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Rezan DEMİRALAY

THE EFFECTS OF POSTTREATMENT BY ERDOSTEİNE AND N-ACETYLCYSTEİNE ON APOPTOTİC AND ANTIAPOPTOTIC MARKERS IN PULMONARY EPITHELIAL CELLS. Rezan DEMİRALAY. INTRODUCTION.

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Rezan DEMİRALAY

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  1. THE EFFECTS OF POSTTREATMENT BY ERDOSTEİNE AND N-ACETYLCYSTEİNE ON APOPTOTİC AND ANTIAPOPTOTIC MARKERS IN PULMONARY EPITHELIAL CELLS Rezan DEMİRALAY

  2. INTRODUCTION Sepsis is a disease that can lead to multiple organ dysfunction and high mortality. It has been established that significant apoptosis (controlled cell death) develops in lymphoid organs of septic patients and that inhibition of apoptosis in these organs is associated with improved survival. In sepsis, the organ primarily targeted for injury is the lung, and acute lung injury (ALI) leading to acute respiratory distress syndrome (ARDS) is resulted often a complication of sepsis.

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

  6. Bcl-2 family Anti-apoptotic (Bcl-2) Pro-apoptotic (Bax) Mitochondria

  7. Bcl-2 Localized to the outer mitchondrial membrane Mitochondrial membrane permeability ↓ Prevents the release of cytochrome c and AFI Bax Localized in the cytosol Mitochondrial membrane permeability ↑ Causes the release of cytochrome c and AFI

  8. Apoptosome complex formation and activation

  9. Caspase activation in the apoptosis-signaling pathways

  10. Reactive oxygen species (ROS) play a major role in mediating injury to the lung in sepsis.

  11. ROS Bax Mitochondria Cytochrome c release Cell death The generation of ROS is required for bax activation at the mitochondria, cytochrome c release, and cell death.

  12. The modulation of apoptosis with agents known to augment the cellular antioxidant defense system and neutralize ROS thus seems to control the course of sepsis and the development of acute lung injury (ALI).

  13. The frequency of apoptosis in rat pulmonary epithelial cells after intraperitoneally endotoxin (LPS) injection , The effects of LPS on apoptotic and antiapoptotic markers (bax, caspase-3, and bcl-2) in lung damage The protective effects of two known antioxidant agents, erdosteine 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; intraperitoneally saline plus oral distilled water ■ Negative control group; intraperitoneally saline plus oral sodium bicarbonat ■ Positive control group; intraperitoneally LPS plus oral distilled water ■ Positive control group; intraperitoneally LPS plus oral sodium bicarbonate ■ Erdosteine-treated group; Intraperitoneally LPS plus erdostein at a dose of 150mg/kg ■ NAC-treated group; Intraperitoneally LPS plus asetilsistein 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 LPS (E.coli 055:B5; Sigma) was dissolved in 1 ml of sterile saline solution and injected intraperitoneally at a dosage of 20 mg/kg. Following LPS injection, the antioxidants were administered orally. The rats were killed 24 h after LPS administration and a thoracotomy was performed, and the lungs were explored. The lung tissue was processed for the analysis of apoptosis, bcl-2, bax, and caspase 3. CONTROL GROUP Control rats were intraperitoneally administered isotonic saline solution at a volume equal to that of the LPS injection, and distilled water at a volume equal to that of the NΑC or a molar quantity of sodium bicarbonate equivalent to that of the erdosteine treatment dissolved in distilled water was given orally

  16. ANALYSIS OF APOPTOSIS The apoptosis level in the lung bronchiolar and alveolar epithelium was determined by using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) kit. 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 of bcl-2 (SantaCruz, USA), bax (Abcam, USA), and caspase 3 (Neomarkers, USA) 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.

