THE EFFECT OF MONTELUKAST AND
MK-886 ON HEMORRHAGIC
SHOCK/RESUSCITATION-INDUCED
ACUTE LUNG INJURY IN MALE RATS
A THESIS
SUBMITTED TO THE COLLEGE OF MEDICINE AND THE
COMMITTEE OF POSTGRADUATE STUDIES OF KUFA
UNIVERSITY IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF MASTER OF
SCIENCE IN PHARMACOLOGY AND THERAPEUTICS
BY
Ali Mohsin Hashim
B.Sc. Pharmacy
Supervised by
Dr. Najah R. AL-Mousawi
Professor of Clinical
Pharmacology and Therapeutics
Ph.D, FRCP, FACP, Post Doc.
1431 A.H
Dr. Fadhil Ghaly Yousif AL-Amran
Assistant Prof. of Cardiothoracic
Surgery
MD, FRCS, FACS, Post Doc.
2010 A.D.
Ali Mohsin
Acknowledgements
Praise is to our Almighty Allah, the Gracious who gives me the power and
motivation to perform and present this work.
I wish to express my heartfelt gratitude and appreciation to my honorable
supervisor Prof. Dr. Najah R. Al Mousawi for his guidance, valuable
advices and support.
I am especially grateful & indebted to my supervisor Assist. Prof. Dr.
Fadhil Ghaly Yousif for his great efforts, guidance, valuable advices in this
research.
I am deeply grateful to Kufa College of Medicine represented by the Dean
and the staff for providing facilities required for this work.
I am so thankful to Assist. Prof. Dr. Ali Al Mohana & Dr. Ahmed Al
Mohana for their kind help.
Also I would like to express my deepest thanks to Dr. Liwaa H. Mahdi AlKulabi who help me in the field of histopathology.
I highly appreciate Assist. Prof. Dr Abdul-Kareem Al Mayah for his help in
statistical analysis.
Special gratitude is also forwarded to Dr. Bassim I Mohammad & Dr.
Hussein Abed Al-khahdam for their kind help.
My gratitude and appreciation to pharmacist Ayad Ali for his kind help.
My deepest thanks to Dr. Sabah Al-Fatlawy for his help in ELISA technique.
Finally, my heartfelt appreciation and gratitude to my family and my wife
for their infinite support and patience during the time of the study.
Ali Mohsin
List of Contents
Subject
Page
Dedication
І
Acknowledgments
ІІ
List of Contents
ІІІ
List of Tables
VIII
List of Figures
IX
List of Abbreviations
XI
Summary
XV
Chapter One: Introduction and Literature Review
1. Introduction
1.1. Background
1
1
1.2. Acute Respiratory Distress Syndrome (ARDS) and Acute Lung
Injury (ALI)
1.2.1 Mortality/Morbidity
2
1.2.2. Pathogenesis
2
1.2.2.1. Endothelial and Epithelial Injury
2
1.2.2.2. Neutrophil-Dependent Lung Injury
5
1.3. Pathogenesis of SIRS Leading to MODS and ARDS
5
1.3.1. The “Two-Hit” Theory of Immune Cell Priming
6
1.3.2. The Influence of Gut Hypoperfusion
2
7
1.3.3. Oxidative Stress
8
1.3.3.1. Oxidative Stress & Free Radicals: Definition and
Mechanisms
8
1.3.3.2. Sources of Oxidants in ALI/ARDS
9
1.3.3.3. Oxidant-Antioxidant Balance
9
1.3.3.4. Potential Targets of Oxidants in ALI/ARDS
1.3.3.5. Oxidants as Mediators of Ischemia/Reperfusion Injury
1.3.4. Proinflammatory Mediators
1.3.4.1. Tumour Necrosis Factor-Alpha (TNF-α)
10
11
14
15
1.3.4.2. Interleukin-6 (IL-6)
16
1.3.4.3. Leukotrienes (LTs)
17
1.3.4.3.1. Leukotriene Biosynthesis
18
1.3.4.3.2. Leukotriene B4 (LTB4)
20
1.3.4.3.3. The Cysteinyl-Leukotrienes (CysLTs)
21
1.3.4.3.4. Leukotrienes and Acute Lung Injury
23
1.4. Some Aspects of the Drugs Used in This Study
24
1.4.1. MK-886
1.4.1.1. Pharmacological Action of MK-886
1.4.1.2. Pharmacokinetics of MK-886
24
24
26
1.4.1.3. Side Effect of MK-886
26
1.4.2. Montelukast
26
1.4.2.1. Montelukast Pharmacology
26
1.4.2.2. Protective Potential of Montelukast
27
1.4.2.3. Adverse Effects
28
1.4.2.4. Drug Interaction
29
1.5. Aim of the Study
30
Chapter Two: Material and Methods
2.1. Materials
2.2. Animals and Study Design
2.3. Drugs
31
33
2.3.1. MK-886
34
2.3.2. Montelukast
34
2.4. Hemorrhagic Shock Protocol
34
2.5 Preparation of Sample
35
2.5.1 Blood Sampling
35
2.5.2. Preparation of Bronchoalveolar Lavage Fluid
2.5.3. Tissue Preparation for Oxidative Stress Measurement
2.5.4. Tissue Sampling for Histopathology
34
35
35
36
2.6. Measurement of Serum IL-6
37
2.7. Measurement of Serum TNF-α
40
2.8. Measurement of BALF LTB4
44
2.9. Measurement of BALF LTC4
49
2.10 Measurement of Lung Reduced Glutathione (GSH)
54
2.11. Measurement of Lung Malondialdehyde (MDA)
55
2.12. Measurement of BALF Total Protein (A Measure of Lung
Leak/Injury)
56
2.13. Histopathological Procedure
57
2.14. Statistical Analysis
58
Chapter Three: Results
3.1. Effect on Serum TNF-α Level
59
3.2. Effect on Serum IL-6 Level
61
3.3. Effect on Lung MDA Level
63
3.4. Effect on Lung GSH Level
65
3.5. Effect on BALF LTB4 Level
67
3.6. Effect on BALF LTC4 Level
69
3.7. Effect on BALF Total Protein
3.8. Histopathological Findings
71
73
3.8.1. Sham Group
73
3.8.2. Control (Induced Untreated) Group
3.8.3. Montelukast Treated Group
73
3.8.4. MK-886 Treated Group
3.9. Correlation Coefficient between Study Parameters
3.9.1. Correlation between Inflammatory Parameter (IL-6 & TNF-α)
and Oxidative Parameter (MDA & GSH)
3.9.2. Correlation between Leukotrienes (LTB4 & LTC4) and
Oxidative Parameter (MDA & GSH)
73
73
78
78
78
3.9.3. Correlation between Leukotrienes (LTB4 & LTC4) and
Inflammatory Parameter (IL-6 & TNF-α)
79
3.9.4. Correlation between Leukotrienes (LTB4 & LTC4) and ALI
Score
79
Chapter Four : Discussion
4. Discussion
4.1. Effect of Hemorrhagic Shock on Study Parameters
81
81
4.1.1. Effect of Hemorrhagic Shock on Leukotrienes
81
4.1.2. Effect of Hemorrhagic Shock on Inflammatory Markers (IL-6
& TNF-α)
82
4.1.3 Effect of Hemorrhagic Shock on Oxidative Stress
83
4.1.4. Effect of Hemorrhagic Shock on Total Protein
4.1.5. Effect of Hemorrhagic Shock on Lung Parenchyma
4.2. Effects of Drug Treatment
4.2.1. Effects of Montelukast
4.2.1.1. Effect of Montelukast on Leukotrienes
4.2.1.2. Effect of Montelukast on Inflammatory Markers (IL-6 &
TNF-α)
4.2.1.3. Effect of Montelukast on Oxidative Stress
85
86
87
87
87
88
89
4.2.1.4. Effect of Montelukast on BALF Total Protein
91
4.2.1.5. Effect of Montelukast on Lung Parenchyma
92
4.2.2 Effects of MK-886
92
4.2.2.1 Effect of MK-886 on Leukotrienes
4.2.2.2. Effect of MK-886 on Inflammatory Markers (IL-6 & TNF-α)
4.2.2.3. Effect of MK-886 on Oxidative Stress
4.2.2.4. Effect of MK-886 on Total Protein
4.2.2.5. Effect of MK-886 on Lung Parenchyma
92
93
94
95
95
Chapter five : Conclusions and Recommendations
5.1: Conclusions
5.2: Recommendations
97
98
References
References
99
List of Tables
Table
Table (1): Serum TNF-α level (pg/ml) of the four experimental
groups at the end of the experiment (N = 6 in each group).
Table (2): Multiple comparisons among different group mean values
of serum TNF-α level (pg/ml) using ANOVA TEST.
Table (3): Serum IL-6 level (pg/ml) of the four experimental groups
at the end of the experiment (N = 6 in each group).
Table (4): Multiple comparisons among different group mean values
of serum IL-6 level (pg/ml) using ANOVA TEST.
Table (5): Lung MDA level (nmol/g tissue) of the four experimental
groups at the end of the experiment (N = 6 in each group).
Table (6): Multiple comparisons among different group mean values
of lung MDA level (nmol/g) using ANOVA TEST.
Table (7): Lung GSH level (μmol/g tissue) of the four experimental
groups at the end of the experiment (N = 6 in each group).
Page
59
60
61
62
63
64
65
Table (8): Multiple comparisons among different group mean values
of lung GSH level (μmol/g) using ANOVA TEST.
Table (9): BALF LTB4 level (pg/ml) of the four experimental groups
at the end of the experiment (N = 6 in each group).
Table (10): Multiple comparisons among different group mean
values of BALF LTB4 level (pg/ml) using ANOVA
TEST.
Table (11): BALF LTC4 level (pg/ml) of the four experimental
groups at the end of the experiment (N = 6 in each
group).
Table (12): Multiple comparisons among different group mean
values of BALF LTC4 level (pg/ml) using ANOVA
TEST.
Table (13): BALF total protein level (g/l) of the four experimental
groups, at the end of experiment (N = 6 in each group).
Table (14): Multiple comparisons among different group mean
values of BALF total protein level (g/l) using ANOVA
TEST.
Table (15): The differences in histopathological grading of abnormal
lung changes among the four experimental groups.
Table (16): Acute lung injury score.
66
67
68
69
70
71
72
74
75
List of Figures
Figure
Figure (1): The normal alveolus (left-hand side) and the injured
alveolus in the acute phase of acute lung injury and the
acute respiratory distress syndrome (right-hand side)
Figure (2): Leukotriene Synthesis, Receptors, and Signaling
Figure (3): Standard curve of IL-6 obtained by Bio-ELISA reader
Elx800 at wave length 405 nm.
Figure (4): Standard curve of TNF-α obtained by Bio-ELISA reader
Elx800 at wave length 405 nm.
Page
4
19
40
44
Figure (5): Standard curve of LTB4 obtained by Bio-ELISA reader
Elx800 at wave length 412 nm.
Figure (6): Standard curve of LTC4 obtained by Bio-ELISA reader
Elx800 at wave length 650 nm.
Figure (7): The mean of serum TNF-α level (pg/ml) in the four
experimental groups at the end of the experiment.
Figure (8): The mean of serum IL-6 level (pg/ml) in the four
experimental groups at the end of the experiment.
Figure (9): The mean of lung MDA level (nmol/g) in the four
experimental groups at the end of the experiment.
Figure (10): The mean of lung GSH level (μmol/g) in the four
experimental groups at the end of the experiment.
Figure (11): The mean of BALF LTB4 level (pg/ml) in the four
experimental groups at the end of the experiment.
Figure (12): The mean of BALF LTC4 level (pg/ml) in the four
experimental groups at the end of the experiment.
Figure (13): The mean of BALF total protein level (g/l) in the four
experimental groups at the end of the experiment.
Figure (14): Component bar chart shows the relative frequency of
different histopathological grading of abnormal lung
changes among the four experimental groups.
Figure (15): Photomicrograph of lung section of normal rats shows
the normal architecture. The section stained with
Haematoxylin and Eosin (X 10). A: alveoli, S: alveolar
septae.
Figure (16): Photomicrograph of lung section with mild injury. The
section stained with Haematoxylin and Eosin (X 40).
Figure (17): Photomicrograph of lung section with moderate injury.
The section stained with Haematoxylin and Eosin (X
10).
Figure (18): Photomicrograph of lung section with severe injury. The
section stained with Haematoxylin and Eosin (X 40).
Figure (19): Correlation of lung MDA level with serum TNF-α level
in control group and sham group.
49
53
60
62
64
66
68
70
72
75
76
76
77
77
80
List of Abbreviations
μg
microgram
μl
Microliter
μM
Micromolar
AAA
Abdominal Aortic Aneurism
ALI
Acute Lung Injury
ANOVA
Analysis of Variance
APACHE
[Acute
score:
Evaluation] a widely-used method for assessing severity of
Physiological
Assessment
and
Chronic
Health
illness in acutely ill patients in intensive care units, taking into
account a variety of routine physiological parameters
ARDS
Acute Respiratory Distress Syndrome
BAL
Broncho-Alveolar Lavage
BALF
Broncho-Alveolar Lavage Fluid
BBB
Blood Brain Barrier
BLT1
B Leukotriene Receptor 1
BLT2
B Leukotriene Receptor 2
C5a
Complement Protein C5a
cAMP
cyclic Adenosine Monophosphate
CINC
Cytokine Induced Neutrophil Chemoattractant
CNS
Central Nervous System
CRP
C Reactive Protein
CysLTs
cysteinyl-leukotrienes
CysLT1
Cysteinyl Leukotriene Receptor Type 1
CysLT2
Cysteinyl Leukotriene Receptor Type 2
CXC
Chemokines
DNA
Deoxyribonucleic Acid
DW
Distill Water
EDTA
Ethylenediaminetetraacetic Acid
ELISA
Enzyme-Linked Immunosorbent Assay
eNOS
Endothelial Nitric Oxide Synthase
FiO2
Inspired Fraction of Oxygen
5-LO
5-Lipoxygenase
FLAP
5-Lipoxygenase Activating Protein
GM-CSF
Granulocyte Macrophage Colony Stimulating Factor
GPCRs
Seven-Transmembrane G-Protein Coupled Receptors
hr
hour
HS
Hemorrhagic Shock
HSR
Resuscitated Hemorrhagic Shock
H2O2
Hydrogen Peroxide
HUVEC
Human Umbilical Vein Endothelial Cells
ICU
Intensive Care Unit
IL-1
Interleukin 1
IL-4
Interleukin 4
IL-6
Interleukin 6
IL-10
Interleukin 10
IL-13
Interleukin 13
IL1-β
Interleukin 1 beta
IFN-γ
-gamma
IL-8
Interleukin 8
i.p.
Intraperitonial
i-NOS
Inducible Nitric Oxide Synthase
I/R
Ischemia/Reperfusion
i.v.
Intravenous
kD
Kilo Dalton
kg
Kilogram
LPS
Lipopolysaccharide
LTB4
Leukotriene B4
LTC4
Leukotriene C4
LTD4
Leukotriene D4
LTE4
Leukotriene E4
LTRA
Leukotriene Receptor Antagonist
MDA
Malondialdehyde
MIF
Macrophage Inhibitory Factor
mL
Millilitre
mmHg
Millimetres of Mercury
MPO
Myeloperoxidase
MODS
Multiple Organ Dysfunction Syndrome
MOF
Multiple Organ Failure
mRNA
Messenger Ribonucleic Acid
NADPH
Nicotinamide Adenine Dinucleotide Phosphate-Oxidase
NF-қB
Nuclear Factor-kappa B
ng
Nanogram
NO2-
Nitrite
NO3-
Nitrate
NO
Nitric Oxide
NOS
Nitric Oxide Synthase
O2
Oxygen
O2–
Superoxide Anion Radical
OH-
Hydroxyl Radical
ONOO-
Peroxynitrate
PAF
Platelet-Activating Factor
PaO2
Partial Pressure Of Arterial Oxygen
PBS
Phosphate Buffered Saline
PGE2
Prostaglandin E2
PLA2
Phospholipase A2
PMNs
Polymorphonuclear cells, neutrophils
PSML
Postshock Mesenteric Lymph
RL
Ringer's Lactate
ROI
Reactive Oxygen Intermediates
ROS
Reactive Oxygen Species
RT
Room Temperature
SIRS
Systemic Inflammatory Response Syndrome
SRS-A
Slow-Reacting Substance of Anaphylaxis
STAT
Signal Transducers and Activators Of Transcription
TBI
Traumatic Brain Injury
T/HS
Trauma/Hemorrhagic Shock
TH2
T Helper Type 2
TNF-α
Tumour Necrosis Factor- Alpha
US
United States
XD
Xanthine Dehydrogenase
XO
Xanthine Oxidase
Summary
Background
Acute lung injury following hemorrhagic shock/resuscitation is an
important contributor to late morbidity and mortality in traumatic patients.
Hemorrhagic shock followed by resuscitation is conceived as an insult
frequently induces a systemic inflammatory response syndrome and
oxidative stress that results in multiple-organ dysfunction syndrome
including acute lung injury. MK-886 (5-lipoxygenase inhibitor) and
montelukast (cysteinyl leukotriene receptor antagonist) exert an anti
inflammatory and antioxidant activity.
Objective
The objective of present study is to assess the possible protective effect of
MK-886 and montelukast against hemorrhagic shock-induced acute lung
injury via interfering with inflammatory and oxidative pathways.
Materials and Methods
24 adult Albino rats were assigned to four groups: group I (n = 6), sham
group, rats underwent all surgical instrumentation but neither hemorrhagic
shock nor resuscitation was done; group II (n = 6), Rats underwent
hemorrhagic shock (HS) for 1hr then resuscitated with Ringer’s lactate (1hr)
(induced untreated group, HS); group III (n = 6), HS + montelukast (7
mg/kg i.p. injection 30 min before the induction of HS, and the same dose
was repeated just before reperfusion period); group IV (n = 6), HS + MK886 (0.6 mg/kg intraperitonially 30 min before the induction of HS, and the
same dose was repeated just before reperfusion period). HS was induced by
subjecting rats to a 50% blood loss (30 ml/kg) via intracardiac puncture from
the left side of the chest over 2 min and left in shock state for 1hr, then
resuscitated with two times blood loss (60 ml/kg) using intravenous lactated
Ringers via tail over 1 hr. At the end of experiment (2 hr after completion of
resuscitation), blood samples were collected for measurement of serum
tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). The trachea was
then isolated and bronchoalveolar lavage fluid (BALF) was carried out for
measurement of leukotriene B4 (LTB4), leukotriene C4 (LTC4) and total
protein. The lungs were harvested, excised and the left lung was
homogenized for measurement of malondialdehyde (MDA) and reduced
glutathione (GSH) and the right lung was fixed in 10% formalin for
histological examination.
Results
Compared with the sham group, levels of serum TNF-α & IL-6; lung
MDA; BALF LTB4, LTC4 & total protein were increased and lung GSH
were decreased in the animals with HS (P < 0.05). Histologically, all
induced untreated rats showed significant lung injury (P < 0.05). Both
montelukast and MK-886 significantly counteract the increase in serum
TNF-α & IL-6; lung MDA; BALF LTB4, LTC4 & total protein (P < 0.05).
Both montelukast and MK-886 significantly prevent the decrease in lung
GSH level (P < 0.05). Morphologic analysis revealed that both montelukast
and MK-886 markedly reduced (P < 0.05) the severity of lung injury in the
shocked rats.
Conclusions
The results of the present study reveal that both montelukast and MK-886
may ameliorate lung injury in shocked rats via interfering with inflammatory
and oxidative pathways implicating the role of leukotrienes in the
pathogenesis of hemorrhagic shock-induced lung inflammation.
1. Introduction
1.1. Background
Hemorrhagic shock (HS) is a commonly encountered complication within
a blunt traumatic or surgical injury. Hemorrhagic shock followed by
resuscitation (HSR) is conceived as an insult frequently induces a systemic
inflammatory response syndrome (SIRS) that results in multiple-organ
dysfunction syndrome (MODS)
(1, 2)
including acute lung injury (ALI),
which is a major clinical problem, leading to significant mortality and
morbidity (1, 3).
The mechanism of pathogenesis of SIRS in the field of HS is complex and
a variety of mechanisms are implicated. The most widely recognized
mechanisms are ischemia and reperfusion (I/R) and stimulation of cells of
the innate immune system
(4)
. Ischemia and reperfusion is mainly
participating in oxidative stress and SIRS arising during post-ischemic
resuscitation. Hemorrhagic shock/resuscitation can be viewed as a global
ischemia/reperfusion injury insult
(5)
. The extent of tissue ischemia, that
defines the degree of oxygen debt, correlates with a systemic inflammatory
response that renders the injured patient at risk for post-resuscitation
multiple organ failure (MOF)
(6)
. I/R injury is, by itself, a potent
inflammatory trigger, increasing cytokine release, reactive oxygen species
generation, and endothelial activation, with consequent nitric oxide
production and expression of adhesion molecules
(7)
. In addition, I/R may
prime the organism for an exaggerated inflammatory response (massive
tissue-cellular infiltration and edema) to a secondary stimulus such as
infection, with devastating consequences
(8)
. These so-called two-hit injuries
occur with remarkable frequency in traumatic patients who suffered
hemodynamic instability (9). The lungs are, most commonly, the target of this
second injury
(10)
, morphologically characterized by alveolar and interstitial
fluid accumulation, alveolar hemorrhage, fibrin deposition, and lung
neutrophil sequestration
(11)
. Accumulation of neutrophils in the lung
vasculature, interstitium and alveolar space is considered to be a critical
event in the pathophysiologic process and has been the target of various
preventive strategies (12).
1.2. Acute Respiratory Distress Syndrome (ARDS) and Acute
Lung Injury (ALI)
ALI is defined by the ratio of the partial pressure of arterial oxygen to the
fraction of inspired oxygen (P/F ratio; PaO2 in mmHg/FIO2) of ≤ 300 while
ARDS (most severe form of ALI) is classified as having a ratio of ≤ 200 (13).
Clinically, ALI is characterized by altered gas exchange, dyspnea, decrease
static compliance, and noncardiogenic pulmonary edema (14).
1.2.1 Mortality/Morbidity
The lungs are one of the initially affected and most frequently affected
organs in MODS following hemorrhagic shock. ARDS occurs in up to 50%
of MODS affected patients
(15)
and remains one of the leading causes of
morbidity and mortality in all ICUs. ARDS has been estimated at 190,000
cases per year in the United States with 74 000 deaths annually (16).
1.2.2. Pathogenesis
1.2.2.1. Endothelial and Epithelial Injury
Two
separate
barriers
form
the
alveolar-capillary
barrier, the
microvascular endothelium and the alveolar epithelium (figure 1). The acute
phase of ALI and ARDS is characterized by the influx of protein-rich edema
fluid into the air spaces as a consequence of increased permeability of the
alveolar-capillary barrier
(17)
. The importance of endothelial injury and
increased vascular permeability to the formation of pulmonary edema in this
disorder has been well established (18). Widespread injury to and activation of
both the lung and systemic endothelium with a resultant increase in
permeability and expression of adhesion molecules is characteristic of
ALI/ARDS (19).
The critical importance of epithelial injury to both the development of and
recovery from the disorder has become well recognized
(20,21)
. The degree of
alveolar epithelial injury is an important predictor of the outcome
(22)
. The
normal alveolar epithelium is composed of two types of cells (figure 1). Flat
type I cells make up 90 percent of the alveolar surface area for gas exchange
and are easily injured. Cuboidal type II cells make up the remaining 10
percent of the alveolar surface area and are more resistant to injury; their
functions include surfactant production, ion transport, and proliferation and
differentiation to type I cells after injury (13).
The loss of epithelial integrity in ALI and ARDS has a number of
consequences. First, under normal conditions, the epithelial barrier is much
less permeable than the endothelial barrier
(21)
. Thus, epithelial injury can
contribute to alveolar flooding. Second, the loss of epithelial integrity and
injury to type II cells disrupt normal epithelial fluid transport, impairing the
removal of edema fluid from the alveolar space
(23)
. Third, injury to type II
cells reduces the production and turnover of surfactant
the characteristic surfactant abnormalities
(25)
(24)
, contributing to
. Fourth, loss of the epithelial
barrier can lead to septic shock in patients with bacterial pneumonia
(26)
.
Finally, if injury to the alveolar epithelium is severe, disorganized or
insufficient epithelial repair may lead to fibrosis (27).
False
False
Figure (1). The normal alveolus (left-hand side)
and the injured alveolus in the acute phase of acute lung injury and the acute
respiratory distress syndrome (right-hand side) (13).
In the acute phase of the syndrome (right-hand side), there is sloughing of
both the bronchial and alveolar epithelial cells, with the formation of
protein-rich hyaline membranes on the denuded basement membrane.
Neutrophils are shown adhering to the injured capillary endothelium and
marginating through the interstitium into the air space, which is filled with
protein-rich edema fluid. In the air space, an alveolar macrophage is
secreting cytokines, interleukin-1, 6, 8, and 10, (IL-1, 6, 8, and 10) and
tumor necrosis factor-α (TNF-α), which act locally to stimulate chemotaxis
and activate neutrophils. Macrophages also secrete other cytokines,
including interleukin-1, 6, and 10. Interleukin-1 can also stimulate the
production of extracellular matrix by fibroblasts. Neutrophils can release
oxidants, proteases, leukotrienes, and other proinflammatory molecules,
such as platelet-activating factor (PAF). The influx of protein-rich edema
fluid into the alveolus has led to the inactivation of surfactant. MIF denotes
macrophage inhibitory factor (13).
