Acknowledgment 2
Table of Contents 3
List of Abbreviations 5
List of Figures 6
List of Tables 6
Abstract 7
Résumé 8
Chapter I: Introduction 9
1. Hepatic fibrosis 9
2. Architecture of the normal liver 10
2.1. Anatomy 10
2.2. Function 11
2.3. Cells within the liver 11
2.4. The liver in health and disease 14
3. Pathway of liver fibrosis 14
3.1. Composition and remodeling of ECM 15
3.2. Immune response 15
3.3. Profibrotic mediators 15
4. Regression of fibrosis 15
4.1 Extracellular matrix degradation 15
4.2 HSCs apoptosis 16
4.3 HSCs senescence 16
4.4 HSCs inactivation 17
5. Effect of Statins on hepatic fibrosis 18
Aim of the Project 20
Chapter II: Materials and methods 21
1. Animals 21
2. Experimental Design 21
2.1 Carbon tetrachloride- (CCL4-) induced liver injury 21
2.2 Antifibrotic effect of Pitavastatin on liver fibrosis. 21
2.3 Regression effect of Pitavastatin on liver fibrosis 21
3. Pico Sirius Red staining. 22
4. Immunohistochemistry staining of hepatic ?SMA. 22
5. ALT and AST detection 23
6. RNA Extraction 23
7. Reverse transcription-PCR 24
8. Real-Time PCR 24
9. Statistical analysis 25
Chapter III: Results 26
1. Experimental liver fibrosis model 26
1.1 Serum ALT AST 26
Chapter IV: Discussion and Conclusion 27
Chapter V: Future perspectives 28
References 29


Figure 1: Structure of the healthy liver 5. 10
Figure 2: Cellular modifications in the sinusoid during liver injury 20. 14
Figure 4: Schematic representation of the HSCs fate during liver regression 29. 17
Figure 5: Schematic summary of the signaling pathway by which statins decrease portal pressure and reduce hepatic fibrosis 38. 19
Figure 6: Schematic representation of the liver fibrosis model in male C57BL/6J mice. 22

Table 1: List of primer sequences used for RT-PCR analysis. 21


Liver 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 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 2.
1. Hepatic fibrosis
Hepatic 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 3. However, the continuous unbalanced synthesis of matrix protein and degradation leads to an incomplete matrix remodeling and irreversible cirrhosis 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 3.
2. Architecture of the normal liver
2.1. 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 (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 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 (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 5.

