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

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.

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

AIF

AIF

AIF

AIF

AIF

AIFAIF

AIF

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

AIF

AIFAIF

AIF

AIFAIF

AIF

AIF

Cytchrome c

Cytchrome c

Cytchrome c

Cytchrome c

Cytchrome c

Cytchrome c

Cytchrome c

Cytchrome c

Cytchrome c

Cytchrome c

Cytchrome c

Cytchrome c

mitokondri

Bcl-2 ailesi

Anti-apoptotik(Bcl-2)

Pro-apoptotik (Bax)

Mitokondri

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.

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.

ROS

Initial injury

Redox imbalance

Extracellular matrix

ECM fragments

PULMONARY FIBROSIS

Inflammatory cells

TNF-αVEGF

TGF-β

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.

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.

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.

AIM

• 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 of 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 (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

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.

LUNG HISTOLOGY Determination and distribution of subendothelial and pericapillary collagen fibres were assessed by using the staining methods of hematoxylin and eosin (HE), 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

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

23 Moderate thickening of walls without obvious damage to lung architecture

45 Increased fibrosis with definite damage to lung structure and formation of fibrous bands or small fibrous masses

67 Severe distortion of structure and large fibrous area: “honeycomb lung” is placed in this category

8 Total fibrous obliteration of the field

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.

_______________________________________________________________Treated group Endothelial Subendothelial Pericapillary damage collagen collagen meanSD meanSD meanSD_______________________________________________________________Negative control (Day 3) 0.0 0.0 0.0 0.0 0.0 0.0Negative control (Day 14) 0.0 0.0 0.0 0.0 0.0 0.0Positive 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)

THE EFFECT OF BLM ON LUNG HISTOLOGY

(A). ANALYSIS OF LUNG HISTOLOGY

RESULTS

_______________________________________________________________Treated group Endothelial Subendothelial Pericapillary

damage collagen collagen meanSD meanSD meanSD

_______________________________________________________________Positive control (Day 14) 3.0 0.0 2.7 0.5 2.7 0.5Erdosteine (150 mg/kg) 1.4 0.5 1.2 0.4 1.2 0.4NAC (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)

THE EFFECTS OF TREATED GROUPS ON LUNG HISTOLOGY

_______________________________________________________Treated group Ashcroft criteria meanSD_______________________________________________________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)

THE EFFECTS OF TREATED GROUPS ON ASHCROFT CRITERIA

Negative control group Positive control group

The analysis of Masson trichrom staining (x200)

Negative control group Positive control group

The analysis of reticulin staining (x200)

(B). ANALYSIS OF APOPTOSIS

________________________________________Treated groups Apopitosis index (%) meanSD______________________________________________________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

Apoptosis analysis by TUNEL method (X200)

Negative control group Positive control group

THE EFFECTS OF TREATED GROUPS ON PULMONARY CAPILLARY ENDOTHELIAL CELLS

________________________________________________________Treated group Apopitosis index (%) meanSD________________________________________________________Positive control (Day 14) 64.3 5.8Erdosteine (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)

(C). ANALYSIS OF TNF-α

THE EFFECT OF BLM ON LOCAL PRODUCTION LEVEL OF TNF-α

__________________________________________________________

Treated group Local production level of TNF-α (%) meanSD__________________________________________________________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)

The effect of BLM on local production level of TNF- α (x400)

Negative control group Positive control group

THE EFFECTS OF TREATED GROUPS ON LOCAL PRODUCTION LEVEL OF TNF-α

________________________________________________________

Treated goups Local production level of TNF-α (%)

meanSD

________________________________________________________

Positive control (Day 14) 78.8 4.8

Erdosteine (150 mg/kg) 44.9 4.9NAC (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)

(D). ANALYSIS OF MPO

THE EFFCET OF BLM ON VASCULAR MPO ACTIVITY

__________________________________________________Treated group Vascular MPO activity (%) meanSD__________________________________________________Negative control (Day 3) 10.8 5.3 Negative control (Day 14) 6.0 1.1Positive 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)

The effect of BLM on vascular MPO activity (x400)

Negative control group Positive control group

THE EFFECTS OF TREATED GROUPS ON VASCULAR MPO ACTIVITY

_________________________________Treated group Vascular MPO activity (%)

meanSD___________________________________________________________

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)

(E). ANALYSIS OF VEGF

THE EFFECT OF BLM ON LOCAL PRODUCTION LEVEL OF VEGF

________________________________________________________Treated group Local productıon level of VEGF (%) meanSD_______________________________________________________________Negative control (Day 3) 6.1 2.6 Negative control (Day 14) 3.4 1.1Positive 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)

The effect BLM on local productıon level of VEGF (x400)

Negative control group Positive control group

THE EFFECTS OF TREATED GROUPS ON LOCAL PRODUCTION LEVEL OF VEGF

____________________________________________________Treated group Local production level of VEGF (%) meanSD_____________________________________________________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)

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.