1.2.2.2. Neutrophil-Dependent Lung Injury
Neutrophils are the major cellular elements involved in acute lung
inflammation after resuscitated hemorrhagic shock
that neutrophils are activated following HS
(29)
(28)
. Studies have shown
and that lung injury is
associated with an increased neutrophils accumulation in the lungs after HS
(30)
. The activated neutrophils appear to infiltrate the injured lung in parallel
with increased expression of adhesion molecules on endothelial cells and
elevated local chemokines/cytokines levels following HS
(29)
. Histologic
studies of lung specimens obtained early in the course of the disorder show a
marked accumulation of neutrophils
(31)
. Neutrophils predominate in the
pulmonary edema fluid and bronchoalveolar-lavage fluid (BALF) obtained
from affected patients
dependent
(32)
(20)
, and many animal models of ALI are neutrophil-
. Some of the mechanisms of the sequestration and activation
of neutrophils and of neutrophil-mediated lung injury are summarized in
(figure1).
1.3. Pathogenesis of SIRS Leading to MODS and ARDS
Multiple mechanisms are at play during the development of MODS and
ARDS, they can be conceptually organized into several components and
these components occur simultaneously and interactively and are part of one
pathophysiological process. Some of the main components are:
1) the “Two-Hit” theory of immune cell priming, 2) the influence of gut
hypoperfusion, 3) the influence of oxidative stress, 4) the influence of
proinflammatory mediators.
1.3.1. The “Two-Hit” Theory of Immune Cell Priming
A concept of a “Two-Hit” hypothesis has emerged from various animal
and clinical studies, suggesting that HS renders trauma victims more
susceptible to the development of SIRS and subsequently MODS (including
ARDS)
by
priming
neutrophils
and
macrophages
for
increased
responsiveness to subsequent, even small, inflammatory stimuli, such as a
line infection or a gastrointestinal bleed
(33,34)
. Immune priming has been
observed in animal models and patients sustaining I/R from trauma or
rupture of an abdominal aortic aneurysm (AAA)
(35)
. In the “Two-Hit”
hypothesis animal models, primed leukocytes are characterized by excessive
release of proinflammatory mediators (superoxidants, TNF-α, and IL-1β, IL8 or CINC) which are thought to play an important role in the propagation of
SIRS
(36,37)
. In rodents, I/R was demonstrated to induce earlier and greater
macrophage release of proinflammatory cytokines such as Cytokine-Induced
Neutrophil Chemoattractant (CINC), which correlated with augmented and
earlier signaling through the proinflammatory intracellular pathway via the
transcription factor, nuclear factor-kappa B (NF-κB)
(37)
. Studies also
showed that I/R could augment PMN-mediated tissue injury via the NADPH
oxidase system (35).
1.3.2. The Influence of Gut Hypoperfusion
Recent studies indicate that during HS, splanchnic ischemia reperfusion
plays a central role in the pathogenesis of shock induced SIRS and MODS
(38)
. Early investigation focused on translocation of intestinal flora to the
systemic circulation following gut I/R with the portal vein as the conduit.
Although initial animal models offered supportive evidence
corroboration in the critically injured patient was lacking
(39)
(40)
, clinical
. Attention
shifted from the bacterial translocation hypothesis when Deitch and
colleagues
following
(41)
began investigating the role of the mesenteric lymphatics
trauma/hemorrhagic
shock.
Cytotoxicity
associated
with
postshock mesenteric lymph (PSML) was not seen in corresponding portal
venous
blood
(41,42)
.
However,
lymphatic
diversion
before
trauma/hemorrhagic shock (T/HS) abrogated polymorphonuclear neutrophil
(PMN)-mediated lung injury
(43)
. Furthermore, Magnotti et al. (1998)
demonstrated that hemorrhagic shock-induced lung injury was completely
prevented by the division of mesenteric lymphatics, indicating that this lung
injury is produced by intestinal I/R
(41)
. It has been demonstrated that the
non-ionic lipid fraction extracted from PSML primed PMNs for increased
superoxide (O2–) production, increased
adhesion
expression, and also inhibited PMN apoptosis
(44,45)
molecule
surface
. Phospholipase A2
(PLA2), a proximal enzyme in the arachidonic acid (AA) cascade found in
the gut, contributes in the generation of proinflammatory lipids via the
lipoxygenase and cyclooxygenase pathways
(46,47)
. Increased activity of this
enzyme has been established following gut I/R
(48)
. Clinically, mortality
related to multiple organ failure correlates with increased plasma levels of
the secretory isoform of PLA2 (sPLA2: represents the major culprit in
(49,50)
inflammation)
. Additionally, the administration of a PLA2 inhibitor
prevents lung injury related to splanchnic hypoperfusion following T/HS,
and PLA2 blockade abrogates PMN priming activity associated with the lipid
fraction of PSML (51). Splanchnic I/R activates gut PLA2-mediated release of
AA into the lymph where it is delivered to the lungs, provoking LTB4
production and subsequent PMN-mediated lung injury
(52)
. Furthermore,
mesenteric lymph produced after hemorrhagic shock potentiates lung injury
by the upregulation of endothelial cell adhesion molecule expression and IL6 production
(53)
. IL-6 is an important autocrine factor produced by
endothelial cells that contributes to the increase in endothelial permeability
during hypoxia
(54)
, so that T/HS lymph induces an increase in endothelial
permeability by triggering the release of IL-6.
1.3.3. Oxidative Stress
1.3.3.1. Oxidative Stress & Free Radicals: Definition and
Mechanisms
Oxidative stress can be defined as imbalance between oxidants and
antioxidants in favor of the oxidants, potentially lead to damage. A free
radical is defined as “any atomic or molecular species capable of
independent existence that contains one or more unpaired electrons in one of
its molecular orbitals”
(55)
. Free radicals are generally reactive oxygen or
nitrogen species. Biologically important reactive oxygen species (ROS)
include superoxide anion radical (O2-), hydrogen peroxide (H2O2), hydroxyl
radical (OH-), and hypohalous acids such as HOCl. Reactive nitrogen
species, including nitric oxide (NO•), peroxynitrate (ONOO-), nitrite (NO2-)
and nitrate (NO3-) have also been implicated in oxidation (nitration) of
proteins and lipids (56). Reactive oxygen and nitrogen species can lead to cell
injury by various mechanisms, including: (i) direct damage to DNA resulting
in strand breaks and point mutations; (ii) lipid peroxidation with formation of
vasoactive and proinflammatory molecules such as thromboxane; (iii)
oxidation of proteins (primarily at sulfhydryl groups) that alter protein
activity, leading to release of proteases and inactivation of antioxidant and
antiprotease enzymes; and (iv) alteration of transcription factors such as
activator protein-1 and NF-қB, leading to enhanced expression of
proinflammatory genes (56).
1.3.3.2. Sources of Oxidants in ALI/ARDS
In the context of ALI/ARDS, there are many potential sources of ROS,
including itinerant and resident leukocytes (neutrophils, monocytes, and
macrophages),
parenchymal
cells
(endothelial and
epithelial
cells,
fibroblasts, and myocytes), circulating oxidant-generating enzymes (xanthine
oxidase), and inhaled gases with high concentrations of oxygen that are often
used during mechanical ventilation. Leukocytes, principally neutrophils and
macrophages, are generally considered to be the most prodigious source of
ROS in ALI/ARDS. The large numbers of activated neutrophils in the lung
in ALI/ARDS has focused attention on these phagocytes as a major source of
ROS (56).
1.3.3.3. Oxidant-Antioxidant Balance
Since oxygen radicals are a normal by-product of aerobic metabolism, all
aerobic
organisms
developed
mechanisms
to
achieve
an
oxidation/antioxidation balance in order to prevent death by oxidation.
Antioxidant systems work to prevent oxidation of lipids, proteins, and
nucleic acids by ROS. Mechanisms also exist for either repair or removal of
any damaged species. Oxidative stress occurs when antioxidants cannot
prevent oxidative injury by overabundant oxidant production. The burden of
oxidative stress occurs in various disease processes such as during
resuscitation of a massive hemorrhagic shock, atherosclerosis, pulmonary
fibrosis, cancer, neurodegenerative diseases, as well as in a natural process
of aging
(57)
. The antioxidant systems can be divided into enzymatic and
non-enzymatic. The non-enzymatic systems include the water soluble
ascorbic acid, and the lipid soluble alpha tocopherol, glutathione and
cysteine containing proteins such as albumin. Another way of removing
ROS is by catalysis with enzymatic antioxidants such as superoxide
dismutase, catalase and glutathione peroxidase which can break down H2O2
to oxygen and water and prevent harmful effects of ROS (58).
1.3.3.4. Potential Targets of Oxidants in ALI/ARDS
Various cellular components can be subject to oxidative modifications
including membrane, cytosolic and nuclear lipids and proteins. Cellular
membranes and especially plasma membranes are primary targets of ROS.
The fatty acid side chains of membrane phospholipids undergo peroxidation
under oxidative stress
(59)
. Membrane fluidity is dependent on lipid
composition of the plasma membrane, and alterations in this composition,
including those by oxidation, profoundly influence diverse aspects of
membrane function. In the context of acute inflammation, oxidation of
components of the endothelial or epithelial plasma membrane could facilitate
neutrophil recruitment into the lung by compromising the barrier function of
these cells,
thereby
allowing
leakage
of
chemokines
and
other
chemoattractant molecules into the vascular space. Additionally, oxidant
exposure can lead to enhanced leukocyte adhesion either by direct oxidative
modification of components of the endothelial plasma membrane generating
lipid mediators or by "inside-out" signaling leading to enhanced surface
expression and affinity of adhesion molecules
(60)
. Cytosolic and nuclear
events initiated by oxidants might also contribute to inflammatory injury and
be amenable to pharmacologic intervention. For example, elevation of
cytokines and chemokines such as TNF-α, IL-1ß, IL-2, IL-6, and IL-8 is a
feature common to lung injury of diverse etiologies. Cytokine expression is
primarily regulated at a transcriptional level. NF-кB is a DNA-binding factor
that stimulates transcription of many different cytokines involved in acute
inflammation, and is activated in ALI and ARDS (61,62,63). NF-кB is normally
a heterodimer that is sequestered in the cytosol in the quiescent state by
association with the IкB family of inhibitors. Upon stimulation, IкB becomes
phosphorylated and dissociates from NF-кB, allowing the free NF-кB to
translocate to the nucleus, where it binds to promoter regions of specific
genes and induces transcription. There is an evidence that NF-кB is activated
in the context of ALI/ARDS and may be regulated by changes in IкB
expression that are in turn dependent on oxidants produced by, for example,
xanthine oxidase (61,62,63).
1.3.3.5. Oxidants as Mediators of Ischemia/Reperfusion Injury
I/R injury refers to tissue damage when blood flow is restored after an
ischemic period and is common to pathophysiology of many clinical
conditions including myocardial infarcts, peripheral vascular insufficiency,
stroke, trauma, and hypovolemic shock. Despite its vital function in
restoring the body’s hemodynamics, reperfusion leads to ROS generation
and results in activation of the proinflammatory signalling pathways.
Specifically, following resuscitation from HS, production of ROS
contributes to the development of SIRS and later to MODS and ARDS.
ARDS patients have been shown to have increased levels of ROS such as
H2O2 in their expired air
(64)
. In addition, alveolar epithelial lining fluid in
ARDS patients is deficient in glutathione and contains high levels of
peroxynitrite
(65)
. Markers of oxidative stress, such as malondialdehyde
(MDA) and 4-hydroxynonenal, nitrites and nitrates, as well as lipid
peroxidation products F2-isoprostanes have been found in increased
amounts in ARDS subjects especially in those with higher APACHE scores
(66)
. The imbalance of the redox state has been observed in SIRS patients and
the process of continued oxidative stress in SIRS is thought to promote the
development of MODS and ARDS in ICU patients
(67)
. There are multiple
sources of ROS during I/R. The most well studied source is the reactions
catalyzed by the enzymes xanthine oxidase (XO) and nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase. These superoxide-generating
reactions occur in many cells, macrophages, PMNs and the endothelium
being some of them. However, any cell is equipped with potential to produce
ROS if injury to the mitochondria or intracellular membranes occurs. The
mechanism of ROS generation during I/R has been studied by Granger et
al. in the 1980s
(68)
. They concluded that ROS are mediators of reperfusion
injury and the enzyme xanthine oxidase, which is known to be present in
high concentrations in the intestinal mucosa, is the initial triggering agent of
ROS formation at reperfusion
(69)
. Granger et al. (1988) described that XO
is generated during ischemia from its precursor xanthine dehydrogenase
(XD), a constitutively expressed 150 kDa protein. An important site of this
conversion has been shown to be the intestinal microvasculature as XD/XO
are especially abundant in the small intestine, where it is expressed
predominantly in the villous epithelium
(70)
. During ischemia, catabolic
processes were found to generate increased levels of purine metabolites:
hypoxanthine and xanthine, from catabolism of ATP. XD is converted to
XO, and once oxygen is reintroduced during reperfusion, superoxide is
generated. Superoxide triggers a free radical chain reaction that results in
further ROS generation such as H2O2, the hydroxyl radical, peroxynitrite,
hypochlorous acid, and the chloramines. Systemic oxidative stress ensues
through a direct release of oxidants as well as xanthine oxidase into the
systemic circulation
(71)
with resulting concomitant tissue damage.
Accordingly, increased plasma levels of XO have been reported after
hypovolemic shock, released from organs rich in XO, such as the liver and
intestine, into the systemic circulation (72).
The second important source of the “respiratory burst” in both phagocytic
and nonphagocytic cells is the NADPH oxidase system that catalyses oneelectron reduction of oxygen to form superoxide using NADPH as an
electron donor (73). When a macrophage or PMN is activated via a number of
signals, the NADPH enzyme complex assembles in the cytosol in a
membrane bound vesicle, once the vesicle fuses with the plasma membrane
superoxide is released. NADPH oxidase can be regulated by inflammatory
cytokines such as TNF-α, IL1-β and lipopolysaccharide (LPS), although its
expression and regulation differs in different cell types.
NADPH is also structurally different in different cell types especially in
the endothelium (74). Recent studies suggest that it may serve not only as an
ROS generator but also as a sensor of oxygen tension and an iron uptake
system (75). The superoxide generation by the XO or NADPH systems is not
the final path to tissue injury. As outlined above, superoxide can trigger
further generation of free radicals from H2O2. In addition, in phagocytes
another enzyme, myeloperoxidase (MPO), can also react with H2O2 to form
hypochlorous acid. Finally, another free radical in ischemia/reperfusion
injury is nitric oxide (NO.). NO. is a microbicidal product, a diatomic free
radical, derived from L-arginine. The synthesis of NO. is catalyzed by a
family of enzymes called nitric oxide synthases (NOS) comprised of
endothelial NOS (eNOS), neuronal cell NOS (neuronal NOS) and a third
inducible form of NOS (iNOS) which is induced in response to
inflammatory-like stimuli
(76)
. NO. production is part of the host defense
mechanism against inflammatory insults and it serves to regulate vascular
tone, maintain vessel patency by helping to prevent platelet aggregation and
to down-regulate adhesion molecule expression. Under normal conditions,
NO. acts to regulate normal vascular permeability and it also scavenges ROS
thus preventing formation of H2O2. Excessive production of NO., however,
can potentially induce tissue damage via formation of ROS such as
peroxynitrite when NO reacts with superoxide
(77)
. Therefore, NO. can be
protective or harmful following I/R a balance that is not yet fully elucidated.
i-NOS inhibition has been shown to prevent endothelial dysfunction,
suggesting that NO may be cytotoxic in certain circumstances
(78)
.
Contrasting studies indicate the therapeutic potential of administering
exogenous (NO.). During I/R, the dysfunctional endothelium decreases its
production of NO. resulting in superoxide accumulation, H2O2 formation and
decreased vasodilation contributing to increased leukocyte adhesion and noreflow phenomenon
(7,79)
, NO. may protect against it
(80)
. In summary, any
insult capable of activating leukocytes is capable of triggering the release of
reactive oxygen species. The imbalance in the redox state of an organism,
therefore, represents a common pathway for many life-threatening
conditions including the proinflammatory state observed following shock
resuscitation.
1.3.4. Proinflammatory Mediators
There are cellular and humoral mediators of the innate immune response
in SIRS. From activated inflammatory cells a wide variety of chemical
mediators are released into the systemic circulation. These mediators act in
an autocrine, paracrine, and endocrine fashion and include proinflammatory
peptides or cytokines, bioactive lipids including the eicosanoids and plateletactivating factor (PAF), toxic oxygen metabolites including Nitric Oxide,
and neutrophil-derived tissue proteases. The discussion here focuses on
those mediators that have been clearly implicated in the pathogenesis of
ARDS.
1.3.4.1. Tumour Necrosis Factor-Alpha (TNF-α)
TNF-α is a 17-kd, 157-amino-acid cytokine that is secreted by a wide
spectrum of cells, including macrophages, monocytes, T cells, natural killer
cells, and neutrophils (81). TNF-α is proinflammatory cytokine predominantly
secreted by macrophages in response to a variety of pathologic processes,
especially in endotoxin-induced lung injury
(82)
.
Investigators have
demonstrated that TNF-α compromises the endothelial barrier and produces
experimental
pulmonary
edema
(83,84)
.
TNF-α, as
well
as
other
proinflammatory cytokines, is detected in the BALF obtained from patients
with ARDS
(85)
and has been shown to increase pulmonary vascular
resistance, resulting in alveolar edema because of the release of thromboxane
A2 in response to activation of polymorphonuclear leukocytes
(84)
. TNF-α
also increases the neutrophil population in the lung caused by inducing
endothelium-derived polymorphonuclear chemotactic and adherent factors
(86)
. These findings indicate that TNF-α is a very important inflammatory
mediator and plays critical roles in mediating neutrophil recruitment to
inflammation sites, both directly and indirectly. In addition to its role in
facilitating neutrophil transendothelial migration
(87)
, TNF-α also stimulates
endothelial cells and neutrophils to generate large amounts of reactive
oxygen intermediates (ROI)
(88)
, which are involved in TNF-α-induced
endothelial cell apoptosis. Hemorrhagic shock induces pulmonary expression
of TNF-α
(89)
, and neutralization of TNF-α with a monoclonal antibody
attenuates lung injury after hemorrhagic shock (90). TNF-α and IL-1β are both
released within the first 30-60 minutes after exposure to LPS and are capable
of initiating the proinflammatory cascade with up-regulation of adhesion
molecules on neutrophils and stimulating the release of IL-6, eicosanoids,
and PAF among others (91). As well, TNF-α stimulates IL-1β release and vice
versa, thus initiating inflammatory cell migration into tissues. Further, TNFα upregulates PLA2, cyclo-oxygenase (COX), and NOS as well as a variety
of inhibitors, which include plasminogen activator inhibitor 1
(1)
. Both TNF-
α and IL-1β propagate the inflammatory response and are primary mediators
of MODS. Finally, a low level of pulmonary TNF-α is sufficient to mediate
hemorrhagic shock-induced ALI during HS (92).
1.3.4.2. Interleukin-6 (IL-6)
IL-6 is a 21-kDa cytokine that is produced by a variety of cells including
fibroblasts,
endothelial
cells, mononuclear
hepatocytes, and T and B lymphocytes
(93)
phagocytes,
neutrophils,
. IL-6 production is increased by
macrophages/monocytes, endothelial cells and smooth muscle cells in
response to endotoxin, TNF-α and IL-1β
(91)
. It has been demonstrated that
IL-6 is an important autocrine factor produced by endothelial cells that
contributes to the increase in endothelial permeability during hypoxia
(54)
.
IL-6 has been demonstrated to increase in post burn and surgery (94). In acute
inflammatory responses, it induces fever and increases acute phase protein
expression by the liver
(95)
. The systemic response to inflammation includes
the production of IL-6, which signals through activation of proteins that
serve the dual function of signal transducers and activators of transcription
(STAT)
(96)
. Activation of STAT proteins, particularly Stat3, in the lungs of
animals subjected to HS and that level of Stat3 activity increased with
increasing severity of shock
(97)
. IL-6 has been demonstrated to significantly
contribute to Stat3 activation, neutrophil recruitment, and lung injury in a rat
hemorrhagic shock model
(98)
. Experimental studies demonstrated that IL-6
mRNA and protein are produced in the lungs, liver, and intestinal tracts of
rats subjected to resuscitated hemorrhagic shock
(98,99)
and that both the
ischemic and reperfusion phases of resuscitated HS were required for their
production
(99)
. Others have demonstrated elevated circulating levels of IL-6
in humans and animals following T/HS
(100,101)
. It has also been shown that
IL-6 plays an important role in mediating lung leakage, neutrophil
infiltration, and chemokine expression in lung injury following kidney I/R
(102)
.
Sustained elevations of IL-6 in the plasma of patients suffering from
ARDS have been demonstrated and negatively correlated with disease
outcome (103).
1.3.4.3. Leukotrienes (LTs)
Leukotrienes are potent mediators of inflammatory and immune reactions
and have been shown to play a major role in the mediation of
pathophysiological processes in some human diseases. The measurement of
such mediators in body fluids provides a better understanding of disease
pathogenesis and a semi-quantitative means of assessing disease activity
(104)
.
Leukotrienes (LTs), compose of cysteinyl LT (CysLT; LTC4, LTD4, and
LTE4) and LTB4, are potent lipid mediators enhancing the vascular
permeability and recruitment of neutrophils, which are common features of
hemorrhagic shock-induced lung injury.
1.3.4.3.1. Leukotrienes Biosynthesis
Leukotriene synthesis can be activated in a cell (e.g., a leukocyte) by a
variety of stimuli. The enzymatic machinery for PLA2-catalyzed
arachidonate hydrolysis and leukotriene synthesis is localized primarily at or
near the nuclear membrane, necessitating that leukotriene B4 (LTB4) and
leukotriene C4 (LTC4) be transported by carrier proteins out of the cell; the
LTC4 transporter is multidrug resistance protein 1; the LTB4 transporter is
unknown. In the extracellular milieu, LTC4 is converted to leukotriene D4
(LTD4) and LTD4 to leukotriene E4 (LTE4). Collectively, these molecules
make up the cysteinyl leukotrienes
(105)
. Leukotrienes act on target cells,
which may be leukocytes, epithelial cells, smooth-muscle cells, or
endothelial cells, by interacting with one or both classes of their cognate
receptors. B leukotriene receptor 1 (BLT1) is expressed primarily on
leukocytes and is a high-affinity receptor, whereas B leukotriene receptor 2
(BLT2) is expressed more ubiquitously, has a somewhat lower affinity for
LTB4, and can bind other lipids. The two cysteinyl leukotriene receptors
have a broad distribution. All leukotriene receptors activate the Gq class of
G proteins, resulting in increased intracellular calcium, the Gi class,
resulting in decreased intracellular cyclic AMP (cAMP), or both. These
effects, which activate downstream protein kinases, culminate in myriad
cellular and tissue responses (105). The sites of action of antileukotriene drugs
(5-lipoxygenase [5-LO] for zileuton and CysLT1 for montelukast,
zafirlukast, and pranlukast) are shown in figure (2). FLAP denotes 5lipoxygenase-activating protein.
Figure (2): Leukotriene Synthesis, Receptors, and Signaling (105).
The cysteinyl-leukotrienes (CysLTs, i.e. LTC4, LTD4 and LTE4) mediate
bronchoconstriction, mucus secretion and vasoconstriction which are central
components of asthmatic airway inflammation, whereas LTB4 is one of the
most potent chemotactic and proinflammatory agent so far described
(106,107)
.
LTC4 is primarily produced by eosinophils, mast cell and basophil, but also
by macrophage/monocyte
(105)
. LTB4 is predominantly produced by
neutrophils, but also by macrophage/monocyte and dendritic cells. In
addition, leukotrienes are produced in transcellular reactions involving
inflammatory cells and surrounding structural elements. The LTs play
important roles in both the innate and adaptive immune responses (105).
1.3.4.3.2. Leukotriene B4 (LTB4)
LTB4
is
chemotactic
for
neutrophils,
monocytes/macrophages,
eosinophils, fibroblasts, dendritic cells and activated CD4 + and CD8+ T
lymphocytes
(108,109,110,111)
. In addition to its chemotactic properties, LTB4
converts leukocytes from rolling on endothelium to firm adhesion, and
LTB4-treated
human
endothelial
cells
were
shown
to
promote
transendothelial neutrophil migration (112,113).
LTB4 activates leukocytes by enhancing macrophage and neutrophil
phagocytosis
(114)
. LTB4 also augments phagocyte microbial killing by
stimulating lysosomal enzyme release, and generation of defensins, ROS,
and NO. In addition, LTB4 stimulates production of cytokines and
chemokines (e.g. TNF-α, IL-8 and monocyte chemoattractant protein-1
[MCP-1]), and also secretion of immunoglobulins. LTB4 can be produced by
neutrophils, mast cells, eosinophils, and macrophages stimulated by
proinflammatory cytokines such as TNF-α, IL-1β and chemoattractants such
as CXCL8, C5a, PAF, and itself
(115,116)
. LTB4 increases the expression of
CD11b/CD18 β2-integrin on neutrophils, which can facilitate neutrophil
adherence and migration
(115)
, even though LTB4 is reported to be able to
stimulate β2-integrin-independent migration of PMN across human
pulmonary endothelial cells in vitro
(117)
. In addition, in vitro experiments
indicate that human umbilical vein endothelial cells (HUVEC) express LTB4
receptors (BLT1 and BLT2), which are more highly expressed after LPS and
TNF-α stimulation and more responsive to LTB4. LTB4 can augment nitric
oxide and MCP-1 production from HUVEC stimulated by LPS
(118)
. This is
an important feature of LTB4, because MCP-1 facilitates PMN
transmigration across endothelial cells barriers
(119)
. This data suggests that
LTB4 interaction with LPS (or TNF-α)-stimulated endothelial cells through
the BLT1 and the BLT2 can enhance PMN transmigration across endothelial
cells. LTB4 facilitates PMN transepithelial cell migration through the ROSextracellular signal-regulated kinase-linked cascade (120).