Figure 1: Structure of the healthy liver 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. Function
The 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 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 6.
2.3. Cells within the liver
There are four major cell types that play different roles in order to allow the proper functioning of the liver.
2.3.1. Hepatocytes
Hepatocytes are parenchymal cells, consisting 70% of the liver population 7, with an average life expectancy of 5 to 6 months. They are characterized by round nuclei with dispersed chromatin and prominent nucleoli. The cytoplasm comprises numerous mitochondria, rough ER and free ribosomes. Hepatocytes, regulated by the NPCs released factor, have a main role in the basic functions of the liver, and in the metabolic activities mentioned before 8. NPCs also stimulate the hepatocyte capacity to replicate mentioning that hepatocyte only secrete TGF a as an autocrine growth factor 9. Regeneration is not the only property that specialize the hepatocyte, 50% of the population possess more than two paired sets of chromosomes, a condition known as Anisokaryosis 8.
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 10, 11.
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 12.
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 13. Also, filtration, recruitment of lymphocytes and antigen presentation are main physiological, scavenger and immunological functions of LSECs. Intrahepatic vasoconstriction and fibrosis progression are inhibited by the LSECs, since they prevent the activation of hepatic stellate cells. Thus, the immune homeostasis within the liver is maintained. Add to the mentioned above, LESCs have their role in liver regeneration following partial hepatectomy and liver injury. However, in case of pathology, they can boost angiogenesis and vasoconstriction by becoming capillarized and dropping their protective features. 14
2.3.4. Hepatic Stellate Cells (HSCs)
Hepatic stellate cells, also called fat-storing cells or perisinusoidal lipocytes are considered the main source of ECM during hepatic disease. Having a star-like shape, they originate from mesenshymal lineage and represent 10% of all resident liver cells. HSCs display two distinctive phenotypes; in the normal liver they express a quiescent phenotype to be activated in case of injury 3.
Having in their cytoplasm plentiful lipid droplets, containing retinoid, triglyceride, cholesterol, and free fatty acids is the most typical feature of these cells. Furthermore, HSCs present a microfilament cluster of actin and cytoskeletal proteins such as the desmin, vimentin, and synemin. The expression of hepatocyte growth factor (HGF), TGF-b, insulin-like growth factor-I (IGF-I) is also regulated by these cells.
Quiescent HSCs are characterized by myofibroblastic and neurondocrine markers such as PDGFR?, LRAT, GFAP, NGF, NT-3, NCAM and BDNF. They also express synaptophysin, Lhx2 to maintain quiescent and PPAR-g; a transcriptional regulator for adipogenesis and transcriptional inhibitor of type I collagen. Yet, the phenotype misses the expression of fatty acid synthase (FAS) and receptor CD95.
Following liver injury, activated HSCs become the major source of ECM deposition, by trans-differentiating from vitamin-A storing cells to myofibroblasts, causing increased production of ?SMA (?-smooth muscle actin). Activated HSCs express markers such reelin, the protease P100, cytoglobin, -2 macroglobin. This phenotype’s gene expression is controlled by several transcription factors such as FoxO, ILK and PPAR-?. They are characterized by the loss of retinoids and lipid droplets, increased ability to proliferate, changing the expression of L-type type voltage-operated Ca2+ channels resulting cellular contraction. Finally, HSCs stimulate the chemotactic and inflammatory process within the liver 11, 15–17.
Besides HSCs, portal fibroblastes (PFs) have also a mesenshymal origin. PFs multiply around bile ducts during fibrosis through biliary and cholestatic liver diseases and appear in portal areas and in newly formed fibrous septa. Both cell types show common characteristics in terms of fibrogenic functions also they express similar markers however, there are specific markers that could distinguish between HSCs and PFs. yet, proteomic analysis confirm that cytoglobin is the best over expressed marker in HSCs that differentiate these cells. In addition, PFs have elevated resistance to apoptosis and important proliferative capacity 18. Additionally, Myofibroblasts of bone marrow origin have been earlier discovered in many parenchymal organs including the liver and has been suggested based on the detection of Y chromosome-positive cells in fibrotic areas 19.
2.4. The liver in health and disease
Generally, 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 (Figure 2) 20.

Figure 2: Cellular modifications in the sinusoid during liver injury 20.
3. Pathway of liver fibrosis
Hepatic fibrosis is a consequence of an inflammatory process that influences hepatocytes or biliary cells. Danger-associated molecular patterns (DAMPs), pathogen-associated molecular patterns (PAMPs) and reactive oxygen species (ROS) are released after hepatocytes apoptosis or necrosis and recognized by the immune system 21. Pro-inflammatory cells starts to secrete cytokines and growth factors such as TGF? which leads to the activation of effector cells, resulting in extracellular matrix deposition. Even that hepatic stellate cell is not the only effector that undergo a transformation into a myofibroblast in response to injury but it appears as the primary source of extracellular matrix synthesize in the liver 22.
3.1 Activation of HSCs

