Open Access
Open Peer Review

This article has Open Peer Review reports available.

How does Open Peer Review work?

Leukotriene biosynthesis inhibition ameliorates acute lung injury following hemorrhagic shock in rats

Journal of Cardiothoracic Surgery20116:81

https://doi.org/10.1186/1749-8090-6-81

Received: 21 February 2011

Accepted: 7 June 2011

Published: 7 June 2011

Abstract

Background

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 is a leukotriene biosynthesis inhibitor exerts an anti inflammatory and antioxidant activity.

Objectives

The objective of present study was to assess the possible protective effect of MK-886 against hemorrhagic shock-induced acute lung injury via interfering with inflammatory and oxidative pathways.

Materials and methods

Eighteen adult Albino rats were assigned to three groups each containing six rats: group I, sham group, rats underwent all surgical instrumentation but neither hemorrhagic shock nor resuscitation was done; group II, Rats underwent hemorrhagic shock (HS) for 1 hr then resuscitated with Ringer's lactate (1 hr) (induced untreated group, HS); group III, HS + 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). 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

MK-886 treatment significantly reduced the total lung injury score compared with the HS group (P < 0.05). MK-886 also significantly decreased serum TNF-α & IL-6; lung MDA; BALF LTB4, LTC4 & total protein compared with the HS group (P < 0.05). MK-886 treatment significantly prevented the decrease in the lung GSH levels compared with the HS group (P < 0.05).

Conclusions

The results of the present study reveal that 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.

Keywords

MK-886hemorrhagic shockacute lung injuryoxidative stressinflammatory markers

1. Introduction

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. 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 [5]. Neutrophils are the major cellular elements involved in acute lung inflammation after resuscitated hemorrhagic shock [6]. Studies have shown that neutrophils are activated following HS [7] and that lung injury is associated with an increased neutrophils accumulation in the lungs after HS [8]. 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 [7].

MK-886 (investigational compound) is a highly potent inhibitor of leukotriene formation in vivo and in vitro [9]. This compound inhibits leukotriene biosynthesis indirectly by a mechanism through the binding of a membrane bound 5-lipoxygenase-activating protein (FLAP), thereby inhibiting the translocation and activation of 5-lipoxygenase [10, 11]. The 5-lipoxygenase inhibition by MK-886 prevents stimulated neutrophil adherence and chemotaxis and neutrophil mediated lung injury in vitro [12]. MK-886 has been shown to reduce the extravasation of plasma [13] and prevent the leukocyte adhesion to the endothelium [14] 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 MK-886 could be protective for these organs in I/R injury [15]. 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 [16]. Recently, treatment of mice with MK-886 significantly abolished the increase in the BALF total protein level in a model of acute lung injury following hemorrhagic shock [17].

2. Materials and methods

2.1. Animals and Study Design

A total of eighteen 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 with alternating 12-h light/12-h dark cycles and were allowed free access to water and chow diet until the start of experiments. All experiments were approved by the Animal Care and Research Committee of the University of Colorado Denver, and this investigation conforms with the Guide for the Care and Use of Laboratory Animals (National Research Council, revised 1996).

After the 1st week of acclimatization the rats were randomized into three groups as follow:
  1. I.

    Sham group: this group consisted of 6 rats; 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.

     
  2. II.

    Control group: (induced untreated group): this group consisted of six rats; rats underwent hemorrhagic shock (for 1 hr) then resuscitated with Ringer's lactate (RL) (for 1 hr), and left until the end of the experiment.

     
  3. III.

    MK-886 treated group: this group consisted of 6 rats; 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.

The drug was purchased from (Cayman chemical, USA) and prepared immediately before use as a homogenized solution in 2% ethanol [15]. Ethanol was used to form a homogenized drug. Each dose was homogenized in 1ml ethanol and injected via i.p [15].

2.2. Hemorrhagic Shock Protocol

Animals were intraperitoneally anesthetized with 80 mg/kg ketamine and 8 mg/kg xylazine [18] 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 1 hr. The animals were then resuscitated with two times blood loss (60 ml/kg) using i.v lactated Ringers via tail over 1 hr [19].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.