LTB4 augments IL-6 production in human monocytes by increasing both
IL-6 gene transcription and messenger RNA (mRNA) stabilization
(121,122)
;
activation of NF-κB and NF-IL-6 transcriptional factors may be important in
this enhancement of IL-6 release (122).
LTB4 exerts its effects by binding to two receptors, termed BLT1, high
affinity receptor and BLT2, low-affinity receptor
(123)
. BLT1 is primarily
expressed on leukocytes, and mediates most of the LTB4 mediated functions.
BLT2 is more ubiquitously expressed, and although its functional role is not
clear enough, a recent study showed that LTB4 mediates dendritic cell
chemotaxis via BLT2 (110).
1.3.4.3.3. The Cysteinyl-Leukotrienes (CysLTs)
The CysLTs were previously known as slow-reacting substance of
anaphylaxis (SRS-A)
(105,106,124,125)
(105,106,124,125)
. They are potent bronchoconstrictors
, with LTC4 and LTD4 being more potent than E4
(124,125)
. In
human subjects, the bronchoconstrictor effect of inhaled CysLTs was shown
to be around 100 to 10,000-fold more potent than inhaled histamine
addition, the CysLTs are potent vasoconstrictors
(124,125)
(126)
. In
. Moreover, CysLTs
mediate increased permeability (leading to leukocyte extravasation, plasma
exudation and edema), and mucus hypersecretion (106,124,125). They may play a
role in airway remodeling, e.g. by stimulating bronchial smooth muscle
proliferation (both hyperplasia and hypertrophy), fibroblast proliferation,
increased collagen deposition, and mucus gland hyperplasia
(127)
. The
CysLTs promote the recruitment of eosinophils, neutrophils, dendritic cells,
and T lymphocytes
(105,124,125)
. The CysLTs stimulate proliferation and
differentiation of bone marrow eosinophil hematopoietic progenitors, and
also their subsequent migration to blood (105,125). In addition, a study suggests
that CysLTs are important in prolonging eosinophil survival. LTD4 appeared
to be of similar potency to granulocyte macrophage colony stimulating
factor (GM-CSF) in that regard
(128)
. In addition, the CysLTs were shown to
induce cell adhesion proteins and thereby promote leukocyte adhesion to
vascular endothelial cells
(105,106,125)
. The CysLTs were also shown to have
some antimicrobial effects, although these actions are narrower than those of
LTB4
(114)
. Studies have demonstrated a capacity for CysLTs to induce ROS
and NO formation. Furthermore, CysLTs promote leukocyte survival
(105,125)
.
The CysLTs stimulate the production of T helper type 2 (TH2) cytokines
(e.g. IL-4, 5 and 13), which in turn stimulate the production of the CysLTs.
IL-4 upregulates LTC4 synthase gene expression, whereas IL-4 and IL-13
upregulates CysLT1 receptor gene expression
(105,125)
. LTD4 enhances IL-1
production by human monocytes and can replace IL-2 in the induction of
interferon-γ secretion by T lymphocytes (129).
There are at least two receptors for CysLTs, termed Cysteinyl leukotriene
receptor type 1 and type 2 (CysLT1 and CysLT2), with a wide distribution
(130)
. The CysLT1 receptor is mainly expressed in the spleen peripheral blood
leukocytes (including eosinophils), and less strongly expressed in the lung
(smooth muscle cells and interstitial macrophages), small intestine, pancreas
and placenta. The CysLT2 receptor is mainly expressed in the heart, adrenal
medulla, placenta, peripheral blood leukocytes (including eosinophils),
spleen and lymph nodes, and some expression throughout the CNS.
All LT receptors (CysLT1, CysLT2, BLT1, and BLT2) are seventransmembrane G-protein coupled receptors (GPCRs) and the LTs either
activate the Gq subtype (resulting in an increased intracellular calcium
concentration) and/or the Gi subtype (resulting in decrease intracellular
cAMP) (123,129).
1.3.4.3.4. Leukotrienes and Acute Lung Injury
Studies in humans confirm elevated levels of LTB4, LTC4 and LTD4 in
BAL, pulmonary edema fluid, and plasma in patients with ALI compared
with “at-risk” group or those with hydrostatic edema
(131,132)
. Studies in
animals and humans have suggested a role for cysteinyl LTs in ALI.
Increased lung levels of LTC4 and LTD4 have been detected in various
animal models of ALI
(133,134,135)
. Animal studies have verified the important
role of LTB4 in the pathogenesis of ALI following splanchnic I/R
(136,137)
.
Leukotriene B4 showed the best correlation with lung-injury severity and
outcome in patients with ARDS (138).
The
5-lipoxygenase
hemorrhagic shock
pathway
(139,140)
products
meditate
ALI
following
. Intrinsic 5-lipoxygenase activity is required for
neutrophil adherence and chemotaxis and neutrophil-mediated lung injury
(141)
. Infusion of LTs into animals produces ALI resembling the clinical
presentation of endotoxemia and ARDS, including pulmonary hypertension
and increased vascular permeability resulting in pulmonary edema and
hypoxemia (142).
1.4. Some Aspects of the Drugs Used in This Study
1.4.1. MK-886 (Investigational compound)
1.4.1.1. Pharmacological Action of MK-886
MK-886 is a highly potent inhibitor of leukotriene formation in vivo and
in vitro (143). This compound inhibits leukotriene biosynthesis indirectly by a
mechanism through the binding of a membrane bound 5-lipoxygenaseactivating protein (FLAP), thereby inhibiting the translocation and activation
of 5-lipoxygenase (144,145). In vivo, it was shown that oral application of MK886 in humans significantly inhibits leukotriene B4 biosynthesis
blocks allergen-induced airway responses
(147)
(146)
and
. MK-886 inhibits leukotriene
biosynthesis and antigen induced bronchoconstriction in animal models and
in asthmatic men
(148)
. In vitro, MK-886 exerts many effects, including
preventing the translocation and activation of 5-lipoxygenase in human
leukocytes
(149)
, and inhibiting DNA synthesis in leukemia cells
(150)
.
Moreover, MK-886 was found to be a potent and specific inhibitor of both
LTB4 and LTC4 synthesis in human phagocytes
potent PPARα antagonist
(152)
(143,151)
and moderately
. MK-886 was found to have novel antitumor
pharmacologic mechanism
(153)
and recently Mk-886 was found to be
effective against human prostate cancer cells
(154)
(by a Ca2+ independent
manner) and human malignant glioma cells (155) (by induction of apoptosis).
Inhibition of endogenous CysLT production by MK-886 significantly
attenuated the generation of TNF-α by mast cells activated by Fc RI crosslinkage
(156)
. Additionally, treatment with MK-886 abrogated cytokine-
elicited PGE2 release including IL-1β in a dose-dependent manner in vitro
(157)
.
MK886 pretreatment attenuated subsequent pulmonary expression of
TNF-α in a mouse model of bronchial inflammation and hyperreactivity (158).
Guidot et al. (1994) found that 5-lipoxygenase inhibition by MK-886
prevents stimulated neutrophil adherence and chemotaxis and neutrophil
mediated lung injury in vitro
extravasation of plasma
(159)
(141)
. MK-886 has been shown to reduce the
and prevent the leukocyte adhesion to the
endothelium (160) in experimental animals.
MK-886 was found to be effective in prevention of liver and intestine
injury by reducing apoptosis and oxidative stress in a hepatic I/R model.
Anti-inflammatory properties and inhibition of lipid peroxidation by MK886 could be protective for these organs in I/R injury (161).
Lehr et al. (1991) revealed that selective inhibition of leukotriene
biosynthesis (by MK-886) prevents postischemic leukotrienes accumulation
and the microcirculatory changes after ischemia-reperfusion
(160)
. The
inhibition of 5-LO by MK-886 was found to be effective in decreasing the
level of fibrosis in experimental cirrhosis
(162)
. MK-886 was also able to
reduce the cortical infarct size by 30% in a model of focal cerebral ischemia
in rats (163). Furthermore, pharmacological reduction of cysteinyl leukotriene
formation after experimental traumatic brain injury, using MK-886, resulted
in reduction of brain lesion volumes (164).
MK-886 inhibits early I/R-induced increase in intestinal P-selectin
expression, where the selectins have been implicated in the recruitment of
leukocytes into tissues exposed to I/R
(165)
. MK-886 significantly reduces
acute colonic mucosal inflammation in animals with colitis when the
treatment is performed during the early phase of the inflammatory response
(166)
. Experimental studies have been shown that MK-886 attenuates ALI
following hemorrhagic shock (139,140).
1.4.1.2. Pharmacokinetics of MK-886
Depre et al. (1993) show that, in double-blind, placebo-controlled,
randomized single-dose study in 12 healthy male subjects, the mean C max
value was 3.9 μg/ml, mean tmax value was 2hr and mean AUC (0-24) value
was 20.5 μg/ml.hr for the 500 mg oral dose of MK-886 (146).
1.4.1.3. Side Effect of MK-886
Depre et al. (1993) show that, in two studies (single and multiple dose
study) in human, no clinically significant changes in the results of laboratory
tests, physical examinations, pulmonary function tests, or vital signs were
observed. In both of these studies, the primary adverse experience was mild
gastrointestinal effects (upset stomach and loose stools) at higher doses (750
mg single dose and 250 mg three times a day) (146).
1.4.2. Montelukast
1.4.2.1. Montelukast Pharmacology
Montelukast is an orally active, highly selective leukotriene receptor
antagonist (LTRA) that inhibits the CysLT1 receptor. Montelukast inhibits
physiologic actions of LTD4 at the CysLT1 receptor without any agonist
activity. It is rapidly absorbed after administration reaching peak plasma
concentration (Cmax) in 3 to 4 hr with a mean bioavailability of 64%
following a 10 mg oral administration. More than 99% is bound to plasma
proteins with minimal distribution across the blood-brain barrier.
Metabolism occurs via liver P450 (CYP) 3A4 and 2CP microsomes, with
potent inhibition of P450 2C8 (in vitro). Excretion occurs almost exclusively
in bile with a half-life from 2.7 to 5.5 hr in healthy adults. The
pharmacokinetic profile is similar in females and males, young and elderly.
It has been found to be effective in the treatment of allergic rhinitis and
asthma (167).
1.4.2.2. Protective Potential of Montelukast
Cysteinyl leukotriene receptor 1 antagonists exert part of their antiinflammatory effects through the suppression of TH2 cells and thus through
the subsequent inhibition of the production of allergen-specific IgE, mass
cell degranulation, leukocyte trafficking, eosinophilia, T cell activation,
release of histamines, leukotrienes, and pro-inflammatory cytokines such as
interleukins 4, 5 and 13
(168,169,170)
. High doses of montelukast modulate the
production of IL-6, TNF-α and MCP-1 through the inhibition of NF-κB
activation (171).
Montelukast was found to be effective in prevention of liver and intestine
injury by reducing apoptosis and oxidative stress in a hepatic I/R model and
anti-inflammatory properties and inhibition of lipid peroxidation by
montelukast could be protective for these organs in I/R injury
(161)
. Lehr et
al. (1991) revealed that blocking of CysLTs inhibits postischemic
macromolecular leakage (160). Şener et al. (2005) found that montelukast has
an anti-inflammatory effect on sepsis-induced hepatic and intestinal damage
and protects against oxidative injury by a neutrophil-dependent mechanism,
and it has also ameliorated burn and sepsis-induced multiple-organ damage
by the same mechanism
(172,173)
. Montelukast also has protective effects on
I/R-induced tissue damage in the kidneys by inhibiting neutrophil
infiltration, balancing oxidant-antioxidant status, and regulating the
generation of inflammatory mediators
(174)
. The inhibitory action of CysLTs
receptor blockers on the generation of pro-inflammatory cytokines was
shown in intestinal I/R injury model, where the treatment with CysLTs
receptor antagonist significantly abolished the increase in vascular
permeability in the intestine and lung tissues and markedly suppressed IL-6
levels (175). Montelukast has also been used as an effective agent to decrease
fibrosis and oxidative stress in lungs in some animal studies
(176,177)
.
Leukotriene receptor antagonist treatment, particularly with montelukast,
may reduce the fibrotic phase of ALI due to sepsis (178).
Montelukast may have beneficial effects against traumatic brain injury
(TBI)-induced oxidative stress of the brain
(179)
. Montelukast treatment
reduced BBB permeability and decreased lipid peroxidation and MPO
activity (marker of tissue PMN accumulation)
(179)
. On the other hand,
montelukast was shown to improve neurological deficits and reduced infarct
volume in cerebral ischemic rats and mice
substantial
reduction
of
inflammatory
(180,181)
. Montelukast produced a
events
including
neutrophil
infiltration, TNF-α and apoptosis that associated with experimental spinal
cord injury (182).
Montelukast has gastroprotective effect on indomethacin-induced
ulcerations and alendronate-induced lesions of the rat gastric mucosa
attributed to its ameliorating effect on oxidative damage and MPO activity
(183,184)
.
1.4.2.3. Adverse Effects
Adverse effects have been described as mild and most often include
headache,
gastrointestinal
disturbances,
fatigue,
pharyngitis,
upper
respiratory tract infection and rash. Isolated reports of Churg-Strauss
syndrome (CSS), a rare systemic vasculitis associated with asthma, have
been described in asthma patients treated with montelukast. Although
LTRAs are well tolerated, particular attention should be given to the
consideration of periodically monitoring liver function tests during treatment
with LTRA. There have been reported cases of liver dysfunction going from
mild to severe in patients treated with these medications. Most recently, the
FDA has published reports of agitation, aggression, anxiousness, dream
abnormalities, hallucinations, depression, insomnia, irritability, restlessness,
suicide, suicidal ideation, and tremor associated with the use of montelukast
and other LTRAs (167).
1.4.2.4. Drug Interaction
The recommended clinical dose of montelukast did not have clinically
important effects on the pharmacokinetics of the following drugs:
theophylline, prednisolone, oral contraceptives, digoxin and warfarin.
Phenobarbital, which induces hepatic metabolism, decreased the plasma
concentration of montelukast approximately 40% following a single 10 mg
dose of montelukast, yet no dosage adjustment for montelukast is
recommended in conjunction with phenobarbital usage (167).
1.5. Aim of the Study
The main objective of the present study is to assess the possible protective
effect of montelukast (a selective antagonist of cysteinyl leukotriene receptor
1) and MK-886 (a leukotriene synthesis inhibitor) against hemorrhagic
shock-induced ALI in male rats.
2.1. Materials
The chemicals, drugs and instruments used in the present study with their
supplier are listed in the following tables
2.1 Table list of chemicals pharmaceuticals, reagent and their supplier
Pharmaceuticals and reagents
Supplier
Pure montelukast powder
Cayman chemical, USA
Pure MK-886 powder
Cayman chemical, USA
Ketamine
HIKMA. Syria
Xylazine (RompunTM) 2% vial
(Rompun 2%; Bayer AG,
Leverkusen, Germany)
TNF-α enzyme
ELISA) kit
immunoassay
(TNF-α
IMMUNOTECH. France
IL-6 enzyme immunoassay (IL-6 ELISA) IMMUNOTECH. France
kit
LTB4 BioAssayTM ELISA kit
USBiological. USA
LTC4 BioAssayTM ELISA kit
USBiological. USA
PBS
Sanofi Diagnostic Pasteur.
France
QuantichromTM
(DIGT-250)
Glutathione
assay
Ethylene
Diamineteteraacetic
Disodium (Na2 -EDTA)
Ethanol
Kit BioAssay Systems. USA
acid BDH,U.K
Fluka, Switzerland
Trichloroacetic acid (TCA)
Merck, Germany
Absolute Methanol
Fluka, Switzerland
Thiobarbaturic acid (TBA)
Merck Co. Ltd
Phosphate buffer
Sigma
Ringer’s lactate (RL)
KSA
Table 2.2 Instruments and their supplier
Equipment
Company
Sensitive electrical balance
Sartorius /Germany
High intensity ultrasonic liquid
Sonics & materials, Inc.USA
processor (sonics vibra csll)
pH meter
Inolab / Germany
spectrophotometer
ShimadzuUV-1650(UVvisible)/Japan
spectrophotometer
Apple UK
Centrifuge
Hettich/Germany
Water bath
Memmert /Germany
Bio-Elisa Reader
Bio-Tek
Instruments.
Inc.,
USA
Deep freeze (-86°C)
GFL, Germany
2.2. Animals and Study Design
A total of 24 adult male Albino rats weighing 150-220 g were purchased
from Animal Resource Center, the Institute of embryo research and
treatment of infertility, Al-Nahrain University. They were housed in the
animal house of Kufa College of Medicine in a temperature-controlled
(25°C) room (humidity was kept at 60–65%) with alternating 12-h light/12-h
dark cycles and were allowed free access to water and chow diet until the
start of experiments. After the 1st week of acclimatization the rats were
randomized into four groups (6 rats in each group) as follow:
I.
Sham group: rats underwent the same anesthetic and surgical procedures
for an identical period of time as shock animals, but neither hemorrhage
nor fluid resuscitation was performed.
II.
Control group: (induced untreated group): rats underwent hemorrhagic
shock (for 1hr) then resuscitated with Ringer’s lactate (RL) (for 1hr), and
left until the end of the experiment.
III.
Montelukast treated group: rats received montelukast (7 mg/kg) i.p.
injection 30 min before the induction of HS, and the same dose was
repeated just before reperfusion period.
IV.
MK-886 treated group: rats received MK-886 (0.6 mg/kg) i.p. injection
30 min before the induction of HS, and the same dose was repeated just
before reperfusion period.
Both sham and induced untreated rats received the same volume of the
vehicle.
2.3. Drugs
2.3.1. MK-886
MK-886 was given in dose of 0.6 mg/kg
(161)
dissolved in 2% ethanol via
i.p route 30 min before the induction of HS, and the same dose was repeated
just before the reperfusion period. The drug prepared immediately before
use.
2.3.2. Montelukast
Montelukast was given in dose of 7 mg/kg
(161)
dissolved in 2% ethanol
via i.p route 30 min before the induction of HS, and the same dose was
repeated just before the reperfusion period. The drug prepared immediately
before use.
 Ethanol was used to form a homogenized drug. Each dose was
homogenized in 1 cc ethanol and injected via i.p (161).
2.4. Hemorrhagic Shock Protocol
Animals were intraperitoneally anesthetized with (80 mg/kg) ketamine
and (8 mg/kg) xylazine (185) and subjected to a 50% blood loss (30 ml/kg) via
intracardiac puncture from the left side of the chest over 2 min and left in
shock state for 1hr. The animals were then resuscitated with two times blood
loss (60 ml/kg) using i.v lactated Ringers via tail over 1 hr (186). The sham
group underwent all instrumentation procedures, but neither hemorrhage nor
resuscitation was carried out. Animals were allowed to breathe
spontaneously throughout the experiment. Two hour after the completion of
resuscitation, rats were again anesthetized and sacrificed by exsanguinations,
where the chest cavity was opened and blood samples were taken directly
from the heart. The trachea was then isolated and bronchoalveolar lavage
fluid (BALF) was carried out. The lungs were harvested, excised and the left
lung was homogenized and stored until use for the study and the right lung
was fixed in 10% formalin for histological examination.
2.5. Preparation of Sample
2.5.1. Blood Sampling
About 3 ml of blood was collected from the heart of each rat. The blood
sampling was done at the end of the experiment (2hr after the completion of
resuscitation). The blood samples were allowed to clot at 37oC and then
centrifuged at 3000 rpm for 15 min; Sera were removed, and analyzed for
determination of serum TNF-α and IL-6.
2.5.2. Preparation of Bronchoalveolar Lavage Fluid
The trachea was then isolated, and bronchoalveolar lavage fluid was
obtained by washing the airways four times with 5 ml of phosphate buffered
saline. The bronchoalveolar lavage fluid was centrifuged at 1200 × g for
10 min at 4°C. The supernatant was collected and stored at -70°C until
analyzed for LTB4, LTC4 and total protein (187).
2.5.3. Tissue Preparation for Oxidative Stress Measurement
The lung specimens were homogenized with a high intensity ultrasonic
liquid processor and sonicated in phosphate buffered saline containing
0.1mmol/L EDTA (pH7.4) (10%). The homogenate was centrifuged at 10
000 rpm for 15 min at 4oC and supernatant was used for determination of
GSH and MDA (185).
2.5.4. Tissue Sampling for Histopathology
At the end of the experiment, rats were sacrificed and the lung was
harvested. All specimens were immediately fixed in 10% buffered formalin.
After fixation they were processed in usual manner. The sections were
examined by microscope under magnification power of (×10 and ×40) then
the histological changes were determined.
The degree of lung injury was assessed using the scoring system described
by Matute-Bello et al. (2001) that graded congestion of alveolar septae,
intra-alveolar cell infiltrates, and alveolar hemorrhage
was graded on a scale of 0–3, as follows:
(188)
. Each parameter
Parameter
alveolar
septae
intra-alveolar
cell infiltrates
Alveolar
hemorrhage
0
septae thin
and delicate
<5 intraalveolar cells
per field
no
hemorrhage
1
2
3
congested
congested
alveolar septae
alveolar septae
in <1/3 of the
in 1/3–2/3 of the
field
field
5 to 10 intra10 to 20 intraalveolar cells per alveolar cells per
field
field
congested
alveolar septae
in >2/3 of the
field
>20 intraalveolar cells
per field
at least 5
erythrocytes per
alveolus in 1 to 5
alveoli
at least 5
erythrocytes in
>10 alveoli
at least 5
erythrocytes in 5
to 10 alveoli
The total lung injury score was calculated be adding the individual scores
for each category and lung injury was categorized according to the sum of
the score to normal (0), mild (1-3), moderate (4-6) and severe injury (7-9).
The histological sections were evaluated by a pathologist without prior
knowledge of the treatment given to the animals.
2.6. Measurement of Serum IL-6 (189)
 Principle of the assay
This ELISA is a one immunological step sandwich type assay. Samples
and calibrators are incubated in the microtiter plate coated with the first
monoclonal antibody anti-IL-6, in presence of the second anti-IL-6
monoclonal antibody linked to acetylcholinesterase (ACE). After incubation,
the wells are washed and the bound enzymatic activity is detected by
addition of a chromogenic substrate. The intensity of the coloration was
proportional to the IL-6 concentration in the sample or calibrator.
 Reagents provided
Plate: 12 x 8 wells (ready-to-use)
Unused strips are stored at 2-8°C in the self-lock bag provided until
expiration date of the kit.
Calibrator: one vial (lyophilized)
The vial contains bovine serum albumin. Reconstitute the calibrator with
the volume of DW stated on the vial label. After reconstitution, use
immediately the calibrator or store at <–18°C until the expiration date. The
calibrator is calibrated by reference to the WHO IL-6 (89/548) standard.
IL-6 ACE conjugate: one vial (lyophilized)
The vial contains bovine serum albumin. Reconstitute the conjugate with
the volume of DW stated on the vial label. After reconstitution, the
conjugate is stable one week at 2-8°C or at <-18°C until the expiration date
of the kit.
Diluent 1: one 25 ml vial (ready to use)
The vial contains bovine serum albumin. The diluent 1 is stable at 2-8°C
until expiration date of the kit.
Diluent 2: one vial (lyophilized)
The vial contains material of human origin. Reconstitute the diluent 2
with the volume of DW stated on the vial label. After reconstitution, the
diluent 2 is stable at <-18°C until the expiration date of the kit.
Wash solution (20x): one 50 ml vial
Concentrated solution has to be diluted before use. Dilute 50 ml in 950 ml
of DW. Stability after dilution: one month at 2-8°C or at <-18°C until the
expiration date of the kit.
Substrate: one vial (lyophilized)
Reconstitute the substrate with the volume of DW stated on the vial label.
The substrate is stable one week at 2-8°C or at <-18°C until expiration date
of the kit.
Stop solution: one 6 ml vial (ready-to-use)
Stop solution is a tacrine solution. The solution is stable at 2-8°C until the
expiration date of the kit.
 Preparation of reagents
- Dilute 50 ml of the wash solution (20 x) with 950 ml of DW.
- Reconstitute the lyophilized calibrator with the volume of DW stated on
the vial label. Wait at least one-half hour after solubilization before
dispensing. Mix gently to avoid foaming. Do not use a vortex system. This
will result in a 10 ng/ml IL-6 solution, stable if aliquoted at <-18°C.
- Reconstitute the lyophilized conjugate with the volume of DW stated on
the vial label.
- Reconstitute the diluent 2 with the volume of DW stated on the vial label.
- Reconstitute the substrate with the volume of DW stated on the vial label
10 minutes before the end of the immunological step.
- From the 1000 pg/ml calibrator solution and the diluent 2 IL-6 for serum,
prepare a fresh dilution series in plastic tubes prior to each assay as indicated
below. This dilution series cannot be stored.
