 21324931
PMCID: PMC3046088″,”shortTitle”:”Remodeling and homeostasis of the extracellular matrix”,”journalAbbreviation”:”Dis Model Mech”,”author”:{“family”:”Cox”,”given”:”Thomas R.”},{“family”:”Erler”,”given”:”Janine T.”},”issued”:{“date-parts”:”2011″,3}}},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 4.

Cirrhosis is a late stage condition in which the architecture of the liver becomes abnormal, the function of hepatocytes is reduced, and the hepatic blood ?ow is altered due to vascularized ?brotic septa surrounding regenerating nodules. Liver cirrhosis results in multiple complications such as coagulation defect and portal hypertension, including ascites, variceal bleeding, renal failure, hepatic encephalopathy, bacterial peritonitis and finally hepatocellular carcinoma ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”d9qH8zwQ”,”properties”:{“formattedCitation”:”3″,”plainCitation”:”3″,”noteIndex”:0},”citationItems”:{“id”:78,”uris”:”http://zotero.org/users/local/dX36zxw6/items/6PR3LU25″,”uri”:”http://zotero.org/users/local/dX36zxw6/items/6PR3LU25″,”itemData”:{“id”:78,”type”:”article-journal”,”title”:”Cellular mechanisms of tissue fibrosis. 5. Novel insights into liver fibrosis”,”container-title”:”American Journal of Physiology. Cell Physiology”,”page”:”C789-799″,”volume”:”305″,”issue”:”8″,”source”:”PubMed”,”abstract”:”Liver fibrosis is the common scarring reaction associated with chronic liver injury that results from prolonged parenchymal cell injury and/or inflammation. The fibrogenic response is characterized by progressive accumulation of extracellular matrix components enriched in fibrillar collagens and a failure of matrix turnover. This process is driven by a heterogeneous population of hepatic myofibroblasts, which mainly derive from hepatic stellate cells and portal fibroblasts. Regression of fibrosis can be achieved by the successful control of chronic liver injury, owing to termination of the fibrogenic reaction following clearance of hepatic myofibroblasts and restoration of fibrolytic pathways. Understanding of the complex network underlying liver fibrogenesis has allowed the identification of a large number of antifibrotic targets, but no antifibrotic drug has as yet been approved. This review will highlight recent advances regarding the mechanisms that regulate liver fibrogenesis and fibrosis regression, with special focus on novel signaling pathways and the role of inflammatory cells. Translation of these findings to therapies will require continued efforts to develop multitarget therapeutic approaches that will improve the grim prognosis of liver cirrhosis.”,”DOI”:”10.1152/ajpcell.00230.2013″,”ISSN”:”1522-1563″,”note”:”PMID: 23903700″,”journalAbbreviation”:”Am. J. Physiol., Cell Physiol.”,”language”:”eng”,”author”:{“family”:”Mallat”,”given”:”Ariane”},{“family”:”Lotersztajn”,”given”:”Sophie”},”issued”:{“date-parts”:”2013″,10,15}}},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 3.

2. Architecture of the normal liver2.1. Anatomy
The liver is the heaviest visceral organ in the body, expressing 2–5% of body weight and exhibits an iterative, multicellular architecture. The organ is divided into four lobes; yet, the liver lobule represents its functional units.

