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Department of Cardiothoracic Surgery, Jinling Hospital, Clinical Medicine School of Nanjing University, Nanjing, China
Accepted for publication July 9, 2007.
* Address correspondence to Dr Jing, Department of Cardiothoracic Surgery, Jinling Hospital, Clinical Medicine School of Nanjing University, 305 East Zhongshan Rd, Nanjing, 210002, China (Email: jing_hua_1{at}yahoo.com.cn).
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Abstract |
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Methods: Male Sprague-Dawley rats were divided into four groups (n = 6 each): sham group; CPB (control group); CPB plus low-dose simvastatin (5 mg/kg daily [L-Sim group]); and CPB plus high-dose simvastatin (10 mg/kg daily [H-Sim group]). Blood samples were collected at the beginning and at the termination of CPB, and at 1, 2, 4, and 24 hours after operation. The bronchoalveolar lavage fluid and lungs were harvested 24 hours postoperatively.
Results: The simvastatin-treated groups had significantly higher ratios of PaO2/FiO2 and lower values of respiratory index than the control group. We observed that simvastatin reduced CPB-induced toll-like receptor 4 and nuclear factor-
B expressions in CPB groups (p < 0.01, versus control group). The levels of interleukin-6, tumor necrosis factor-
, and monocyte chemotactic protein-1 in serum, bronchoalveolar lavage fluid, and lung tissues increased in CPB groups, whereas pretreatment with simvastatins reduced these inflammatory marks in a dose-dependent manner (p < 0.01, versus control group). Furthermore, pretreatment with simvastatin had a lower lung injury score (p < 0.05, versus control group).
Conclusions: These findings suggest that simvastatin inhibited CPB-induced toll-like receptor 4 upregulation and nuclear factor-B activation, efficaciously reduing the pulmonary inflammatory response after CPB.
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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(TNF-) could be explored as predicting factors of organ dysfunction.
Infiltration of the lung with neutrophil is an important feature of the inflammatory response that characterizes acute lung injury. Data suggested that toll-like receptor 4 (TLR4) played a particular role in the regulation of neutrophil life span [3, 4]. One of the major pathways activated by TLR4 is the activation of the nuclear factor-B (NF-B), which acts as a master switch for inflammation, regulating the transcription of many genes that encode proteins involved in immunity and inflammation. Studies have clearly shown the upregulation of TLR4 and NF-B in sepis [5] and ischemia/reperfusion models [6]. Dybdahl and colleagues [7] have demonstrated that heat shock protein 70, an endogenous ligand for TLR4, released and regulated TLR4 expression on monocytes after CPB. On the basis of the above findings, it seems reasonable to speculate that the transduction pathways of TLRs and NF-B should be for the pathophysiologic progress of CPB.
Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase. In addition to lipid-lowering effects, these drugs are shown to have pleiotropic effects such as immunomodulation, anti-inflammation, and modulation of endothelial nitric oxide synthase. A number of studies have demonstrated that pretreatment with simvastatin reduced neutrophil adhesion to the venous endothelium in patients undergoing CPB [8], downstreamed signaling in human CD14+ monocytes [9], reduced circulating markers of inflammation, increased neutrophil apoptosis [10], and attenuated vascular leak in murine lung [11]. However, the anti-inflammation mechanism of statins and their effect on the proinflammation cytokines levels, TLR4 and NF-kB expression in lung tissue after CPB, are largely unknown.
We hypothesis that TLR4 upregulation could be inhibited by treatment of simvastatin in a rat CPB model, and thus result in the reduction of cytokines expression. Therefore, it follows that simvastatin might suppress lung inflammatory response after CPB.
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Material and Methods |
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Surgical Procedure
We modified the rat model of CPB described in our previous publication [12, 13]. We supplied the oxygen by a specific veil with the oxygen flow at 10 L/min from beginning till 4 hours after the CPB. Rats were anesthetized with intraperitoneal administration of butaylone (60 mg/kg) at the beginning. Anesthesia was maintained with additional butaylone. The right femoral artery was cannulated for arterial pressure monitoring (24G catheter). Mean arterial pressures were recorded during the experiment. After administration of heparin (250 U/kg), a modified 16G catheter was inserted into the right atrium through the right jugular vein approach. Tail artery cannulation was achieved with a 22G catheter, which served as the arterial infusion line. The mini-CPB circuit comprised a venous reservoir (10 mL), a specially designed membrane oxygenator (Micro-1; Kewei Medical Instrument, Guangzhou, China), and a roller pump (BT00-300M; Lange, Shanghai, China). All components were connected with polyethylene tubing. We used three circuits in each CPB group, and every circuit was used twice. The reused circuits were all sterilized by ethylene oxide before operations. Body central temperature was monitored with a rectal probe and kept at 37°C to 38°C by a heat lamp placed around the animal and the CPB equipments. The CPB circuit was primed with 10 mL consisting of heparin, 1 mL (250 U/kg), synthetic colloid, 8 mL, and sodium bicarbonate, 1 mL. The flow rate was gradually added to 100 mL · kg-1 · min-1 and maintained for 60 minutes. Throughout the experiment, mean arterial pressure was maintained at 60 to 80 mm Hg. The rats that died during the procedure were excluded and were replaced by a new one from the corresponding group. The right jugular vein was decannulated as soon as CPB finished. According to the blood pressure, a part of the remaining priming volume was infused through the tail artery. The tail and femoral artery catheters were removed, and the incisions were sutured 4 hours after CPB. The rats were given water and food 6 hours after the operation, and they were monitored for 24 hours postoperatively.
