Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are caused by a variety of direct or indirect insults that lead to pulmonary inflammation (1). Because lung inflammation plays a key role in the pathogenesis of this devastating clinical syndrome, many investigators have focused on therapeutic interventions that might modify or minimize this process (1,2).
Statins are a well established class of drugs that effectively decrease serum cholesterol levels by inhibiting the action of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (3). These drugs have also been shown to reduce the risk of cardiovascular and cerebrovascular events in patients with atherosclerosis (4–6). However, in addition to their cholesterol-decreasing activity, studies have revealed pleiotropic properties of these drugs, including promotion of vasculogenesis, stimulation of bone formation, and antiinflammatory and immunomodulatory effects (7–12). The anti-inflammatory effects of statins are believed to result from inhibition of leukocyte adhesion and decreased production of cytokines (13,14). Infiltration of the lung with neutrophils and high levels of proinflammatory cytokines in serum and bronchoalveolar lavage fluid (BALF) are important features of the inflammatory response that characterizes ALI (15,16). Therefore, it follows that simvastatin might reduce ALI. The hypothesis for this rat model study was that pretreatment with simvastatin reduces the severity of ALI induced by intestinal ischemia-reperfusion (I/R).
Methods
The Başkent University Animal Research Committee approved the experimental protocol and animal care methods. A pool of male Wistar rats weighing 250–300 g was used. The animals were acclimatized to the university's Animal Research Laboratory for 7 days before experiment and were given free access to food and water during this time.
Rats were randomly allocated to one of three groups. The simvastatin group was pretreated with simvastatin 10 mg · kg−1 · day−1 in 1 mL of distilled water for 3 days. This was administered via an orogastric tube that was inserted each day, and the last dose was given 1 h before the experiment. Each animal in the control group and sham group received 1 mL/day of distilled water via the same route. Eight animals from each group completed the experiment.
Before introduction of anesthesia for the experiment, each rat was subjected to 12 h of fasting with no restriction of water access. Anesthesia was induced with intraperitoneal ketamine 85 mg/kg. A tail vein was cannulated to administer 10 mg · kg−1 · h−1 of lactated Ringer's solution as the maintenance fluid and 50 mg · kg−1 · h−1 of ketamine for anesthesia maintenance. The right carotid artery was also cannulated to monitor arterial blood pressure and to obtain blood samples. All animals spontaneously breathed room air throughout the experiment. Once these initial steps were completed, a midline laparotomy was performed under aseptic conditions. After the superior mesenteric artery (SMA) was visualized, the animal was subjected to 1 of 2 procedures: a sham operation that involved clamping the SMA for 1 min followed by 90 min of reperfusion (sham group); or intestinal I/R injury induced by occluding the SMA for 60 min, followed by 90 min of reperfusion (simvastatin group and control group). Intestinal ischemia was verified by the deep purple color of the small intestine at the end of the 60 min of SMA occlusion. Reperfusion was confirmed by the return of pulsation to the mesenteric vasculature after clip removal. Systemic mean arterial blood pressure (MAP) and rectal temperature were recorded every 5 min throughout the experiment. The mean values for MAP before SMA occlusion (baseline), during the ischemia period, and during the reperfusion period were calculated and used for comparisons. At the end of each experiment, the anesthetized rat was killed by sternotomy and exsanguination.
Throughout the experiment, each animal was required to meet the following criteria to continue: normothermia (rectal temperature: 37°C–38°C), hematocrit >30%, and MAP >50 mm Hg. If these criteria were not fulfilled, the rat was excluded from the protocol and a replacement animal was acquired from the corresponding group in the randomization pool, such that there were 8 animals per group for final analysis.
Arterial blood samples (0.7 mL) were drawn from each animal at baseline (0 min), at the end of ischemia (60 min), and at the end of reperfusion (90 min). After collection, specimens were immediately centrifuged at 3000g for 10 min and stored at –80°C until they were assayed. For each sample, arterial blood gas analysis was performed (measurement of partial oxygen pressure [Pao2], partial carbon dioxide pressure [Paco2], arterial oxygen saturation [O2Sat], bicarbonate [HCO3−], and pH) and serum levels of interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, and P-selectin were measured.