Each lobule is composed of hexagonal cords of hepatocytes arranged around a central vein that drain into the large hepatic vein. The corners of the hexagon constitute the portal triad consisting of a portal vein, hepatic artery and biliary duct ( REF _Ref522294612 h REF _Ref522294640 h Figure 1-A). Within a lobule, two afferent vessels supply hepatic blood: the hepatic artery and the portal vein, and flows in specialized sinusoidal vessels towards the central vein ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”ALq4Badn”,”properties”:{“formattedCitation”:”1″,”plainCitation”:”1″,”noteIndex”:0},”citationItems”:{“id”:66,”uris”:”http://zotero.org/users/local/dX36zxw6/items/A29872Z6″,”uri”:”http://zotero.org/users/local/dX36zxw6/items/A29872Z6″,”itemData”:{“id”:66,”type”:”article-journal”,”title”:”Cell and Tissue Engineering for Liver Disease”,”container-title”:”Science translational medicine”,”page”:”245sr2″,”volume”:”6″,”issue”:”245″,”source”:”PubMed Central”,”abstract”:”Despite the tremendous hurdles presented by the complexity of the liver’s structure and function, advances in liver physiology, stem cell biology and reprogramming, and the engineering of tissues and devices are accelerating the development of cell-based therapies for treating liver disease and liver failure. This State of the Art Review discusses both the near and long-term prospects for such cell-based therapies and the unique challenges for clinical translation.”,”DOI”:”10.1126/scitranslmed.3005975″,”ISSN”:”1946-6234″,”note”:”PMID: 25031271
PMCID: PMC4374645″,”journalAbbreviation”:”Sci Transl Med”,”author”:{“family”:”Bhatia”,”given”:”Sangeeta N.”},{“family”:”Underhill”,”given”:”Gregory H.”},{“family”:”Zaret”,”given”:”Kenneth S.”},{“family”:”Fox”,”given”:”Ira J.”},”issued”:{“date-parts”:”2014″,7,16}},”label”:”page”},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 1.

The hepatic sinusoid is a complex vascular channel built from specialized fenestrated endothelial cells of the liver also it is the residence of the hepatic macrophages named Kupffer cells. Stellate cells are located in the sub-endothelial space known as the space of Disse that separates the hepatocyte cords from the blood and the sinusoids ( REF _Ref522294640 h Figure 1-B). Bile, that is produced and excreted by hepatocytes into the bile canaliculi, flows in the opposite direction to sinusoidal blood flow towards the intrahepatic bile duct, which is lined by epithelial cells called cholangiocytes ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”74bJOJza”,”properties”:{“formattedCitation”:”5″,”plainCitation”:”5″,”noteIndex”:0},”citationItems”:{“id”:63,”uris”:”http://zotero.org/users/local/dX36zxw6/items/8VD79RWL”,”uri”:”http://zotero.org/users/local/dX36zxw6/items/8VD79RWL”,”itemData”:{“id”:63,”type”:”article-journal”,”title”:”Orchestrating liver development”,”container-title”:”Development (Cambridge, England)”,”page”:”2094-2108″,”volume”:”142″,”issue”:”12″,”source”:”PubMed Central”,”abstract”:”The liver is a central regulator of metabolism, and liver failure thus constitutes a major health burden. Understanding how this complex organ develops during embryogenesis will yield insights into how liver regeneration can be promoted and how functional liver replacement tissue can be engineered. Recent studies of animal models have identified key signaling pathways and complex tissue interactions that progressively generate liver progenitor cells, differentiated lineages and functional tissues. In addition, progress in understanding how these cells interact, and how transcriptional and signaling programs precisely coordinate liver development, has begun to elucidate the molecular mechanisms underlying this complexity. Here, we review the lineage relationships, signaling pathways and transcriptional programs that orchestrate hepatogenesis., Summary: This review summarises the complex interplay between cellular lineages, signalling pathways, and transcriptional programs necessary to form a vertebrate liver.”,”DOI”:”10.1242/dev.114215″,”ISSN”:”0950-1991″,”note”:”PMID: 26081571
PMCID: PMC4483763″,”journalAbbreviation”:”Development”,”author”:{“family”:”Gordillo”,”given”:”Miriam”},{“family”:”Evans”,”given”:”Todd”},{“family”:”Gouon-Evans”,”given”:”Valerie”},”issued”:{“date-parts”:”2015″,6,15}}},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 5.