2.3. Preparation of Blood Samples and Cytokine Assays

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 37°C and then centrifuged at 3000 rpm for 15 min; Sera were removed, and analyzed for determination of serum TNF-a and IL-6. Serum TNF-a and IL-6 were quantified according to the manufacturer's instructions and guidelines using enzyme-linked immunosorbent assay (ELISA) kits (IMMUNOTECH. France).

2.4. Preparation of Bronchoalveolar Lavage Fluid and determination of leukotrienes and total protein

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 [20]. The BALF levels of LTB4 and LTC4 were quantified according to the manufacturer's instructions and guidelines using ELISA kits (USBiological. USA). Cell free BALF was evaluated for total protein content using Biuret method (photometric colorimetric test total proteins) [21].

2.5. 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 4°C and supernatant was used for determination of GSH and MDA [18]. The MDA levels were assayed for products of lipid peroxidation by monitoring thiobarbituric acid reactive substance formation according to the method of Buege and Aust in 1978 [22]. Lipid peroxidation was expressed in terms of MDA equivalents using an extinction coefficient of 1.56 × 105 M−1 cm −1 and results were expressed as nmol MDA/g tissue. GSH measurements were performed using a colorimetric method at 412nm (BioAssay Systems' QuantiChrom™ Glutathione Assay Kit).

2.6. 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 then the histological changes were determined.

The degree of lung injury was assessed using the scoring system described by Matute-Bello et al. that graded congestion of alveolar septae, intra-alveolar cell infiltrates, and alveolar hemorrhage [23]. Each parameter was graded on a scale of 0-3, as follows: alveolar septae, 0: septae thin and delicate, 1: congested alveolar septae in < 1/3 of the field, 2: congested alveolar septae in 1/3-2/3 of the field, 3: congested alveolar septae in > 2/3 of the field; intra-alveolar cell infiltrates, 0: < 5 intra-alveolar cells per field, 1: 5 to 10 intra-alveolar cells per field, 2: 10 to 20 intra-alveolar cells per field, 3: > 20 intra-alveolar cells per field; Alveolar hemorrhage, 0: no hemorrhage, 1: at least 5 erythrocytes per alveolus in 1 to 5 alveoli, 2: at least 5 erythrocytes in 5 to 10 alveoli, 3: at least 5 erythrocytes in > 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.

2.7. Statistical Analysis

Statistical analyses were performed using SPSS 12.0 for windows.lnc. Data were expressed as mean ± SEM. Analysis of Variance (ANOVA) was used for the multiple comparisons among all groups followed by post-hoc tests using LSD method. 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. Results

3.1. Effect on Proinflammatory Cytokines (TNF-α and IL-6)

At the end of the experiment, the serum TNF-α and IL-6 levels were significantly higher in the HS group when compared with the sham group (P < 0.05). Treatment with MK-886 significantly decreased the serum TNF-α and IL-6 levels when compared with the HS group (P < 0.05). The TNF-α and IL-6 values for the different groups are shown in table 1 and Figures 1&2.
Table 1

Serum TNF-α and IL-6 levels (pg/ml) of the three experimental groups at the end of the experiment

Group

TNF-α (pg/ml)

IL-6 (pg/ml)

1. Sham

19.4 ± 2.12

21.16 ± 2.61

2. Control (HS)

93.3 ± 6.48*

44.84 ± 2.33*

3. MK-886 treated group

49.4 ± 3.81

29.78 ± 1.27

The data expressed as mean ± SEM (N = 6 in each group).

P < 0.05 vs. sham group, P < 0.05 vs. HS (induced untreated) group

Figure 1

The mean of serum TNF-α level (pg/ml) in the three experimental groups at the end of the experiment.

Figure 2

The mean of serum IL-6 level (pg/ml) in the three experimental groups at the end of the experiment.

3.2. Effect on Lung MDA and GSH Levels

The MDA levels, measured as a major degradation product of lipid peroxidation in the pulmonary tissue, were found to be significantly higher in HS group as compared to those of the sham group (P < 0.05), while treatment with MK-886 abolished these elevations (P < 0.05). The HS caused a significant decrease in lung GSH level (P < 0.05) when compared with the sham group, while in the MK-886 treated group, the lung GSH level was found to be preserved (P < 0.05) and not significantly different from that of the sham group. The MDA and GSH values for the different groups are shown in table 2 and Figure 3, 4.
Table 2

Lung MDA and GSH levels of the three experimental groups at the end of the experiment

Group

Lung MDA (nmol/g)

Lung GSH (μmol/g)

1. Sham

95 ± 2.78

4.36 ± 0.27

2. Control (HS)

157 ± 6.15*

2.12 ± 0.25*

3. MK-886 treated group

107.2 ± 3.76

3.7 ± 0.35

The data expressed as mean ± SEM (N = 6 in each group).