Specimen Collection
Blood samples were obtained immediately after heparinization (T0), at the end of CPB (T1), and at 1, 2, 4, and 24 hours after operation (T2 to T5). A blood sample, 0.3 mL, was used to detected blood gas analyses. At the same time, PaO2/FiO2 and respiratory index (RI) were calculated. The RI was calculated as follows: RI = alveolar minus arterial oxygen tension gradient divided by arterial oxygen tension.
Plasma was separated by centrifuge at 4°C for 10 minutes and was stored at –70°C until analyzed. They were used to detect serum levels of TNF-, IL-6, and monocyte chemotactic protein-1 (MCP-1).
At 24 hours after CPB, the right ventricle was cannulated with a 25G needle, and the lung was flushed with 20 mL normal saline. The lung was removed, and the right main bronchus was clamped. After injection with four sequential 0.5-mL aliquots of Hanks’ balanced salt solution intratracheally, the bronchoalveolar lavage fluid was harvested from the left lung and then used to test IL-6, TNF-, and MCP-1. The right lung was used for microscopic examination and NF-B and TLR4 determination.
Assays of Inflammatory Markers
Serum, bronchoalveolar lavage fluid, and tissue levels of TNF-, IL-6, and MCP-1 were measured by enzyme-linked immunosorbent assay kits specific for rats according to the manufacturer’s instruction (Biosource, Camarillo, California). The lung samples were homogenized in 0.5 mL phosphate-buffered saline solution containing protease inhibitor, sonicated, and then centrifuged at 10,000 rpm for 20 minutes. Protein measurements were analyzed according to the method of Lowry and coworkers [14]. Values were expressed as pictogram per milliliter for serum and bronchoalveolar lavage fluid, and pictogram per milligram protein for tissue.
Electrophoretic Mobility Shift Assays
An oligonucleotide containing the consensus sequence motifs for NF-B binding (5’-AGTTGAGGGGACTTTCCCAGGC-3’) was labelled with [
-32P] adenosine triphosphate (Free Biotech, Beijing, China) with T4 polynucleotide kinase. Equal amounts of nuclear extract (60 µg) were added to 9 µL gel shift binding buffer (in mM: Tris–HCl 10, pH 7.5, NaCl 50, EDTA 0.5, MgCl2 1, DTT 0.5, 4% glycerol, and 0.05 mg/mL Poly dIdC [15 minutes, room temperature]). The mixture was incubated for 30 minutes with 1 µL 32P-labelled oligonucleotide probe. Loading buffer, 1 µL, was added, and the sample electrophoresed in a 4% polyacrylamide gel at 390 V for 1 hour. The dried gel was exposed to x-ray film (Fuji Hyperfilm) at –70°C. The intensity of the NF-B complex was quantified by densitometry.
Western Immunoblot Analysis
The lungs’ protein levels of TLR4 were determined by Western blot analysis with specific polyclonal antibodies (Kangchen KC-415; Kangchen, Shanghai, China). Proteins were separated on a 15% sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The protein membrane was blocked in washing solution with 5% nonfat dry milk for 1 hour at 37°C. The membrane was then incubated with 1:3000 dilution of primary antibody overnight at 4°C, and subsequently with a peroxidase-conjugated secondary antibody for 1 hour at 37°C. The bands were detected by a chemiluminescent system (Amersham Bioscience, Piscataway, New Jersey) and quantified by scanning densitometry using a GS-710 Imaging Densitometer (Bio-Rad, Hercules, California).
