1 / 36

Rezan DEMİRALAY

THE EFFECTS OF ERDOSTEINE AND N-ACETYLCYSTEINE TREATMENT FOLLOWING LUNG INJURY ON THE REGULATION OF APOPTOSİS OF CAPILLARY ENDOTHELIAL CELLS IN THE MODEL OF EXPERIMENTAL PULMONARY FIBROSIS INDUCED BY BLOEMYCIN. Rezan DEMİRALAY. INTRODUCTION.

elom
Download Presentation

Rezan DEMİRALAY

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. THE EFFECTS OF ERDOSTEINE AND N-ACETYLCYSTEINE TREATMENT FOLLOWING LUNG INJURY ON THE REGULATION OF APOPTOSİS OF CAPILLARY ENDOTHELIAL CELLS IN THE MODEL OF EXPERIMENTAL PULMONARY FIBROSIS INDUCED BY BLOEMYCIN Rezan DEMİRALAY

  2. INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is characterized by epithelial and endothelial cell injury leading to destruction of normal lung archiecture. Endothelial cell injury and apoptosis (controlled cell death) is considered to play an important role in the initiation of fibrogenic response.

  3. Apoptosis signaling pathways;Some central components of the apoptotic response mediated via either “extrensic” death receptor or “intrinsic” mitochondrial and/or endoplasmic reticular (ER) pathways.

  4. Death receptor-mediated caspase activation

  5. mitokondri Cytchrome c Cytchrome c Cytchrome c Cytchrome c Cytchrome c Cytchrome c Cytchrome c Cytchrome c Cytchrome c Cytchrome c Cytchrome c Cytchrome c AIF AIF AIF AIF AIF AIF AIF AIF AIF AIF AIF AIF AIF AIF AIF AIF Mitochondrial (intrinsic) pathway involves the release of apoptogenic factors such as cytochrome c and apoptosis-inducing factor (AIF) from the mitochondria.

  6. Bcl-2 ailesi Anti-apoptotik (Bcl-2) Pro-apoptotik (Bax) Mitokondri

  7. The apoptosis of endothelial and epithelial cells in the lungs increases the permeability of the air-blood barrier and enhances the infiltration of inflammatory cells. The progresssion of pulmonary fibrosis is closely related to a complicated network consisting of many cytokines such as TNF-α and VEGF, mediators, growth factors, and peptides derived from inflammatory immune cells, endothelial cells, and alveolar cells.

  8. On the other hand, it has been proposed that the cellular redox state and the balance of oxidant /antioxidants play a significant role in the progression of pulmonary fibrosis.

  9. Initial injury Redox imbalance ROS TNF-α VEGF TGF-β Inflammatory cells Extracellular matrix ECM fragments PULMONARY FIBROSIS

  10. The regulation of apoptosis with agents known to augment the cellular antioxidant defense system and neutralize ROS thus seems to control the course of IPF.

  11. BLM is a commonly used chemotherapeutic agent that can cause dose-dependent pulmonary fibrosis. The animal model of bleomycin-induced pulmonary fibrosis in rats has been used extensively in the investigation of the pathogenesis of human pulmonary fibrosis because of its close histopathological similarities to human idiopathic pulmonary fibrosis.

  12. BLM can bind metal ions and DNA at the same time at two different sites. The interaction of BLM with DNA appears to initiate inflammatory and fibroproliferative changes leading to accumulaton of collagen in the lung. The lung is selectively affected because this tissue lacks an enzyme that hydrolyzes the b-aminoalanine moiety of BLM, which prevents its metabolite from binding metals such as iron.

  13. The frequency of apoptosis in pulmonary capillary endothelial cells in the model of experimental pulmonary fibrosis induced by bleomycin (BLM) The role of inflammatory markers [myeloperoxidase (MPO), tumor necrosis factor alpha (TNF-α), and vascular endothelial growth factor (VEGF)] in endothelial damage The protective effects oferdosteine and N-acetylcysteine (NAC) AIM

  14. MATERIALS AND METHODS EXPERIMENTAL GROUPS The rats were divided into six groups, each composed of nine rats: ■ Negative control group (Day 3); intratracheally saline plus oral sodium bicarbonat ■ Negative control group (Day 14); intratracheally saline plus oral sodium bicarbonat ■ Positive control group; intratracheally BLM plus oral sodium bicarbonat ■ Positive control group; intratracheally BLM plus oral sodium bicarbonat ■ Intratracheally BLM plus erdostein at a dose of 150mg/kg ■ Intratracheally BLM plus n-acetylcysteine at a dose of 150mg/kg

  15. DRUGS Erdosteine (Sandoz Drug Industries; İstanbul, Turkey) was dissolved with an equivalent molar quantity of sodium bicarbonate in distilled water and NAC (Bılım Drug Industries; Istanbul,Turkey) was dissolved in distilled water. EXPERIMENTAL PROTOCOL BLM (5mg/kg) was instilled intratracheally into rats to induce pulmonary fibrosis. Oral antioxidants were initiated 3 days after BLM-induced lung injury. The rats were killed 14 days after BLM administration, and the lungs were explored. The lung tissue was processed for the analysis of histopathological, apoptosis, TNF-α, MPO, and VEGF. CONTROL GROUP Control rats were intratracheally administered isotonic saline solution at a volume equal to that of the BLM, and a molar quantity of sodium bicarbonate equivalent to that of the erdosteine treatment dissolved in distilled water was given orally.

