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].
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 LTB
4, LTC
4, LTD
4 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 (LTB
4 & LTC
4) 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 PLA
2-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 PLA
2 that can engage the LTB
4 receptor and initiate LTB
4 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 PLA
2 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 (LTB
4 & LTC
4) 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 LTB
4 and LTC
4 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]. LTB
4 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 LTC
4 and LTD
4[
45]. So that, inhibition of LTB
4 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, LTB
4 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].
Competing interests
The authors participated in the design of the study and performed the statistical analysis declare that they have no competing interests.
Authors' contributions
FG carried out the surgical experimental work and gives the outline of research. NR participated in the design of the study and performed the statistical analysis and supervised main skeleton. AM participated in the sequence alignment and drafted the manuscript and did all the biochemical and histopathological tests.
All authors read and approved the final manuscript.