Assay procedure
STEP1: Add 100 μL of calibrator or sample per well and 100 μL conjugate,
then incubate 2 hr at 18-25°C while shaking, and then wash the wells (Plate
washing: This step is essential to obtain the expected kit performance. After
washing, wells must not dry prior to the addition of the next reagent).
STEP2: Add 200 μL of substrate, and then incubate 30 min at 18-25°C in
the dark, while shaking.
STEP3: Add 50 μL of stop solution & read absorbance at 405 nm.
 Calculation of the result
The sample results are calculated by interpolation from a calibrator curve
that is performed in the same assay as that of the sample. Draw the curve,
plotting on the horizontal axis the IL-6 concentration of the calibrators and
on the vertical axis the corresponding absorbance. Locate the absorbance for
each sample on the vertical axis and read off the corresponding IL-6
concentration on the horizontal axis.
 Calibrator curve
The results of a typical standard run with absorbance reading at 405nm
shown on Y axis against the IL-6 concentration (pg/ml) shown on the X
axis.
[IL-6] pg/ml
Absorbance
1000
1.5
333
0.56
111
0.2
37
0.07
12.3
0.03
0
0.01
1.6
1.4
Absorbance
1.2
1
0.8
0.6
0.4
0.2
0
0
200
400
600
800
1000
1200
IL-6 CONC. (pg/ml)
Figure (3): Standard curve of IL-6 obtained by Bio-ELISA reader Elx800 at
wave length 405 nm.
2.7. Measurement of Serum TNF-α (190)
 Principle of the assay
This ELISA is a one immunological step sandwich type assay. Samples
and calibrators are incubated in the microtiter plate coated with the first
monoclonal antibody anti-TNF-α, in presence of the second anti-TNF-α
monoclonal antibody linked to alkaline phosphatase. After incubation, the
wells are washed and the bound enzymatic activity is detected by addition of
a chromogenic substrate. The intensity of the coloration is proportional to
the TNF-α concentration in the sample or calibrator.
 Reagents provided
Plate: 12 x 8 wells (ready-to-use)
Unused strips are stored at 2-8°C in the self-lock bag provided until
expiration date of the kit.
Calibrator: one vial (lyophilized)
The vial contains bovine serum albumin. Reconstitute the calibrator with
the volume of DW stated on the vial label. After reconstitution, use
immediately the calibrator or store at <-18°C until the expiration date of the
kit. The calibrator is calibrated by reference to the WHO TNF-α (87/650)
standard.
TNF-α conjugate: one vial (lyophilized)
The vial contains bovine serum albumin. Reconstitute the conjugate with
the volume of diluent 1 stated on the vial label. After reconstitution the
conjugate is stable one week at 2-8°C or at <-18°C until the expiration date
of the kit.
Diluent 1: one 25 ml vial (ready to use)
The vial contains bovine serum albumin. The diluent 1 is stable at 2-8°C
until expiration date of the kit.
Diluent 2: one vial (lyophilized)
The vial contains material of human origin. Reconstitute the diluent 2
with the volume of DW stated on the vial label. After reconstitution the
diluent 2 is stable until the expiration date of the kit at <-18°C.
Wash solution (20x): one 50 ml vial
Concentrated solution has to be diluted before use. Dilute 50 ml in 950 ml
of DW. Stability after dilution: one month at 2-8°C or at <-18°C until the
expiration date of the kit.
Substrate buffer: one 30 ml vial (ready to use)
Substrate buffer is a diethanolamine-HCl solution, classified irritant at this
concentration. The substrate is stable at 2-8°C until expiration date of the kit.
Substrate: two tablets
Dissolve 1 tablet in 15 ml of substrate buffer. The substrate is stable 24 hr
at 2-8°C or at <-18°C until expiration date of the kit.
Stop solution: one 6 ml vial (ready-to-use)
Stop solution is a NaOH 1N solution. The solution is stable at 2-8°C until
the expiration date of the kit.
 Preparation of reagents
- Dilute 50 ml of the wash solution (20 x) with 950 ml of DW.
- Reconstitute the lyophilized conjugate with the volume of diluent 1 stated
on the vial label.
- Reconstitute the diluent 2 with the volume of DW stated on the vial label.
- Dissolve 1 tablet of substrate in 15 ml of substrate buffer.
- Reconstitute the lyophilized calibrator with the volume of DW stated on
the vial label. Wait at least one-half hour after solubilization before
dispensing. Mix gently to avoid foaming. Do not use a vortex system. This
will result in a 10 ng/ml TNF-α solution, stored at <-18°C if not immediately
used.
- From the 10 ng/ml calibrator solution, diluent 1 for culture supernatant or
diluent 2 for plasma and serum, prepare a fresh dilution series in plastic
tubes prior to each assay as indicated below. This dilution series cannot be
stored.
 Assay procedure
STEP1: Add 100 μL of conjugate per well and 100 μL of calibrator, or
sample, then incubate 2 hr at 18-25°C while shaking, & then wash the wells.
STEP2: Add 200 μL of substrate, and then incubate 45 min at 18-25°C in
the dark, while shaking.
STEP3: Add 50 μL of stop solution, and then read absorbance at 405 nm.
 Calculation of the result
The sample results are calculated by interpolation from a calibrator curve
that is performed in the same assay as that of the sample. Draw the curve,
plotting on the horizontal axis the TNF-α concentration of the calibrator and
on the vertical axis the corresponding absorbance. Locate the absorbance for
each sample on the vertical axis and read off the corresponding TNF-α
concentration on the horizontal axis.
 Calibrator curve
The results of a typical standard run with absorbance reading at 405nm
shown on Y axis against the TNF-α concentration (pg/ml) shown on the X
axis.
[TNF-α] pg/ml
Absorbance
1000
2.1
250
0.52
62.5
0.14
15.6
0.047
0
0.02
2.5
Absorbance
2
1.5
1
0.5
0
0
200
400
600
800
1000
1200
TNF-α CONC. (pg/ml)
Figure (4): Standard curve of TNF-α obtained by Bio-ELISA reader Elx800
at wave length 405 nm.
2.8. Measurement of BALF LTB4 (191)
 Principle of the assay
This is an ELISA kit for the quantitative analysis of leukotriene B4 levels
in biological fluid. This test kit operates on the basis of competition between
the enzyme conjugate and the LTB4 in the sample for a limited number of
binding sites. First, the sample or standard solution is added to the
microplate. Next, the diluted enzyme conjugate is added and the mixture is
shaken and incubated at room temperature (RT) for 1hr. During the
incubation, competition for binding takes place. The plate is then washed,
removing all the unbound material. The bound enzyme conjugate is detected
by the addition of substrate, which generates an optimal color after 30
minutes. Quantitative test results may be obtained by measuring and
comparing the absorbance reading of the wells of the samples against the
standards with a microplate reader at 412nm. The extent of color
development is inversely proportional to the amount of LTB4 in the sample
or standard. For example, the absence of LTB4 in the sample will result in a
bright yellow color, whereas the presence of LTB4 will result in decreased or
no color development.
 Kit Components
L2046-10A: Microtiter Plate,1x96 wells
L2046-10B: LTB4 Standard, 1x1vial
L2046-10C: LTB4 Antiserum, 1x1vial
L2046-10D: LTB4 (AchE) Tracer 1x1vial
L2046-10E: Buffer (10X) 2x10ml
L2046-10F: Wash Buffer (400X) 1x5ml
L2046-10G: Tween-20 1x3ml
L2046-10H: Ellman’s Reagent 3x1vial
L2046-10J: Tracer Dye 1x1vial
L2046-10K: Antiserum Dye 1x1vial
 Reagent preparation
 Buffer preparation
1. Dilute the contents of one vial of ELISA buffer (10X) (L2046-10E) with
90ml of DW. Be certain to rinse the vial to remove any salts that might have
precipitated.
2. Dilute 5ml vial Wash Buffer Concentrate (400X) (L2046-10F) to a total
volume of 2 liters with DW and add 1ml of Tween20 (L2046-10G).
 Standard preparation
Equilibrate a pipette tip in ethanol by repeatedly filling and expelling the
tip with ethanol several times. Using the equilibrated pipette tip,
transfer100μl of the LTB4 standard (L2046-10B) into a clean test tube, then
dilute with 900μl DW (the bulk standard) will be 5ng/ml.
To prepare standards: obtain eight clean test tubes and number them #1
through #8. Aliquot 900μl ELISA buffer to tube #1 and 500μl ELISA buffer
to tubes #2-8. Transfer 100μl of the bulk standard (0.5ng/ml) to tube #1 and
mix thoroughly. Serially dilute the standard by removing 500μl from tube #1
and placing in tube #2; mix thoroughly. Next remove 500μl from tube #2
and place it in tube #3; mix thoroughly. Repeat this process for tube #4-8.
These diluted standards should not be stored for more than 24 hr.
 LTB4 (AchE) Tracer preparation
Reconstitute the LTB4 (AchE) Tracer (L2046-10D) with 6ml ELISA buffer.
 LTB4 Antiserum preparation
Reconstitute the LTB4 EIA Antiserum (L2046-10C) with 6ml ELISA buffer.
 Procedure
1. Add 100μl ELISA buffer to Non-Specific Binding (NSB) wells. Add 50μl
EIA buffer to Maximum Binding (Bo) wells. If culture medium was used to
dilute the standard curve, substitute 50μl of culture medium for ELISA
buffer in the NSB and Bo wells (i.e., add 50μl culture medium to NSB and
Bo wells).
2. Add 50μl from LTB4 standard tube #8 to both of the lowest standard wells
(S8). Add 50μl from tube #7 to each of the next two standard wells (S7).
Continue with this procedure until all the standards are aliquoted. The same
pipette tip should be to aliquot all the standards.
3. Add 50μl of the sample per well. Each sample should be assayed at a
minimum of two dilutions. Each dilution should be assayed in duplicate.
4. Add 50μl of LTB4 (AchE) Tracer to each well except the Total Activity
(TA) and the Blank (Blk) wells.
5. Add LTB4 Antiserum 50μl to each well except the Total Activity (TA),
the Non-Specific Binding (NSB), and the Blank (Blk) wells.
6. Cover each plate with plastic film and incubate overnight at 4◦C.
7. Reconstitute Ellman’s Reagent (L2046-10H) immediately before use with
20 ml of DW.
8. Empty the well and rinse 5X with Wash Buffer.
9. Add 200μl of Ellman’s Reagent to each well.
10. Add 5μl of tracer to the Total Activity wells.
11. Cover the plate with plastic film. Optimum development is obtained by
using an orbital shaker equipped with a large, flat cover to allow plate to
develop in the dark. This assay typically develops in 90-120 minutes.
12. Wipe the bottom of the plate with a clean tissue to remove fingerprints,
dirt, etc.
13. Remove the plate cover being careful to keep Ellman’s Reagent from
splashing on the cover.
14. Read the plate at a wavelength between 405-420nm.
 Calculations
1. Subtract the absorbance readings of the blank wells from the absorbance
reading of the rest of the plate.
2. Average the absorbance readings from the NSB wells.
3. Average the absorbance readings from the Bo wells.
4. Subtract the NSB average from the Bo average. This is the corrected Bo
or corrected maximum binding.
5. Calculate the B/Bo (sample or Standard Bound/Maximum Bound) for the
remaining wells. To do this, subtract the average NSB absorbance from the
S1 absorbance and divide by the corrected Bo (from step 4). Repeat for S2S8 and all sample wells. (To obtain %B/Bo for a logistic four-parameter fit,
multiply these values by 100).
6. Plot %B/Bo for standards S1-S8 versus LTB4 concentration using linear
(y) and log (x) axes and perform a 4-parameter logistic fit.
7. Calculate the B/Bo (or %B/Bo) value for each sample. Determine the
concentration of each sample by identifying the %B/Bo on the standard
curve and reading the corresponding values on the x-axis.
[LTB4] pg/ml
%B/Bo
50
7.7
25
13.1
12.5
27.7
6.25
44.1
3.1
59.1
1.55
74.7
0.75
87.5
0.37
100
120
100
%B/Bo
80
60
40
20
0
0
10
20
30
40
50
60
LTB4 CONC. (pg/ml)
Figure (5): Standard curve of LTB4 obtained by Bio-ELISA reader Elx800
at wave length 412 nm.
2.9. Measurement of BALF LTC4 (192)
 Principle of the assay
This test kit operated on the basis of competition between the enzyme
conjugate and the LTC4 in the sample for a limited number of binding sites.
First, the sample or standard solution is added to the microplate. Next, the
diluted enzyme conjugate is added and the mixture is shaken and incubated
at RT for 1hr. During the incubation, competition for binding sites is taking
place. The plate is then washed, removing all the unbound material. The
bound enzyme conjugate is detected by the addition of substrate, which
generates an optimal color after 30 minutes. Quantitative test results may be
obtained by measuring and comparing the absorbance reading of the wells of
the samples against the standards with a microplate reader at 650nm. The
extent of color development is inversely proportional to the amount of LTC4
in the sample or standard. For example, the absence of LTC4 in the sample
will result in a bright blue color, whereas the presence of LTC 4 will result in
decreased or no color development.
 Kit Components
L2047-10A ELISA Buffer: 1x30ml. To be used to dilute enzyme conjugate
and LTC4 standards
L2047-10B Wash Buffer 10X: 1x20ml. To be diluted 1X with deionized
water. This is used to wash all unbound enzyme conjugate, samples and
standards from the plate after the 1hr incubation.
L2047-10C Substrate: 1x20ml. Stabilized 3, 3’, 5,5' Tetramethylbenzidine
(TMB) plus hydrogen peroxide (H2O2) in a single bottle. It is used to
develop the color in the wells after they had been washed.
L2047-10D Extraction Buffer 5X: 1x30ml. To be diluted 1X with
deionized water. This is used for diluting extracted and non-extracted
samples.
L2047-10E leukotriene C4 (HRP): 2x75ul. Lyophilized LTC4 HRP
conjugate. Reconstitute with 75ul of deionized water and then add to 5.5ml
of L2047-10A.
L2047-10F leukotriene Standard C4: 1x100ul leukotriene C4 standard at
the concentration of 1ng/ml.
L2047-10G leukotriene C4 Microtiter Plate: 1x96 well MaxiSorp™ Nunc
microplate with anti-LTC4 rabbit antibody precoated on each well. The plate
is ready to use as is. Do not wash.
 Test procedure
1. Prepare standard as follows:
Standard preparation
A stock solution 1ng/ml (this is provided)
B take 20μl of A, add to 980μl of ELISA Buffer and mix=20pg/ml
C take 200μl of B, add to 1.8ml of ELISA Buffer and mix=2pg/ml
D take 200μl of C, add to 1.8ml of ELISA Buffer and mix=0.2pg/ml
Continue standard preparation following Scheme 1.
Scheme 1
Standards
pg/ml
ELISA buffer (μl added)
B/C/D standard μl
S0
0
1000
S1
0.02
800
D, 200
S2
0.04
500
D, 500
S3
0.10
---
D, 1000
---
S4
0.20
800
C, 200
S5
0.40
500
C, 500
S6
S7
1.00
2.00
--800
C, 1000
B, 200
2. Determine the number of wells to be used.
3. Dilute the leukotriene C4 enzyme conjugate. Add 1μl of enzyme conjugate
into 50μl total volume of ELISA buffer for each well assayed. Mix the
solution thoroughly (avoid foaming).
4. Add 50μl of standards (S) or unknown (U) to the appropriate wells in
duplicate.
5. Add 50μl of the diluted enzyme conjugate to each well. Incubate at RT for
1hr.
6. Wash the plate with 300μl of the wash buffer. Repeat for a total of three
washings.
7. Add 150μl of TMB substrate to each well. Incubate for 30 min at RT.
8. Read plate in a microplate reader at 650nm.
9. Alternately, add 50μl of 1N HCl or stop solution to each well to stop
enzyme reaction. Read plate at 450nm if 1M HCl solution was used.
 Calculations
1. After the substrate background has been subtracted from all absorbance
values, average all of your duplicate well absorbance values.
2. The average of your two S0 values is now your Bo value. (S1 now
become B1, etc.)
3. Next, find the percent of maximal binding (%B/Bo value). To do this,
divide the averages of each standard absorbance value (now known as B1
through B7) by the Bo absorbance value and multiply 100 to achieve
percentages
4. Graph your standard curve by plotting the %B/Bo for each standard
concentration on the ordinate (y) axis against concentration on the abscissa
(x) axis. Draw a curve by using a curve fitting routine.
5. If the samples were diluted, the concentration determined from the
standard curve must be multiplied by the dilution factor.
[LTC4] pg/ml
%B/Bo
0
100
0.02
97
0.04
94
0.1
86
0.2
72
0.4
53
1
32
2
26
120
Absorbance
100
80
60
40
20
0
0
0.5
1
1.5
2
2.5
LTC4 CONC. (pg/ml)
Figure (6): Standard curve of LTC4 obtained by Bio-ELISA reader Elx800
at wave length 650 nm.
2.10. Measurement of Lung Reduced Glutathione (GSH) (193)
 Principle of the assay
Glutathione is a tripeptide of glycine, glutamic acid and cysteine. In the
red blood cell, the reduced form of glutathione is vital in maintaining
hemoglobin in a reduced state and hence protecting the cells from oxidative
damage. Glutathione is involved in detoxification of hydrogen peroxide
through glutathione oxidase.
BioAssay Systems' QuantiChromTM Glutathione Assay Kit is designed to
accurately measure reduced glutathione in biological samples. The improved
5,5'-dithiobis
(2-nitrobenzoic
acid)
(DTNB)
method
combines
deproteination and detection (Reagent A) into one reagent. DTNB reacted
with reduced glutathione to form a yellow product. The optical density,
measured at 412 nm, is directly proportional to glutathione concentration in
the sample.
 Reagents and materials provided
* Reagent A: 30 ml
* Reagent B: 30 ml
* Calibrator: 10 ml (equivalent to100 μM glutathione)
 Assay procedure
* Important: equilibrate Reagents to RT. Shake Reagent A before use.
Mix 400 μL sample with 400 μL Reagent A, centrifuge sample tubes if
precipitation occurs. Transfer 600 μL supernatant and mix with 400 μL
Reagent B. Incubate 25 min at RT. Measure OD412nm against water. Transfer
400 μL Calibrator and 800 μL Water into a clean cuvet and measure OD 412nm
against water.
 Calculation
The glutathione concentration of Sample is calculated as
= (ODSAMPLE – ODBLANK) / (ODCALIBRATOR – ODBLANK) X 100 X n (μM)
* ODSAMPLE, ODSTD and ODBLANK are optical density values of the sample,
Calibrator and sample “Blank” (water or buffer in which the sample was
dissolved). n is the dilution factor
2.11. Measurement of Lung Malondialdehyde (MDA) (194)
 Principle of the assay
Malondialdehyde (MDA), the end product of lipid peroxidation, was
analyzed according to the method of Buege and Aust in 1978
(194)
which
based on the reaction of MDA with thiobarbituric acid (TBA) to form
MDA/TBA complex, a red chromophore, which can be quantitated
spectrophotometrically according to this method.
 Preparation of TBA reagent:
1- Measure 75 ml of DW.
2- Add 0.375 gm of TBA to DW.
3- Add 15 gm of trichloroacetic acid (TCA) to DW.
4- Add 2.1 ml of 11.9 N Hydrochloric acid (HCL) to DW.
5- Complete the solution up to 100ml of DW.
 Procedure:
1- Add (1 ml) of lung homogenate to (2 ml) of TBA reagent and mixing.
2- Heat the mixture in water bath at (100 o C) for (15 min).
3- Cooled and then centrifuged at 3000 rpm for (10 min).
4- Light absorbance of clear supernatant was determined at 535 nm against
blank using spectrophotometer.
 Calculation
The concentration of MDA =
𝜀: Extinction coefficient =1.56
𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑎𝑡 535 𝑛𝑚
𝜀
×𝐷
105 M-1 Cm-1
D: Dilution factor
The results were expressed as nmol MDA/g tissue.
2.12. Measurement of BALF Total Protein (A Measure of Lung
Leak/Injury) (195)
Cell-free bronchoalveolar lavage fluid was evaluated for total protein
content using Biuret method (photometric colorimetric test total proteins).
 Principle of the assay
Cupric ions react with protein in alkaline solution to form a purple
complex. The absorbance of this complex is proportional to the protein
concentration in the sample.
 Contents of the test
-Reagent (RGT) 4x100 or 1x1000 ml color reagent (Sodium hydroxide 200
mmol/l, Potassium sodium tartrate 32 mmol/l, Copper sulphate 12 mmol/l,
Potassium iodide 30 mmol/l, Irritant R 36/38).
-Standard (STD) 1x3 ml standard (protein 80 g/l, sodium azide 0.095%)
 Reagent preparation and stability
Reagent and standard are ready for use. They are stable even after opening
up to the given expiry date when stored at 2-25 ◦C.
 Assay
Wavelength: 546 nm, 520-580 nm, optical path: 1 cm, temperature: 20-25◦C
Measurement: Against reagent blank. Only one reagent blank per series is required.
Procedure
Pipette into cuvettes
Sample / STD
RGT
Reagent blank
Sample / STD
--1000 μl
20 μl
1000 μl
Mix, incubate for 10 min at 20-25◦C. Measure the absorbance of the sample
and STD against reagent blank within 30 min (ΔA).
Calculation of the protein concentration
1. With factor:
C = 190 x ΔA [g/l]
2. With standard:
C = 80 x (ΔAsample / ΔASTD) [g/l]
2.13. Histopathological Procedure (196)
The lung was fixed in 10% formalin for 24hr, then washed and dehydrated
in ascending series of ethanol solution (70, 80, 90, 95%) for 2hr in each
concentration and 4hr divided on two changes for 100% (v/v) , cleared in
two changes of xylen, 15 min for each, then embedded in paraffin wax.
Thin section about (6mm) was dewaxed in xylen for 6 minutes, hydrated in
descending series of ethanol (2 min for each 2 change in 100% ethanol, then
2 min in each of
95, 90, 80 and 70% (v/v) of ethanol solution, then
transferred to DW for 2 min). The sections were stained in haematoxylin for
2-5 min, washed in tap water for 2-3 min, discolored in 0.5-1% HCL in 70%
alcohol for few seconds, washed in tap water for at least 5 min and stained in
1% aqueous eosin for 1-2 min., then the section washed in tap water and
dehydrated in ascending series of ethanol (70, 80, 90, and 100% v/v ) for the
same period of hydration, then cleared in xylen and mounted in DPX.
2.14. Statistical Analysis (197)
Statistical analyses were performed using SPSS 12.0 for windows.lnc.
Data were expressed as mean ± SEM unless otherwise stated. Analysis of
Variance (ANOVA) was used for the multiple comparisons among all
groups followed by post-hoc tests using LSD method. Pearson correlation
coefficient was used to assess the associations between two variables of
study parameters. Spearman correlation coefficient was used for non
parametric correlations. The histopathological grading of lung changes is a
non-normally distributed variable measured on an ordinal level of
measurement; therefore non-parametric tests were used to assess the
statistical significance involving this variable. The statistical significance of
difference in total score between more than 2 groups was assessed by
Kruskal-Wallis test, while Mann-Whitney U test was used for the difference
between 2 groups. In all tests, P < 0.05 was considered to be statistically
significant.
3.1. Effect on Serum TNF-α Level
At the end of the experiment; the level of serum TNF-α was significantly
(P < 0.05) increased in induced untreated group (II) as compared with sham
group (І).
The serum TNF-α level of both Montelukast treated group (III) and MK886 treated group (IV) was significantly (P < 0.05) lower than that of
induced untreated group (II). The serum TNF-α level of Montelukast treated
group (III) was insignificantly (P > 0.05) lower than that of MK-886 treated
group (IV).
For both Montelukast treated group (III) & MK-886 treated group (IV), at
the end of the experiment, there was a significant increase (P < 0.05) in the
TNF-α level as compared with sham group (І). The changes in the serum
TNF-α level are summarized in table (1) & (2) and figure (7).
Table (1): Serum TNF-α level (pg/ml) of the four experimental groups at the
end of the experiment (N = 6 in each group).
Group
1.Sham
2.Control
3.Montelukast treated group
4.MK-886 treated group
* vs. sham group, † vs. control group.
TNF-α (pg/ml)
19.4±2.12
93.3±6.48
42.66±2.42
49.4±3.81
P value
< 0.05*
< 0.05†
< 0.05†
Table (2): Multiple comparisons among different group mean values of
serum TNF-α level (pg/ml) using ANOVA TEST.
Groups
MK-886
Montelukast
Control
Sham
-30*
-23.26*
-73.9*
Control
43.9*
50.63*
Montelukast
-6.73
* P < 0.05
Serum TNF-α (pg/ml)
120
Serum TNF-α (pg/ml)
100
80
60
40
20
0
1. Sham
2. Control
3. Montelukast
treated group
4. MK-886 treated
group
Figure (7): The mean of serum TNF-α level (pg/ml) in the four
experimental groups at the end of the experiment.
3.2. Effect on Serum IL-6 Level
At the end of the experiment; the level of serum IL-6 was significantly (P
< 0.05) increased in induced untreated group (II) as compared with sham
group (І).
The serum IL-6 level of both Montelukast treated group (III) and MK-886
treated group (IV) was significantly (P < 0.05) lower than that of induced
untreated group (II). The serum IL-6 level of MK-886 treated group (IV)
was insignificantly (P > 0.05) lower than that of Montelukast treated group
(III).
