الفهرس 
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Abstract
In The Present Investigation, The Effect Of Phosphodiesterase-5 (PDE-5) Inhibitor Vardenafil And The PDE-3 Inhibitor Cilostazol Against Rheumatoid Arthritis/Diabetes Mellitus (RA/DM) Comorbidity-Induced Defects In Vascular Reactivity In Rats In Vivo And In Vitro Was Studied. To Achieve This Goal, An RA/DM Comorbidity Model Induced In Adult Female Albino Rats Was Performed.
For Setting The In Vivo And The In Vitro RA/DM Comorbidity Model, Two Sets Of Experiments Were Performed; The First Was Performed To Set The Model Of RA/DM Comorbidity, But The Second Was Done To Test The Effects Of Vardenafil And Cilostazol On Vascular Abnormalities Induced By RA/DM Comorbidity. In The First Set, RA Was Induced Using Three Subcutaneous Doses Of Complete Freund’s Adjuvant (CFA), Each Of 0.4 Ml, Injected In Three Different Limbs, With 3 Days Interval Between Every Two Doses, While DM Was Induced Via Feeding The Rats For 3 Weeks With A High Fat Diet (HFD) Then Injected With A Single I.P. Dose Of Streptozotocin (STZ; 45 Mg/Kg) Dissolved In Sodium Citrate Buffer. RA/DM Comorbidity Model Was Done Via Feeding The Rats For 3 Weeks On HFD, Induction Of DM Was Performed Using STZ As Described And On Day 3 After STZ Administration, Confirmation Of DM Induction Was Performed, Then CFA Was Administered On Days 4, 7 And 10. Induction Of RA Was Reported To Take Place Within 10-12 Days Of The First Dose Of CFA.
Based On The Published Literature As Well As On Pilot Trials, Test Agents Vardenafil (10 Mg/Kg/Day) And Cilostazol (30 Mg/Kg/Day) Were Administered Orally For Twenty-One Consecutive Days; Starting from Day 16 After Induction Of RA/DM Comorbidity.
Endothelial Dysfunction Was Assessed Via Estimating The Pro-Inflammatory Vasoconstrictor Molecules Angiotensin-II (Ang II) And Endothelin-1 (ET-1), The Adhesion Molecules P-Selectin And Vascular Cell Adhesion Molecule-1 (VCAM-1), And The Vasodilator Anti-Inflammatory Molecule Vasoactive Intestinal Peptide (VIP) Using Enzyme-Linked Immunosorbent Assay (ELISA) And The Energy Sensor Phosphorylated-Adenosine-5’-Monophosphate-Activated Protein Kinase (P-AMPK) Using Western Blot Analysis. Immunohistochemical Estimations Of Endothelial Nitric Oxide Synthase (Enos) And Matrix Metalloproteinase (MMP)-2 Were Also Performed Coupled With Histopathological Examination Using Routine Hematoxylin And Eosin (H&E) And Special Masson Trichrome Staining. The In Vitro Study Was Conducted Using Rat Aortic Strips where Cumulative Concentration Response Curves Were Done For The Endothelium-Dependent Relaxing Factor Acetylcholine (Ach) And The Endothelium-Independent Relaxing Factor Sodium Nitroprusside (SNP) After Submaximal Contraction With Phenylephrine (PE).
The Chief Findings Of The Present Study Can Be Summed Up As Follows:
6.1. FIRST SET OF EXPERIMENT:
6.1.1. Effect Of CFA On Normal Adult Female Albino Rats:
Complete Freund’s Adjuvant Significantly Increased Serum ACPA, Fasting Serum Insulin Levels And Homeostatic Model Assessment For Insulin Resistance (HOMA-IR), But Not Fasting Blood Glucose Levels, As Compared To Normal Control Rats. Moreover, CFA Significantly Increased Aortic Tissue ET-1, P-Selectin And VIP Levels, As Compared To Normal Control Rats.
6.1.2. Effect Of STZ On Normal Adult Female Albino Rats:
Streptozotocin Significantly Increased Serum Anti-Citrullinated Peptide Antibody (ACPA), Fasting Serum Insulin, Fasting Blood Glucose Levels And HOMA-IR As Compared To Normal Control Rats. STZ Also Significantly Increased Aortic Tissue Levels Of ET-1, P-Selectin And VIP.
