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.

Death receptor-mediated caspase activation

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 family

Anti-apoptotic(Bcl-2)

Pro-apoptotic (Bax)

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

Apoptosome complex formation and activation

Caspase activation in the apoptosis-signaling pathways

Reactive oxygen species (ROS) play a major role in mediating

injury to the lung in sepsis.

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

Apoptosis analysis of negative control group (X200)

Negative control group Positive control group

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.