At the end of each experiment, a median sternotomy was performed and BALF and lung samples were collected immediately after death by exsanguination. For this collection, the superior and inferior vena cavae were ligated. The right ventricle was cannulated with a 25-gauge needle and the lungs were flushed with 20 mL normal saline. The lungs were then removed and the right main bronchus was clamped. The left lung was lavaged with 10 mL/kg of normal saline to obtain BALF. Testing was done to determine the levels of IL-1, IL-6, TNF-α, and P-selectin in each BALF sample. The upper lobe of the right lung was stored at –80°C for later analysis of lung tissue malondialdehyde (MDA) level. The middle lobe of the right lung was placed in 10% formaldehyde and subjected to routine processing for histopathologic examination. A pathologist microscopically examined the hematoxylin & eosin-stained sections in blinded fashion, recording results for each of the following tissue-injury criteria: 1) neutrophil infiltration, 2) airway epithelial-cell damage, 3) interstitial edema, 4) hyaline membrane formation, 5) hemorrhage, and 6) total lung injury score (that is, the sum of the scores for criteria 1 through 5). Each criterion was scored on a scale of 0 to 4, where 0 = normal, 1 = minimal change, 2 = mild change, 3 = moderate change, and 4 = severe change. The lower lobe of the right lung was dried at 80°C for 8 h to calculate the lung wet-to-dry weight (W/D) ratio.
Analyses of serum and BALF IL-1, IL-6, TNF-α and P-selectin levels were performed in duplicate and in blinded fashion using respective commercially available polyclonal sandwich antibody enzyme-linked immunosorbent assay kits for the rat (Biosource International Immunoassay Kits for Rat TNF-α, IL-1, and IL-6, Camarillo, CA, and sP-Selectin Immunoassay Kit; R&D Systems; Minneapolis, MN). The MDA content of lung tissue was also determined in duplicate and in blinded fashion using the original method described by Buege and Aust (17).
Data are expressed as mean ± sd. The Friedman and Wilcoxon tests were used to analyze dependent variables, and the Kruskal-Wallis and Mann-Whitney U-test were used to compare independent variables. The level of significance was set at P < 0.05.
Results
As specified above, 8 animals from each group completed the experiment protocol. Ten control group rats and 8 simvastatin group rats did not survive reperfusion and were therefore excluded from the study (overall model mortality, 42%).
In the sham group, there were no significant changes in MAP throughout the experiment. However, compared with their respective baseline values, the control and simvastatin groups had significantly higher MAP during ischemia (P = 0.017 and P = 0.036, respectively) and significantly lower MAP during reperfusion (P = 0.012 for both). The sham group had significantly higher reperfusion MAP than the other groups (P = 0.001 for both) (Fig. 1).
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Figure 1.:
Mean arterial blood pressure (MAP) findings in the three groups. a P = 0.017 for comparison with baseline in the control group; b P = 0.036 for comparison with baseline in the simvastatin group; c P = 0.012 compared with baseline for both groups; d P = 0.001 compared with sham for both groups.
Compared with their respective baseline arterial blood gas findings, the control and simvastatin groups had significantly lower Pao2, O2Sat, pH, and HCO3− values at the end of reperfusion (P < 0.05 for all) (Table 1). The control and simvastatin groups both showed significantly lower Pao2, O2Sat, pH, and HCO3− than the sham group at the end of reperfusion (Table 1) (P ≤ 0.017 for all). Further, there were significantly lower Pao2 and O2Sat values in the control group than in the simvastatin group at the reperfusion stage (Table 1) (P ≤ 0.01 for Pao2 and O2Sat).
Table 1:
Results of Arterial Blood Gas Analysis for the Three Groups
Regarding serum levels of cytokines and P-selectin, the control and simvastatin groups had similar baseline values, but both had significantly higher serum IL-1, IL-6, and TNF-α values during reperfusion compared with their respective baseline values (P ≤ 0.017 for all comparisons) and compared with corresponding sham group findings during reperfusion (P ≤ 0.002 for all comparisons) (Fig. 2A–C). Serum soluble P-selectin levels remained steady throughout the experiment in each group, and there were no differences among the groups at any of the three stages investigated (Fig. 2D).
Figure 2.:
Serum cytokine and P-selectin levels at baseline, end of ischemia, and end of reperfusion. IL, interleukin; TNF, tumor necrosis factor.