6.1.3. Effect Of CFA And STZ Combination On Normal Adult Female Albino Rats:
Both STZ And CFA Significantly Increased Aortic Tissue Levels Of ET-1 And Ang II, As Compared To Normal Control Rats. STZ And CFA Together, Also Significantly Increased Aortic Tissue VCAM-1 And P-Selectin Levels, As Compared To Normal Control Rats. Moreover, Aortic Tissue VIP Levels Were Significantly Increased. In Addition, P-AMPK/T-AMPK Expression Was Significantly Decreased. Aortic Tissue H&E Histopathology Was Found To Show That The Aortic Wall Was With Intimal Destruction, Sub-Intimal Clefting And Edema, Medial Clefting With Marked Medial Destruction And Smooth Muscle Cells Showing Small Pyknotic Nuclei And Vacuolated Cytoplasm, Together With Marked Sub-Medial Separation. Additionally, Aortic Tissue Masson Trichrome Histopathology Showed That The Aortic Wall Was With Excess Collagen In The Media. Concerning Immunohistochemical Changes, Mild Enos Reactivity, As Well As Strong MMP-2 Reactivity Was Found In The Endothelial Cells And The Smooth Muscle Cells. In Vitro, Exaggerated Contraction Was Observed With PE, But A Decreased Relaxation Was Observed With Ach And SNP, As Compared To Normal Control Rats.
6.2. SECOND SET OF EXPERIMENT:
6.2.1. Effect Of Vardenafil On Rats With RA/DM Comorbidity:
Vardenafil-Treated Rats Showed A Significant Decrease In Aortic Tissue Levels Of ET-1 And Ang II. Both Aortic Tissue VCAM-1 And P-Selectin Levels Were Significantly Decreased After Vardenafil Administration. Rats Treated With Vardenafil Showed A Significant Decrease In VIP Levels. The Expression Of P-AMPK/T-AMPK Was Significantly Increased After Treatment With Vardenafil. Vardenafil-Treated Rats Aortic H&E Histopathological Sections Showed Aortic Wall With Average Intima, Minimal Medial Clefting With Average Smooth Muscle Cells And Average Elastic Lamina, And Minimal Sub-Medial Separation While Masson Trichrome Histopathological Sections Showed Aortic Wall With Excess Collagen In The Sub-Intima And In The Media. Concerning Immunohistochemical Aortic Changes, Aortic Wall Of Vardenafil-Treated Rats Showed Mild Enos Cytoplasmic Reactivity In The Endothelial Cells, But No Reactivity In The Smooth Muscle Cells While Mild MMP-2 Cytoplasmic Reactivity In The Endothelial Cells And In The Smooth Muscle Cells Was Observed. In Vitro, A Significant Decreased Contraction Was Observed With PE, But A Significant Increased Relaxation Was Observed With Ach And SNP Restoring Relaxation Back To Normal.
6.2.2. Effect Of Cilostazol On Rats With RA/DM Comorbidity:
Rats Treated With Cilostazol Showed A Significant Decrease In Aortic Tissue Levels Of ET-1 And Ang II. Cilostazol Treated Rats Significantly Decreased Aortic Tissue VCAM-1 And P-Selectin Levels. A Significant Decrease In Aortic VIP Levels Was Observed After Cilostazol Administration. Daily Oral Administration With Cilostazol Significantly Increased The Expression Of P-AMPK/T-AMPK. Cilostazol-Treated Rats Aortic H&E Histopathological Sections Showed Intact Intima, Minimal Medial Clefting With Average Smooth Muscle Cells And Average Elastic Lamina And Sub-Medial Separation While Masson Trichrome Histopathological Sections Showed Aortic Wall With Minimal Collagen In Sub-Intima And Excess Collagen In The Media. Concerning Immunohistochemical Aortic Changes, Aortic Wall After Cilostazol-Treated Rats Showed Mild Enos Cytoplasmic Reactivity In Endothelial Cells And No Reactivity In Smooth Muscle Cells While Mild MMP-2 Cytoplasmic Reactivity In The Endothelial Cells And In The Smooth Muscle Cells Was Observed. In Vitro, After Treatment With Cilostazol, A Significant Decreased Contraction Was Observed With PE, While A Significant Increased Relaxation Was Observed With Ach And SNP.
Depending On The Aforementioned Findings, It Can Be Concluded That:
1- Diabetes Mellitus/ Rheumatoid Arthritis Comorbidity Is A Model Of Endothelial Dysfunction And Vascular Reactivity Defects.
2- The PDE-5 Inhibitor Vardenafil And The PDE-3 Inhibitor Cilostazol Seem To Be Promising Agents For Correcting Vascular Integrity And Reactivity Defects Induced By Experimental RA/DM Comorbidity.
3- Apart from Their Known PDE Inhibition, Their Useful Effects In RA/DM Comorbidity-Induced Endothelial Dysfunction And Vascular Reactivity Defects May Be Due To Up-Regulation Of Vascular AMPK And Enos Coupled With Down-Regulation Of Ang II, ET-1, P-Selectin, VCAM-1 And MMP-2.
Further Clinical Trials Are Needed To Prove These Effects And Apply The Use Of These Drugs Clinically.