الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 187 |  |  |
| | | from | 187 | | |
Abstract
In The Present Study, The Role Of Blood Coagulation System Activation In The Pathogenesis Of Liver Fibrosis Induced Experimentally In Rats By Carbon Tetrachloride (Ccl4) Was Investigated. In Addition, We Investigated The Possible Protective Role Of Different Anti-Coagulant Drugs In The Protection Against Ccl4-Induced Liver Fibrosis. In This Study, Liver Fibrosis Was Induced By 50% Solution, V/V, In Olive Oil Via The Subcutaneous (S.C.) Route Twice Weekly For 6 Weeks. In The First 2 Weeks, Ccl4 Solution Was Given In A Dose Of 5 Ml/Kg. In The Remaining 4 Weeks, The Dose Was Reduced To 3 Ml/Kg.
The Study Was Performed As Two Sets; In The First Set, A 6-Week Time-Course Study Was Conducted To Investigate The Time Relationship Between Coagulation Cascade Initiation And Commencement Of Ccl4-Induced Liver Fibrosis. Animals Were Classified Into 7 Groups. One group Served As Normal Control, While Groups 2-7 Received Ccl4 And Were Sacrificed After 1 Week, 2 Weeks, 3 Weeks And 4 Weeks, 5 Weeks And 6 Weeks Respectively. In The Second Set; In The Second Set, The Effect Of selected Anticoagulants Was Studied Against Experimental Liver Fibrosis. Dabigatran Etexilate (20 Mg/Kg/Day), Rivaroxaban (5 Mg/Kg/Day) And Clopidogrel (20 Mg/Kg/Day) Against Ccl4-Induced Liver Fibrosis Were Administered On Daily Basis Starting 3 Days Before The First Ccl4 Dose Until The End Of The Experiment (6 Weeks). Then, Blood And Tissue Samples Were Collected For Molecular And Biochemical Estimations.
Estimation Of Liver Function Markers Was Performed By Detection Of Serum Levels Of Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Bilirubin And Albumin Spectrophotometrically. In Addition, We Detected Animal Weight, Relative Liver Weight And Hepatic Histological Changes.
Hepatic Glutathione (GSH) Content, Malondialdehyde (MDA) Content And Nitric Oxide (Nox) Production Were Determined As Oxidative Stress Markers. In Addition, Tumor Necrosis Factor Alpha (TNF-Α), Interleukin 1-Beta (IL-1β) And Hydroxyl Proline (HYP) Also Assessed Using ELISA Technique.
Side By Side, Hematological Parameters As Coagulation Profile Including Prothrombin Time (PT), Prothrombin Concentration (PC), And International Normalized Ratio (INR) Were Estimated. In Addition, Immunofluorescence Detection Of Tissue Factor (TF), Fibrin And Transforming Growth Factor-Β (TGF-Β1) Proteins Was Performed. Moreover, Immunohistochemical Estimation Of Alpha Smooth Muscle Actin (Α-SMA) Positive Cells As Well As Routine Histopathological Study (H&E) And Special Staining (Masson Trichrome) Were Also Carried Out.
6.1. THE MAIN FINDINGS OF THE PRESENT INVESTIGATION CAN BE SUMMARIZED AS FOLLOW:
6.1.1. Effect Of Ccl4 And Test Agents On Histological characters:
Histologically, Liver Tissue In The First And The Third Week Of Ccl4-Administration Showed More Or Less Normal Histological Structure Of Showed Slightly Congested Central Vein And Dilated Blood Sinusoids. Moreover, Mild Degenerative Changes Of Hepatocytes With Fatty Degeneration Were Also Observed. Carbon Tetrachloride Administration For 6 Weeks Showed Severe Degenerative Changes where Cytoplasmic Vacuolations Were Observed. Pyknotic Nuclei Were Also Observed In Which There Are Irreversible Condensation Of chromatin Of A Cell Undergoing Necrosis Or Apoptosis.