Figure 1: Structure of the healthy liver ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”zxQ3C0uJ”,”properties”:{“formattedCitation”:”5″,”plainCitation”:”5″,”noteIndex”:0},”citationItems”:{“id”:63,”uris”:”http://zotero.org/users/local/dX36zxw6/items/8VD79RWL”,”uri”:”http://zotero.org/users/local/dX36zxw6/items/8VD79RWL”,”itemData”:{“id”:63,”type”:”article-journal”,”title”:”Orchestrating liver development”,”container-title”:”Development (Cambridge, England)”,”page”:”2094-2108″,”volume”:”142″,”issue”:”12″,”source”:”PubMed Central”,”abstract”:”The liver is a central regulator of metabolism, and liver failure thus constitutes a major health burden. Understanding how this complex organ develops during embryogenesis will yield insights into how liver regeneration can be promoted and how functional liver replacement tissue can be engineered. Recent studies of animal models have identified key signaling pathways and complex tissue interactions that progressively generate liver progenitor cells, differentiated lineages and functional tissues. In addition, progress in understanding how these cells interact, and how transcriptional and signaling programs precisely coordinate liver development, has begun to elucidate the molecular mechanisms underlying this complexity. Here, we review the lineage relationships, signaling pathways and transcriptional programs that orchestrate hepatogenesis., Summary: This review summarises the complex interplay between cellular lineages, signalling pathways, and transcriptional programs necessary to form a vertebrate liver.”,”DOI”:”10.1242/dev.114215″,”ISSN”:”0950-1991″,”note”:”PMID: 26081571
PMCID: PMC4483763″,”journalAbbreviation”:”Development”,”author”:{“family”:”Gordillo”,”given”:”Miriam”},{“family”:”Evans”,”given”:”Todd”},{“family”:”Gouon-Evans”,”given”:”Valerie”},”issued”:{“date-parts”:”2015″,6,15}}},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 5.(A) Geometric organization of the hepatic lobule, the functional unit of the liver. (B) A schematic representation of a sinusoid within the liver and the corresponding location of different hepatic cells.
2.2. FunctionThe liver exhibits many functions in the body, including filtration of the blood, endocrine control of growth signaling pathways and biliary excretion (bile salts and bicarbonate) that facilitates digestion of fats and lipids ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”UW5CgcX4″,”properties”:{“formattedCitation”:”1″,”plainCitation”:”1″,”noteIndex”:0},”citationItems”:{“id”:66,”uris”:”http://zotero.org/users/local/dX36zxw6/items/A29872Z6″,”uri”:”http://zotero.org/users/local/dX36zxw6/items/A29872Z6″,”itemData”:{“id”:66,”type”:”article-journal”,”title”:”Cell and Tissue Engineering for Liver Disease”,”container-title”:”Science translational medicine”,”page”:”245sr2″,”volume”:”6″,”issue”:”245″,”source”:”PubMed Central”,”abstract”:”Despite the tremendous hurdles presented by the complexity of the liver’s structure and function, advances in liver physiology, stem cell biology and reprogramming, and the engineering of tissues and devices are accelerating the development of cell-based therapies for treating liver disease and liver failure. This State of the Art Review discusses both the near and long-term prospects for such cell-based therapies and the unique challenges for clinical translation.”,”DOI”:”10.1126/scitranslmed.3005975″,”ISSN”:”1946-6234″,”note”:”PMID: 25031271
PMCID: PMC4374645″,”journalAbbreviation”:”Sci Transl Med”,”author”:{“family”:”Bhatia”,”given”:”Sangeeta N.”},{“family”:”Underhill”,”given”:”Gregory H.”},{“family”:”Zaret”,”given”:”Kenneth S.”},{“family”:”Fox”,”given”:”Ira J.”},”issued”:{“date-parts”:”2014″,7,16}}},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 1. The liver also provides immune system support, detoxifies chemicals such as xenobiotics, and metabolizes drugs and macronutrient supplying the body with the needed energy.

