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Eur J Cardiothorac Surg 2006;30:77-84
© 2006 Elsevier Science NL

P38 mitogen-activated protein kinase inhibition attenuates pulmonary inflammatory response in a rat cardiopulmonary bypass model

Xiao Dong * , Yinglong Liu, Ming Du, Qiang Wang, Cun Tao Yu, Xiangming Fan

Research Center for Congenital Heart Disease in FuWai Hospital, The Ministry of Health and Department of Cardiovascular Surgery, Cardiovascular Institute and FuWai Hospital, CAMS and PUMC, Beijing 100037, PR China

Received 14 September 2005; received in revised form 15 January 2006; accepted 14 February 2006.

* Corresponding author. Tel.: +86 10 86369800; fax: +86 10 88398496. (Email: dongshi22000{at}yahoo.com.cn).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Cardiopulmonary bypass (CPB) produces an inflammatory response associated with pulmonary dysfunction. P38 mitogen-activated protein kinase (P38MAPK) have been shown to mediate pulmonary inflammatory response after CPB, we examined the effect of SB203580, a specific p38 MAPK inhibitor, on CPB-induced pulmonary inflammatory response. Methods: Sprague–Dawley rats (n = 54) were randomized into three groups (each n = 18): (1) S group, rats underwent sham CPB; (2) CPB group, rats underwent CPB; (3) SB group, rats underwent CPB plus pretreatment with SB203580 (10 mg/kg, i.v., 30 min before CPB). The lung samples were collected after 10 min, 60 min, and 150 min lung reperfusion (each n = 6) in CPB group and SB group, and after 70 min, 120 min, and 210 min observation in S group as the control. Results: The level of lung phospho-I{kappa}B{alpha}, nuclear factor (NF)-{kappa}B activity and activating protein (AP)-1 activity in CPB group was increased than S group. CPB resulted in increased pulmonary tissue tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1ß expression and production, increased pulmonary inflammatory response. The in vivo administration of SB203580 prevented up-regulation of lung-phosphorylated p38 MAP kinase, decreased pulmonary tissue level of proinflammatory cytokines expression and production, and reduced lung inflammation. Conclusions: These findings suggested that (1) p38 MAP kinase activation is one of the important aspects of the signaling event that mediate the release of TNF-{alpha} and IL-1ß and contributes to CPB-induced pulmonary inflammatory response, (2) SB203580 selectively inhibiting p38 MAP kinase activation efficaciously reduces pulmonary inflammatory response after CPB, and (3) p38 MAP kinase influence the activation of NF-{kappa}B in the lung during and after CPB.

Key Words: Cardiopulmonary bypass (CPB) • p38 mitogen-activated protein (MAP) kinase • Tumor necrosis factor-{alpha} • Interleukin-1ß • Pulmonary inflammatory response • Nuclear factor (NF)-{kappa}B


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Lung injury secondary to cardiopulmonary bypass (CPB) is probably caused by a combination of ischemia–repefusion injury and systemic inflammatory response. The mechanisms that mediate lung injury after CPB are likely multiple and include inflammatory elements of leukocyte activation and proinflammatory cytokine release [1]. The p38 kinases are members of the mitogen-activated protein (MAP) kinases, which are important signal transducers. They modulate the production of proinfammatory cytokines (IL-1ß, tumor necrosis factor (TNF)-{alpha}, IL-6, IL-8) and other mediators such as nitric oxide [2,3]. Several studies showed that p38 MAPK be activated during and after CPB [4], but the role of p38 MAPK activation in response to pulmonary inflammatory response after CPB remains unclear. NF-{kappa}B system is another major signaling pathway responsible for proinflammatory cytokine release after cardiopulmonary bypass [5]. Whether p38 MAP kinase can activate NF-{kappa}B directly remains controversial. Many studies suggested that p38 MAP kinase does not activate NF-{kappa}B directly in response to LPS or cytokines [6,7]. But Madrid et al. [8] found that the p38 MAP kinase promoted direct transactivation of NF-{kappa}B through I{kappa}B kinase. The relationship between p38 MAP kinase and NF-{kappa}B system in the proinflammatory cytokine release following cardiopulmonary bypass has not yet been addressed. In this study, we have examined the effect of SB203580, a specific p38 MAP kinase inhibitor, on CPB-induced pulmonary inflammatory response as well as the activation of NF-{kappa}B in rat CPB model, as to characterize the role of p38 MAP kinase in the pathogenesis of CPB-induced pulmonary inflammatory response.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
Adult male Sprague–Dawley rats (250 ± 10 g) were used for all experiments. The animals were obtained from Beijing Weitonglihua Experimental Animal Inc. [Beijing, China, certificate number: SCXK (jing) 2005]. All animals received humane care in compliance with ‘Regulation to the Care and Use of Experimental Animals’ (1996) of the Beijing Council on Animal Care study. The rats were housed and fed at the Animal Center of Beijing fuwai Hospital for at least 7 days before surgery to allow them to adjust to the environment.