Concerning BALF cytokine results, the control and simvastatin groups both had significantly higher levels of IL-1, IL-6, and TNF-α in their BALF than the sham group (P ≤ 0.006 for all comparisons) (Fig. 3A–C). However, the control and simvastatin rats had similar levels of the measured cytokines in their BALF. Testing also showed that the control and simvastatin groups both had significantly higher BALF P-selectin levels than the sham group (P = 0.001 and P = 0.015, respectively). Further, the level of P-selectin in the BALF from the control animals was significantly higher than the level in the BALF from the simvastatin animals (P = 0.006) (Fig. 3D).
Figure 3.:
Bronchoalveolar lavage fluid levels of cytokines and p-selectin. BALF, bronchoalveolar lavage fluid; IL, interleukin; TNF, tumor necrosis factor.
Microscopic findings in the lung specimens revealed normal lung parenchyma in the sham group but severe lung injury in the simvastatin and control groups. Further, animals in the simvastatin group had significantly less severe lung injury than those in the control group, as indicated by less neutrophil infiltration and lower total lung injury scores (P = 0.003 for both) (Table 2).
Table 2:
Mean Histopathologic Scores for the Lung Tissue from Each Group
The lung-tissue MDA assays (Fig. 4) and the results for W/D ratio (Fig. 5) revealed negligible lung injury in the sham group; however, these same variables reflected severe lung injury in the animals that underwent I/R (P ≤ 0.002 for all comparisons). Comparisons of group MDA and W/D ratio results also showed significantly less severe lung injury in the simvastatin group than in the control group (P = 0.016 and P = 0.009, respectively).
Figure 4.:
Lung tissue malondialdehyde levels in the three groups.
Figure 5.:
Lung wet-to-dry weight ratio in the three groups.
Discussion
This study examined whether pretreatment with simvastatin, a drug with potential antiinflammatory and immunomodulatory effects, might attenuate the ALI that follows intestinal I/R. The data clearly demonstrate that simvastatin pretreatment reduces the severity of ALI in this setting in rats.
Statins competitively inhibit HMG-CoA reductase, the enzyme that catalyzes the production of mevalonate from acetyl-CoA, which is the rate-limiting step of the cholesterol-synthesis pathway in the liver and other tissues (3,18). The pleiotropic effects of statins are also thought to be linked to inhibition of mevalonate production, which results in reduced levels of products further along this pathway, such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate (9,11). These downstream products are necessary for the isoprenylation of a variety of proteins, including the small Ras and Rho guanosine triphosphate (GTP)-binding proteins (19). These small proteins play a key role in signal transduction pathways that regulate cell proliferation, cell differentiation, vesicular transport, and apoptosis. Studies indicate that statins have antiinflammatory effects that are also based, in part, on inhibition of the prenylation pathway (19,20). Geranylgeranylation of Rho GTPase is critical to several steps of the leukocyte adhesion cascade (21). Statins reduce cell adhesion by blocking expression of monocyte chemotactic protein-1 (MCP-1) (22). They also inhibit both expression and activation of integrin, which is essential for leukocyte adhesion (23). Other research has indicated that statins interfere with the expression of intercellular adhesion molecule-1 (ICAM-1) and cytokines in endothelial cells and monocytes (24). Another consequence of the inhibition of geranylgeranylation of Rho GTPase by statins is increased expression and activity of endothelial nitric oxide synthase (eNOS) (20). The resultant increase in nitric oxide (NO) production causes reduced expression of adhesion molecules, including selectins and ICAM-1 (25,26).