Pre-Treatment Of Animals With Dabigatran (20 Mg/Kg), Rivaroxaban (5 Mg/Kg) And Clopidogrel (20 Mg/Kg) Significantly Restored Hepatic Architecture.
6.1.2. Effect Of Ccl4 And Test Agents On Liver Function Tests:
Rats Injected With Ccl4 Showed Significant Increases In Serum ALT, AST As Well As Total Bilirubin Levels After 3 And 4 Weeks. Further Increases In Serum ALT, AST And Total Bilirubin Levels Was Evident At Weeks 5 And 6 As Compared To Normal Control Rats. On The Other Hand, A DROP In Albumin Level Was Significant In Week 5 And Further Declined In Week 6 As Compared To Normal Control Rats.
Pre-Treatment Of Animals With Dabigatran (20 Mg/Kg), Rivaroxaban (5 Mg/Kg) And Clopidogrel (20 Mg/Kg) Significantly Normalized These Values.
6.1.3. Effect Of Ccl4 And Test Agents On Coagulation Profile:
In The Current Study, PT And INR Significantly Increased Starting from Week 1 After Ccl4 Administration As Compared To Normal Control Values. In Addition, A Significant Reduction In PC Was Induced As Compared To Normal Control Group.
Mean PT And INR Values Showed Further Significant Elevations Compared With Previous Week Recordings Starting from Week 4 And The Increase Continued Till Week 6 As Compared With Normal Control. Moreover, Rats Showed A Significant Decrease In PC After 2 And 3 Weeks. The DROP Continued Till Week 6 As Compared To Normal Control Rats.
Pre-Treatment Of Animals With Dabigatran (20 Mg/Kg), Rivaroxaban (5 Mg/Kg) And Clopidogrel (20 Mg/Kg) Significantly Normalized These Values As Compared To Ccl4-Fibrotic Control Group.
6.1.4. Effect Of Ccl4 And Test Agents On Oxidative Stress Markers:
Hepatic MDA Content Started To Increase Significantly In Week 3 With Further Significant Rises Every Subsequent Week Until Week 6 Compared With Normal Control Level. Nitrate/Nitrite Production Started To Increase Significantly In A Successive Manner Starting from Week 4 Through Weeks 5 And 6 As Compared With Normal Control Level.
Similarly, Hepatic GSH Depletion Caused By Ccl4 Administration Became Significant Compared With Normal Control Level Starting Mildly from Week 2.
Pre-Treatment Of Animals With Dabigatran (20 Mg/Kg), Rivaroxaban (5 Mg/Kg) And Clopidogrel (20 Mg/Kg) Significantly Decreased MDA And Nox Production. On The Other Hand, Treatments Significantly Increased GSH Content As Compared To Ccl4-Fibrotic Control Group.
6.1.5. Effect Of Ccl4 And Test Agents On Inflammatory Markers:
Administration Of Ccl4 Significantly Increased Relative Liver Weight (RLW) Starting from Week 1 Compared To Normal Control. The Increase Continued Till Week 6 Compared With Normal Control.
On The Other Hand, Neutrophil Count Showed Significant Reduction At Week 1, Reaching About Half Of Its Normal Control Value. Starting from Week 2, Neutrophil Count Showed Significant Increase Compared With Week 1. Further Significant Increase In Neutrophil Count In The Subsequent Weeks Continued Successively Until Week 6 Which Compared With Normal Value.
Side By Side, TNF-Α And IL-1β Showed Significant Elevations In Liver Tissue Starting from Week 2 And Week 3, Respectively, Exceeding 2-Fold Increases Regarding Both Markers. The Elevation Continued chronologically Until Week 6.
Pre-Treatment Of Animals With Dabigatran (20 Mg/Kg), Rivaroxaban (5 Mg/Kg) And Clopidogrel (20 Mg/Kg) Significantly Decreased TNF-Α And IL-1β Values As Compared To Ccl4-Fibrotic Control Group.