2.1 Experimental design
Rats were randomized into three groups: (1) S group, rats underwent sham CPB; (2) CPB group, rats underwent CPB; (3) SB group, rats underwent CPB plus pretreatment with SB203580 [(Alexis Biochemicals, Switzerland) 10 mg/kg SB203580 in 0.5 ml saline intravenously 30 min before the CPB established], C and S groups received 0.5 ml saline only, The relative selectivity of 10 mg/kg of SB203580 to P38 MAPK was shown in previous studies [9]. Animals in the CPB group and SB203580 group underwent CPB for 90 min. After 10 min of CPB, the pulmonary artery was cross-clamped for 50 min, then the cross-clamp was removed, the lung was reperfused for 30 min on CPB, followed by a period of post-CPB reperfusion for 120 min. The sham group was subjected to sternotomy, heparinization, and insertion of all cannula only. Arterial pressure, heart rate, electrocardiogram, oxygen saturation, and temperature were monitored continuously throughout the experiment. Water balance was kept according to Haematocrit, mean arterial pressure, blood level in reservoir, and central venous pressure. During CPB, arterial blood gases were monitored every 30 min and as needed using a blood gas analyzer (AVL Scientific Corp., Roswell, GA, USA), arterial blood gas parameters were maintained according to the following: pH 7.35–7.45, Formula 35–45 mmHg, Formula 100–200 mmHg, and HCO3 – 22–28 mmol/l.

2.2 Surgical procedure
Each animal was anesthetized intraperitoneally with 50 mg/kg of pentobarbital sodium and placed in the supine position. After insertion of a 16-gauge cannula into the trachea, mechanical ventilation was performed, except the period that the pulmonary artery was clamped, by a jianwan animal ventilator (Jianwan, China) with 10 ml/kg of tidal volume, 50 breaths/min of respiratory rate, 100% of inspired oxygen fraction, and a 24-gauge cannula was placed in the caudal artery to monitor arterial blood pressure. For CPB, a 22-gauge cannula for arterial return was introduced into the right femoral artery, a 16-gauge catheter, modified to a multiside-orifices cannula in the forepart, was inserted into the right jugular vein and advanced to the right atrium and ventricule, the blood was drained from the right atrium and ventricule to a 10 ml sterile open reservoir by gravity and siphon following median sternotomy. The CPB circuit comprising a membranous oxygenator (Xijin Medical Co. Ltd, China) [membrane surface area of the oxygenator = 0.04 m2], and double head roller pump (Stöckert, Germany). Priming was composed of 10 ml heparinized homologous blood obtained from a donor rat immediately before the experiments and 5 ml synthetic colloid (HAES-steril), 5% NaHCO3 0.5 ml, 20% mannitol 0.5 ml. During the CPB, maintained the mean arterial pressure above 60 mmHg and the hematocrit about 30–35%. After 300 IU/kg of heparin was intravenously administered, CPB was established and maintained at 120–150 ml/(kg min) of flow rate for 90 min except in the S group rats. Pure oxygen was delivered to the membrane oxygenator at a flow of 50 ml/min adjusted to blood gases. In C and SB groups, when the rectal temperature reduces to 28 °C at 10 min after CPB started, pulmonary artery was cross-clamped for 50 min by a vascular clip and the ventilation was stopped. CPB ceased at 30 min after pulmonary artery declamped and rectal temperature rewarmed to 37°C. The lung was excised immediately after being sacrificed at three time points (reperfusion 10 min, 60 min, and 150 min) after pulmonary artery declamped (each n = 6) in C and SB groups, and after 70 min, 120 min, and 210 min observations (each n = 6) in S group as the control. Rats in which the hemoglobin level declined to less than 6 g/dl at any point were excluded from the study.