P < 0.05 vs. sham group, P < 0.05 vs. HS (induced untreated) group

Figure 3

The mean of lung MDA level (nmol/g) in the three experimental groups at the end of the experiment.

Figure 4

The mean of lung GSH level (μmol/g) in the three experimental groups at the end of the experiment.

3.3. Effect on Leukotrienes (LTB4 & LTC4)

At the end of the experiment; the LTB4 and LTC4 levels in the BALF were significantly increased in the HS group as compared with the sham group (P < 0.05). Treatment with MK-886 significantly decreased the BALF LTB4 and LTC4 levels when compared with the HS group (P < 0.05). The LTB4 and LTC4 values for the different groups are shown in table 3 and Figure 5, 6.
Table 3

BALF LTB4 and LTC4 level (pg/ml) of the three experimental groups at the end of the experiment

Group

BALF LTB4 (pg/ml)

BALF LTC4 (pg/ml)

1. Sham

0.42 ± 0.02

0.33 ± 0.05

2. Control (HS)

1.84 ± 0.03*

8.64 ± 0.31*

3. MK-886 treated group

0.37 ± 0.04

0.28 ± 0.05

The data expressed as mean ± SEM (N = 6 in each group).

P < 0.05 vs. sham group, P < 0.05 vs. HS (induced untreated) group

Figure 5

The mean of BALF LTB 4 level (pg/ml) in the three experimental groups at the end of the experiment.

Figure 6

The mean of BALF LTC 4 level (pg/ml) in the three experimental groups at the end of the experiment.

3.4. Effect on BALF Total Protein

At the end of the experiment; the total protein level of the BALF was significantly increased in HS group as compared with sham group (P < 0.05). Treatment with MK-886 significantly decreased the BALF total protein levels when compared with the HS group (P < 0.05). The total protein values for the different groups are shown in table 4 and Figure 7.
Table 4

BALF total protein level (g/l) of the three experimental groups, at the end of the experiment

Group

BALF total protein (g/l)

1. Sham

7.2 ± 0.5

2. Control (HS)

14.7 ± 0.57*

3. MK-886 treated group

8 ± 0.3

The data expressed as mean ± SEM (N = 6 in each group).

P < 0.05 vs. sham group, P < 0.05 vs. HS (induced untreated) group

Figure 7

The mean of BALF total protein level (g/l) in the three experimental groups at the end of the experiment.

3.5. Histological finding

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) Figure 8. All rats in this group showed normal lung appearance (100%) as shown in table 5.
Figure 8

Photomicrograph of lung section of normal rats shows the normal architecture. The section stained with Haematoxylin and Eosin (X 10).

Table 5

The differences in histopathological grading of abnormal lung changes among the three experimental groups

Histopathological grading

Study group

 

Sham

Control (HS)

MK-886

 

N

%

N

%

N

%

Normal

6

100

0

0

1

16.7

Mild

0

0

0

0

5

83.3

Moderate

0

0

4

66.7

0

0

Severe

0

0

2

33.3

0

0

Total

6

100

6

100

6

100

There was statistically significant difference between induced untreated (HS) group and sham group (P < 0.05) and the total score mean of the HS 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 5, 6 and Figures 9, 10.
Table 6

Acute lung injury score

Study group

Congestion of alveolar septae

Intra-alveolar cell infiltrates

Alveolar hemorrhage

Total score

Total score grade

Sham

0

0

0

0

Normal

HS

1.5 ± 0.34

2.5 ± 0.22

1.83 ± 0.16

5.83 ± 0.60*

Moderate

MK-886 treated group

0.5 ± 0.22

0.66 ± 0.21

0.17 ± 0.16

1.33 ± 0.42

Mild

The data expressed as means ± SEM.

* P < 0.05 vs. sham group, P < 0.05 vs. HS (induced untreated) group

Figure 9

Photomicrograph of lung section with moderate injury. The section stained with Haematoxylin and Eosin (X 10).

Figure 10

Photomicrograph of lung section with severe injury. The section stained with Haematoxylin and Eosin (X 40).