  16. LUNG HISTOLOGY Determination and distribution of subendothelial and pericapillary collagen fibres were assessed by using the staining methods of hematoxylin and eosin (HE), masson trichrome, and reticulin. The evaluation parameters of pulmonary artery endothelial injury: Endothelial damage Subendothelial collagen deposition Pericapillary collagen deposition The judging categories of severity of vascular injury: 0= no injury 1= mild injury 2= moderate injury 3= severe injury

  17. Severity of fibrosis of pericapillary paranchyma was evaluated according to Ashcroft criteria. Criteria for grading lung fibrosis; 0 Normal lung 1 Minimal fibrous thickening of alveolar or bronchiolar walls 2 3 Moderate thickening of walls without obvious damage to lung architecture 4 5 Increased fibrosis with definite damage to lung structure and formation of fibrous bands or small fibrous masses 6 7 Severe distortion of structure and large fibrous area: “honeycomb lung” is placed in this category 8 Total fibrous obliteration of the field

  18. ANALYSIS OF APOPTOSIS The apoptosis level in the pulmonary capillary endothelial cells was determined by using a TUNEL method (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling). The apoptosis index (AI) was expressed as a percentage of TUNEL-positive cells in 1000 cells counted in the same section. ANALYSIS OF IMMUNOHISTOCHEMISTRY The local production level of TNF-α (Histopathology Ltd.; Akác, Hungary) and VEGF (NeoMarkers Inc.; Portsmouth, NH, USΑ) in the pulmonary capillary endothelial cells, and vascular MPO activity (NeoMarkers Inc.; Portsmouth, NH, USΑ) was evaluated immunohistochemically The results were expressed as the percentage of bronchial and alveolar epithelial cells cytoplasmically stained positive in 1000 cells counted in the same section.

  19. RESULTS (A). ANALYSIS OF LUNG HISTOLOGY THE EFFECT OF BLM ON LUNG HISTOLOGY _______________________________________________________________ Treated group Endothelial Subendothelial Pericapillary damage collagen collagen meanSD meanSD meanSD _______________________________________________________________ Negative control (Day 3)0.0  0.0 0.0  0.00.0  0.0 Negative control (Day 14) 0.0  0.0 0.0  0.0 0.0  0.0 Positive control (Day 3) 2.4  0.5 ††2.1  0.3 †† 2.2  0.4 †† Positive control (Day 14) 3.0  0.0 ††2.7  0.5 †† 2.1  0.8 †† _______________________________________________________________ Statistical analysis: Significantly higher compared with the negative control group (††p=0.000)

  20. THE EFFECTS OF TREATED GROUPS ON LUNG HISTOLOGY _______________________________________________________________ Treated group Endothelial Subendothelial Pericapillary damage collagen collagen meanSD meanSD meanSD _______________________________________________________________ Positive control (Day 14) 3.0  0.0 2.7  0.5 2.7  0.5 Erdosteine (150 mg/kg) 1.4  0.5 1.2  0.4  1.2  0.4 NAC (150 mg/kg) 2.3  0.52.0  0.7  2.0  0.8 _______________________________________________________________ İStatistical analysis: Significantly lower compared with the positive control group (p=0.05) Significantly lower compared with the positive control group (p=0.000)

  21. THE EFFECTS OF TREATED GROUPS ON ASHCROFT CRITERIA _______________________________________________________ Treated group Ashcroft criteria meanSD _______________________________________________________ Negative control (Day 3) 0.0  0.0 Negative control (Day 14) 0.0  0.0 Positive control (Day 3) 5.0 1.4 †† Positive control (Day 14) 5.7  1.0 †† Erdosteine (150 mg/kg) 1.9  0.9  NAC (150 mg/kg) 2.6  2.4  _______________________________________________________Statistical analysis: Significantly different compared with the positive control group (††p=0.000) Significantly different compared with the positive control group (p=0.000)

  22. Negative control group Positive control group The analysis of Masson trichrom staining (x200)

  23. Negative control group Positive control group The analysis of reticulin staining (x200)

  24. (B). ANALYSIS OF APOPTOSIS ________________________________________ Treated groups Apopitosis index (%) meanSD ______________________________________________________ Negative control (3. gün)2.7  1.5 Negative control (14. gün) 1.6  1.0 Positive control (3. gün) 76.7  4.3 †† Positive control (14. gün) 64.3  5.8 †† ________________________________________ Statistical analysis: Significantly higher compared with the negative control group (††p=0.000) THE EFFECTS OF BLM ON APOPTOSIS IN PULMONARY CAPILLARY ENDOTHELIAL CELLS