2.3 Measurements
2.3.1 Western blot analysis for p38 MAP kinase, phosphorylated p38 MAP kinase and phosphorylated I{kappa}B{alpha}
Lung tissue samples were analyzed for levels of the p38 MAP kinase and the activated, phosphorylated forms of p-I{kappa}B{alpha} and p38 by immunoblotting. Total lysate from lung tissue was obtained by homogenization using a PowerGen 125 Homogenizer (Fischer, Brightwaters, NY, USA) for 30 s on ice in lysis buffer containing 1% NP-40 surfactant, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate, and by centrifugation at 12,000 x g for 10 min at 4 °C to separate solubilized from unsolubilized proteins. The supernatant protein concentration was measured spectrophotometrically at 595 nm wavelength using a BCA protein assay kit (Pierce, USA). Total protein was fractionated by 10% sodium dodocyl sulfate–polyacrylamide gel electropheresis (SDS–PAGE) and electrotransferred to a nitrocellulose membrane. After the blocking procedure, the membrane was then immunoblotted for 2 h with rabbit polyclonal primary antibodies. P38 MAP kinase, phosphorylated p38 MAP kinase, and phosphorylated I{kappa}B{alpha} were probed with a rabbit polyclonal anti-p38 MAP kinase antibody, a rabbit polyclonal anti-phosphorylated p38 MAP kinase antibody, a rabbit polyclonal anti-phosphorylated I{kappa}B{alpha} antibody, and a rabbit polyclonal anti-phospho-c-Jun NH2-terminal kinase (JNK) antibody, respectively. After being washed, the membrane was incubated with horseradish peroxidase-conjugated antirabbit secondary antibody for 1 h at room temperature. The immunoblot was washed; the immunocomplexes then were detected by using chemiluminescence and were exposed to radiograph. Immunoblottings were analyzed after digitalization of X-ray films using a BioSenSC300 (Shanghai Bio-Tech Co. Ltd, China) and NIH Image package (National Institutes of Health, Bethesda, MD, USA). The band density for the target protein in each sample was normalized to ß-actin expression. All antibodies and the chemiluminescent reagents were purchased from Sigma (USA). The values were expressed as fold change from the S group rats in each comparison.

2.3.2 Evaluation of lung TNF-{alpha} and IL-1ß expression and production
The frozen lung tissue was cut into small slices on a block of dry ice and transferred to a prechilled (liquid nitrogen) capsule containing Teflon-coated tungsten ball. The capsule was kept in liquid nitrogen for 2 min and thereafter shaken in a dismembranation apparatus (Retsch KG, Germany) at full speed for 2 min. The procedure was repeated after intermediated freezing in liquid nitrogen until the tissue became powder. Hereafter followed either RNA extraction or measurement of cytokine concentrations.

Changes in levels of lung TNF-{alpha} and IL-1ß mRNA were quantified by real-time PCR. Total RNA was isolated from lung samples using RNeasy Mini kit (Qiagenw, Germany). All RNA samples had an A260/A280 ratio 1.8/2.0 at pH > 7.5. The levels of TNF-{alpha} and IL-1ß mRNA expression were measured by reverse transcriptase polymerase chain reaction (RT-PCR) (Super Scripte Firststrand Synthesis System for RT-PCR, Gibco BRL, USA) and Taq Man Universal PCR Master Mix (Applied Biosystems, USA) at GeneAmp 5700 Sequence Detection System (Applied BioSystems) using specific rat TNF-{alpha} and IL-1ß primer and probe. Results were expressed relative to the amount of GAPDH mRNA present in each specimen and presented as mean ± SD in six individual samples after 150 min reperfusion (each n = 6) in CPB and SB groups, as well as after 210 min observation in S group.