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 (Figure 11). 16.7% of the group had normal histopathological appearance and 83.3% of the group had mild lung injury as shown in table 5.
Figure 11

Photomicrograph of lung section with mild injury. The section stained with Haematoxylin and Eosin (X 40).

Discussion

The present study demonstrates that HS causes ALI, as evidenced by biochemical and histologic changes. MK-886 prevented the biochemical changes and protected the lung morphology after HS. Although leukotrieneshave been known to be associated with the I/R injury in other tissues, including intestine [24]kidney [25], myocardium [26] and liver [27], there are only a few studies describing the correlation between hemorrhagic shock-induced lung injury and 5-lipoxygenase pathway products, where two studies demonstrated that the 5-lipoxygenase pathway products meditate acute lung injury following hemorrhagic shock [28, 29]. And it has been demonstrated that LTB4 levels were significantly increased in the rat lungs following T/HS [30]. Studies in humans confirm elevated levels of LTB4, LTC4, LTD4 in BAL, pulmonary edema fluid, and plasma in patients with ALI compared with "at-risk" group or those with hydrostatic edema [31, 32]. In the present study a significant increase in BALF leukotriene (LTB4 & LTC4) levels were found in the shocked rats as compared with sham group. The increased leukotriene level in shocked rats might be due to the associated splanchnic I/R, which activates gut PLA2-mediated release of AA into the lymph where it is delivered to the lungs [33]. 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 [34]. Arachidonic acid also promotes 5-lipoxygenase translocation to the nucleus, a key step in leukotrienes production [35]. 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 [36]. In addition to neutrophils, alveolar macrophages and circulating macrophages aggravate lung injury and alveolar neutrophil sequestration in hemorrhagic shock [37] and might contribute to further release of leukotrienes. In this study we have demonstrated that treatment with MK-886 appeared to have a significant decrease in BALF leukotrienes (LTB4 & LTC4) level in the shocked rats in comparison with the induced untreated group. It is reported 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 [14]. Furthermore, MK-886 was found to be a potent and specific inhibitor of both LTB4 and LTC4 synthesis in human phagocytes [9, 38].

Hemorrhagic shock is considered as an insult frequently leading to systemic inflammatory response syndrome including the systemic release of proinflammatory cytokines which is central in the inflammatory response. Previous studies have shown that levels of IL-6 and TNF-α significantly increased following trauma-hemorrhage and remain elevated for several hours [39]. The results in present study are consistent with that reported by Vincenzi et al. [40] Who 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. In this study MK-886 significantly reduced the elevation of IL-6 and TNF-α level 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 FcεRI cross-linkage [41]. MK-886 pretreatment attenuated subsequent pulmonary expression of TNF- α in a mouse model of bronchial inflammation and hyperreactivity [42]. LTB4 augments IL-6 production in human monocytes by increasing both IL-6 gene transcription and mRNA stabilization [43, 44]. activation of NF-κB and NF-IL-6 transcriptional factors may be important in this enhancement of IL-6 release [44]. Furthermore, TNF-α production is enhanced by LTC4 and LTD4[45]. So that, inhibition of LTB4 and CysLTs synthesis by MK-886 might result in lowering TNF-α and IL-6 levels.

Through examination of metabolic processes, GSH has been shown to be important in host defenses against oxidative stress [46]. Another important agent showing oxidative stress is MDA, a marker of free radical activity [4]. It was reported that oxidative stress significantly elevated MDA levels and reduced GSH levels [47]. Oxidative stress has been implicated as an important cause of HSR pathogenesis [2, 46]. The result in present study are consistent with other study 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 [18]. In this study MK-886 significantly reduced the elevation of lung MDA level and significantly elevates the lung GSH level 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. But they 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 [18]. The antioxidant effect of MK-886 might be largely due to its inhibitory action on leukotrienes synthesis.