Carbohydrate storage as glycogen and glucose manufacture via the gluconeogenic pathway is the most critical liver function, in addition to cholesterol homeostasis, lipids oxidation, and storage of excess lipid in other tissues, such as adipose. Finally, the liver is a major producer of the proteins secreted in the blood, their conversion into amino acids, and removal of nitrogen in the form of urea metabolism ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”DLiHW4RK”,”properties”:{“formattedCitation”:”6″,”plainCitation”:”6″,”noteIndex”:0},”citationItems”:{“id”:86,”uris”:”http://zotero.org/users/local/dX36zxw6/items/7LMTCX9Y”,”uri”:”http://zotero.org/users/local/dX36zxw6/items/7LMTCX9Y”,”itemData”:{“id”:86,”type”:”article-journal”,”title”:”The liver”,”container-title”:”Current Biology”,”page”:”R1147-R1151″,”volume”:”27″,”issue”:”21″,”source”:”ScienceDirect”,”abstract”:”Summary
The liver is a critical hub for numerous physiological processes. These include macronutrient metabolism, blood volume regulation, immune system support, endocrine control of growth signaling pathways, lipid and cholesterol homeostasis, and the breakdown of xenobiotic compounds, including many current drugs. Processing, partitioning, and metabolism of macronutrients provide the energy needed to drive the aforementioned processes and are therefore among the liver’s most critical functions. Moreover, the liver’s capacities to store glucose in the form of glycogen, with feeding, and assemble glucose via the gluconeogenic pathway, in response to fasting, are critical. The liver oxidizes lipids, but can also package excess lipid for secretion to and storage in other tissues, such as adipose. Finally, the liver is a major handler of protein and amino acid metabolism as it is responsible for the majority of proteins secreted in the blood (whether based on mass or range of unique proteins), the processing of amino acids for energy, and disposal of nitrogenous waste from protein degradation in the form of urea metabolism. Over the course of evolution this array of hepatic functions has been consolidated in a single organ, the liver, which is conserved in all vertebrates. Developmentally, this organ arises as a result of a complex differentiation program that is initiated by exogenous signal gradients, cellular localization cues, and an intricate hierarchy of transcription factors. These processes that are fully developed in the mature liver are imperative for life. Liver failure from any number of sources (e.g. viral infection, overnutrition, or oncologic burden) is a global health problem. The goal of this primer is to concisely summarize hepatic functions with respect to macronutrient metabolism. Introducing concepts critical to liver development, organization, and physiology sets the stage for these functions and serves to orient the reader. It is important to emphasize that insight into hepatic pathologies and potential therapeutic avenues to treat these conditions requires an understanding of the development and physiology of specialized hepatic functions.”,”DOI”:”10.1016/j.cub.2017.09.019″,”ISSN”:”0960-9822″,”journalAbbreviation”:”Current Biology”,”author”:{“family”:”Trefts”,”given”:”Elijah”},{“family”:”Gannon”,”given”:”Maureen”},{“family”:”Wasserman”,”given”:”David H.”},”issued”:{“date-parts”:”2017″,11,6}}},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 6.
2.3. Cells within the liverThere are four major cell types that play different roles in order to allow the proper functioning of the liver.

2.3.1. Hepatocytes
2.3.2. Kuppfer cells
Kupffer cells are non-parenchymal, resident macrophages which are different from in?ltrating macrophages. They are positioned through the sinusoidal endothelial cells and represent 15% of the total hepatic cells. Kupffer cells are important phagocytes in the liver; they help the innate immune response by scavenging microorganisms that reach the sinusoidal vessels, regulating of inflammatory processes, and finally by removing immune complexes, blood debris and toxic substances. Moreover, kupffer cells regulate iron, bilirubin and cholesterol metabolism.