The above-mentioned antiinflammatory mechanisms of statins are likely responsible for the protective effect of simvastatin that we observed in this rat model of ALI induced by intestinal I/R. Specifically, we noted better Pao2 and O2Sat values, less neutrophil infiltration in the lungs, better histopathologic lung injury scores, lower lung tissue MDA levels, and lower W/D ratios in the animals pretreated with simvastatin than in the control animals pretreated with distilled water. We also detected significantly lower BALF P-selectin levels in the simvastatin group than in the control group. Although these findings clearly demonstrate that simvastatin has protective effects in this model, we cannot fully explain the mechanism or mechanisms that underlie these effects. Our suspicion is that the protective effect of simvastatin against ALI might be at least partially mediated through reduced expression of P-selectin, which is central to the leukocyte adhesion cascade. Other investigators have also demonstrated that pretreatment with statins protects against I/R-induced lung injury in lung and lower torso I/R models in rats (27,28). The same researchers have also shown increased expression of eNOS with statins, and have related the beneficial effects of these drugs to this finding. Although hypothetical, we believe that increased eNOS activity is the mechanism that leads to reduced expression of P-selectin in statin-treated animals. However, although we observed significantly lower levels of BALF P-selectin in the simvastatin group than in the control group, despite a trend towards lower serum P-selectin levels in the simvastatin group than in the control group at the end of reperfusion (Fig. 2D), the serum P-selectin levels in these groups were statistically similar. Although this finding is likely a result of the wide variation of animals' serum P-selectin levels in this study, further studies are needed to elucidate whether lower BALF P-selectin levels reflect a specific protective effect of simvastatin for lungs.
Inhibition of free radical production is another potential mechanism by which statins protect against I/R injury. MDA is a marker of the generation of free radicals and subsequent lipid peroxidation associated with reperfusion injury. In our study, we found significantly lower lung-tissue MDA levels in the rats pretreated with simvastatin than in the control animals. This indicates that pretreatment with simvastatin effectively inhibits lipid peroxidation in this model of ALI induced by intestinal I/R. In other words, our evidence suggests that this mechanism of protection by statins was functioning in our model. It has been suggested that these drugs inhibit lipid peroxidation and scavenge free oxygen radicals independently of their cholesterol-decreasing activity (29–32). Naidu et al.'s (27) work with an experimental model of lung I/R showed that simvastatin reduces the expression of nicotinamide adenine dinucleotide phosphate oxidase in lung tissue. This, in turn, might decrease the production of mitochondrial free oxygen radicals. Reactive oxygen metabolites can directly stimulate the activity of the proinflammatory nuclear transcription factor known as nuclear factor-κB (NF-κB), which plays a key role in initiation and propagation of the inflammatory response (33). Therefore, by inhibiting production of free oxygen radicals, statins not only reduce direct tissue injury but also potentially attenuate the inflammatory cascade by blocking the transcription of NF-κB.
Proinflammatory cytokines, including TNF-α, IL-1, and IL-6, are crucial to the initiation and propagation of the inflammatory response that leads to pulmonary injury in ALI and ARDS (1,16). Cytokines mediate the bi-directional interaction between leukocytes and endothelial cells. The inflammatory cytokines IL-1 and TNF-α modulate the extravasation of leukocytes and their localization at inflammatory sites, steps that involve adhesion of leukocytes to vessel walls and passage through the endothelial lining in response to tissue-derived signals (34). Studies have suggested that statins might partially block the translocation of NF-κB to the nucleus, a step that is essential for chemokine expression (35).
We found no significant differences between our control group animals and statin-treated animals with respect to serum or BALF levels of IL-1, IL-6, and TNF-α at the end of reperfusion. The findings for these variables might be a reflection of the I/R model we used, which is associated with a generalized severe inflammatory response. This type of reaction could potentially overwhelm the inhibitory effects of simvastatin on chemokine expression. Another possible explanation for our BALF cytokine findings is that BALF cytokine levels are actually determined by the amount of cytokines released from a variety of cell populations, including alveolar macrophages, endothelial cells, and epithelial cells, and these multiple sources mean that reduced neutrophil infiltration of the lungs may not directly translate to a decreased level of cytokines in the BALF (36,37). However, it is important to stress that, although the serum and BALF cytokine levels in the control and simvastatin groups were similar, simvastatin pretreatment was associated with less severe lung injury. Our interpretation is that the beneficial effects of statins in this model reflect direct inhibition of the leukocyte adhesion cascade and direct inhibition of free radical production, as opposed to a nonspecific immunosuppressive mechanism.
In conclusion, our results show that pretreatment with simvastatin attenuates the severity of ALI induced by intestinal I/R in the rat. This protective effect of simvastatin might be attributed to decreased production of free oxygen radicals and reduced leukocyte infiltration of the lungs. However, the exact mechanism or mechanisms by which simvastatin protects against I/R-induced ALI need to be addressed in future experiments.
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