In the present study a significant increase in the BALF total protein level 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 [48]

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 [49]. 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 [50, 51]. Two studies showed that hemorrhagic shock significantly increases BALF total protein in the rats and mice [20, 29]. CysLTs mediate increased permeability leading to leukocyte extravasation, plasma exudation and edema[52, 53, and 54]. Furthermore, LTB4 increases the expression of CD11b/CD18 β2-integrin (Mac-1) on neutrophils, which can facilitate neutrophil adherence and migration [55] and enhanced leukocyte adhesivity accounts for capillary obstruction after I/R [56]. T/HS lymph induces an increase in endothelial permeability by triggering the release of IL-6 [57]. 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 [58]. Free radicals are implicated to damage biomembranes, thereby compromising cell integrity and function [59]. Besides increasing pulmonary arterial pressure [60], the free radical production under hypoxic environment may cause oxidative injury of the endothelium [61], resulting in increased pulmonary capillary permeability. In this study treatment with MK-886 appeared to have a significant decrease in BALF total protein level in the shocked rats in comparison with the induced untreated group. MK-886 has been shown to reduce the extravasation of plasma [13] and prevent the leukocyte adhesion to the endothelium [14] in experimental animals. It was 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 [29].

Morphologically, there was a statistically significant difference between induced untreated group and sham group and the total score mean of the HS group shows moderate lung injury. 66.7% of the HS group had moderate lung injury and 33.3% had severe lung injury. Treatment of rats with MK-886 ameliorates the lung injury significantly 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 MK-886 on the lung parenchyma in HS rats, but they found that MK-886 significantly reduces the histological changes in the liver and small intestine of rats that underwent hepatic I/R model (15). MK-886 was able to reduce the cortical infarct size by 30% in a model of focal cerebral ischemia in rats [62]. Furthermore, a separate research work found that treatment of rats with MK-886 reduces brain lesion volume in experimental traumatic brain injury model [63].

Declarations

Authors’ Affiliations

(1)
Department of Surgery, Colorado Denver university
(2)
Department of pharmacy, Kufa university