Furthermore, to be activated, these cells express several receptors; for instance receptor-mediated endocytosis, Fc receptor and Toll-like receptor 4 (TLR4). They also express CD14 and CD68 as surface markers, yet they are negative for CX3CR1. Activation by LPS, DAMPs or complement component leads kypffer cells to release cytokines and chemokines such as CCL2, CCL5, TNF-?, IL-1, IL-6, and reactive oxygen species, promoting the recruitment and activation of other pro-inflammatory cells. In addition, kupffer cells stimulate anti-inflammatory cells by secreting IL-10 specially at the acute phase of liver damage ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”qOG5t765″,”properties”:{“formattedCitation”:”7, 8″,”plainCitation”:”7, 8″,”noteIndex”:0},”citationItems”:{“id”:106,”uris”:”http://zotero.org/users/local/dX36zxw6/items/JT3XLU9U”,”uri”:”http://zotero.org/users/local/dX36zxw6/items/JT3XLU9U”,”itemData”:{“id”:106,”type”:”article-journal”,”title”:”Liver macrophages in tissue homeostasis and disease”,”container-title”:”Nature Reviews Immunology”,”page”:”306-321″,”volume”:”17″,”issue”:”5″,”source”:”www.nature.com”,”abstract”:”Macrophages represent a key cellular component of the liver, and are essential for maintaining tissue homeostasis and ensuring rapid responses to hepatic injury. Our understanding of liver macrophages has been revolutionized by the delineation of heterogeneous subsets of these cells. Kupffer cells are a self-sustaining, liver-resident population of macrophages and can be distinguished from the monocyte-derived macrophages that rapidly accumulate in the injured liver. Specific environmental signals further determine the polarization and function of hepatic macrophages. These cells promote the restoration of tissue integrity following liver injury or infection, but they can also contribute to the progression of liver diseases, including hepatitis, fibrosis and cancer. In this Review, we highlight novel findings regarding the origin, classification and function of hepatic macrophages, and we discuss their divergent roles in the healthy and diseased liver.”,”DOI”:”10.1038/nri.2017.11″,”ISSN”:”1474-1741″,”language”:”en”,”author”:{“family”:”Krenkel”,”given”:”Oliver”},{“family”:”Tacke”,”given”:”Frank”},”issued”:{“date-parts”:”2017″,5}},”label”:”page”},{“id”:110,”uris”:”http://zotero.org/users/local/dX36zxw6/items/AYWRY4PP”,”uri”:”http://zotero.org/users/local/dX36zxw6/items/AYWRY4PP”,”itemData”:{“id”:110,”type”:”article-journal”,”title”:”Understanding the Mechanism of Hepatic Fibrosis and Potential Therapeutic Approaches”,”container-title”:”Saudi Journal of Gastroenterology : Official Journal of the Saudi Gastroenterology Association”,”page”:”155-167″,”volume”:”18″,”issue”:”3″,”source”:”PubMed Central”,”abstract”:”Hepatic fibrosis (HF) is a progressive condition with serious clinical complications arising from abnormal proliferation and amassing of tough fibrous scar tissue. This defiance of collagen fibers becomes fatal due to ultimate failure of liver functions. Participation of various cell types, interlinked cellular events, and large number of mediator molecules make the fibrotic process enormously complex and dynamic. However, with better appreciation of underlying cellular and molecular mechanisms of fibrosis, the assumption that HF cannot be cured is gradually changing. Recent findings have underlined the therapeutic potential of a number of synthetic compounds as well as plant derivatives for cessation or even the reversal of the processes that transforms the liver into fibrotic tissue. It is expected that future inputs will provide a conceptual framework to develop more specific strategies that would facilitate the assessment of risk factors, shortlist early diagnosis biomarkers, and eventually guide development of effective therapeutic alternatives.”,”DOI”:”10.4103/1319-3767.96445″,”ISSN”:”1319-3767″,”note”:”PMID: 22626794
PMCID: PMC3371417″,”journalAbbreviation”:”Saudi J Gastroenterol”,”author”:{“family”:”Ahmad”,”given”:”Areeba”},{“family”:”Ahmad”,”given”:”Riaz”},”issued”:{“date-parts”:”2012″}},”label”:”page”},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 7, 8.
2.3.3. Sinusoidal endothelial cells (SEC)
Liver sinusoidal endothelial cells (LSECs) form the wall of liver sinusoid that separate hepatocytes from the blood. These cells have the highest percentage of the non-parenshymal hepatic cells; comprising about 15% of liver cells and 3% of hepatic volume. Upon their differentiation into adult LSECs, they gain markers such as CD4, CD32 and ICAM-1. Yet, some of these markers are similar to other cells including endothelial and hematopoietic cells but none of them is specific for LSECs ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”md3xeUip”,”properties”:{“formattedCitation”:”9″,”plainCitation”:”9″,”noteIndex”:0},”citationItems”:{“id”:121,”uris”:”http://zotero.org/users/local/dX36zxw6/items/YCZHRX2M”,”uri”:”http://zotero.org/users/local/dX36zxw6/items/YCZHRX2M”,”itemData”:{“id”:121,”type”:”article-journal”,”title”:”Liver sinusoidal endothelial cells: Physiology and role in liver diseases”,”container-title”:”Journal of Hepatology”,”page”:”212-227″,”volume”:”66″,”issue”:”1″,”source”:”www.journal-of-hepatology.eu”,”DOI”:”10.1016/j.jhep.2016.07.009″,”ISSN”:”0168-8278, 1600-0641″,”note”:”PMID: 27423426″,”shortTitle”:”Liver sinusoidal endothelial cells”,”journalAbbreviation”:”Journal of Hepatology”,”language”:”English”,”author”:{“family”:”Poisson”,”given”:”Johanne”},{“family”:”Lemoinne”,”given”:”Sara”},{“family”:”Boulanger”,”given”:”Chantal”},{“family”:”Durand”,”given”:”François”},{“family”:”Moreau”,”given”:”Richard”},{“family”:”Valla”,”given”:”Dominique”},{“family”:”Rautou”,”given”:”Pierre-Emmanuel”},”issued”:{“date-parts”:”2017″,1,1}}},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 9.