For both Montelukast treated group (III) & MK-886 treated group (IV), at
the end of the experiment, there was a significant increase (P < 0.05) in the
IL-6 level as compared with sham group (І). The changes in the serum IL-6
level are summarized in table (3) & (4) and figure (8).
Table (3): Serum IL-6 level (pg/ml) of the four experimental groups at the
end of the experiment (N = 6 in each group).
Group
1.Sham
2.Control
3.Montelukast treated group
4.MK-886 treated group
* vs. sham group, † vs. control group.
IL-6 (pg/ml)
21.16±2.61
44.84±2.33
31.88±1.65
29.78±1.27
P value
< 0.05*
< 0.05†
< 0.05†
Table (4): Multiple comparisons among different group mean values of
serum IL-6 level (pg/ml) using ANOVA TEST.
Groups
MK-886
Montelukast
Control
Sham
-8.62*
-10.72*
-23.68*
Control
15.06*
12.96*
Montelukast
2.1
* P < 0.05
Serum IL-6 (pg/ml)
50
45
Serum IL-6 (pg/ml)
40
35
30
25
20
15
10
5
0
1.Sham
2.Control
3.Montelukast
treated group
4.MK-886 treated
group
Figure (8): The mean of serum IL-6 level (pg/ml) in the four experimental
groups at the end of the experiment.
3.3. Effect on Lung MDA Level
At the end of the experiment; the MDA level of the lung was significantly
(P < 0.05) increased in induced untreated group (II) as compared with sham
group (І).
The lung MDA level of both Montelukast treated group (III) and MK-886
treated group (IV) was significantly (P < 0.05) lower than that of induced
untreated group (II). The lung MDA level of MK-886 treated group (IV) was
insignificantly (P > 0.05) lower than that of Montelukast treated group (III).
For Montelukast treated group (III), at the end of the experiment, there
was a significant increase (P < 0.05) in the MDA level as compared with
sham group (І) while for MK-886 treated group (IV), there was no
significant change (P > 0.05) in comparison with sham group. The changes
in the lung MDA level are summarized in table (5) & (6) and figure (9).
Table (5): Lung MDA level (nmol/g tissue) of the four experimental groups
at the end of the experiment (N = 6 in each group).
Group
Lung MDA (nmol/g)
1.Sham
95±2.78
2.Control
157±6.15
3.Montelukast treated group
115.1±5.18
4.MK-886 treated group
107.2±3.76
* vs. sham group, † vs. control group.
P value
< 0.05*
< 0.05†
< 0.05†
Table (6): Multiple comparisons among different group mean values of lung
MDA level (nmol/g) using ANOVA TEST.
Groups
MK-886
Montelukast
Control
Sham
-12.2
-20.1*
-62*
Control
49.8*
41.9*
Montelukast
7.9
* P < 0.05
Lung MDA (nmol/g)
180
160
Lung MDA (nmol/g)
140
120
100
80
60
40
20
0
1. Sham
2. Control
3. Montelukast
treated group
4. MK-886 treated
group
Figure (9): The mean of lung MDA level (nmol/g) in the four experimental
groups at the end of the experiment.
3.4. Effect on Lung GSH Level
At the end of the experiment; the GSH level of the lung was significantly
(P < 0.05) declined in induced untreated group (II) as compared with sham
group (І).
The lung GSH level of both Montelukast treated group (III) and MK-886
treated group (IV) was significantly (P < 0.05) higher than that of induced
untreated group (II). The lung GSH level of MK-886 treated group (IV) was
insignificantly (P > 0.05) higher than that of Montelukast treated group (III).
For both Montelukast treated group (III) & MK-886 treated group (IV), at
the end of the experiment, there was no significant change (P > 0.05) in the
GSH level as compared with sham group (І). The changes in the lung GSH
level are summarized in table (7) & (8) and figure (10).
Table (7): Lung GSH level (μmol/g tissue) of the four experimental groups
at the end of the experiment (N = 6 in each group).
Group
1.Sham
2.Control
3.Montelukast treated group
4.MK-886 treated group
Lung GSH (μmol/g)
4.36±0.27
2.12±0.25
3.54±0.4
3.7±0.35
* vs. sham group, † vs. control group.
P value
< 0.05*
< 0.05†
< 0.05†
Table (8): Multiple comparisons among different group mean values of lung
GSH level (μmol/g) using ANOVA TEST.
Groups
Mk-886
Montelukast
Control
Sham
0.66
0.82
2.24*
Control
-1.58*
-1.42*
Montelukast
-0.16
* P < 0.05
Lung GSH (μmol/g)
5
4.5
Lung GSH (μmol/g)
4
3.5
3
2.5
2
1.5
1
0.5
0
1.Sham
2.Control
3.Montelukast
treated group
4.MK-886 treated
group
Figure (10): The mean of lung GSH level (μmol/g) in the four experimental
groups at the end of the experiment.
3.5. Effect on BALF LTB4 Level
At the end of the experiment; the LTB4 level of the BALF was
significantly (P < 0.05) increased in induced untreated group (II) as
compared with sham group (І).
The BALF LTB4 level of Montelukast treated group (III) and MK-886
treated group (IV) was significantly (P < 0.05) lower than that of induced
untreated group (II). The BALF LTB4 level of MK-886 treated group (IV)
was significantly (P < 0.05) lower than that of Montelukast treated group
(III).
For Montelukast treated group (III), at the end of the experiment, there
was a significant increase (P < 0.05) in the BALF LTB4 level as compared
with sham group (І) while for MK-886 treated group (IV), there was no
significant (P > 0.05) change in comparison with sham group. The changes
in the BALF LTB4 level are summarized in table (9) & (10) and figure (11).
Table (9): BALF LTB4 level (pg/ml) of the four experimental groups at the
end of the experiment (N = 6 in each group).
Group
1.Sham
2.Control
3.Montelukast treated group
4.MK-886 treated group
BALF LTB4 (pg/ml)
0.42±0.02
1.84±0.03
1.42±0.14
0.37±0.04
* vs. sham group, † vs. control group.
P value
< 0.05
< 0.05
< 0.05
Table (10): Multiple comparisons among different group mean values of
BALF LTB4 level (pg/ml) using ANOVA TEST.
Groups
MK-886
Montelukast
Control
Sham
0.05
-1*
-1.42*
Control
1.47*
0.42*
Montelukast
1.05*
* P < 0.05
BALF LTB4 (pg/ml)
2
1.8
BALF LTB4 (pg/ml)
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
1. Sham
2. Control
3. Montelukast
treated group
4. MK-886 treated
group
Figure (11): The mean of BALF LTB4 level (pg/ml) in the four
experimental groups at the end of the experiment.
3.6. Effect on BALF LTC4 Level
At the end of the experiment; the LTC4 level of the BALF was
significantly (P < 0.05) increased in induced untreated group (II) as
compared with sham group (І).
The BALF LTC4 level of Montelukast treated group (III) and MK-886
treated group (IV) was significantly (P < 0.05) lower than that of induced
untreated group (II). The BALF LTC4 level of MK-886 treated group (IV)
was significantly (P < 0.05) lower than that of Montelukast treated group
(III).
For Montelukast treated group (III), at the end of the experiment, there
was a significant change (P < 0.05) in the BALF LTC4 level as compared
with sham group (І) while for MK-886 treated group (IV), there was no
significant (P > 0.05) change in comparison with sham group. The changes
in the BALF LTC4 level are summarized in table (11) & (12) and figure
(12).
Table (11): BALF LTC4 level (pg/ml) of the four experimental groups at the
end of the experiment (N = 6 in each group).
Group
1.Sham
2.Control
3.Montelukast treated group
4.MK-886 treated group
BALF LTC4 (pg/ml)
0.33±0.05
8.64±0.31
7.03±0.3
0.28±0.05
* vs. sham group, † vs. control group.
P value
0.05*
0.05†
0.05†
Table (12): Multiple comparisons among different group mean values of
BALF LTC4 level (pg/ml) using ANOVA TEST.
Groups
MK-886
Montelukast
Control
Sham
0.04
-6.7*
-8.31*
Control
8.35*
1.61*
Montelukast
6.74*
* P < 0.05
BALF LTC4 (pg/ml)
10
9
BALF LTC4 (pg/ml)
8
7
6
5
4
3
2
1
0
1.Sham
2.Control
3.Montelukast
treated group
4.MK-886 treated
group
Figure (12): The mean of BALF LTC4 level (pg/ml) in the four
experimental groups at the end of the experiment.
3.7. Effect on BALF Total Protein
At the end of the experiment; the total protein level of the BALF was
significantly (P < 0.05) increased in induced untreated group (II) as
compared with sham group (І).
The BALF total protein level of both Montelukast treated group (III) and
MK-886 treated group (IV) was significantly (P < 0.05) lower than that of
induced untreated group (II). The BALF total protein level of MK-886
treated group (IV) was significantly (P < 0.05) lower than that of
Montelukast treated group (III).
For Montelukast treated group (III), at the end of the experiment, there
was a significant increase (P < 0.05) in the BALF total protein level as
compared with sham group (І) while for MK-886 treated group (IV), there
was no significant change (P > 0.05) in comparison with sham group. The
changes in the BALF total protein level are summarized in table (13) & (14)
and figure (13).
Table (13): BALF total protein level (g/l) of the four experimental groups,
at the end of the experiment (N = 6 in each group).
Group
BALF total protein (g/l)
1.Sham
7.2±0.5
2.Control
14.7±0.57
3.Montelukast treated group
11.2±0.61
4.Mk-886 treated group
8±0.3
* vs. sham group, † vs. control group.
P value
< 0.05*
< 0.05†
< 0.05†
Table (14): Multiple comparisons among different group mean values of
BALF total protein level (g/l) using ANOVA TEST.
Groups
MK-886
Montelukast
Control
Sham
-0.8
-4*
-7.5*
Control
6.7*
3.5*
Montelukast
3.2*
* P < 0.05
BALF total protein (g/l)
18
16
BALF total protein (g/l)
14
12
10
8
6
4
2
0
1.Sham
2.Control
3.Montelukast
treated group
4.Mk-886 treated
group
Figure (13): The mean of BALF total protein level (g/l) in the four
experimental groups at the end of the experiment.
3.8. Histopathological Findings
Acute lung injury was assessed in the rat's lung of the four experimental
groups at the end of the study and the results are as follow:
3.8.1. Sham Group
A cross section of sham rat's lung showed the normal appearance of all
three parameters (thin and delicate alveolar septae, no intra-alveolar cell
infiltrates and no alveolar hemorrhage). All rats in this group showed normal
lung appearance (100%) as shown in table (15) & (16) and figure (14).
3.8.2. Control (Induced Untreated) Group
There was statistically significant difference between induced untreated
group and sham group (P < 0.05) and the total score mean of the control
group showed moderate lung injury. 66.7% of the group had moderate lung
injury and 33.3% had severe lung injury as shown in table (15) & (16) and
figure (14).
3.8.3. Montelukast Treated Group
Treatment of rats with montelukast ameliorated the lung injury
significantly (P < 0.05) as compared with induced untreated group and the
total score mean of this group showed mild lung injury. 16.7% of the group
had normal histopathological appearance, 66.6% of the group had mild lung
injury and 16.7% of the group had moderate lung injury as shown in table
(15) & (16) and figure (14).
3.8.4. MK-886 Treated Group
Treatment of rats with MK-886 ameliorated the lung injury significantly
(P < 0.05) as compared with induced untreated group and the total score
mean of this group showed mild lung injury. 16.7% of the group had normal
histopathological appearance and 83.3% of the group had mild lung injury as
shown in table (15) & (16) and figure (14).
Table (15): The differences in histopathological grading of abnormal lung
changes among the four experimental groups.
Study groups
Histopathological
grading
Sham
Control (HS)
Montelukast
MK-886
N
%
N
%
N
%
N
%
Normal
6
100
0
0
1
16.7
1
16.7
Mild
0
0
0
0
4
66.6
5
83.3
Moderate
0
0
4
66.7
1
16.7
0
0
Severe
0
0
2
33.3
0
0
0
0
Total
6
100
6
100
6
100
6
100
100%
16.7
90%
33.3
80%
70%
60%
83.3
50%
100
Severe
66.6
Moderate
40%
Mild
66.7
30%
Normal
20%
10%
16.7
16.7
montelukast
treated group
MK-886 treated
group
0%
Sham
Control
Figure (14): Component bar chart shows the relative frequency of different
histopathological grading of abnormal lung changes among the four
experimental groups.
Table (16): Acute lung injury score.
Study group
Congestion of
alveolar
septae
Intraalveolar cell
infiltrates
Alveolar
hemorrhage
Total score
Total score
grade
Sham
0
0
0
0
Normal
Control
1.5±0.34
2.5±0.22
1.83±0.16
5.83±0.60*
Moderate
Montelukast
treated group
1±0.25
1.33±0.42
0.5±0.34
2.33±0.56†
Mild
MK-886
treated group
0.5±0.22
0.66±0.21
0.17±0.16
1.33±0.42†
Mild
* P < 0.05 vs. sham group, † P < 0.05 vs. control group.
S
A
S
A
A
Figure (15): Photomicrograph of lung section of normal rats shows the
normal architecture. The section stained with Haematoxylin and Eosin (X
10). A: alveoli, S: alveolar septae.
Congested alveolar
septae
S
A
Figure (16): Photomicrograph of lung section with mild injury. The section
stained with Haematoxylin and Eosin (X 40).
Congested alveolar
septae
Intra-alveolar cell
infiltrate
Figure (17): Photomicrograph of lung section with moderate injury. The
section stained with Haematoxylin and Eosin (X 10).
Intra-alveolar cell
infiltrate
alveolar hemorrhage
Figure (18): Photomicrograph of lung section with severe injury. The
section stained with Haematoxylin and Eosin (X 40).
3.9. Correlation Coefficient between Study Parameters
3.9.1. Correlation between Inflammatory Parameter (IL-6 &
TNF-α) and Oxidative Parameter (MDA & GSH)
The mean increase in serum of TNF-α significantly associated with
oxidative parameter (MDA and GSH). There was strong positive correlation
with lung MDA (r = 0.964, P < 0.01). While, there was statistically
significant strong negative correlation with lung GSH (r = -0.858, P < 0.01).
The mean increase in serum of IL-6 significantly associated with
oxidative parameter (MDA and GSH). There was strong positive correlation
with lung MDA (r = 0.865, P < 0.01).While, there is statically significant
negative strong correlation with lung GSH (r = -0.831, P < 0.01).
3.9.2. Correlation between Leukotrienes (LTB4 & LTC4) and
Oxidative Parameter (MDA & GSH)
The mean increase in BALF LTB4 significantly associated with oxidative
parameter (MDA and GSH). There was strong positive correlation with lung
MDA (r = 0.922, P < 0.01). While, there was statistically significant strong
negative correlation with lung GSH (r = -0.901, P < 0.01).
The mean increase in BALF LTC4 significantly associated with oxidative
parameter (MDA and GSH). There was strong positive correlation with lung
MDA (r = 0.930, P < 0.01). While, there was statistically significant strong
negative correlation with lung GSH (r = -0.865, P < 0.01).
3.9.3. Correlation between Leukotrienes (LTB4 & LTC4) and
Inflammatory Parameter (IL-6 & TNF-α)
The mean increase in BALF LTB4 significantly associated with
inflammatory parameter (IL-6 & TNF-α). There was strong positive
correlation with serum TNF-α (r = 0.940, P < 0.01) and serum IL-6 (r =
0.898, P < 0.01).
The mean increase in BALF LTC4 significantly associated with
inflammatory parameter (IL-6 & TNF-α). There was strong positive
correlation with serum TNF-α (r = 0.942, P < 0.01) and serum IL-6 (r =
0.884, P < 0.01).
3.9.4. Correlation between Leukotrienes (LTB4 & LTC4) and
ALI score
The total lung injury score showed statistically significant and strong
positive correlation with BALF LTB4 (r = 0.723, P < 0.01) and BALF LTC4
(r = 0.847, P < 0.01).
200
180
Lung MDA (nmol/g)
160
140
120
R = 0.964
P < 0.01
N = 12
100
80
60
40
20
0
0
20
40
60
80
100
120
Serum TNF-α (pg/ml)
Figure (19): Correlation of lung MDA level with serum TNF-α level in
control group and sham group.
4. Discussion
4.1 Effect of Hemorrhagic Shock on Study Parameters
4.1.1 Effect of Hemorrhagic Shock on Leukotrienes
In the present study a significant increase in BALF leukotriene (LTB 4 &
LTC4) levels (P < 0.05) were found in the shocked rats as compared with
sham group.
Although leukotrienes have been known to be associated with the I/R
injury in other tissues, including brain
myocardium
(201)
, hind limb
(202)
and liver
(198)
(203)
, intestine
(199)
, kidney
(200)
,
, there are only a few studies
describing the correlation between hemorrhagic shock-induced lung injury
and 5-lipoxygenase pathway products, where Eun et al. (2009 & 2010) in
two studies demonstrated that the 5-lipoxygenase pathway products meditate
acute lung injury following hemorrhagic shock (139,140).
Takamatsu et al. (2004) demonstrated that CysLTs production in the
liver was increased following hepatic I/R associated with the development of
hepatic edema and dysfunction
(203)
. Jordan et al. (2008) demonstrated that
LTB4 levels were significantly increased in the lungs following T/HS in the
rats compared with the sham group (52).
Studies in humans confirm elevated levels of LTB4, LTC4, LTD4 in BAL,
pulmonary edema fluid, and plasma in patients with ALI compared with “atrisk” group or those with hydrostatic edema
(131,132)
. Increased lung levels of
LTC4 and LTD4 have been detected in various animal models of acute lung
injury (133,134,135).
The increased leukotriene level in shocked rats might be due to the
associated splanchnic ischemia/reperfusion, which activates gut PLA2mediated release of AA into the lymph where it is delivered to the lungs
(45)
.
Arachidonic acid is a biologically active lipid released from the cellular
membrane by PLA2 that can engage the LTB4 receptor and initiate LTB4
production with autocrine effects
(204)
. Arachidonic acid also promotes 5-
lipoxygenase translocation to the nucleus, a key step in leukotrienes
production
(46)
. Additionally, it is known that ischemia elevates cytosolic
calcium concentration, which in turn elevates PLA2 and lipoxygenase
activity, generating leukotrienes. Furthermore, increased leukotriene level
might be due to the leukocytes accumulated in the lungs as observed in the
histological section of the shocked rat lung where activated neutrophils
following hemorrhagic shock are capable of releasing cytotoxic products
including leukotrienes, and the intrinsic 5-lipoxygenase activity is required
for neutrophil adherence and chemotaxis and neutrophil-mediated lung
injury (141).
In addition to neutrophils, alveolar and circulating macrophages aggravate
lung injury and alveolar neutrophil sequestration in hemorrhagic shock
(37)
might contribute to further release of leukotrienes.
4.1.2 Effect of Hemorrhagic Shock on Inflammatory Markers
(IL-6 & TNF-α)
In the present study a significant increase in inflammatory markers (IL-6
&TNF-α) level (P < 0.05) was found in the shocked rats as compared with
sham group.
Hemorrhagic shock is conceived as an insult frequently leading to
systemic inflammatory response syndrome including the systemic release of
proinflammatory cytokines which is central in the inflammatory response.
Experimental studies demonstrated that IL-6 mRNA and protein are
produced in the lungs, liver, and intestinal tracts of rats subjected to
resuscitated hemorrhagic shock
(98,99)
and that both the ischemic and
reperfusion phases of resuscitated HS were required for their production
(99)
.
Others have demonstrated elevated circulating levels of IL-6 in humans and
animals following trauma and HS (100,101).
Hemorrhagic shock induces pulmonary expression of TNF-α
(89)
. Angele
et al. (2000) showed that injury, such as trauma-hemorrhagic shock, can
induce proinflammatory cytokine production including IL-6 and TNF-α (205).
Furthermore, previous studies have shown that levels of IL-6 and TNF-α
significantly increased following trauma-hemorrhage and remain elevated
for several hours (206).
Moreover, Teng et al. (2004), who studied the role of cytokines in I/R
injury of the lung tissue, suggested that I/R led to the expression of TNF-α
and IL-6 mRNAs in the isolated-perfused rat lung model, and the expressed
cytokines are expected to aggravate I/R injury (207).
The results in present study are in agreement with that reported by
Fernandes et al. (2009) (208) and Vincenzi et al. (2009) (209) they found that a
significant increase in the TNF-α and IL-6 levels in shocked rats in
comparison with sham group. Activated inflammatory cells, especially
macrophages and neutrophils have been shown to play a pivotal role in the
propagation of SIRS following resuscitated shock and could be considered
the main source of inflammatory cytokines including TNF-α and IL-6.
4.1.3 Effect of Hemorrhagic Shock on Oxidative Stress
In the present study a significant increase in the lung level of the lipid
peroxidation product MDA (P < 0.05) indicating the presence of enhanced
lipid peroxidation due to I/R injury and significant decrease in the level of
GSH (P < 0.05) demonstrating the depletion of antioxidant pool were found
in the shocked rat in comparison with sham group, suggesting an increase in
the levels or activity of oxygen radicals.
Through examination of metabolic processes, GSH has been shown to be
important in host defenses against oxidative stress
(210)
. Another important
agent showing oxidative stress is MDA, a marker of free radical activity (210).
It was reported that oxidative stress significantly elevated MDA levels and
reduced GSH levels
(211)
. Oxidative stress has been implicated as an
important cause of HSR pathogenesis (2,210).
The results in present study are in agreement with that reported by
Kilicoglu et al. (2006) who found that a significant increase in lung MDA
level and significant decrease in lung GSH level were found in hemorrhagic
shock group as compared to sham group in a rat model of hemorrhagic
shock-induced acute lung injury (185).
Ischemia/Reperfusion injury refers to tissue damage when blood flow is
restored after an ischemic period and is common to pathophysiology of
many clinical conditions including trauma, and hypovolemic shock. Despite
its vital function in restoring the body’s hemodynamics, reperfusion leads to
ROS generation and results in activation of the proinflammatory signalling
pathways. Specifically, following resuscitation from hemorrhagic shock,
production of ROS contributes to the development of SIRS and later to
MODS and ARDS. The imbalance in the redox state of an organism,
therefore, represents a common pathway for many life-threatening
conditions including the proinflammatory state observed following shock
resuscitation. Furthermore, the imbalance of the redox state has been
observed in SIRS patients and the process of continued oxidative stress in
SIRS is thought to promote the development of MODS and ARDS in ICU
patients (67).
Leukocytes, parenchymal cells and circulating oxidant-generating
enzymes (xanthine oxidase) are potential source of ROS. Leukocytes,
principally neutrophils and macrophages, are generally considered to be the
most prodigious source of ROS in ALI/ARDS
(56)
. The large numbers of
activated neutrophils in the lung in hemorrhagic shock were considered as a
major source of ROS and different proinflammatory compounds including
cytokines, chemokines, complement fragments, clotting fragments, and lipid
mediators that are elevated in hemorrhagic shock, are capable of priming
and/or activating neutrophils to generate ROS.
4.1.4. Effect of Hemorrhagic Shock on Total Protein
In the present study a significant increase in the BALF total protein level
(P < 0.05) was found in the shocked rats as compared with sham group,
suggesting that hemorrhagic shock induces lung injury in rats. Increased
protein concentration in BALF is an important marker of damage to the
alveolar-capillary barrier of lung. Furthermore, the increase in BALF total
protein concentration may be due to increased lung permeability and lung
edema during acute lung injury (212).
The acute phase of acute lung injury and the acute respiratory distress
syndrome is characterized by the influx of protein-rich edema fluid into the
air spaces as a consequence of increased permeability of the alveolarcapillary barrier (17).
As previously reported, T/HS caused lung injury as reflected in increased
permeability to Evans blue dye, BALF protein levels and the BALF to
plasma protein ratio
(41,43)
. Yu et al. (2009) showed that hemorrhagic shock
significantly increases BALF total protein in the rats
(188)
and similar result
has been reported by Eun et al. (2010) in mice (140).
Furthermore, various studies support the result of the present study and
indicate that hemorrhagic shock induces lung injury reflected by an increase
in the BALF protein concentration (213,214,215).
CysLTs
mediate
increased
permeability
extravasation, plasma exudation and edema
leading
(106,124,125)
to
leukocyte
. Furthermore, LTB4
increases the expression of CD11b/CD18 β2-integrin (Mac-1) on
neutrophils, which can facilitate neutrophil adherence and migration (115) and
enhanced leukocyte adhesivity accounts for capillary obstruction after I/R
(216)
.
T/HS lymph induces an increase in endothelial permeability by triggering
the release of IL-6
(53)
. It has been demonstrated that IL-6 is an important
autocrine factor produced by endothelial cells that contributes to the increase
in endothelial permeability during hypoxia (54).
Free radicals are implicated to damage biomembranes, thereby
compromising cell integrity and function (217). Besides increasing pulmonary
arterial pressure (218), the free radical production under hypoxic environment
may cause oxidative injury of the endothelium
(219)
, resulting in increase
pulmonary capillary permeability.
4.1.5. Effect of Hemorrhagic Shock on Lung Parenchyma
There was statistically significant difference between induced untreated
group and normal control group (P < 0.05) and the total score mean of the
control group shows moderate lung injury. 66.7% of the control group had
moderate lung injury and 33.3% had severe lung injury.