Light Microscope Examination
Formalin-fixed lung samples were embedded in paraffin, and 4-µm sections were stained with hematoxylin and eosin. Criteria to evaluate the degree of lung damage included (1) neutrophil infiltration, (2) airway epithelial cell damage, (3) hemorrhage, (4) hyaline membrane formation, and (5) interstitial edema. Each criterion was scored on a scale of 0 to 4, that is, 0 = normal, 1 = minimal change, 2 = mild change, 3 = moderate change, and 4 = severe change. Total scores represented lung injury. The examination was performed by a pathologist who was blinded to the grouping.
Statistical Analysis
All values were expressed as mean ± SD. Data were analyzed using a commercially available statistics software package (SPSS for Windows 14.0; SPSS, Chicago, Illinois). Comparisons between the groups were analyzed by one-way or two-way repeated-measures analysis of variance. Time-dependent changes and post-hoc comparisons were performed using the Tukey test or Dunnett’s T3 test. All p values less than 0.05 were considered as statistically significant.
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Results |
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Physiologic data before and after operation are illustrated in Table 1. There was no difference among the four groups before operation. Both simvastatin groups had a significantly higher ratio of PaO2/FiO2 and lower RI value than the control group (p < 0.001; Fig 1A, B).
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were below minimum detectable levels in all groups. Plasma MCP-1 levels before CPB were not significantly different among the four groups. After the termination of CPB, they all increased, and there were significant time-dependent changes in the CPB groups. There was no time-dependent change in the sham group. Compared with corresponding findings in the control group, pretreatment with simvastatin inhibited IL-6, TNF-, and MCP-1 production (p < 0.001 for comparisons, Fig 2A–C). Moreover, the inhibitory effects of IL-6 and TNF- were in a dose-dependent manner (compared with L-Sim and H-Sim,p = 0.006 for IL-6, p = 0.038 for TNF-). However, there was no different between the groups L-Sim and H-Sim (p = 0.611) for MCP-1.
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, and MCP-1 than the simvastatin groups (p < 0.001 for all comparisons; Table 2). No difference was found between the L-Sim and H-Sim groups.
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, and MCP-1 increased after CPB operation. Compared with corresponding control group, the levels of IL-6, TNF-, and MCP-1 in simvastatin groups were significantly lower (p < 0.01 for all comparisons; Table 2). Furthermore, there was no difference between simvastatin groups.
Expression of NF-B in Lung Tissues
Activation of NF-B was observed after CPB. However, by densitometric analysis of the gels, with normalization of band density to total protein loaded, the apparent amount of NF-B was significantly reduced in the simvastatin groups compared with the control group (p < 0.01; Fig 3). Simvastatin modified NF-B activation in a dose-dependent way (p = 0.032, comparing the L-Sim and H-Sim groups).
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Comment |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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, and MCP-1 expression that followed CPB in a rat model. Additionally, simvastatin showed the effects on downregulation of TLR4 and NF-B expressions, which should be considered as the protective mechanisms of simvastatin.
There are many studies concentrating on the organ-protective effect of statins. Owing to ethics restrictions, however, it is difficult to obtain both the morphologic data and the level of proinflammatory cytokines from tissues having clinical status. Our study detected the levels of TNF-, IL-6, and MCP-1 not only in serum but also in bronchoalveolar lavage fluid and lung tissues; additionally, NF-B and TLR4 expressions in lung tissues were detected. This is the first report of TLR4 protein expression in lung tissue after CPB.
The underlying mechanisms of inflammatory response after CPB are multifactorial, and include operative trauma, endotoxins released from the temporarily ischemic intestine, heart and lung ischemia-reperfusion injury, and blood contact with the foreign surfaces of the CPB system. With respect to these mechanisms, endotoximia is a salient feature. Aydin and colleagues [15] found that endotoxin levels increased after start of CPB and remained elevated for as long as 12 hours after surgery. Emerging evidence from in-vivo and in-vitro studies has shown that lipopolysaccharide challenge upregulates TLR4 expression [7]. On the cell surface, especially on monocytes, CD14 and myeloid differentiation-2 recognize lipopolysaccharide by TLR4 and form the complex, which is thought to induce dimerization and activation of TLR4. Through a series of intracellular transduction, NF-B translocated into the nucleus, which is responsible for synthesis of chemokines (MCP-1) and proinflammatory cytokines (TNF-, IL-1, and IL-6) [2, 16].
Our study demonstrated that TLR4 protein expressions markedly increased in lung tissues after CPB and could be suppressed by simvastatin. As far as the involved mechanisms are concerned, most researchers considered that statins might influence TLR4 expression in a cholesterol-dependent fashion [17, 18]. By altering cholesterol-rich membrane domains, trafficking of TLR4 on cellular membrane may be interfered with. Also, the synthesis of isoprenoid intermediates, which are essential for the posttranslational modification, may influence TLR4 expression [9]. Additionally, a recent study reported by Niessner and coworkers [17] demonstrated that simvastatin did not influence the increase in TLR transcripts after lipopolysaccharide administration measured in mRNA isolated from whole blood of 20 healthy subjects. Based on those in-vitro researches noted above, we have reason to believe that simvastatin inhibits TLR4 expression during the CPB procedure potentially on a posttranslational level.