LSECs represent a permeable barrier which displays distinctive structural features that make them different from other endothelial cells. In fact, not having a basal membrane neither a diaphragm yet possessing of fenestrae make these cells the most permeable cells with the highest endocytosis capacity of any cell in the body ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”tPG3AZsk”,”properties”:{“formattedCitation”:”10″,”plainCitation”:”10″,”noteIndex”:0},”citationItems”:{“id”:126,”uris”:”http://zotero.org/users/local/dX36zxw6/items/S6NVGWQJ”,”uri”:”http://zotero.org/users/local/dX36zxw6/items/S6NVGWQJ”,”itemData”:{“id”:126,”type”:”article-journal”,”title”:”Pathological process of liver sinusoidal endothelial cells in liver diseases”,”container-title”:”World Journal of Gastroenterology”,”page”:”7666-7677″,”volume”:”23″,”issue”:”43″,”source”:”www.wjgnet.com”,”abstract”:”Pathological process of liver sinusoidal endothelial cells in liver diseases”,”DOI”:”10.3748/wjg.v23.i43.7666″,”language”:”en”,”author”:{“family”:”Ni”,”given”:”Yao”},{“family”:”Li”,”given”:”Juan-Mei”},{“family”:”Liu”,”given”:”Ming-Kun”},{“family”:”Zhang”,”given”:”Ting-Ting”},{“family”:”Wang”,”given”:”Dong-Ping”},{“family”:”Zhou”,”given”:”Wen-Hui”},{“family”:”Hu”,”given”:”Ling-Zi”},{“family”:”Lv”,”given”:”Wen-Liang”},”issued”:{“date-parts”:”2017″,11,21}}},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 10. Also, LSECs have critical physiological, scavenger and immunological functions
2.3.4. Hepatic Stellate Cells (HSCs)
2.4. The liver in health and diseaseGenerally, disturbance of liver’s morphology and function initiate with the injured hepatocytes, once they stimulate the pro-inflammatory pathway. Activated kupffer cells release pro-fibrotic mediators that change the phenotype of HSCs from quiescent to activated cells, resulting in scar formation. The accumulation of extracellular matrix proteins is responsible for the disappearance of endothelial fenestrae and the loss of hepatocytes microvilli ( REF _Ref522498307 h Figure 2) ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”0jm5zeRM”,”properties”:{“formattedCitation”:”11″,”plainCitation”:”11″,”noteIndex”:0},”citationItems”:{“id”:88,”uris”:”http://zotero.org/users/local/dX36zxw6/items/X79E99UB”,”uri”:”http://zotero.org/users/local/dX36zxw6/items/X79E99UB”,”itemData”:{“id”:88,”type”:”article-journal”,”title”:”Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ”,”container-title”:”Journal of Clinical Investigation”,”page”:”539-548″,”volume”:”117″,”issue”:”3″,”source”:”PubMed Central”,”abstract”:”Models of liver fibrosis, which include cell culture models, explanted and biopsied human material, and experimental animal models, have demonstrated that liver fibrosis is a highly dynamic example of solid organ wound healing. Recent work in human and animal models has shown that liver fibrosis is potentially reversible and, in specific circumstances, demonstrates resolution with a restoration of near normal architecture. This Review highlights the manner in which studies of models of liver fibrosis have contributed to the paradigm of dynamic wound healing in this solid organ.”,”DOI”:”10.1172/JCI30542″,”ISSN”:”0021-9738″,”note”:”PMID: 17332881
PMCID: PMC1804370″,”shortTitle”:”Models of liver fibrosis”,”journalAbbreviation”:”J Clin Invest”,”author”:{“family”:”Iredale”,”given”:”John P.”},”issued”:{“date-parts”:”2007″,3,1}}},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 11.

