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THE EFFECTS OF POSTTREATMENT BY ERDOSTEİNE AND N-ACETYLCYSTEİNE ON APOPTOTİC AND ANTIAPOPTOTIC MARKERS IN PULMONARY EPITHELIAL CELLS. Rezan DEMİRALAY. INTRODUCTION. - PowerPoint PPT Presentation
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THE EFFECTS OF POSTTREATMENT BY ERDOSTEİNE AND
N-ACETYLCYSTEİNE ON
APOPTOTİC AND ANTIAPOPTOTIC MARKERS
IN PULMONARY EPITHELIAL CELLS
Rezan DEMİRALAY
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.
INTRODUCTION
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.
Cytchrome cCytchrome c
Cytchrome c
Cytchrome c
Cytchrome c
Cytchrome c
Cytchrome c
Cytchrome c
AIF
AIFAIFAIF
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.
Cytchrome c
Cytchrome c
Cytchrome c
Cytchrome c
AIF
AIF
AIF
AIFAIF
AIF
mitochondria
Bcl-2
Localized to the outer mitchondrial membraneMitochondrial membrane permeability ↓ Prevents the release of cytochrome cand AFI
Bax
Localized in the cytosolMitochondrial membrane permeability ↑Causes the release of cytochrome c and AFI
Mitochondria
Bax
Cytochrome c release
Cell death
ROS
The generation of ROS is required for bax activation at the mitochondria, cytochrome c release, and cell death.
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).
AIM
• 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)
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
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
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.
(A). ANALYSIS OF APOPTOSIS
______________________________________Treated group Apoptosis index (%) meanSD_________________________________________________________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
RESULTS
THE EFFECTS OF ANTIOXIDANT TREATMENTS ON APOPTOSIS IN PULMONARY EPITHELIAL CELLS
________________________________________________________Treated groups Apopitosis index (%) MeanSD________________________________________________________Negative control 10.6 8.5 Positive control 84.1 7.0Erdosteine (150mg/kg) 19.4 9.8N-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)
(B). ANALYSIS OF BCL-2
THE EFFECTS OF LPS ON LOCAL PRODUCTION LEVEL OF BCL-2
____________________________________________Treated group Local production level of bcl (%) meanSD_______________________________________________________Negative control 21.1 3.3 Positive control 8.8 3.5
____________________________________________Statistical analysis: Statistically different compared with negative control group (p=0.000)
The effects of LPS on local production level of bcl-2 (x400)
Negative control group Positive control group
THE EFFECTS OF ANTIOXIDANT TREATMENTS ON LOCAL PRODUCTION LEVEL OF BCL-2
________________________________________________________
Treated groups Local production level of bcl-2 (%)
meanSD
________________________________________________________
Positive control 8.8 3.5
Erdosteine (150mg/kg) 43.8 12.2N-acetylcysteine (150mg/kg) 45.6 5.3
________________________________________________________Statistical analysis: Statistically different compared with positive control group ( p=0.000)
(D). ANALYSIS OF BAX
THE EFFECTS OF LPS ON LOCAL PRODUCTION LEVEL OF BAX
__________________________________________________Treated group Local production level of bax (%) meanSD________________________________________________________Negative controll 10.8 5.3 Positive control 77.5 5.5
_____________________________________________Statistical analysis: Statistically different compared with negative control group ( p=0.000)
The effects of LPS on local production level of bax (x400)
Negative control group Positive control group
THE EFFECTS OF ANTIOXIDANT TREATMENTS ON LOCAL PRODUCTION LEVEL OF BAX
________________________Treated groups Local production level of bax (%)
meanSD
_____________________________________________________
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)
(D). ANALYSIS OF CASPASE 3
THE EFFECTS OF LPS ON LOCAL PRODUCTION LEVEL OF CASPASE 3
________________________________________________________Tedavi grubu Lokal kaspaz 3 oluşum seviyesi (%) ortalamaSD_______________________________________________________________Negatif kontrol 10.8 5.3 Pozitif kontrol 80.0 8.01 ________________________________________________________Statistical analysis: Statistically different compared with negative control group ( p=0.000)
The effects of LPS on local production level of caspase 3 (x400)
Negative control group Positive control group
THE EFFECTS OF ANTIOXIDANT TREATMENTS ON LOCAL PRODUCTION LEVEL OF CASPASE 3
__________________________________________________Treated groups Local production level of caspase 3 (%) meanSD________________________________________________________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)
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.