3.1. Composition and remodeling of ECM
3.2.2 Hepatocytes apoptosis
3.3. Profibrotic mediators
4. Regression of fibrosis
Liver fibrosis was thought to be irreversible. However, it was proven wrong. In fact, after the cause of injury is removed, regression of hepatic fibrosis happens 23. Liver can revert to a normal architecture although fibrogenesis is not fully reversible in patients with cirrhosis 24. Fibrosis regression occurs if activated HSCs undergo apoptosis, senescence and inactivation and finally if the extracellular matrix is degraded. In order to attain a normal or near normal liver structure and function, the restitution of a functional hepatocellular mass is required. Accordingly, hepatocytes possess critical regenerative capacity, which compensate for the organ homeostatic turnover and respond in the context of acute damage 21.
4.1 Extracellular matrix degradation
The fundamentally step for attaining resolution of fibrosis is the degradation of the extracellular matrix. Yet, this mechanism depends on the activity of ECM degrading MMPs, however, continued and prolonged expression of TIMPs inhibits MMPs function. During liver regression the balance of MMP-TIMP is altered resulting an increased MMP activity, reduced TIMP level and simultaneously degradation of extracellular matrix 21. In vivo studies of the reversibility of hepatic fibrosis in rats, demonstrated that levels of TIMP1 is reduced after the cause of injury was removed. This decrease comes along with an increase of hepatic collagenase activity. This turnover indicates that the liver has significant permanent protease activity 24. However, resistance to matrix degradation during advanced fibrosis may be caused by collagen cross-linking preventing proteolytic cleavage of collagens, and by deposition of elastin 25.
4.2 HSCs apoptosis
Apoptosis of hepatic stellate cells is mediated by either death receptors-mediated pathway or by pro-apoptotic proteins increased expression, while the expression of pro-survival proteins is decreased (Figure 4). Indeed, HSCs express several death receptors such as FAS, TNFR1, P75 and TRAIL as well as their ligands; FASL, TNF, NGF (nerve growth factor) and TRAIL respectively. Death receptors induce apoptosis by classical caspase activation. Furthermore, caspase-9-mediated programmed cell death results from significant expression of pro-apoptotic proteins such as Bcl, Bax and p53. Immune cells also contribute to the HSCs removal. Natural killer (NK) cells and ?? T (NKT) are involved in the restoration of hepatic fibrosis by killing HSCs when activated by interferon-? (IFN-?). Finally, deprevation of fibrogenic and anti-apoptotic factors is a key mechanism in fibrosis resolution. In fact, Resistance to apoptosis is an important feature of activated HSCs which may due to the survival signals expressed by protein kinases and NF-?B cascade, in addition to anti-apoptotic cytokines such as TIMP1 and TGF? 21, 24, 26.
4.3 HSCs senescence
Senescence is a phenotype associated with irreversible permanent cell-cycle arrest by which cells stop dividing without undergoing cell death,. Interestingly, studies have confirmed that some of activated HSCs in rodent and human hepatic ?brosis express senescence markers like p53.
P53 knockout mice had increased HSCs proliferations and worsened liver fibrosis in response to CCl4. In addition, cessation of CCl4 did not limit HSCs activation, preventing scar resolution. Suggesting that during hepatic fibrosis, senescence is essential in the removal of hepatic stellate cells. Also, in vitro study showed a decreased level of ECM components associated with an up regulation of MMPs expression and genes of immune surveillance, when HSCs access a senescent phenotype. Other studies indicate that senescent HSCs are deleted by NK cells (Figure 4) 27, 28.

Figure 4: Schematic representation of the HSCs fate during liver regression 29.

4.4 HSCs inactivation
Recently, lineage tracing of transgenic mice model revealed that HSCs can return to the space of Disse and also revert to a near-quiescent phenotype following the elimination of injury .This observation confirms earlier in vitro studies, where activated HSCs were able to revert to an inactivated phenotype in cell culture. However, the signals responsible for this transformation have not been known yet (Figure 4) 24. Kisseleva et al induced liver ?brosis in transgenic mice with ?uorescently labeled cells expressing collagen I, followed by a period of ?brosis resolution. After no signi?cant residual liver ?brosis was present, ?uorescent quiescent HSCs were identified, indicating an origin from a previously activated myofibroblast. In the same time, Troeger et al used the same technique and also identi?ed ?uorescent quiescent HSCs following ?brosis resolution. Both groups estimated that half of hepatic myofibroblasts have escaped apoptosis by reversion to a quiescent phenotype 29. These HSCs are distinct from pre-injury quiescent HSCs, and seemed to remain in a “primed” state with an augmented response to further ?brogenic stimuli 26.
5. Effect of Statins on hepatic fibrosis
Statins, also known as HMG-CoA reductase inhibitors, are potent cholesterol-lowering drug which inhibit the mevalonate pathway. The target of statins is the liver and more specifically the hepatocytes which are the only cells capable of transforming cholesterol to bile salts 30.
The enzyme 3-hydroxy 3-methylglutaryl CoA (HMG-CoA) reductase, catalyze a rate-limiting step, the conversion of HMG-CoA to mevalonate, a central component of cholesterol biosynthesis. Blocking this pathway by statins has revolutionized the treatment of hypercholesterolemia 31. In other way, statins do not only compete for the binding site of the substrate, but they also change the conformation of the enzyme thus, preventing the reach of a functional structure. However this binding is reversible 32.
Reducing cholesterol level is not the only effect of statins, they are broadly known for their pleotropic effects on cardiovascular diseases and many more including, anti-oxidative, immune modulatory, antibacterial, antithrombotic 33, anti-inflammatory, improvement or restoration of endothelial function as well as the stability of atherosclerotic plaques 34. Furthermore, studies have shown that statins can control cell proliferation by reducing the DNA synthesis of normal and tumor cells in vitro, this is due to the reduction of mevalonate- derived metabolites synthesis such as isoprenoids 35. Isoprenoids are essential for the prenylation of proteins that facilitates their anchoring in the cell membrane, such as small GTPases families of Ras and Rho. Hence, intracellular signaling pathways are modified by statins 36.
Regarding the effect of statins on hepatic fibrosis, many studies have demonstrated that this drug inhibits the activation and proliferation of HSCs as well as induces their apoptosis. Hence, decreases the production of ECM. Therefore, Statin can be an efficient anti?brotic agent in liver ?brosis 37. For example a study on cirrhotic rats showed that atorvastatin inhibits HSCs activity and reduction of collagen deposition as well as decreases portal hypertension by inhibiting the RhoA/Rho kinase pathway 36. In fact, geranylgeranyl pyrophosphate reduction alters RhoA activity and its downstream effector Rho-kinase in activated hepatic stellate cells. Furthermore, portal pressure reduction and intrahepatic resistance in vivo were proven to be mediated by the upregulation of endothelial NO synthase (eNOS). Thus enhancing NO synthesis 38. Indeed, NO is normally produced by LSECs mediated by eNOS and applies exerts paracrine effects on HSCs. However, NO produced by inducible NO synthase (iNOS), contributes to tissue damage in case of inflammation. In vivo study confirmed that simvastatin ameliorate hepatic fibrosis by mediating the expression eNOS of and inhibiting iNOS expression 39. Other in vivo and in vitro studies demonstrated that decreasing and increasing level of collagen production as well as activation and contraction of HSCs is dependent on the nuclear receptor KLF2 a downstream effector of RhoA 38. Statins play a major role in up-regulating KLF2; this will not only improve the phenotype of HSCs but also inhibit the paracrine interaction with LSECs and finally decrease fibrosis levels ( Figure 5) 40.