References

  1. Bhatia M, Moochhala S: Role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome. J Pathol. 2004, 202: 145-56. 10.1002/path.1491.View ArticlePubMedGoogle Scholar
  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.PubMedGoogle Scholar
  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.View ArticlePubMedGoogle Scholar
  4. Keel M, Trentz O: Pathophysiology of polytrauma. Injury. 2005, 36: 691-709. 10.1016/j.injury.2004.12.037.View ArticlePubMedGoogle Scholar
  5. Anaya-Prado R, Toledo-Pereyra LH, Lentsch AB, Ward PA: Ischemia/reperfusion injury. J Surg Res. 2002, 105: 248-258. 10.1006/jsre.2002.6385.View ArticlePubMedGoogle Scholar
  6. 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.PubMedGoogle Scholar
  7. Yu HP, Shimizu T, Hsieh YC, Suzuki T, Choudhry MA, Schwacha MG: 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. 10.1189/jlb.1005596.View ArticlePubMedGoogle Scholar
  8. Yu HP, Hsieh YC, Suzuki T, Shimizu T, Choudhry MA, Schwacha MG: Salutary effects of estrogen receptor-β agonist on lung injury after trauma-hemorrhage. Am J Physiol Lung Cell Mol Physiol. 2006, 290: L1004-L1009. 10.1152/ajplung.00504.2005.View ArticlePubMedGoogle Scholar
  9. Gillard J, Ford-Hutchinson AW, Chan C, Charleson S, Denis D, Foster A: Full-size imageL-663,536 (MK-886) (3-1-(4-chlorobenzyl)-3-t-butyl-thio-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. 10.1139/y89-073.View ArticlePubMedGoogle Scholar
  10. Dixon RAF, Diehl RE, Opas E, Rands E, Vickers PJ, Evans JF: Requirement of a 5-lipoxygenase activating protein for leukotriene synthesis. Nature. 1990, 343: 282-284. 10.1038/343282a0.View ArticlePubMedGoogle Scholar
  11. Rouzer CA, Ford-Hutchinson AW, Morton HE, Gillard JW: MK-886, 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.PubMedGoogle Scholar
  12. Guidot DM, Repine MJ, Westcott JY, Repine JE: Intrinsic 5-lipoxygenase activity is required for neutrophil responsivity. Proc Natl Acad Sci USA. 1994, 91: 8156-8159. 10.1073/pnas.91.17.8156.View ArticlePubMedPubMed CentralGoogle Scholar
  13. 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.View ArticlePubMedPubMed CentralGoogle Scholar
  14. 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: 2036-10.1172/JCI115233.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Daglar G, Karaca T, Yuksek YN, Gozalan U, Akbiyik F, Sokmensuer C: Effect of Montelukast and MK-886 on Hepatic Ischemia-Reperfusion Injury in Rats. Journal of surgical research. 2009, 153 (1): 31-38. 10.1016/j.jss.2008.02.052.View ArticlePubMedGoogle Scholar
  16. 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.PubMedGoogle Scholar
  17. Eun JC, Moore EE, Mauchley DC, Meng X, Banerjee A: The 5-Lipoxygenase Pathway Meditates Acute Lung Injury Following Hemorrhagic Shock. Journal of Surgical Research. 2010, 158 (2): 215-216.View ArticleGoogle Scholar
  18. 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.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Rhee P, Waxman K, Clark L, Kaupke CJ, Vaziri ND, Tominaga G: Tumor necrosis factor and monocytes are released during hemorrhagic shock. Resuscitation. 1993, 25 (3): 249-255. 10.1016/0300-9572(93)90122-7.View ArticlePubMedGoogle Scholar
  20. 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. 10.1016/j.bcp.2009.06.008.View ArticlePubMedGoogle Scholar
  21. Josephson B, Gyllenswärd C: Scand J Clin Lab Invest. 1957, 9: 29-10.3109/00365515709088110.View ArticlePubMedGoogle Scholar
  22. Beuge JA, Aust SD: Microsomal lipid peroxidation. Meth Enzymol. 1978, 52: 302-311.View ArticleGoogle Scholar
  23. 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: 153-10.1016/S0002-9440(10)63953-3.View ArticlePubMedPubMed CentralGoogle Scholar
  24. 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: 121-10.1016/S0014-2999(00)00574-4.View ArticlePubMedGoogle Scholar
  25. 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: 823-10.1073/pnas.97.2.823.View ArticlePubMedPubMed CentralGoogle Scholar
  26. Rossoni G, Sala A, Berti F, Testa T, Buccellati C, Molta C: Myocardial protection by the leukotriene synthesis inhibitor BAY X1005: Importance of transcellular biosynthesis of cysteinyl-leukotrienes. J Pharmacol Exp Ther. 1996, 276: 335-PubMedGoogle Scholar
  27. 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. 10.1016/j.jss.2003.07.004.View ArticlePubMedGoogle Scholar
  28. 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): S35-Suppl 1View ArticleGoogle Scholar
  29. Eun JC, Moore EE, Mauchley DC, Meng X, Banerjee A: The 5-Lipoxygenase Pathway Meditates Acute Lung Injury Following Hemorrhagic Shock. Journal of Surgical Research. 2010, 158 (2): 215-216.View ArticleGoogle Scholar
  30. Jordan JR, Moore EE, Damle SS, Kashuk SB, Silliman CC: Arachidonic acid in postshock mesenteric lymph induces pulmonary synthesis of leukotriene B4. J Appl Physiol. 2008, 104: 1161-1166. 10.1152/japplphysiol.00022.2007.View ArticlePubMedGoogle Scholar