  25. Negative control group Positive control group Apoptosis analysis by TUNEL method (X200)

  26. THE EFFECTS OF TREATED GROUPS ON PULMONARY CAPILLARY ENDOTHELIAL CELLS ________________________________________________________ Treated group Apopitosis index (%) meanSD ________________________________________________________ Positive control (Day 14) 64.3  5.8 Erdosteine (150 mg/kg) 25.2  10.6  NAC (150 mg/kg) 40.9  7.5  ¶¶ ________________________________________________________ Statistical analysis: Significantly different compared with the negative control group (¶¶p=0.000) Significantly different compared with the positive control group (p=0.000)

  27. (C). ANALYSIS OF TNF-αTHE EFFECT OF BLM ON LOCAL PRODUCTION LEVEL OF TNF-α __________________________________________________________ Treated group Local production level of TNF-α (%) meanSD __________________________________________________________ Negative control (Day 3) 9.2  3.8 Negative control (Day 14) 4.0  1.0 Positive control (Day 3) 86.7  7.5‍‍†† Positive control (Day 14) 78.8  4.8†† __________________________________________________________ Statistical analysis: Significantly different compared with the negative control group(†† p=0.000)

  28. Negative control group Positive control group The effect of BLM on local production level of TNF-α(x400)

  29. THE EFFECTS OF TREATED GROUPS ON LOCAL PRODUCTION LEVEL OF TNF-α ________________________________________________________ Treated goups Local production level of TNF-α (%) meanSD ________________________________________________________ Positive control (Day 14) 78.8  4.8 Erdosteine (150 mg/kg) 44.9  4.9 NAC (150 mg/kg) 58.4  3.3 ¶ ¶ ________________________________________________________ Statistical analysis: Significantly different compared with the negative control group (¶¶p=0.000) Significantly different compared with the positive control group (p=0.000)

  30. (D). ANALYSIS OF MPO THE EFFCET OF BLM ON VASCULAR MPO ACTIVITY __________________________________________________ Treated group Vascular MPO activity (%) meanSD __________________________________________________ Negative control (Day 3) 10.8  5.3 Negative control (Day 14) 6.0 1.1 Positive control (Day 3) 77.5  5.5 †† Positive control (Day 14) 73.1  7.8 †† _______________________________________________ Statistical analysis: Significantly different compared with the negative control group (†† p=0.000)

  31. Negative control group Positive control group The effect of BLM on vascular MPO activity (x400)

  32. THE EFFECTS OF TREATED GROUPS ON VASCULAR MPO ACTIVITY _________________________________ Treated group Vascular MPO activity(%) meanSD ___________________________________________________________ Positive control (Day 14) 73.1  7.8 Erdosteine (150mg/kg) 38.1  8.5  NAC (150mg/kg) 51.7  12.8  ¶ _____________________________________________________ Statistical analysis: Significantly different compared with the negative controLgroup (¶p 0.05) Significantly different compared with the positive control group( p=0.000)

  33. (E). ANALYSIS OF VEGF THE EFFECT OF BLM ON LOCAL PRODUCTION LEVEL OF VEGF ________________________________________________________ Treated group Local productıon level of VEGF (%) meanSD _______________________________________________________________ Negative control (Day 3) 6.1  2.6 Negative control (Day 14) 3.4  1.1 Positive control (Day 3) 77.2  7.5 †† Positive control (Dy 14) 66.9 7.0 †† ________________________________________________________ Statistical analysis: Significantly different compared with the negative control group (†† p=0.000)

  34. Negative control group Positive control group The effect BLM on local productıon level of VEGF (x400)

  35. THE EFFECTS OF TREATED GROUPS ON LOCAL PRODUCTION LEVEL OF VEGF ____________________________________________________ Treated group Local production level of VEGF (%) meanSD _____________________________________________________ Positive control (Day 14) 66.9  7.0 Erdosteine (150mg/kg) 34.6  8.2 NAC (150mg/kg) 58.1  6.2 ¶¶ ____________________________________________________________ Statistical analysis: Significantly different compared with the negative control group (¶¶p=0.000) Significantly different compared with the positive control group (p=0.05) Significantly different compared with the positive control group (p=0.000)

  36. CONCLUSION In conclusion, Treatment with erdosteine and NAC significantly reduced the rate of BLM-induced capillary endothelial cell apoptosis. Treatment with erdosteine and NAC significantly reduced the increases in the local production of TNF-α and VEGF, and MPO activity. The effects of NAC on apoptosis regulation, the increases in the local production of TNF-α and VEGF, and endothelial MPO activity were weaker than that of erdosteine.

More Related