  17. (A). ANALYSIS OF APOPTOSIS RESULTS ______________________________________ Treated group Apoptosis index (%) meanSD _________________________________________________________ Negative control 10.6  8.5 Positive control 85.6  6.2 ______________________________________ Statistical analysis: Significantly higher compared with the negative control group (p=0.000) THE EFFECTS OF LPS ON APOPTOSIS IN PULMONARY EPITHELIAL CELLS

  18. Negative control group Positive control group Apoptosis analysis of negative control group (X200)

  19. THE EFFECTS OF ANTIOXIDANT TREATMENTS ON APOPTOSIS IN PULMONARY EPITHELIAL CELLS ________________________________________________________ Treated groups Apopitosis index (%) MeanSD ________________________________________________________ Negative control 10.6  8.5 Positive control 84.1  7.0 Erdosteine (150mg/kg) 19.4  9.8 N-acetylcysteine (150mg/kg) 36.1  4.2 ¶ ________________________________________________________ Statistical analysis: Significantly different compared with the negative control group (¶p<0.05) Significantly different compared with the positive control group (p=0.000)

  20. (B). ANALYSIS OF BCL-2THE EFFECTS OF LPS ON LOCAL PRODUCTION LEVEL OF BCL-2 ____________________________________________ Treated group Local production level of bcl (%) meanSD _______________________________________________________ Negative control 21.1  3.3 Positive control 8.8  3.5 ____________________________________________ Statistical analysis: Statistically different compared with negative control group (p=0.000)

  21. Negative control group Positive control group The effects of LPS on local production level of bcl-2 (x400)

  22. THE EFFECTS OF ANTIOXIDANT TREATMENTS ON LOCAL PRODUCTION LEVEL OF BCL-2 ________________________________________________________ Treated groups Local production level of bcl-2 (%) meanSD ________________________________________________________ Positive control 8.8  3.5 Erdosteine (150mg/kg) 43.8  12.2 N-acetylcysteine (150mg/kg) 45.6  5.3 ________________________________________________________ Statistical analysis: Statistically different compared with positive control group ( p=0.000)

  23. (D). ANALYSIS OF BAXTHE EFFECTS OF LPS ON LOCAL PRODUCTION LEVEL OF BAX __________________________________________________ Treated group Local production level of bax (%) meanSD ________________________________________________________ Negative controll 10.8 5.3 Positive control 77.5  5.5_____________________________________________Statistical analysis: Statistically different compared with negative control group ( p=0.000)

  24. Negative control group Positive control group The effects of LPS on local production level of bax (x400)

  25. THE EFFECTS OF ANTIOXIDANT TREATMENTS ON LOCAL PRODUCTION LEVEL OF BAX ________________________ Treated groups Local production level of bax(%) meanSD _____________________________________________________ Positive control 77.5  5.5 Erdosteine (150mg/kg) 48.8  7.9  N-asetylscysteine (150mg/kg) 42.8  2.6  _____________________________________________________ Statistical analysis: Statistically different compared with positive control group ( p=0.000)

  26. (D). ANALYSIS OF CASPASE 3 THE EFFECTS OF LPS ON LOCAL PRODUCTION LEVEL OF CASPASE 3 ________________________________________________________ Tedavi grubu Lokal kaspaz 3 oluşum seviyesi (%) ortalamaSD _______________________________________________________________ Negatif kontrol 10.8  5.3 Pozitif kontrol 80.0  8.01  ________________________________________________________ Statistical analysis: Statistically different compared with negative control group ( p=0.000)

  27. Positive control group Negative control group The effects of LPS on local production level of caspase 3 (x400)

  28. THE EFFECTS OF ANTIOXIDANT TREATMENTS ON LOCAL PRODUCTION LEVEL OF CASPASE 3 __________________________________________________ Treated groups Local production level of caspase 3 (%) meanSD ________________________________________________________ Positive control 80.0  8.01 Erdosteine (150mg/kg) 51.3  6.9 N-acetylcysteine (150mg/kg) 43.9  3.3 ________________________________________________________ Statistical analysis: Statistically different compared with positive control group ( p=0.000)

  29. CONCLUSION In conclusion, Posttreatment with erdosteine and NAC significantly reduced the rate of LPS-induced pulmonary epithelial cell apoptosis. The effect of NAC on apoptosis in the bronchiolar and alveolar epithelial cells was weaker than that of erdosteine. Posttreatment with erdosteine and NAC significantly reduced the increases in the local production of bax and caspase-3, and significantly increased the decrease in the local production of bcl-2.

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