Figure 5: Schematic summary of the signaling pathway by which statins decrease portal pressure and reduce hepatic fibrosis 38.

1. 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 Design
2.1 Carbon tetrachloride- (CCL4-) induced liver injury
Each mouse was given two intraperitoneal injections of 0.6ml/kg CCl4 (**) mixed with mineral oil (**) at the ratio of 1:10, for 6 consecutive weeks. Mice were sacrificed at one and four days after the last injection. The control group was injected with mineral oil (Figure 6-A).
2.2 Antifibrotic effect of Pitavastatin on liver fibrosis.
Liver fibrosis model was generated as mentioned before. Mice were injected by CCl4. Starting from the third week, the mice were divided into two groups and beside CCl4 injection, they were injected daily by either 10mg/kg of Pitavastatin (**) or by the same volume of DMSO (***) as a vehicle for 13 or 15 days until their sacrifice at 1 or 3 days after the last injection of CCl4 (Figure 6–B).
2.3 Regression effect of Pitavastatin on liver fibrosis
As mentioned previously, fibrosis induction was done by injecting for 6 weeks in a row, 0.6 ml/kg CCl4. After the last injection of CCl4, the mice were divided into two groups and daily treated by either 10mg/kg of Pitavastatin or DMSO until their sacrifice at 1, 2, 3 or 4 days post treatment (Figure 6-C).