In the present work, we found that NF-B expression increased markedly after CPB and could be inhibited by simvastatin. As mentioned above, this effect may be the result from inhibiting TLR4 expression. On the other hand, decreased free oxygen radicals by statins play an important role. Schlensak and coworkers [19] showed that pulmonary blood flow significantly diminished during CPB, leading to the production of the free oxygen radicals, which result in the activity of NF-B in the lung [5]. An ischemia/reperfusion lung model [20] showed that simvastatin reduced the expression of nicotinamide adenine dinucleotide phosphate oxidase, thus in turn decreasing the production of mitochondrial free oxygen radicals. Consequently, statins not only reduce direct tissue injury by inhibiting production of free oxygen radicals, but also potentially attenuate the inflammatory cascade by blocking the activation of NF-B.
Interleukin-6 is responsible for the coordination of the acute phase response and plays a positive role in the local inflammatory reaction by amplifying leukocyte accumulation. Clinical investigation showed that plasma levels of IL-6 were associated with the severity of the inflammatory response to CPB and also with postoperative morbidity in adults [21]. The effect of statin therapy on IL-6 plasma levels is still debated [21-25]. Inhibitory effects have been shown in several studies [21, 22], whereas others have shown fair [23] or even no effects [24], perhaps depending on the populations studied. In the present work, we noted that treatment of rats with simvastatin led to a significant decrease in levels of IL-6 in serum, bronchoalveolar lavage fluid, and lung tissue. An interesting aspect of the present study was that maximum level of IL-6 was detected at 2 hours after CPB, earlier than what has been shown by others.
Monocyte chemotactic protein-1 is known to induce leukocyte migration and activation on target cells during inflammation [26]. Moreover, MCP-1 can induce respiratory burst activity and stimulate lysosomal enzyme release from monocytes, which contribute to lung damage. Present data showed that circulating level of MCP-1 increased significantly in response to the CPB procedure and the maximum level occurred at 3 hours after operation. This finding was in line with a study by De Mensonca-Fiho and colleagues [2]. In-vitro research found that simvastatin could suppress MCP-1 expression, and this effect was reversed by mevalonate [4]. On the basis of in-vitro findings, we speculate that mevalonate-derived products are required for MCP-1 production.
Tumor necrosis factor- is an important proinflammatory factor to activate other cytokines and to induce the expression of adhesive molecules in endothelial cells [27]. We documented that the level of TNF- in lung tissues in the control group was 20 times higher than that of the simvastatin groups, and there were significant differences between plasma levels of TNF- in the simvastatin-treated groups and the control group. These results disagreed with those in clinical studies [1, 28], which failed to demonstrate a clear rise in TNF- level during and after CPB. There are some explanations for this discrepancy. First, ventilation continued during CPB in clinical studies, possibly reducing hypoxia-induced inflammatory reaction. Second, inflammation was enlarged because of the reused circuits in our study. The observed anti-inflammatory action of simvastatin treatment on TNF- level shown in this study may be explained in part through down-regulation of TLR4 and NF-B.
An intriguing find in our study was the immense protective effect of simvastatin on PaO2. Possible reasons were that pretreatment with simvastatin not only blocks activation of calcium-dependent focal-adhesion tyrosine kinase, necessary for angiotensin II receptor–mediated inflammatory signaling in pulmonary vein endothelial cells [29], but also improves endothelial nitric oxide synthase expression during hypoxia in CPB, leading to increased production of nitric oxide.
Finally, compared with human dosing regiments, the concentration of simvastatin used in our study were obviously higher (ie, 1 mg/kg daily is equivalent to a 75-mg single dose in a human subject) [30]. Our study showing the partly dose-dependent anti-inflammatory effects of simvastatin revealed that 10 mg/kg daily may be more effective. Recently, a clinical study [31] has used simvastatin at 80 mg per day for 4 days, and no side effects have been observed, suggesting that a high dose of simvastatin could be utilized before emergency operation.
In summary, pretreatment with simvastatin inhibited TLR4 expression in lung in a rat CPB model. This effect translated into inhibited NF-B production. As a result, the productions of TNF- IL-6 and MCP-1 decreased. Our investigation into the role of simvastatin in attenuating inflammation and its influence on innate immunity holds the promise of a profound clinical use for this class of drugs.
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Acknowledgments |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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