Consistently Yang et al. (2009) demonstrated that moderate lung injury in
hemorrhagic shocked
rats with regard to alveolar wall oedema,
haemorrhage, vascular congestion, and PMN infiltration
(215)
. Furthermore,
Wang et al. (2009) showed that lung specimens from those hemorrhagic
shock rats resuscitated with RL developed significant histological changes
including cellular infiltration, edema, and alveolar-capillary membrane
thickening in comparison with sham group
(220)
. Moreover, Vincenzi et al.
(2009) revealed that accentuated congestion and inflammatory cell
infiltration in the lung of rats that underwent HS/R (209).
Lee et al. (2007) demonstrated that most severe pulmonary edema,
congestion, and inflammatory cell accumulation in rats that underwent HS
with delayed resuscitation (60 min)
(221)
. In the present study HS was
associated with increases in the lung MDA suggesting an increase in the
levels or activity of ROS and increases in serum TNF-α and IL-6 suggesting
an increase in the systemic inflammatory response where both oxidative
stress and systemic inflammatory response are involved lung injury
following hemorrhagic shock.
The findings of present study support the notion that oxidative stress and
5-lipoxygenase pathway are involved in hemorrhagic shock.
4.2 Effects of Drug Treatment
4.2.1 Effects of Montelukast
To the best of our knowledge, montelukast has not been studied before in
a hemorrhagic shock-induced acute lung injury model.
4.2.1.1 Effect of Montelukast on Leukotrienes
In this study we have demonstrated that treatment with montelukast
appeared to have a significant decrease in BALF leukotrienes (LTB4 &
LTC4) level (P < 0.05) in the shocked rats in comparison with the induced
untreated group.
Şener et al. (2006) demonstrated that montelukast significantly decreases
the plasma LTB4 level in the rats as compared with induced untreated group
in renal I/R injury model (174). Furthermore, Şener et al. (2007) demonstrated
that montelukast significantly decreases the plasma LTB4 level in the rats
that underwent chronic renal failure-induced multiple-organ injury in
comparison with induced untreated group (222).
Genovese et al. (2008) showed that the spinal cord level of LTB4 was
significantly attenuated in mice treated with montelukast in a model of
experimental spinal cord injury (182).
Moreover, Volovitz et al. (1999) demonstrated that montelukast reduces
the concentration of leukotrienes in the respiratory tract of children with
persistent asthma (223).
Although montelukast is a CysLT1 receptor antagonist and from its
mechanism of action has no effect on leukotriene level but the result of
present study may be largely due to its inhibitory effect on neutrophil
activation and infiltration that have been shown in various studies
(172,173,174)
in addition to the above mentioned studies where neutrophils are considered
a potential source of leukotrienes.
4.2.1.2 Effect of Montelukast on Inflammatory Markers (IL-6
& TNF-α)
In this study montelukast significantly reduced the elevation of
proinflammatory markers (IL-6 & TNF-α) level (P < 0.05) in the shocked
rats as compared with induced untreated group suggesting that montelukast
has protective effect in hemorrhagic shock-induced acute lung injury.
Maeba et al. (2005) showed that high doses of montelukast modulate the
production of IL-6 and TNF-α through the inhibition of NF-κB activation
(171)
.
Şener et al. (2006) demonstrated that montelukast significantly decreases
the plasma TNF-α and IL-6 level in the rats as compared with induced
untreated group in a renal I/R injury model
(174)
. Furthermore, Şener et al.
(2007) demonstrated that montelukast significantly decreases the plasma
TNF-α and IL-6 levels in the rats that underwent chronic renal failure-
induced multiple-organ injury in comparison with induced untreated group
(222)
.
The inhibitory action of CysLTs receptor blockers on the generation of
pro-inflammatory cytokines was shown in intestinal I/R injury model, where
the treatment with CysLTs receptor antagonist markedly suppressed IL-6
levels (175).
Montelukast was found to decrease serum TNF-α level in burn and sepsis
-induced multiple-organ injury in the rats (172,173). Furthermore, Kabasakal et
al. (2005) demonstrated that montelukast significantly decreases the serum
TNF-α level in the rats that underwent burn-induced oxidative injury of the
gut (224).
Thus, it seems likely that the amelioration of hemorrhagic shock-induced
acute lung injury by montelukast involves the suppression of a variety of
pro-inflammatory mediators produced by the leukocytes and macrophages.
4.2.1.3 Effect of Montelukast on Oxidative Stress
In this study montelukast significantly reduced the elevation of lung MDA
level (P < 0.05) and significantly elevates the lung GSH level (P < 0.05) in
the shocked rats as compared with induced untreated group suggesting that
montelukast has protective effect in hemorrhagic shock-induced oxidative
injury of the lung.
Şener et al. (2005, 2007) found that montelukast significantly reduces
lung MDA level and elevates lung GSH level in the rats that underwent burn
and chronic renal failure-induced oxidative injury of remote organs
(173,222)
.
Montelukast has also been used as an effective agent to decrease fibrosis and
oxidative stress in lungs in some animal studies (176,177).
Furthermore, montelukast has been shown to reduce I/R-induced
oxidative damage in the rat kidneys, bladder and liver, through its antiinflammatory and antioxidant properties (by balancing oxidant-antioxidant
status)
(225,226)
. Moreover, montelukast has antioxidant properties in various
animal studies (183,184,224).
The antioxidant effect of montelukast was further supported by its action
and largely based on its anti-inflammatory effect, where proinflammatory
cytokines, chemokines, and activated complement factors are responsible for
neutrophil recruitment and the subsequent neutrophil-induced oxidant stress
during the reperfusion phase
(227)
. Wang et al. (2008) demonstrated that
LTC4 affects the GSH/GSSG ratio by activating signals to increase IL-8
production while pretreatment with a leukotriene receptor antagonist,
montelukast,
significantly
suppressed
LTC4-induced
time-dependent
changes in the intracellular redox state, and also suppressed upregulation of
IL-8 production by suppressing NF-κB activation
(228)
. On the other hand,
CysLTs have been implicated as inflammatory mediators in various studies
(174,203)
based on their potent chemotactic and chemokinetic properties
(including recruitment of neutrophils), and because of their ability to
increase vascular permeability which are common features of I/R injury.
Thus, in the ischemic lung tissue, CysLT receptor antagonist montelukast
may have acted as an antioxidant by blocking the recruitment of neutrophils
and macrophages.
4.2.1.4. Effect of Montelukast on BALF Total Protein
In this study treatment with montelukast appeared to have a significant
decrease in BALF total protein level (P < 0.05) in the shocked rats in
comparison with the induced untreated group.
Lehr et al. (1991) revealed that blocking of CysLTs inhibits postischemic
macromolecular leakage
(160)
, where CysLTs mediate increased permeability
leading to leukocyte extravasation, plasma exudation and edema (106,124,125).
Furthermore, montelukast treatment reduced BBB permeability in traumatic
brain injury (TBI)-induced oxidative stress of the brain (179).
Souza et al. (2002) demonstrated that treatment with CysLTs receptor
antagonist significantly abolished the increase in vascular permeability in the
intestine and lung tissues in intestinal I/R injury model (175).
Previous evidence has reported that another CysLTs receptor antagonist,
pranlukast, blocked LTD4-induced plasma extravasation in small size
Guinea-pig bronchi
, thus suggesting that CysLT receptors contribute to
(229)
increase microvascular leakage in peripheral airways. The ability of CysLT 1
receptor antagonism to reduce plasma protein extravasation in the trachea,
bronchi and intra-pulmonary airways of antigen-sensitized rats has been
previously reported (230).
Montelukast treatment led to a reduction in hemorrhagic shock-induced
increase of protein content in alveoli, suggesting that montelukast
ameliorates the alveolar-capillary barrier damage induced by hemorrhagic
shock. In the present study, the antioxidant and anti-inflammatory properties
of montelukast may have contributed to its ability to prevent HS-induced
increases in lung permeability and pulmonary leukosequestration.
4.2.1.5. Effect of Montelukast on Lung Parenchyma
Treatment of rats with montelukast ameliorates the lung injury
significantly (P < 0.05) as compared with induced untreated group and the
total score mean of the control group shows mild lung injury.
Although there is no data available about the protective effect of
montelukast on the lung parenchyma in HS rats, but Şener et al. (2007)
showed that treatment with montelukast ameliorated the degenerated lung
epithelium and significantly decreased the number of inflammatory cells in
the lung of rats that underwent chronic renal failure-induced multiple-organ
injury
(222)
. Furthermore, montelukast protects against burn induced lung
injury reflected by amelioration of massive alveolar structural disturbance
and disappearance of interstitial hemorrhage (173).
In the context of I/R models, various studies showed that montelukast can
retard histopathological changes in different organs including liver, kidney
and testes (161,174,231).
In the present study, the amelioration effect of montelukast can be
attributed to its ability to balance oxidant-antioxidant status and to reduce
the generation of pro-inflammatory mediators as well as its inhibitory effect
on neutrophil activation and infiltration that have been reported in various
studies (172,173,174).
4.2.2 Effects of MK-886:
4.2.2.1 Effect of MK-886 on Leukotrienes
In this study treatment with MK-886 appeared to have a significant
decrease in BALF leukotrienes (LTB4 & LTC4) level (P < 0.05) in the
shocked rats in comparison with the induced untreated group.
Lehr et al. (1991) revealed that selective inhibition of leukotriene
biosynthesis (by MK-886) prevents postischemic leukotrienes accumulation
and the microcirculatory changes after I/R in the striated muscle in vivo (160).
Furthermore,
pharmacological
reduction
of
cysteinyl
leukotriene
formation after experimental traumatic brain injury, using MK-886, resulted
in reduction of brain lesion volumes
(164)
. Moreover, MK-886 was found to
be a potent and specific inhibitor of both LTB4 and LTC4 synthesis in human
phagocytes (143,151).
In the present study, we have found that the BALF leukotrienes level of
MK-886 treated group significantly lower than montelukast treated group,
furthermore, no significant change was found between MK-886 treated
group and sham group. These results could be explained by the mechanism
of action of MK-886, which is a leukotriene biosynthesis inhibitor, inhibits
5-LO enzyme via 5-lipoxygenase-activating protein (FLAP) inhibition result
in inhibition of synthesis of LTB4 and CysLTs, while montelukast is a
selective reversible CysLT1 receptor antagonist.
4.2.2.2. Effect of MK-886 on Inflammatory Markers (IL-6 &
TNF-α)
In this study, MK-886 significantly reduced the elevation of
proinflammatory markers (IL-6 & TNF-α) level (P < 0.05) in the shocked
rats as compared with induced untreated group suggesting that MK-886 has
protective effect in hemorrhagic shock-induced acute lung injury.
Inhibition of endogenous CysLT production by MK-886 significantly
attenuated the generation of TNF-α by mast cells activated by Fc RI crosslinkage (156).
MK-886 pretreatment attenuated subsequent pulmonary expression of
TNF- α in a mouse model of bronchial inflammation and hyperreactivity
(158)
. LTB4 augments IL-6 production in human monocytes by increasing
both IL-6 gene transcription and mRNA stabilization
(121,122)
; activation of
NF-κB and NF-IL-6 transcriptional factors may be important in this
enhancement of IL-6 release
(122)
. Furthermore, TNF-α production is
enhanced by LTC4 and LTD4 (232). So that, inhibition of LTB4 and CysLTs
synthesis by MK-886 might result in lowering TNF-α and IL-6 levels.
4.2.2.3. Effect of MK-886 on Oxidative Stress
In this study MK-886 significantly reduced the elevation of lung MDA
level (P < 0.05) and significantly elevates the lung GSH level (P < 0.05) in
the shocked rats as compared with induced untreated group suggesting that
MK-886 has protective effect in hemorrhagic shock-induced oxidative injury
of the lung.
There is no data available about the effect of MK-886 on oxidative lung
injury in HS. Daglar et al. (2009) found that MK-886 significantly reduces
hepatic and intestinal MDA level and elevates GSH level in these organs in
rats that underwent hepatic I/R model and anti-inflammatory properties and
inhibition of lipid peroxidation by MK-886 could be protective for these
organs in I/R injury (161). The antioxidant effect of MK-886 might be largely
due to its inhibitory action on leukotrienes synthesis.
4.2.2.4. Effect of MK-886 on Total Protein
In the present study, treatment with montelukast appeared to have a
significant decrease in BALF total protein level (P < 0.05) in the shocked
rats in comparison with the induced untreated group.
MK-886 has been shown to reduce the extravasation of plasma
prevent the leukocyte adhesion to the endothelium
(160)
(159)
and
in experimental
animals.
Consistently, Eun et al. (2010) demonstrated that treatment of mice with
MK-886 significantly abolished the increase in the BALF total protein level
in acute lung injury following hemorrhagic shock (140).
4.2.2.5. Effect of MK-886 on Lung Parenchyma
Treatment of rats with MK-886 ameliorates the lung injury significantly
(P < 0.05) as compared with induced untreated group and the total score
mean of this group shows mild lung injury. In addition, there is a significant
strong positive correlation between leukotrienes and lung injury score [with
BALF LTB4 (r =0.723, P < 0.01) and with BALF LTC4 (r =0.847, P <
0.01)].
There is no data available on the effect of MK-886 on the lung
parenchyma in HS rats.
Daglar et al. (2009) found that MK-886 significantly reduces the
histological changes in the liver and small intestine of rats that underwent
hepatic I/R model
(161)
. MK-886 was able to reduce the cortical infarct size
by 30% in a model of focal cerebral ischemia in rats
(163)
. Furthermore,
Farias et al. (2009) found that treatment of rats with MK-886 reduces brain
lesion volume in experimental traumatic brain injury model (164).
In the present study we have found that MK-886 treatment exerts antiinflammatory effect by reducing (TNF-α & IL-6) and antioxidant effect by
reducing lipid peroxide (MDA) and enhancing GSH level. So these findings
may provide mechanistic answers how MK-886 reduces lung injury via
number of pathways, including the suppression of systemic inflammatory
response and oxidative stress.
In the present study, the protective effect of MK-886 is better than
montelukast in hemorrhagic shock-induced lung injury considering the
BALF total protein level. This could be explained by the mechanism of MK886, which inhibits the secretion of both LTB4 and CysLTs. Thus, the
antioxidant and anti-inflammatory effects of montelukast and MK-886 might
be applicable to clinical situations to ameliorate hemorrhagic shock-induced
acute lung injury.
5.1. Conclusions
The findings of present study suggest the followings:
1- Both montelukast and MK-886 reduce BALF levels of LTB4, LTC4 and
total protein in hemorrhagic shocked rats.
2- Both montelukast and MK-886 ameliorate lung injury in hemorrhagic
shocked rats.
3- Both montelukast and MK-886 reduce systemic inflammatory response
associated with hemorrhagic shock as indicated by lowering TNF-α and
IL-6 which may provide a mechanistic answer for their protective effect.
4- Both montelukast and MK-886 reduce lipid peroxidation and elevate
GSH level in hemorrhagic shocked rats which may further explain their
protective effect for lungs.
5- The findings of present study further support the hypothesis that
inflammatory, lipoxygenase and oxidative pathways are involved in
hemorrhagic shock-induced acute lung injury and drugs that interfere
with these pathways are protective for lung in hemorrhagic shock.
5.2. Recommendations
In view of present study we recommend the following:
1- This study warrants for clinical study to test the role of montelukast and
MK-886 for the prevention of acute lung injury development in patient at
risk for example surgery and lung transplantation.
2- Measuring of LTB4 and LTC4 in the lung to further support their role in
the pathogenesis of hemorrhagic shock-induced acute lung injury.
3- Measuring of lung myeloperoxidase (MPO) activity to estimate tissue
PMN accumulation in inflamed lungs.
(1) Bhatia M, Moochhala S. Role of inflammatory mediators in the
pathophysiology of acute respiratory distress syndrome. J Pathol 2004;
202: 145-56.
(2) Jarrar D, Chaudry IH, Wang P. Organ dysfunction following
hemorrhage and sepsis: mechanisms and therapeutic approaches. Int J
Mol Med 1999; 4: 575-583.
(3) Hudson LD, Milberg JA, Anardi D, Maunder RJ. Clinical risks for
development of the acute respiratory distress syndrome. Am J Respir
Crit Care Med 1995; 151: 293-301.
(4) Keel M, Trentz O. Pathophysiology of polytrauma. Injury 2005; 36:
691-709.
(5) Li C, Jackson RM. Reactive species mechanisms of cellular hypoxiareoxygenation injury. Am J Physiol Cell Physiol 2002; 282: C227C241.
(6) Claridge JA, Schulman AM, Young JS. Improved resuscitation
minimizes respiratory dysfunction and blunts interleukin-6 and nuclear
factor-kappa B activation after traumatic hemorrhage. Crit Care Med
2002; 30: 1815-1819.
(7) Anaya-Prado R, Toledo-Pereyra LH, Lentsch AB, Ward PA.
Ischemia/reperfusion injury. J Surg Res 2002; 105(2): 248-258.
(8) Rotstein OD. Modeling the two-hit hypothesis for evaluating strategies
to prevent organ injury after shock/resuscitation. J Trauma 2003; 54:
S203-S206.
(9) Nast-Kolb D, Aufmkolk M, Rucholtz S, Obertacke U, Waydhas C.
Multiple organ failure still a major cause of morbidity but not mortality
in blunt multiple trauma. J Trauma 2001; 51: 835-841.
(10) Moore FA, McKinley BA, Moore EE. The next generation in shock
resuscitation. Lancet 2004; 363: 1988-1996.
(11) Fernandes AB, Zin WA, Rocco PR. Corticosteroids in acute
respiratory distress syndrome. Braz J Med Biol Res 2005; 38: 147-159.
(12) Rocco PR, Zin WA. Pulmonary and extrapulmonary acute respiratory
distress syndrome: are they different? Curr Opin Crit Care 2005; 11:
10-17.
(13) Ware LB, Matthay MA. Medical progress: the acute respiratory
distress syndrome. N Engl J Med 2000; 342(18): 1334-1349.
(14) Bernard GR. N-acetylcysteine in experimental and clinical acute lung
injury. Am J Med 1991; 91: S54-59.
(15) Bernard GR. Acute respiratory distress syndrome: a historical
perspective. Am J Respir Crit Care Med 2005; 172(7): 798-806.
(16) Rubenfeld GD, Herridge MS. Epidemiology and outcomes of acute
lung injury. Chest 2007; 131(2): 554-562.
(17) Pugin J, Verghese G, Widmer M-C, Matthay MA. The alveolar
space is the site of intense inflammatory and profibrotic reactions in the
early phase of acute respiratory distress syndrome. Crit Care Med 1999;
27: 304-312.
(18) Ware LB. Pathophysiology of Acute Lung Injury and the Acute
Respiratory Distress Syndrome. Semin Respir Crit Care Med 2006; 27
(4): 337-349.
(19) Zimmerman GA, Albertine KH, Carveth HJ, Gill EA, Grissom
CK, Hoidal JR, et al. Endothelial activation in ARDS. Chest 1999;
116: 18S-24S.
(20) Pittet JF, MacKersie RC, Martin TR, Matthay MA. Biological
markers of acute lung injury: prognostic and pathogenetic significance.
Am J Respir Crit Care Med 1997; 155: 1187-1205.
(21) Wiener-Kronish JP, Albertine KH, Matthay MA. Differential
responses of the endothelial and epithelial barriers of the lung in sheep
to Escherichia coli endotoxin. J Clin Invest 1991; 88: 864-875.
(22) Ware LB, Matthay MA. Alveolar fluid clearance is impaired in the
majority of patients with acute lung injury and the acute respiratory
distress syndrome. Am J Respir Crit Care Med 2001; 163: 1376-1383.
(23) Sznajder JI. Strategies to increase alveolar epithelial fluid removal in
the injured lung. Am J Respir Crit Care Med 1999; 160: 1441-1442.
(24) Greene KE, Wright JR, Steinberg KP, Ruzinski JT, Caldwell E,
Wong WB, et al. Serial changes in surfactant-associated proteins in
lung and serum before and after onset of ARDS. Am J Respir Crit Care
Med 1999; 160: 1843-1850.
(25) Lewis JF, Jobe AH. Surfactant and the adult respiratory distress
syndrome. Am Rev Respir Dis 1993; 147: 218-233.
(26) Kurahashi K, Kajikawa O, Sawa T, Ohara M, Gropper MA, Frank
DW, et al. Pathogenesis of septic shock in Pseudomonas aeruginosa
pneumonia. J Clin Invest 1999; 104: 743-750.
(27) Bitterman PB. Pathogenesis of fibrosis in acute lung injury. Am J Med
1992; 92: 39S-43S.
(28) Rizoli SB, Kapus A, Fan J, Li YH, Marshall JC, Rotstein OD.
Immunomodulatory effects of hypertonic resuscitation on the
development of lung inflammation following hemorrhagic shock. J
Immunol 1998; 161: 6288-6296.
(29) Yu HP, Shimizu T, Hsieh YC, Suzuki T, Choudhry MA, Schwacha
MG, et al. Tissue specific expression and their role in the regulation of
neutrophil infiltration in various organs following trauma-hemorrhage.
J Leukoc Biol 2006; 79: 963- 970.
(30) Yu HP, Hsieh YC, Suzuki T, Shimizu T, Choudhry MA, Schwacha
MG, et al. Salutary effects of estrogen receptor-β agonist on lung
injury after trauma-hemorrhage. Am J Physiol Lung Cell Mol Physiol
2006; 290: L1004-L1009.
(31) Bachofen M, Weibel ER. Structural alterations of lung parenchyma in
the adult respiratory distress syndrome. Clin Chest Med 1982; 3: 35-56.
(32) Matthay MA. Conference summary: acute lung injury. Chest 1999;
116: Suppl: 119S-126S.
(33) Botha AJ, Moore FA, Moore EE, Kim FJ, Banerjee A, Peterson
VM. Postinjury neutrophil priming and activation: an early vulnerable
window. Surgery 1995; 118(2): 358-364.
(34) Moore FA, Moore EE. Evolving concepts in the pathogenesis of
postinjury multiple organ failure. Surg Clin North Am 1995; 75 (2):
257-77.
(35) Partrick DA, Moore FA, Moore EE, Barnett CC Jr., Silliman CC.
Neutrophil priming and activation in the pathogenesis of postinjury
multiple organ failure. New Horiz. 1996; 4(2): 194-210.
(36) Fan J, Kapus A, Li YH, Rizoli S, Marshall JC, Rotstein OD.
Priming for enhanced alveolar fibrin deposition after hemorrhagic
shock: role of tumor necrosis factor. Am J Respir Cell Mol Biol 2000;
22(4): 412-421.
(37) Fan J, Marshall JC, Jimenez M, Shek PN, Zagorski J, Rotstein OD.
Hemorrhagic shock primes for increased expression of cytokine-
induced neutrophil chemoattractant in the lung: role in pulmonary
inflammation following lipopolysaccharide. J Immunol 1998; 161(1):
440-447.
(38) Hassoun HT, Kone BC, Mercer DW, Moody FG, Weisbrodt NW,
Moore FA. Postinjury multiple organ failure: the role of the gut. Shock
2001; 15(1):1-10.
(39) Sori AJ, Rush BF, Lysz TW, Smith S, Machiedo GW. The gut as
source of sepsis after hemorrhagic shock. Am J Surg 1988; 155: 187192.
(40) Moore FA, Moore EE, Poggetti R, McAnena OJ, Peterson VM,
Abernathy CM, et al. Gut bacterial translocation via the portal vein: a
clinical perspective with major torso trauma. J Trauma 1991; 31: 629638.
(41) Magnotti LJ, Upperman JS, Xu DZ, Lu Deitch EA Q. Gut-derived
mesenteric lymph but not portal blood increases endothelial cell
permeability and promotes lung injury after hemorrhagic shock. Ann
Surg 1998, 228: 518-527.
(42) Moore EE, Moore FA, Franciose RJ, Kim FJ, Biffl WL, Banerjee
A. The postischemic gut serves as a priming bed for circulating
neutrophils that provoke multiple organ failure. J Trauma 1994; 37:
881-887.
(43) Deitch EA, Adams C, Lu Q, Xu DZ. A time course study of the
protective effect of mesenteric lymph duct ligation on hemorrhagic
shock-induced pulmonary injury and the toxic effects of shocked rats
on endothelial cell monolayer permeability. Surgery 2001; 129: 39-47.
(44) Gonzalez RJ, Moore EE, Biffl WL, Ciesla DJ, Silliman CC. The
lipid fraction of post hemorrhagic shock mesenteric lymph (PHSML)
inhibits neutrophil apoptosis and enhances cytotoxic potential. Shock
2000; 14: 404-408.
(45) Partrick D, Moore EE, Moore FA, Barnett CC, Silliman CC. Lipid
mediators up-regulate cd11b and prime for concordant superoxide and
elastase release in human neutrophils. J Trauma 1997; 43: 297-303.
(46) Murphy RC, Gijon MA. Biosynthesis and metabolism of leukotrienes.
Biochem J 2007; 405: 379-395.
(47) Nyman KM, Ojala P, Laine VJ, Nevalainen TJ. Distribution of
group II phospholipase A2 protein and mRNA in rat tissues. J
Histochem Cytochem 2000; 48: 1468-1477.
(48) Otamiri T, Franzen L, Lindmark D, Tagesson C. Increased
phospholipase A2 and decreased lysophospholipase activity in the small
intestinal mucosa after ischaemia and revascularisation. Gut 1987; 28:
1445-1453.
(49) Uhl W, Berger H, Hoffman G, Hanisch E, Schild A, Waydhas C, et
al. A multicenter study of phospholipase A2 in patients in intensive care
units. J Am Coll Surg 1995; 180: 323-331.