Figure 6: Schematic representation of the liver fibrosis model in male C57BL/6J mice.
(A) Liver fibrosis was developed by injecting 0.6 ml/kg CCl4 intraperitonealy twice a week for 6 weeks. sacrifice occurred at day 1 and 4 post-treatment. (B) Antifibrotic model was induced after the third week of CCl4 administration by daily injection of 10mg/kg Pitavastatin. Mice were sacrificed after day 1 and 3 post-treatment (C) Liver regression was induced by injecting 10mg/kg pitavastatin after 6 weeks injection of CCl4 followed by harvest at serial time points after the final injection.
3. Pico Sirius Red staining.
Liver fragments were fixed in 10% buffered formaldehyde for at least ***days. Four micrometers sections were cut and stained with Pico Sirius red (reference).
4. Immunohistochemistry staining of hepatic ?SMA.
Liver tissue was deparaffined into 100% xylene followed by a hydration with 100%, 90% and 80% ethanol for 5min each. Sections were rinsed by tape water for 10 minutes and were hydrated in 1x TBS buffer for 30 min at RT. In order to break down the molecular cross links formed by formalin fixation, sections were heated twice in a boiling antigen retrieval buffer in the microwaver for 5 min. After cooling down, the slides were washed by TBS pH=7.6 for 10 minutes then blocked in a normal serum (4% serum, 0.1% TX100) (Serotec Biorad cat #) for 30 min at RT. 3% H2O2 was then used to Block endogenous peroxidase activity. the endogenous unspecific avidin biotin was blocked by Avidin-Biotin blocking kit (Vector Lab, SP-2001) according to the instructions received from the vendor. The sections were then washed twice in 1x TBS. Next, the slides were incubated working solution of MOM mouse Ig blocking reagent (from M.O.M kit (Vector, BMK-2202, 4°C)) for 1h , washed twice by 1x TBS pH=7.6 for 2 min and then incubated by working solution of MOM diluents (from M.O.M kit (Vector, BMK-2202, 4°C)) for 5 min at RT. Mouse monoclonal anti ?SMA antibody diluted 1:10000 was used as primary antibody (Sigma, A2547, clone 1A4). The sections were incubated overnight. Next day, slides were incubated by secondary antibody; MOM biotinylated goat-anti-mouse antibody (from M.O.M kit) diluted 1:500 for 30 min. after several washes tissues were incubated first with working solution of Vectastain ABC reagent (**) for 30 min, then with DAB solution (Dako) and finally counterstained with hematoxyline (Novacastra Leica Biosystem Ref RE7107 Lot 6055893) and mounted by Shandon Immu-mount (Thermo scientific, Ref 9990412). Negative control was performed using MOM diliuent solution instead of the primary antibody which demonstrated no reaction.
5. ALT and AST detection
Blood samples were collected on the day of sacrifice. The samples were centrifuged at 2 xg for 15 minutes at 4 ºC. Serum ALT and AST levels were detected using *** kit (***) by ***
6. RNA Extraction
RNA was isolated from frozen liver tissue. The tissue were disrupted and homogenized with 1 ml QIAzol Lysis Reagent (QIAGEN,79306) and 5mm stainless steel beads (QIAGEN, Ref 69989) using the TissueLyser Qiagen (QIAGEN, II) adjusted to a frequency of 20 Hz for 2 minutes twice. Following homogenization, the lysates were transferred to 1.5 ml eppendorf tubes. 200 ?l chloroform was added to each sample, and shacked vigorously for 15 seconds by inversion. Mixtures were incubated at room temperature for three minutes before being centrifuged at 12,000xg for 15 minutes at 4°C.
After centrifugation, the sample separates into 3 phases: an upper, colorless, aqueous phase containing RNA; a white interphase; and a lower, red, organic phase. The upper aqueous phase of RNA was collected and transferred to the gDNA Removal Column and centrifuged for 30s at 11000 xg. After removing genomic DNA, 100?l of binding solution were added to 350?l lysate, mixed well, transferred to the RNA plus column and centrifuged for 15s at 11000xg. Now the RNA was binding the silica gel membrane, three washes were done in order to removes any remaining impurities from the membrane. First one by adding 200 µl WB1 wash buffer, the second with 600 µl WB2 and the third with 250 µl WB2. Samples were centrifuged after each wash as mentioned before. Finally, in order to eluate the RNA in a new eppendorf tube, 30 µl RNase-free H2O were added to the column then centrifuged for 1 min at 11000 xg. This step was repeated using the remainder of the sample. Total RNA was extracted using the RNeasy Kit MN Nucleospin RNA plus (***, cat # 740984.50).
The resulting RNA was quantified using Nanodrop (Thermo Fisher Scientific) by measuring the absorbance at 260 nm (A260). The ratio of the readings at 260 nm and 280 nm (A260/A280) provides an estimate of the purity of RNA regarding contaminants that absorb in the UV, such as protein. Pure RNA has an A260/A280 ratio of 1.8 to 2.0.
7. Reverse transcription-PCR
Reverse 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 PCR
Real-time PCR reactions were performed using CFX384 system (Bio-Rad Laboratories, California, USA) with iTaq™ Universal SYBR® Green supermix (Bio-Rad Laboratories, California, USA). The plate was run for 56 cycles. The first cycle was run at 94°C for 15 min followed by 55 cycles each at 94°C for 15 seconds, 56°C for 20 seconds, and finally 72°C for 30 seconds. Melting curves were evaluated to check for primer specificity for the PCR product and the results were quantified and analyzed using the Delta-Delta CT method. The primer sequences are listed in Table 1.The housekeeping gene 18S rRNA was used for normalization.
Table 1: List of primer sequences used for RT-PCR analysis.
Target genes Forward primer Reverse primer
MMP13 5′- 5′-
PDGFR? 5′- 5′-

9. Statistical analysis
Animals 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