6.1.6. Effect Of Ccl4 And Test Agents On Fibrosis Markers:
Regarding Hepatic HYP Content, A Mild Significant Elevation Starting from Week 4 After Ccl4 Administration Was Observed. Abrupt Elevation Of Hydroxyproline Level Was Evident In Week 5 And Week 6 As Compared To Normal Control Levels.
Analysis Of Liver Sections Stained With The Special Masson Trichrome Stain Revealed Mild Significant Elevation Of Extracellular Matrix (ECM) Deposition In Peri-Portal Area At Week 3, But Massive Increase Was Shown In Week 6 After Ccl4 Administration
Immunohistochemistry Of Liver Α-SMA Expressing Areas Did Not Show Any Significant Change In Weeks 1 And 3. By Contrast, Massive Expression Of Α-SMA In The Peri-Central Sinusoidal Spaces, where Hscs Is Normally Located Was Detected In Week 6 After Ccl4 Administration As Compared With Basal Level.
Immunofluorescence Of TGF-Β1 Protein Expression Did Not Show Significant Increase In The First Week. On The Other Hand, The Expression Of Protein Started To Show A Significant Expression At Week 3. The Expression Increased Gradually In Hepatocytes And Reached To The Highest Level After 6 Weeks
Pre-Treatment Of Animals With Dabigatran (20 Mg/Kg), Rivaroxaban (5 Mg/Kg) And Clopidogrel (20 Mg/Kg) Significantly Decreased HYP Content. Side By Side, Treatments Decreased The Expression Levels Of Α-SMA And TGF-Β1 And Decreased Collagen Depositions.
6.1.7. Effect Of Ccl4 And Test Agents On Coagulation Proteins Expression (TF And Fibrin):
Immunofluorescence Analysis Of TF And Fibrin Deposition Revealed That Normal Control Rats Showed Low Expression Of Proteins In Liver Tissue. Meanwhile, The Expressions Of These Proteins Were Significantly Increased In In The Peri-Central Areas Compared To The Low Basal Level In Vehicle Treated Animals After 1 Week Of Ccl4 Administration.
The Expression Of Both TF And Fibrin Proteins Was Highly Up Regulated After 3 Weeks Of Ccl4 Administration And Continued To Increase Till Peaked After 6 Weeks Of Ccl4 Administration As Compared To Normal Control.
Pre-Treatment Of Animals With Dabigatran (20 Mg/Kg), Rivaroxaban (5 Mg/Kg) And Clopidogrel (20 Mg/Kg) Significantly Decreased The Expression Of Both TF And Fibrin Proteins.
6.2. DEPENDING ON THE PREVIOUS FINDINGS, IT COULD BE CONCLUDED THAT:
6.2.1. Ccl4 Administration Is A Successful Model For Induction And Development Of Ccl4-Induced Liver Fibrosis In Rats. Activation Of Blood Coagulation Plays A Crucial Role In The Pathophysiology Of Ccl4-Induced Renal Failure. Oxidative Stress And Release Of Inflammatory Mediators Have Important Role In The Progression Of Ccl4-Induced Liver Fibrosis In Rats.
6.2.2. The High Expression Of Coagulation Cascade Proteins Prior To The Hepatotoxicity Induced By Ccl4 May Elucidate The Role Of Activation Of Coagulation System In The Initiation Of Liver Fibrosis.
6.2.3. Inhibition Of Coagulation Cascade By Dabigatran (Thrombin Inhibitor), Rivaroxaban (Fxa Inhibitor) Or Clopidogrel (Platelet Inhibitor) May Protect Against Ccl4-Induced Liver Fibrosis.
6.2.4. These Drugs Seem Promising For Protection Against Ccl4-Induced Liver Fibrosis But Clinical Trials Are Needed To Prove These Findings On Human.