(50) Uhl W, Buchler M, Nevalainen TJ, Deller A, Beger HG. Serum
phospholipase A2 in patients with multiple injuries. J Trauma 1990; 30:
1285-1287.
(51) Koike K, Moore EE, Moore FA, Kim FJ, Carl VS, Banerjee A. Gut
phospholipase A2 mediates neutrophil priming and lung injury after
mesenteric ischemia-reperfusion. Am J Physiol Gastrointest Liver
Physiol 1995; 268: G397-G403.
(52) Jordan JR, Moore EE, SarinEL, Damle SS, Kashuk SB, Silliman
CC, et al. Arachidonic acid in postshock mesenteric lymph induces
pulmonary synthesis of leukotriene B4. J Appl Physiol 2008; 104:
1161-1166.
(53) Dayal SD, Haskó G, Lu Q, Xu DZ, Caruso JM, Sambol JT, et al.
Trauma/Hemorrhagic Shock Mesenteric Lymph Upregulates Adhesion
Molecule Expression and IL-6 Production in Human Umbilical Vein
Endothelial Cells. Shock 2002; 17(6): 491-495.
(54) Ali MH, Schlidt SA, Chandel NS, Hynes KL, Schumacker PT,
Gewertz BL. Endothelial permeability and IL-6 production during
hypoxia: role of ROS in signal transduction. Am J Physiol 1999; 277:
L1057-L1065.
(55) Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine.
New York: Oxford University Press 1989: 22-85.
(56) Chow CW, Abreu MTH, Suzuki T, Downey GP. Oxidative Stress
and Acute Lung Injury. Am. J. Respir. Cell Mol Biol 2003; 29: 427431.
(57) McCord JM. Human disease, free radicals, and the oxidant/antioxidant
balance. Clin Biochem 1993; 26(5): 351-7.
(58) McCord JM. The evolution of free radicals and oxidative stress. Am J
Med 2000; 108(8): 652-659.
(59) Nachbar F, Korting HC. The role of vitamin E in normal and
damaged skin. J Mol Med 1995; 73: 7-17.
(60) Feldhaus MJ, Weyrich AS, Zimmerman GA, Mclntyre TM.
Ceramide generation in situ alters leukocyte cytoskeletal organization
and beta 2-integrin function and causes complete degranulation. J Biol
Chem 2002; 277: 4285-4293.
(61) Schwartz MD, Moore EE, Moore FA, Shenkar R, Moine P, Haenel
JB, et al. Nuclear factor-kappa B is activated in alveolar macrophages
from patients with acute respiratory distress syndrome. Crit Care Med
1996; 24: 1285-1292.
(62) Le Tulzo Y, Shenkar R, Kaneko D, Moine P, Fantuzzi G, Dinarello
CA, et al. Hemorrhage increases cytokine expression in lung
mononuclear cells in mice: involvement of catecholamines in nuclear
factor-kappaB regulation and cytokine expression. J Clin Invest 1997;
99: 1516-1524.
(63) Moine P, Shenkar R, Kaneko D, Le Tulzo Y, Abraham E. Systemic
blood loss affects NF-kappa B regulatory mechanisms in the lungs. Am
J Physiol 1997; 273: L185-L192.
(64) Baldwin SR, Simon RH, Grum CM, Ketai LH, Boxer LA, Devall
LJ. Oxidant activity in expired breath of patients with adult respiratory
distress syndrome. Lancet 1986; 1(8471): 11-14.
(65) Bunnell E, Pacht ER. Oxidized glutathione is increased in the alveolar
fluid of patients with the adult respiratory distress syndrome. Am Rev
Respir Dis 1993; 148(5): 1174-1178.
(66) Schmidt R, Luboeinski T, Markart P, Ruppert C, Daum C,
Grimminger F, et al. Alveolar antioxidant status in patients with acute
respiratory distress syndrome. Eur Respir J 2004; 24(6): 994-999.
(67) Zhang H, Slutsky AS, Vincent JL. Oxygen free radicals in ARDS,
septic shock and organ dysfunction. Intensive Care Med 2000; 26 (4):
474-476.
(68) Granger DN, Hollwarth ME, Parks DA. Ischemia-reperfusion injury:
role of oxygen derived free radicals. Acta Physiol Scand Suppl 1986;
548: 47-63.
(69) Granger DN. Role of xanthine oxidase and granulocytes in ischemiareperfusion injury. Am J Physiol 1988; 255(6 Pt 2): H1269-H1275.
(70) Kooij A, Bosch KS, Frederiks WM, Van Noorden CJ. High levels of
xanthine oxidoreductase in rat endothelial, epithelial and connective
tissue cells. A relation between localization and function? Virchows
Arch B Cell Pathol Incl Mol Pathol 1992; 62(3): 143-50.
(71) Nakamura M, Motoyama S, Saito S, Minamiya Y, Saito R, Ogawa
J. Hydrogen peroxide derived from intestine through the mesenteric
lymph induces lung edema after surgical stress. Shock 2004; 21(2):
160-164.
(72) Meneshian A, Bulkley GB. The physiology of endothelial xanthine
oxidase: from urate catabolism to reperfusion injury to inflammatory
signal transduction. Microcirculation 2002; 9(3): 161-175.
(73) Fink MP. Reactive oxygen species as mediators of organ dysfunction
caused by sepsis, acute respiratory distress syndrome, or hemorrhagic
shock: potential benefits of resuscitation with Ringer's ethyl pyruvate
solution. Curr Opin Clin Nutr Metab Care 2002; 5(2): 167-174.
(74) Souza HP, Laurindo FR, Ziegelstein RC, Berlowitz CO, Zweier JL.
Vascular NAD(P)H oxidase is distinct from the phagocytic enzyme and
modulates vascular reactivity control. Am J Physiol Heart Circ Physiol
2001; 280(2): H658-H667.
(75) Sanders KA, Huecksteadt T, Xu P, Sturrock AB, Hoidal JR.
Regulation of oxidant production in acute lung injury. Chest 1999;
116(1 Suppl): 56S-61S.
(76) Kilbourn RG, Traber DL, Szabo C. Nitric oxide and shock. Dis Mon
1997; 43(5): 277-348.
(77) Paul-Clark MJ, Gilroy DW, Willis D, Willoughby DA, Tomlinson
A. Nitric oxide synthase inhibitors have opposite effects on acute
inflammation depending on their route of administration. J Immunol
2001; 166(2): 1169-1177.
(78) Worrall NK, Chang K, Suau GM, Allison WS, Misko TP, Sullivan
PM, et al. Inhibition of inducible nitric oxide synthase prevents
myocardial and systemic vascular barrier dysfunction during early
cardiac allograft rejection. Circ Res 1996; 78(5): 769-779.
(79) Gauthier TW, Davenpeck KL, Lefer AM. Nitric oxide attenuates
leukocyte-endothelial interaction via P-selectin in splanchnic ischemiareperfusion. Am J Physiol 1994; 267(4 Pt 1): G562-G568.
(80) Okabayashi K, Triantafillou AN, Yamashita M, Aoe M, DeMeester
SR, Cooper JD, et al. Inhaled nitric oxide improves lung allograft
function after prolonged storage. J Thorac Cardiovasc Surg 1996;
112(2): 293-299.
(81) Krishnadasan B, Naidu BV, Byrne K, Fraga C, Verrier ED,
Mulligan MS. The role of proinflammatory cytokines in lung
ischemia-reperfusion injury. J Thorac Cardiovasc Surg 2003; 125: 261272.
(82) Beck-Schimmer B, Schwendener R, Pasch T, Reyes L, Booy C,
Schimmer R C. Alveolar macrophages regulate neutrophil recruitment
in endotoxin-induced lung injury. Respir Res 2005; 6: 61.
(83) Koga S, Morris S, Ogawa S, Liao H, Bilezikian JP, Chen G, et al.
TNF modulates endothelial properties by decreasing cAMP. Am J
Physiol 1995; 268: C1104-1113.
(84) Hocking DC, Phillips PG, Ferro TJ, Johnson A. Mechanism of
pulmonary edema induced by tumor necrosis factor-α. Circ Res 1990;
67: 68-77.
(85) Strieter RM, Kunkel SL, Showell HJ, Remick DG, Phan SH, Ward
PA, et al. Endothelial cell expression of a neutrophil chemotactic factor
by TNF-alpha, LPS, and IL-1-beta. Science 1989; 243: 1467-1469.
(86) Bevilacqua MJ, Prober J, Mendrick D, Cotran R, Gimbrone M.
Identification of an inducible endothelial-leukocyte adhesion molecule.
Proc Natl Acad Sci U S A 1987; 84: 9238-9242.
(87) McHale JF, Harari OA, Marshall D, Haskard DO. TNF-alpha and IL-1
sequentially induce endothelial ICAM-1 and VCAM-1 expression in
MRL/lpr lupus-prone mice. J Immunol 1999; 163 (7): 3993-4000.
(88) Mukhopadhyay S, Hoidal JR, Mukherjee TK. Role of TNF alpha in
pulmonary pathophysiology. Respir Res 2006; 7: 125.
(89) Abraham E, Carmody A, Shenkar R, Arcaroli J. Neutrophils as
early immunologic effectors in hemorrhage- or endotoxemia-induced
acute lung injury. Am J Physiol Lung Cell Mol Physiol 2000; 279:
L1137-L1145.
(90) Abraham E, Jesmok G, Tuder R, Allbee J, Chang Y. Contribution
of tumor necrosis factor-α to pulmonary cytokine expression and lung
injury after hemorrhage and resuscitation. Crit Care Med 1995; 23:
1319-1326.
(91) Cohen J. The immunopathogenesis of sepsis. Nature 2002; 420(6917):
885-891.
(92) Song Y, Ao L, Raeburn CD, Calkins CM, Abraham E, Harken AH,
et al. A low level of TNF-α mediates hemorrhage-induced acute lung
injury via p55 TNF receptor. Am J Physiol Lung Cell Mol Physiol
2001; 281(3): L677-L684.
(93) Meng ZH, Dyer K, Billiar TR, Tweardy DJ. Essential role for IL-6 in
postresuscitation inflammation in hemorrhagic shock. Am J Physiol
Cell Physiol 2001; 280(2): C343-C351.
(94) Nijsten MW, Hack CE, Helle M, ten Duis HJ, Klasen HJ, Aarden
LA. Interleukin-6 and its relation to the humoral immune response and
clinical parameters in burned patients. Surgery 1991; 109 (6): 761-767.
(95) Barton BE. The biological effects of interleukin 6. Med Res Rev 1996;
16(1): 87-109.
(96) Kishimoto T, Taga T, Akira S. Cytokine signal transduction. Cell
1994; 76: 253-262.
(97) Hierholzer C, Kalff J, Billiar T, Tweardy D. Activation of STAT
proteins in the lung of rats following resuscitation from hemorrhagic
shock. Arch Orthop Trauma Surg 1998; 117(6-7): 372-375.
(98) Hierholzer C, Kalff JC, Omert L, Tsukada K, Loeffert JE, Watkins
SC, et al. Interleukin-6 production in hemorrhagic shock is
accompanied by neutrophil recruitment and lung injury. Am J Physiol
Lung Cell Mol Physiol 1998; 275(3): L611-L621.
(99) Hierholzer C, Kalff JC, Bednarski B, Memarzadeh F, Kim YM,
Billiar TR, et al. Rapid and simultaneous activation of Stat3 and
production of interleukin 6 in resuscitated hemorrhagic shock. Arch
Orthop Trauma Surg 1999; 119: 332-336.
(100) Hamano K, Gohra H, Noda H, Katoh T, Fujimura Y, Zempo N, et
al. Increased serum interleukin-8: correlation with poor prognosis in
patients with postoperative multiple organ failure. World J Surg 1998;
22: 1077-1081.
(101) Toda H, Murata A, Tanaka N, Ohashi I, Kato T, Hayashida H, et
al. Changes in serum granulocyte colony-stimulating factor (G-CSF)
and interleukin 6 (IL-6) after surgical intervention. Res Commun Mol
Pathol Pharmacol 1995; 87: 275-286.
(102) Klein CL, Hoke TS, Fang WF, Altmann CJ, Douglas IS, Faubel S.
Interleukin-6 mediates lung injury following ischemic acute kidney
injury or bilateral nephrectomy. Kidney Int 2008; 74(7): 901-909.
(103) Meduri GU, Headley S, Kohler G, Stentz F, Tolley E, Umberger
R, et al. Persistent elevation of inflammatory cytokines predicts a poor
outcome in ARDS. Plasma IL-1 beta and IL-6 levels are consistent and
efficient predictors of outcome over time. Chest 1995; 107: 1062-1073.
(104) Newcombe DS. Chapter 34 Leukotrienes. Principle of medical
biology 1997; 8(2): 655-686.
(105) Peters-Golden M, Henderson WR Jr. Leukotrienes. N Engl J Med
2007; 357(18):1841-1854.
(106) Funk CD. Prostaglandins and leukotrienes: advances in eicosanoid
biology. Science 2001; 294: 1871-1875.
(107) Samuelesson B, Dahlén SE, Lindgren JA, Rouzer CA, Serhan CN.
Leukotrienes and lipoxins: structures, biosynthesis, and biological
effects. Science 1987; 237: 1171-1176.
(108) Goodarzi K, Goodarzi M, Tager AM, Luster AD, von Andrian
UH. Leukotriene B4 and BLT1 control cytotoxic effector T cell
recruitment to inflamed tissues. Nat Immunol 2003; 4: 965-973.
(109) Sampson AP. The role of eosinophils and neutrophils in
inflammation. Clin Exp Allergy 2000; 30 Suppl 1: 22-27.
(110) Shin EH, Lee HY, Bae YS. Leukotriene B4 stimulates human
moncyte-derived dendritic cell chemotaxis. Biochem Biophys Res
Commun 2006; 348: 606-611.
(111) Tager AM, Bromley SK, Medoff BD, Islam SA, Bercury SD,
Friedrich EB, et al. Leukotriene B4 receptor BLT1 mediates early
effector T cell recruitment. Nat Immunol 2003; 4: 982-990.
(112) Dahlén SE, Björk J, Hedqvist P, Arfors KE, Hammarström S,
Lindgren JA, et al. Leukotrienes promote plasma leakage and
leukocyte adhesion in postcapillary venules: in vivo effects with
relevance to the acute inflammatory response. Proc Natl Acad Sci U S
A 1981; 78: 3887-3891.
(113) Tager AM, Luster AD. BLT1 and BLT2: the leukotriene B (4)
receptors. Prostaglandins Leukot Essent Fatty Acids 2003; 69: 123-134.
(114) Peters-Golden M, Canetti C, Mancuso P, Coffey MJ. Leukotrienes:
underappreciated mediators of innate immune responses. J Immunol
2005; 174: 589-594.
(115) Crooks SW, Stockley RA. Leukotriene B4. Int J Biochem Cell Biol
1998; 30(2): 173-178.
(116) Serhan CN. Preventing injury from within, using selective cPLA2
inhibitors. Nat Immunol 2000; 1(1): 13-15.
(117) Mackarel AJ, Russell KJ, Brady CS, FitzGerald MX, O'Connor
CM. Interleukin-8 and leukotriene-B(4), but not formylmethionyl
leucylphenylalanine,
stimulate
CD18-independent
migration
of
neutrophils across human pulmonary endothelial cells in vitro. Am J
Respir Cell Mol Biol 2000; 23(2): 154-161.
(118) Qiu H, Johansson AS, Sjöström M, Wan M, Schröder O,
Palmblad J, et al. Differential induction of BLT receptor expression
on human endothelial cells by lipopolysaccharide, cytokines, and
leukotriene B4. Proc Natl Acad Sci U S A 2006; 103(18): 6913-6918.
(119) Maus UA, Waelsch K, Kuziel WA, Delbeck T, Mack M, Blackwell
TS, et al. Monocytes are potent facilitators of alveolar neutrophil
emigration during lung inflammation: role of the CCL2-CCR2 axis. J
Immunol 2003; 170(6): 3273-3278.
(120) Woo CH, Yoo MH, You HJ, Cho SH, Mun YC, Seong CM, et al.
Transepithelial migration of neutrophils in response to leukotriene B4
is mediated by a reactive oxygen species-extracellular signal-regulated
kinase-linked cascade. J Immunol 2003; 170(12): 6273-6279.
(121) Rola-Pleszczynski M, Stankova J. Leukotriene B4 enhances
interleukin-6 (IL-6) production and IL-6 messenger RNA accumulation
in human monocytes in vitro: transcriptional and posttranscriptional
mechanisms. Blood 1992; 80: 1004-1011.
(122) Brach MA, de Vos S, Arnold C, Gruss HJ, Mertelsmann R,
Herrmann F. Leukotriene B4 transcriptionally activates interleukin-6
expression involving NK-κ B and NF-IL6. Eur J Immunol 1992; 22:
2705-2711.
(123) Toda A, Yokomizo T, Shimizu T. Leukotriene B4 receptors.
Prostaglandins Other Lipid Mediat 2002; 68-69: 575-585.
(124) Dahlén SE. Treatment of asthma with antileukotrienes: first line or
last resort therapy? Eur J Pharmacol 2006; 533: 40-56.
(125) Ogawa Y, Calhoun WJ. The role of leukotrienes in airway
inflammation. J Allergy Clin Immunol 2006; 118: 789-798.
(126) Barnes NC, Piper PJ, Costello JF. Comparative effects of inhaled
leukotriene C4, leukotriene D4, and histamine in normal human
subjects. Thorax 1984; 39: 500-504.
(127) Holgate ST, Peters-Golden M, Panettieri RA, Henderson WR Jr.
Roles of cysteinyl leukotrienes in airway inflammation, smooth muscle
function, and remodeling. J Allergy Clin Immunol 2003; 111: s18-34.
(128) Lee E, Robertson T, Smith J, Kilfeather S. Leukotriene receptor
antagonists and synthesis inhibitors reverse survival in eosinophils of
asthmatic individuals. Am J Respir Crit Care Med 2000; 161: 18811886.
(129) Johnson HM, Russell JK, Torres BA. Second messenger role of
arachidonic acid and its metabolites in interferon-γ production. J
Immunol 1986; 137: 3053-3056.
(130) Evans JF. Cysteinyl leukotriene receptors. Prostaglandins Other
Lipid Mediat 2002; 68-69: 587-597.
(131) Amat M, Barcons M, Mancebo J, Mateo J, Oliver A, Mayoral JF,
et al. Evolution of
leukotriene B4, peptide
leukotrienes, and
interleukin-8 plasma concentrations in patients at risk of
respiratory distress syndrome and with
acute
acute respiratory distress
syndrome: mortality prognostic study. Crit Care Med 2000; 28: 262263.
(132) Matthay
MA,
Eschenbacher
WL,
Goetzl
EJ.
Elevated
concentrations of leukotriene D4 in pulmonary edema fluid of patients
with the adult respiratory distress syndrome. J Clin Immunol 1984; 4:
479-483.
(133) Stephenson AH, Sprague RS, Dahms TE, Lonigro AJ. Increased
leukotriene C4 in ethchlorvynol-induced acute lung injury in dogs. J
Appl Physiol 1987; 62: 732-738.
(134) Mancuso P, Whelan J, DeMichele SJ, Snider CC, Guszcza JA,
Claycombe KJ, et al. Effects of eicosapentaenoic and gamma-linolenic
acid on lung permeability and alveolar macrophage eicosanoid
synthesis in endotoxic rats. Crit Care Med 1997; 25: 523-532.
(135) Fink MP, Kruithoff KL, Antonsson JB, Wang HL, Rothschild
HR. Delayed treatment with an LTD4/E4 antagonist limits pulmonary
edema in endotoxic pigs. Am J Physiol 1991; 260: R1007-R1013.
(136) Karasawa A, Guo JP, Ma XL, Tsao PS, Lefer AM. Protective
actions of a leukotriene B4 antagonist in splanchnic ischemia and
reperfusion in rats. Am J Physiol Gastrointest Liver Physiol 1991; 261:
G191-G198.
(137) Souza DG, Coutinho SF, Silveira MR, Cara DC, Teixeira MM.
Effects of a BLT receptor antagonist on local and remote reperfusion
injuries after transient ischemia of the superior mesenteric artery in rats.
Eur J Pharmacol 2000; 403: 121-128.
(138) Masclans JR, Sabater J, Sacanell J, Chacon P, Sabin P, Roca O, et
al. Possible Prognostic Value of Leukotriene B4 in Acute Respiratory
Distress Syndrome. Respir Care 2007; 52(12): 1695-1700.
(139) Eun JC, Moore EE, Jordan JR, Peltz ED, Banerjee A. Products of
the 5-lipoxygenase pathway are critical for the development of acute
lung injury following hemorrhagic shock. Journal Of the American
College of Surgeons 2009; 209(3), Suppl 1: S35.
(140) Eun JC, Moore EE, Mauchley DC, Meng X, Banerjee A. The 5Lipoxygenase Pathway Meditates Acute Lung Injury Following
Hemorrhagic Shock. Journal of Surgical Research 2010; 158(2): 215216.
(141) Guidot DM, Repine MJ, Westcott JY, Repine JE. Intrinsic 5lipoxygenase activity is required for neutrophil responsivity. Proc Natl
Acad Sci USA 1994; 91: 8156-8159.
(142) Noonan TC, Kern DF, Malik AB. Pulmonary microcirculatory
responses to leukotrienes B4, C4, and D4 in sheep. Prostaglandins 1985;
30: 419-434.
(143) Gillard J, Ford-Hutchinson AW, Chan C, Charleson S, Denis D,
Foster A, et al. L-663,536 (MK-886) (3-1-(4-chlorobenzyl)-3-t-butylthio-5-isopropylindol-2-yl) 2,2-dimethylpro-panoic acid), a novel,
orally active leukotriene biosynthesis inhibitor. Can J Physiol
Pharmacol 1989; 67(5): 456-464.
(144) Dixon RAF, Diehl RE, Opas E, Rands E, Vickers PJ, Evans JF, et
al. Requirement of a 5-lipoxygenase activating protein for leukotriene
synthesis. Nature 1990; 343: 282-284.
(145) Rouzer CA, Ford-Hutchinson AW, Morton HE, Gillard JW. MK886, a potent and specific leukotriene biosynthesis inhibitor, blocks and
reverses the membrane association of 5-lipooxygenase in ionophore
challenged leucocytes. J Biol Chem 1990; 265: 1436-1442.
(146) Depre M, Friedman B, Tanaka W, Van Hecken A, Buntinx A,
DeSchepper
PJ.
Biochemical
activity,
pharmacokinetics,
and
tolerability of MK-886, a leukotriene biosynthesis inhibitor, in humans.
Clin Pharmacol Ther 1993; 53: 602-607.
(147) Friedman BS, Bel EH, Buntinx A, Tanaka W, Han YH, Shingo S,
et al. Oral leukotriene inhibitor (MK-886) blocks allergen-induced
airway responses. Am Rev Res Dis 1993; 147: 839-844.
(148) Young RN. Development of novel leukotriene-based anti-asthma
drugs: MK-886 and MK-571. Agents Actions Suppl 1991; 34: 179-187.
(149) Rouzer CA, Ford-Hutchinson AW, Morton HE, Gillard JW.
MK886, a potent and specific leukotriene biosynthesis inhibitor blocks
and reverses the membrane association of 5-lipoxygenase in ionophorechallenged leukocytes. J Biol Chem 1990; 265: 1436-1442.
(150) Khan MA, Hoffbrand AV, Mehta A, Wright F, Tahami F,
Wickremasinghe RG. MK 886, an antagonist of leukotriene
generation, inhibits DNA synthesis in a subset of acute myeloid
leukaemia cells. Leuk Res 1993; 17: 759-762.
(151) Menard L, Pilote S, Naccache PH, Laviolette M, Borgeat P.
Inhibitory effects of MK-886 on arachidonic acid metabolism in human
phagocytes. Br J Pharmacol 1990; 100: 15-20.
(152) Kehrer JP, Biswal SS, La E, Thuillier P, Datta K, Fischer SM, et
al. Inhibition of peroxisome-proliferator-activated receptor (PPAR)α by
MK886. Biochem J 2001; 356: 899-906.
(153) Mayburd AL, Martlínez A, Sackett D, Liu H, Shih J, Tauler J, et
al. Ingenuity network-assisted transcription profiling: identification of a
new pharmacologic mechanism for MK886. Clin Cancer Res 2006; 12:
1820-1827.
(154) Huang JK, Huang CC, Lu T, Chang HT, Lin KL, Tsai JY, et al.
Effect of MK-886 on Ca2+ Level and Viability in PC3 Human Prostate
Cancer Cells. Basic & Clinical Pharmacology & Toxicology 2009;
104(6): 441-447.
(155) Lim JY, Oh JH, Jung JR, Kim SM, Ryu CH, Kim HT, et al.
MK886-induced apoptosis depends on the 5-LO expression level in
human malignant glioma cells. J Neurooncol 2010; 97(3): 339-346.
(156) Mellor EA, Austen KF, Boyce JA. Cysteinyl leukotrienes and
uridine diphosphate induce cytokine generation by human mast cells
through an interleukin 4-regulated pathway that is inhibited by
leukotriene receptor antagonists. J Exp Med 2002; 195: 583.
(157) Ackerman
WE
IV,
Robinson
JM,
Kniss
DA.
Despite
Transcriptional and Functional Coordination, Cyclooxygenase-2 and
Microsomal Prostaglandin E Synthase-1 Largely Reside in Distinct
Lipid
Microdomains
in
WISH
Epithelial
Cells.