  31. Amat M, Barcons M, Mancebo J, Mateo J, Oliver A, Mayoral JF: Evolution of leukotriene B4, peptide leukotrienes, and interleukin-8 plasma concentrations in patients at risk of acute respiratory distress syndrome and with acute respiratory distress syndrome: mortality prognostic study. Crit Care Med. 2000, 28: 262-263. 10.1097/00003246-200001000-00051.View ArticleGoogle Scholar
  32. 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. 10.1007/BF00916578.View ArticlePubMedGoogle Scholar
  33. 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. 10.1097/00005373-199708000-00015.View ArticlePubMedGoogle Scholar
  34. 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: 1055-1062.PubMedGoogle Scholar
  35. Murphy RC, Gijon MA: Biosynthesis and metabolism of leukotrienes. Biochem J. 2007, 405: 379-395. 10.1042/BJ20070289.View ArticlePubMedGoogle Scholar
  36. Guidot DM, Repine MJ, Westcott JY, Repine JE: Intrinsic 5-lipoxygenase activity is required for neutrophil responsivity. Proc Natl Acad Sci USA. 1994, 91: 8156-8159. 10.1073/pnas.91.17.8156.View ArticlePubMedPubMed CentralGoogle Scholar
  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.PubMedGoogle Scholar
  38. 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.View ArticlePubMedPubMed CentralGoogle Scholar
  39. 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.PubMedGoogle Scholar
  40. Vincenzi R, Cepeda LA, Pirani WM, Sannomyia P, Rocha-e-Silva M, Cruz RJ: 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. 10.1016/j.amjsurg.2009.01.017.View ArticlePubMedGoogle Scholar
  41. 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: 583-10.1084/jem.20020044.View ArticlePubMedPubMed CentralGoogle Scholar
  42. 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.PubMedGoogle Scholar
  43. 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.PubMedGoogle Scholar
  44. Brach MA, de Vos S, Arnold C, Gruss HJ, Mertelsmann R, Herrmann F: Leukotriene B4 transcriptionally activates interleukin-6 expression involving NK-κB and NF-IL6. Eur J Immunol. 1992, 22: 2705-2711. 10.1002/eji.1830221034.View ArticlePubMedGoogle Scholar
  45. 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. 10.1016/0192-0561(94)90027-2.View ArticlePubMedGoogle Scholar
  46. Szabo C: The pathophysiological role of peroxynitrite in shock, inflammation, and ischemia-reperfusion injury. Shock. 1996, 6: 79-88. 10.1097/00024382-199608000-00001.View ArticlePubMedGoogle Scholar
  47. Johnson KJ, Fantone JC, Kaplan J, Ward PA: In vivo damage of rat lungs by oxygen metabolites. J Clin Invest. 1981, 67: 983-993. 10.1172/JCI110149.View ArticlePubMedPubMed CentralGoogle Scholar
  48. Lum H, Roebuck KA: Oxidant stress and endothelial dysfunction. Am J Physiol Cell Physiol. 2001, 280: C719-C741.PubMedGoogle Scholar
  49. 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. 10.1097/00003246-199902000-00036.View ArticlePubMedGoogle Scholar
  50. 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. 10.1097/00000658-199810000-00008.View ArticlePubMedPubMed CentralGoogle Scholar
  51. 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. 10.1067/msy.2001.109119.View ArticlePubMedGoogle Scholar
  52. Funk CD: Prostaglandins and leukotrienes: advances in eicosanoid biology. Science. 2001, 294: 1871-1875. 10.1126/science.294.5548.1871.View ArticlePubMedGoogle Scholar
  53. Dahlén SE: Treatment of asthma with antileukotrienes: first line or last resort therapy?. Eur J Pharmacol. 2006, 533: 40-56. 10.1016/j.ejphar.2005.12.070.View ArticlePubMedGoogle Scholar
  54. Ogawa Y, Calhoun WJ: The role of leukotrienes in airway inflammation. J Allergy Clin Immunol. 2006, 118: 789-798. 10.1016/j.jaci.2006.08.009.View ArticlePubMedGoogle Scholar
  55. Crooks SW, Stockley RA: Leukotriene B4. Int J Biochem Cell Biol. 1998, 30 (2): 173-178. 10.1016/S1357-2725(97)00123-4.View ArticlePubMedGoogle Scholar
  56. Schmid-Schonbein GW: Capillary plugging by granulocytes and the no-reflow phenomenon in the microcirculation. Fed Proc. 1987, 46: 2397-2401.PubMedGoogle Scholar
  57. Dayal SD, Haskó G, Lu Q, Xu DZ, Caruso JM, Sambol JT: 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. 10.1097/00024382-200206000-00009.View ArticlePubMedGoogle Scholar
  58. 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.PubMedGoogle Scholar
  59. 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.Google Scholar
  60. Hoshikawa Y, Sadafumi O, Satoshi S, Tatsuo T, Masayuki C, Chun S: Generation of oxidative stress contributes to the development of pulmonary hypertension induced by hypoxia. J Appl Physiol. 2001, 90: 1299-1306.PubMedGoogle Scholar
  61. Herget J, Wilhelm J, Novotna J, Eckhardt A, Vytasek R, Mrazkova L: A possible role of the oxidant tissue injury in the development of hypoxic pulmonary hypertension. Physiol Res. 2000, 49: 493-501.PubMedGoogle Scholar
  62. 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: 1323-10.1038/sj.bjp.0704189.View ArticlePubMedPubMed CentralGoogle Scholar
  63. 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. 10.1089/neu.2009.0877.View ArticlePubMedPubMed CentralGoogle Scholar

Copyright

© Al-Amran et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.