Figure 2: Cellular modifications in the sinusoid during liver injury ADDIN ZOTERO_ITEM CSL_CITATION {“citationID”:”gNeeDt48″,”properties”:{“formattedCitation”:”11″,”plainCitation”:”11″,”noteIndex”:0},”citationItems”:{“id”:88,”uris”:”http://zotero.org/users/local/dX36zxw6/items/X79E99UB”,”uri”:”http://zotero.org/users/local/dX36zxw6/items/X79E99UB”,”itemData”:{“id”:88,”type”:”article-journal”,”title”:”Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ”,”container-title”:”Journal of Clinical Investigation”,”page”:”539-548″,”volume”:”117″,”issue”:”3″,”source”:”PubMed Central”,”abstract”:”Models of liver fibrosis, which include cell culture models, explanted and biopsied human material, and experimental animal models, have demonstrated that liver fibrosis is a highly dynamic example of solid organ wound healing. Recent work in human and animal models has shown that liver fibrosis is potentially reversible and, in specific circumstances, demonstrates resolution with a restoration of near normal architecture. This Review highlights the manner in which studies of models of liver fibrosis have contributed to the paradigm of dynamic wound healing in this solid organ.”,”DOI”:”10.1172/JCI30542″,”ISSN”:”0021-9738″,”note”:”PMID: 17332881
PMCID: PMC1804370″,”shortTitle”:”Models of liver fibrosis”,”journalAbbreviation”:”J Clin Invest”,”author”:{“family”:”Iredale”,”given”:”John P.”},”issued”:{“date-parts”:”2007″,3,1}}},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”} 11.3. Pathway of liver fibrosis3.1. Composition and remodeling of ECM3.2. Immune response3.2.1 Activation of HSC
3.2.2 Hepatocytes apoptosis
3.3. Profibrotic mediators4. Regression of fibrosis5. Effect of Statins on hepatic fibrosis5.1. Definition5.2. Pathway
5.3. Role in inflammation and fibrosisAim of the Project
Chapter II: Materials and methods1. Animals
Eleven-week-old male C57BL/6J mice weighing 20-30 g were purchased from *** Laboratories and housed in a pathogen-free environment. All experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of the American University of Beirut (AUB) following the ‘Guide for the care and use of laboratory animals’ and the “US Government Principles for the Utilization and Care of Vertebrate Animals used in Testing, Research and Training”.
2. Experimental Design2.1 Carbon tetrachloride- (CCL4-) induced liver injuryEach mouse was given two intraperitoneal injections of 0.6ml/kg CCl4 (**) mixed with mineral oil (**) at the ratio of 1:10, for – consecutive weeks. The control group was injected with mineral oil.

3. SR4. Immunohistochemistry staining of hepatic ?SMA5. ALT and AST detectionBlood samples were collected on the day of sacrifice.  The samples were centrifuged at 15000 xg for 15 minutes at 4 ºC. Serum ALT and AST levels were detected using *** kit (***) by ***
6. RNA Extraction7. Reverse transcription-PCRReverse transcription was performed on 1µg of total RNA in a final 20 µl volume using the *** kit () this included creating a negative RT control without reverse transcriptase. The cycle begins at 25°C for 10 min, 37°C for 2 hours, 85°C for 5 min, and ends at 4°C, using the RT-PCR machine (Bio-Rad Laboratories, California, USA). The cDNA samples were stored at -20°C.

8. Real-Time PCR9. Statistical analysisAnimals were randomly selected for the control and treatment group. All results were expressed as the means ± SEM. Differences between groups were analyzed by the Mann-Whitney test, using “GraphPad Prism” software. The p values for p<0.05 and p<0.01, (*, ** respectively) deemed to be indicative of significance.

Chapter III: Results
Chapter IV: Discussion and Conclusion
Chapter V: Future perspectives
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