Journal
of
Histochemistry & Cytochemistry 2005; 53(11): 1391-1401.
(158) Oliveira SH, Hogaboam CM, Berlin A, Lukacs NW. SCF-induced
airway hyperreactivity is dependent on leukotriene production. Am J
Physiol Lung Cell Mol Physiol 2001; 280: L1242-1249.
(159) Fernandez-gallardo S, Gijon MA, Garcia C, Furio V, Ciu FT,
Crespo
SM.
The
role
of
platelet
activating
factor
and
peptidoleukotrienes in the vascular changes of rat passive anaphylaxis.
Br J Pharmacol 1992; 105: 119-125.
(160) Lehr HA, Guhlmann A, Nolte D, Keppler D, Messmers K.
Leukotrienes as mediators in ischemia-reperfusion injury in a
microcirculation model in the hamster. J Clin Invest 1991; 87: 2036.
(161) Daglar G, Karaca T, Yuksek YN, Gozalan U, Akbiyik F,
Sokmensuer C, et al. Effect of Montelukast and MK-886 on Hepatic
Ischemia-Reperfusion Injury in Rats. Journal of surgical research 2009;
153(1): 31-38.
(162) Titos E, Clària J, Planagumà A, López-Parra M, Villamor N,
Párrizas M, et al. Inhibition of 5-lipoxygenase induces cell growth
arrest and apoptosis in rat Kupffer cells: Implications for liver fibrosis.
FASEB J 2003; 17: 1745.
(163) Ciceri P, Rabuffetti M, Monopoli A, Nicosia S. Production of
leukotrienes in a model of focal cerebral ischemia in the rat. Br J
Pharmacol 2001; 133: 1323.
(164) Farias S, Frey LC, Murphy RC, Heidenreich KA. Injury-Related
Production of Cysteinyl Leukotrienes Contributes to Brain Damage
following
Experimental
Traumatic
Brain
Injury.
Journal
of
Neurotrauma 2009; 26(11): 1977-1986.
(165) Eppihimer MJ, Russell J, Anderson DC, Epstein CJ, Laroux S,
Granger DN. Modulation of P-selectin expression in the postischemic
intestinal microvasculature. Am J Physiol Gastrointest Liver Physiol
1997; 273(6): G1326-G1332.
(166) Wallace JL, Keenan CM. An orally active inhibitor of leukotriene
synthesis accelerates healing in rat model of colitis. Am J Physiol 1990;
258: G527-G534.
(167) Benninger MS, Waters H. Montelukast: Pharmacology, Safety,
Tolerability and Efficacy. Clinical Medicine: Therapeutics 2009; 1:
1253-1261.
(168) Eum SY, Maghni K, Hamid Q, Campbell H, Eidelman DH,
Martin JG. Involvement of the cysteinyl-leukotrienes in allergeninduced airway eosinophilia and hyperresponsiveness in the mouse.
Am J Respir Cell Mol Biol 2003; 28: 25-32.
(169) Henderson WR Jr, Tang LO, Chu SJ, Tsao SM, Chiang GK,
Jones F, et al. A role for cysteinyl leukotrienes in airway remodeling in
a mouse asthma model. Am J Respir Crit Care Med 2002; 165: 108116.
(170) Wu AY, Chik SC, Chan AW, Li Z, Tsang KW, Li W. Antiinflammatory effects of high-dose montelukast in an animal model of
acute asthma. Clin Exp Allergy 2003; 33: 359-360.
(171) Maeba S, Ichiyama T, Ueno Y, Makata H, Matsubara T,
Furukawa S. Effect of montelukast on nuclear factor kappaB
activation and pro-inflammatory molecules. Ann Allergy Asthma
Immunol 2005; 94: 670-674.
(172) Şener G, Şehirli Ö, Çetinel Ş, Ercan F, Yüksel M, Gedik N, et al.
Amelioration of sepsis-induced hepatic and ileal injury in rats by the
leukotriene receptor blocker montelukast. Prostaglandins Leukot Essent
Fatty Acids 2005; 73: 453.
(173) Şener G, Kabasakal L, Çetinel Ş, Contuk G, Gedik N, Yeğen BÇ.
Leukotriene receptor blocker montelukast protects against burn-induced
oxidative injury of the skin and remote organs. Burns 2005; 31: 587.
(174) Şener G, Şehirli Ö, Velioğlu-Öğünç A, Çetinel Ş, Gedik N, Caner
M, et al. Montelukast protects against renal ischemia/reperfusion injury
in rats. Pharmacol Res 2006; 54: 65.
(175) Souza DG, Pinho V, Cassali GD, Poole S, Teixeria MM. Effect of a
BLT receptor antagonist in a model of severe ischemia and reperfusion
injury in the rat. Eur J Pharmacol 2002; 440: 61-69.
(176) Izumo T, Kondo M, Nagai A. Cysteinyl-leukotriene 1 receptor
antagonist attenuates bleomycin-induced pulmonary fibrosis in mice.
Life Sci 2007; 80(20): 1882-1886.
(177) Fireman E, Schwartz Y, Mann A, Greif J. Effect of montelukast, a
cysteinyl receptor antagonist, on myofibroblasts in interstitial lung
disease. J Clin Immunol 2004; 24(4): 418-425.
(178) Abul Y, Eryuksel E, Karakurt S, Celikel T, Ceyhan B.
Montelukast as a Treatment of Acute Lung Injury in Sepsis.
Hypothesis 2010; 8 (1): 1-5.
(179) Bıber N, Toklu HZ, Solakoglu S, Gultomruk M, Hakan T,
Berkman
Z, et
al.
Cysteinyl-leukotriene receptor antagonist
montelukast decreases blood–brain barrier permeability but does not
prevent oedema formation in traumatic brain injury. Brain inj 2009;
23(6): 577-584.
(180) Baba T, Black KL, Ikezaki K, Chen KN, Becker DP. Intracarotid
infusion of leukotriene C4 selectively increases blood–brain barrier
permeability after focal ischemia in rats. J Cereb Blood Flow Metab
1991; 11: 638-643.
(181) Yu GL, Wei EQ, Zhang SH, Xu HM, Chu LS, Zhang WP, et al.
Montelukast, a cysteinyl leukotriene receptor-1 antagonist, dose- and
time-dependently
protects
against
focal
ischemia
in
mice.
Pharmacology 2005; 73: 31-40.
(182) Genovese T, Rossi A, Mazzon E, Paola RD, Muià C, Caminiti R,
et al. Effects of zileuton and montelukast in mouse experimental spinal
cord injury. Br J Pharmacol 2008; 153(3): 568-582.
(183) Dengiz GO, Odabasoglu F, Halici Z, Cadirci E, Suleyman H.
Gastroprotective
and
antioxidant
effects
of
montelukast
on
indomethacin-induced gastric ulcer in rats. J Pharmacol Sci 2007; 105:
94-102.
(184) Şener G, Kapucu C, Çetinel Ş, Cikler E, Ayanoglu G.
Gastroprotective effect of leukotriene receptor blocker montelukast in
alendronat-induced lesions of the rat gastric mucosa. Prostaglandins
Leukot Essent Fatty Acids 2005; 72: 1-11.
(185) Kilicoglu B, Eroglu E, Kilicoglu SS, Kismet K, Eroglu F. Effect of
abdominal trauma on hemorrhagic shock induced acute lung injury in
rats. World J Gastroenterol 2006; 12(22): 3593-3596.
(186) Rhee P, Waxman K, Clark L, Kaupke CJ, Vaziri ND, Tominaga
G, et al. Tumor necrosis factor and monocytes are released during
hemorrhagic shock. Resuscitation 1993; 25(3): 249-255.
(187) Yu HP, Hsieh PW, Chang YJ, Chung PJ, Kuo LM, Hwang TL.
DSM-RX78, a new phosphodiesterase inhibitor, suppresses superoxide
anion production in activated human neutrophils and attenuates
hemorrhagic
shock-induced
lung
injury
in
rats.
Biochemical
pharmacology 2009; 78(8): 983-992.
(188) Matute-Bello G, Winn RK, Jonas M, Chi EY, Martin TR, Liles
WC. Fas (CD95) induces alveolar epithelial cell apoptosis in vivo:
Implications for acute pulmonary inflammation. Am J Pathol 2001;
158: 153.
(189) Literature of kit by IMMUNOTECH. France.
(190) Literature of kit by IMMUNOTECH. France.
(191) Literature of kit by USBiological. USA
(192) Literature of kit by USBiological. USA
(193) Literature of kit by BioAssay Systems. USA
(194) Beuge JA, Aust SD. Microsomal lipid peroxidation. Meth Enzymol
1978; 52: 302-311.
(195) Josephson B, Gyllenswärd C. Scand J Clin Lab Invest 1957; 9: 29.
(196) Drury R, Wallington D(Ed): Carleton's histological technique, 5th
edition, Oxford New York Toronto.
(197) Sorlie DE (ed). Medical biostatistics and epidemiology: Examination
and Board review. First ed, 1995, Norwalk, Connecticut, Appleton and
Lange: 47-88.
(198) Rao AM, Hatcher JF, Kindy MS, Dempsey RJ. Arachidonic acid
and leukotriene C4: Role in transient cerebral ischemia of gerbils.
Neurochem Res 1999; 24: 1225.
(199) Souza DG, Coutinho SF, Silveira MR, Cara DC, Teixeira MM.
Effects of a BLT receptor antagonist on local and remote reperfusion
injuries after transient ischemia of the superior mesenteric artery in rats.
Eur J Pharmacol 2000; 403: 121.
(200) Noiri E, Yokomizo T, Nakao A, Izumi T, Fujita T, Kimura S,
Shimizu T. An in vivo approach showing the chemotactic activity of
leukotriene B (4) in acute renal ischemic-reperfusion injury. Proc Natl
Acad Sci USA 2000; 97: 823.
(201) Rossoni G, Sala A, Berti F, Testa T, Buccellati C, Molta C, et al.
Myocardial protection by the leukotriene synthesis inhibitor BAY
X1005: Importance of transcellular biosynthesis of cysteinylleukotrienes. J Pharmacol Exp Ther 1996; 276: 335.
(202) Chiang N, Gronert K, Clish CB, O’Brien JA, Freeman MW,
Serhan CN. Leukotriene B4 receptor transgenic mice reveal novel
protective roles for lipoxins and aspirin-triggered lipoxins in
reperfusion. J Clin Invest 1999; 104: 309.
(203) Takamatsu Y, Shimada K, Chijiiwa K, Kuroki S, Yamaguchi K,
Tanaka M. Role of leukotrienes on hepatic ischemia/reperfusion injury
in rats. Journal of Surgical Research 2004; 119(1): 14-20.
(204) Surette ME, Krump E, Picard S, Borgeat P. Activation of
leukotriene synthesis in human neutrophils by exogenous arachidonic
acid: inhibition by adenosine A2a receptor agonists and crucial role of
autocrine activation by leukotriene B4. Mol Pharmacol 1999; 56: 10551062.
(205) Angele MK, Schwacha MG, Ayala A, Chaudry IH. Effect of
gender and sex hormones on immune responses following shock. Shock
2000; 14: 81-90.
(206) Ayala A, Wang P, Ba ZF, Perrin MM, Ertel W, Chaudry IH.
Differential alterations in plasma IL-6 and TNF levels after trauma and
hemorrhage. Am J Physiol 1991; 260: R167-R171.
(207) Teng S, Kurata S, Katoh I, Georgieva GS, Nosaka T, Mitaka C, et
al. Cytokine mRNA expression in unilateral ischemic-reperfused rat
lung with salt solution supplemented with low-endotoxin or standard
bovine serum albumin. Am J Physiol Lung Cell Mol Physiol 2004;
286(1): L137–L142.
(208) Fernandes CI, Llimona F, Godoy LC, Negri EM, Pontieri V,
Moretti AI, et al. Treatment of hemorrhagic shock with hypertonic
saline solution modulates the inflammatory response to live bacteria in
lungs. Braz J Med Biol Res 2009; 42(10): 892-901.
(209) Vincenzi R, Cepeda LA, Pirani WM, Sannomyia P, Rocha-e-Silva
M, Cruz RJ Jr. Small volume resuscitation with 3% hypertonic saline
solution decrease inflammatory response and attenuates end organ
damage after controlled hemorrhagic shock. The American Journal of
Surgery 2009; 198(3): 407-414.
(210) Szabo C. The pathophysiological role of peroxynitrite in shock,
inflammation, and ischemia-reperfusion injury. Shock 1996; 6: 79-88.
(211) Johnson KJ, Fantone JC, Kaplan J, Ward PA. In vivo damage of
rat lungs by oxygen metabolites. J Clin Invest 1981; 67: 983-993.
(212) Lum H, Roebuck KA. Oxidant stress and endothelial dysfunction.
Am J Physiol Cell Physiol 2001; 280: C719-C741.
(213) Wu1 CT, Yu HP, Chung CY, Lau YT, Liao SK. Attenuation of
Lung Inflammation and Pro-Inflammatory Cytokine Production by
Resveratrol following Trauma-Hemorrhage. Chinese Journal of
Physiology 2008; 51(6): 363-368.
(214) Osband AJ, Deitch EA, Hauser CJ, Lu Q, Zaets S, Berezina T, et
al. Albumin Protects Against Gut-Induced Lung Injury In Vitro and In
Vivo. Ann Surg 2004; 240: 331-339.
(215) Yang CH, Tsai PS, Wang TY, Huang CJ. Dexmedetomidine–
ketamine combination mitigates acute lung injury in haemorrhagic
shock rats. Resuscitation 2009; 80(10): 1204-1210.
(216) Schmid-Schonbein GW. Capillary plugging by granulocytes and the
no-reflow phenomenon in the microcirculation. Fed Proc 1987; 46:
2397-2401.
(217) Vanita G, Asheesh G, Shalini S, Harish MD, Grover SK, Ratan K.
Anti-stress and adaptogenic activity of L-arginine supplementation.
eCAM 2005; 2: 93-97.
(218) Hoshikawa Y, Sadafumi O, Satoshi S, Tatsuo T, Masayuki C,
Chun S, et al. Generation of oxidative stress contributes to the
development of pulmonary hypertension induced by hypoxia. J Appl
Physiol 2001; 90: 1299-1306.
(219) Herget J, Wilhelm J, Novotna J, Eckhardt A, Vytasek R,
Mrazkova L, et al. A possible role of the oxidant tissue injury in the
development of hypoxic pulmonary hypertension. Physiol Res 2000;
49: 493-501.
(220) Wang P, Li Y, Li J. Hydroxyethyl starch 130/0.4 prevents the early
pulmonary
inflammatory
hemorrhagic
shock
and
response
and
resuscitation
oxidative
stress
in
International
rats.
after
Immunopharmacology 2009; 9(3): 347-353.
(221) Lee CC, Chang IJ, Yen ZS, Hsu CY, Chen SY, Su CP, et al.
Delayed
Fluid
Resuscitation
in
Hemorrhagic
Shock
Induces
Proinflammatory Cytokine Response. Annals of Emergerncy Medicine
2007; 49(1): 37-44.
(222) Şener G, Sakarcan A, Şehirli Ö, Ekşioğlu-Demiralp E, Şener E,
Ercan F, et al. Chronic renal failure-induced multiple-organ injury in
rats is alleviated by the selective CysLT1 receptor antagonist
montelukast. Prostaglandins &Other Lipid Mediators 2007; 83(4): 257267.
(223) Volovitz B, Tabachnik E, Nussinovitch M, Shtaif B, Blau H, GilAd I, et al. Montelukast, a leukotriene receptor antagonist, reduces the
concentration of leukotrienes in the respiratory tract of children with
persistent asthma. Journal of Allergy and Clinical Immunology 1999;
104(6): 1162-1167.
(224) Kabasakal L, Şener G, Çetinel Ş, Contuk G, Gedik N, Yeğen BÇ.
Burn-induced oxidative injury of the gut is ameliorated by the
leukotriene receptor blocker montelukast. Prostaglandins, Leukotrienes
and Essential Fatty Acids 2005; 72(6): 431-440.
(225) Sener G, Sehirli O, Toklu H, Ercan F, Alican I. Montelukast
reduces ischaemia/reperfusion-induced bladder dysfunction and oxidant
damage in the rat. J Pharm Pharmacol 2007; 59: 837.
(226) Özkan E, Yardimci S, Dulundu E, Topaloğlu Ü, Şehirli Ö, Ercan
F, et al. Protective Potential of Montelukast Against Hepatic
Ischemia/Reperfusion Injury in Rats. Journal of Surgical Research.
2010; 159(1): 588-594.
(227) Jaeschke H, Bautista AP, Spolarics Z, Spitzer JJ. Superoxide
generation by neutrophils and Kupffer cells during in vivo reperfusion
after hepatic ischemia in rats. J Leukoc Biol 1992; 52: 377.
(228) Wang J, Mochizuki H, Todokoro M, Arakawa H, Morikawa A.
Does leukotriene affect intracellular glutathione redox state in cultured
human air way epithelial cells?. Antioxid Redox Signal 2008; 10: 821.
(229) Bochnowicz S, Underwood DC. Dose-dependent mediation of
leukotriene
D4-induced
airway
microvascular
leakage
and
bronchoconstriction in the guinea pig. Prostaglandins Leukot Essent
Fatty Acids 1995; 52: 403-411.
(230) Hele DJ, Birrell MA, Webber SE, Foster ML, Belvisi MG.
Mediator
involvement
in
antigen-induced
bronchospasm
and
microvascular leakage in the airways of ovalbumin sensitized Brown
Norway rats. Br J Pharmacol 2001; 132: 481-488.
(231) Ozturk H, Ozturk H, Gideroglu K, Terzi H, Bugdayci G.
Montelukast protects against testes ischemia/reperfusion injury in rats.
Can Urol Assoc J 2010; 4(3): 174-179.
(232) Ben-Efraim B, Bonta IL. Modulation of antitumour activity of
macrophages by regulation of eicosanoids and cytokine production. Int
J Immunopharmacol 1994; 16: 397-399.
‫تأثير المونتيلوكاست واأل م ك ‪ 886‬على ضرر‬
‫الرئة الحاد الناتج عن الصدمة النزفية وإنعاشها‬
‫ف ذكور الجرذان‬
‫رسالة‬
‫مقدمة إلى مجلس كلية الطب‪ /‬جامعة الكوفة‬
‫كجزء من متطلبات نيل درجة الماجستير في األدوية و العالجيات‬
‫من قبل‬
‫علي محسن هاشم‬
‫بكالوريوس علوم صيدلة‬
‫المشرف‬
‫د‪ .‬نجاح رايش الموسوي‬
‫أستاذ في علم األدوية والعالجيات‬
‫المشرف‬
‫د‪.‬فاضل غال يوسف العمران‬
‫أستاذ مساعد في جراحة القلب والصدر‬
‫واألوعية الدموية‬
‫‪1431‬هــ‬
‫‪ 2010‬م‬
‫الخالصة‬
‫ضرر الرئة الحاد الناتج عن الصدمة النزفية وإنعاشها بالسوائل الوريدية يعد مساهم مهم في‬
‫االمراضية ونسبة الوفيات المتأخرة لدى مرضى الصدمة‪ .‬الصدمة النزفية المتبوعة باإلنعاش يعد‬
‫كذريعة تؤدي وبصورة متكررة إلى متالزمة االستجابة االلتهابية الشاملة واإلجهاد ألتأكسدي الذي‬
‫ينتج عنه متالزمة االختالل الوظيفي لألعضاء المتعددة المتضمن ضرر الرئة الحاد‪ .‬األم كي ‪886‬‬
‫(مانع لاليبواوكسيجينيز) والمونتيلوكاست (مثبط لمستقبل السيستينيل ليكوتراين) يبديان فعالية تضاد‬
‫االلتهاب واإلجهاد ألتأكسدي‪.‬‬
‫الطريقة‬
‫أربعة وعشرين جرذا أبرصا بالغا أستخدموا في الدراسة وتم توزيعهم على أربعة مجاميع‪:‬‬
‫المجموعة األولى‪ :‬هي المجموعة المزيفة التي تمر بها جميع ظروف التجربة دون تعريضها الى‬
‫الصدمة النزفية وإنعاشها وعددها ستة جرذان‪.‬‬
‫المجموعة الثانية‪ :‬هي مجموعة السيطرة التي تم تعريضها إلى الصدمة النزفية لمدة ساعة واحدة‬
‫وإنعاشها بالرنگر الكتيت لمدة ساعة أيضا ولم يتم عالجها وعددها ستة جرذان‪.‬‬
‫المجموعة الثالثة‪ :‬وهي المجموعة التي تم إعطائها المونتيلوكاست (‪ )montelukast‬بجرعة ‪7‬‬
‫ملغ\ كغم داخل البريتون نصف ساعة قبل تعريضها للصدمة ومباشرتا قبل إنعاشها وعددها ستة‬
‫جرذان‪.‬‬
‫المجموعة الرابعة‪ :‬وهي المجموعة التي تم إعطائها األم كي ‪ )MK-886( 886‬بجرعة ‪ 0.6‬ملغ\‬
‫كغم داخل البريتون نصف ساعة قبل تعريضها للصدمة ومباشرتا قبل إنعاشها وعددها ستة جرذان‪.‬‬
‫الصدمة النزفية تحدث بواسطة تعريض الجرذان الى ‪ %50‬من فقدان الدم (‪30‬مل\كغم) عن طريق‬
‫سحب الدم مباشرتا من القلب من الجهة اليسرى من الصدر في غضون دقيقتين وتترك بحالة الصدمة‬
‫لمدة ساعة واحدة ثم يتم إنعاشها بالرنگر الكتيت وريديا عن طريق الذيل بحجم يساوي مرتين من‬
‫حجم الدم المفقود أي (‪60‬مل\كغم) خالل ساعة واحدة‪ .‬عند نهاية التجربة (ساعتين بعد إكمال‬
‫اإلنعاش بالسوائل) تم أخذ نماذج من الدم وتم قياس عاملي االلتهاب ‪ TNF-α‬و ‪ .IL-6‬بعد ذلك تم‬
‫عزل القصبة الهوائية وغسل الرئة للحصول على السائل الغسل القصبي الحويصلي (‪ )BALF‬وتم‬
‫فيه قياس ليكوتراين نوع ‪ )LTB4( B4‬و ليكوتراين نوع ‪ )LTC4( C4‬ومجموع البروتين‪ .‬بعد ذلك‬
‫تم إزالة الرئة و أخذ الرئة اليسرى ومجانستها وتم قياس عامل التأكسد المالون ثنائي االلديهايد‬
‫(‪ )MDA‬وكذلك مستوى الكلوتوثايون المختزل (‪ )GSH‬فيها وتم أخذ الرئة اليمنى و فحص‬
‫التغيرات النسيجية الحاصلة فيها‪.‬‬
‫النتائج‬
‫الصدمة النزفية سببت زيادة معنوية (‪ )p> 0.05‬في مستوى عاملي االلتهاب ‪ TNF-α‬و ‪ IL-6‬في‬
‫مصل الدم وكذلك ارتفاع بمستوى عامل التأكسد المالون ثنائي االلديهايد (‪ )MDA‬في الرئة‬
‫باإلضافة إلى ارتفاع بمستوى ليكوتراين نوع ‪ )LTB4( B4‬و ليكوتراين نوع ‪ )LTC4( C4‬ومجموع‬
‫البروتين في السائل القصبي الحويصلي وسببت الصدمة أيضا انخفاض معنوي (‪ )p> 0.05‬بمستوى‬
‫الكلوتوثايون المختزل (‪ )GSH‬في الرئة بالمقارنة مع المجموعة المزيفة‪ .‬أما بالنسبة للنتائج النسيجية‬
‫فان جميع الجرذان التي تعرضت للصدمة أظهرت أضرار معنوية في الرئة (‪.)p> 0.05‬‬
‫إن العالج بكال من المونتيلوكاست واألم كي ‪ 886‬أظهر تأثيرا معنويا (‪ )p> 0.05‬على االلتهاب من‬
‫خالل منع ارتفاع عاملي االلتهاب ‪ TNF-α‬و ‪ IL-6‬وعلى اإلجهاد التأكسدي بالرئة من خالل منع‬
‫ارتفاع عامل التأكسد المالون ثنائي االلديهايد (‪ )p> 0.05‬وعلى مستوى ليكوتراين نوع ‪B4‬‬
‫(‪ )LTB4‬و ليكوتراين نوع ‪ )LTC4( C4‬ومجموع البروتين من خالل منع ارتفاعها في السائل‬
‫القصبي الحويصلي(‪ .)p> 0.05‬كذلك ان العالج بكال من المونتيلوكاست واألم كي ‪ 886‬منع‬
‫النقصان بمستوى الكلوتوثايون المختزل بصورة معنوية (‪ .)p> 0.05‬تحليل النتائج نسيجيا أظهر ان‬
‫كال من المونتيلوكاست واألم كي ‪ 886‬سبب انخفاضا معنويا (‪ )p> 0.05‬من حدة أو شدة ضرر‬
‫الرئة الحاصل بالجرذان المتعرضة للصدمة‪.‬‬
‫االستنتاج‬
‫نتائج دراستنا أظهرت ان كل من المونتيلوكاست واألم كي ‪ 886‬قد يحسن من ضرر الرئة الحاصل‬
‫لدى الجرذان المتعرضة للصدمة النزفية من خالل تثبيط االلتهاب واإلجهاد التأكسدي مبينا دور‬
‫الليكوتراينات في نشوء التهاب الرئة الناتج عن الصدمة النزفية‪.‬‬