Following the tissue homogenization, 1 ml of phosphate-buffered saline (PBS) was added. The lung tissue homogenates was centrifuged at 12,000 x g for 10 min at 4 °C. The supernatant was used for analysis of tissue cytokine concentration. The content of TNF-{alpha} and IL-1ß in the lung tissue was determined by using a rat TNF-{alpha} and IL-1ß ELISA kit (BioSource, USA). These assays were performed according to the manufacturer's instructions. Results p presented as mean ± SD in six individual samples after 150 min reperfusion (each n = 6) in CPB and SB groups and after 210 min observation in S group.

2.3.3 Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
The EMSA method was used to characterize the binding activities of NF-{kappa}B and activating protein (AP)-1 transcription factors in nuclear extracts. Nuclear extracts from lung tissue were prepared following the method of Gabriel et al. [10]. Briefly, frozen tissues were weighed, transferred to Corex tubes and homogenized in four volumes (w/v) of buffer A containing 0.25 M sucrose, 15 mM Tris–HCl pH 7.9, 60 mM KCl, 15 mM NaCl, 5 mM EDTA, 1 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, and a mixture of protease inhibitors (1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 2 mg/ml leupeptin, and 5 mg/ml aprotinin). After centrifugation at 2000 x g for 10 min, the pellet was resuspended in four volumes of buffer B (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, and protease inhibitors as above), and then centrifuged at 4000 x g for 10 min. The supernatant was discarded and the pellet was resuspended in buffer C containing 0.5 M HEPES pH 7.9, 0.75 mM MgCl2, 0.5 M KCl, 12.5% glycerol, and the protease inhibitors. The homogenate was kept for 30 min at 4 °C under continuous rotary shaking, and then centrifuged at 14,000 x g for 30 min. Finally, the resulting supernatant was dialysed overnight at 48 °C with buffer D (10 mM Tris–HCl pH 7.9, 1 mM EDTA, 5 mM MgCl2, 10 mM KCl, 10% glycerol, and the protease inhibitors). Protein concentrations were determined by the Bradford method.

The binding activities of NF-{kappa}B and activating protein-1 transcription factors bands was detected by ‘The LightShiftTM Chemiluminescent EMSA kit’ (Pierce) that uses a non-isotopic method to detect DNA–protein interactions. Biotin end-labeled DNA duplex of sequence 5'-AGT TGA GGG GAC TTT CCC AGG C-3 and 3'-TCA ACT CCC CTG AAA GGG TCC G-5' containing a putative binding site for nuclear factor {kappa}B, 5'-CGC TTG ATG ACT TGG CCG GAA-3' and 3'-GCG AAC TAC TGA ACC GGC CTT-5' for AP-1 was incubated with the nuclear extracts. After the reaction, the DNA–protein complexes were subjected to a 6% native polyacrylamide gel electrophoresis and transferred to a nylon membrane (Biodyne B membrane also supplied by Pierce). After transfer the membrane was immediately cross-linked for 15 min on a UV transilluminator equipped with 312 nm bulbs. A chemiluminescent detection method utilizing a luminol/enhancer solution and a stable peroxide solution (Pierce) was used as described by the manufacturer, and membranes were exposed to X-ray films for 2–5 min before developing. The bands were scanned with a BioSenSC300, and relative intensities were analyzed by using an NIH Image package. The values were expressed as fold change from the S group in each comparison.

2.3.4 MPO activity
MPO is a mammalian enzyme found in granulocytic leukocytes, and its activity is a reliable marker of tissue neutrophil content. The method described by Mullane et al. [11] was used to measure myeloperoxidase (MPO) activity in the lungs. The lungs were frozen in liquid nitrogen, pulverized, and then homogenized in 10% hexadecyltrimethyl ammonium bromide (HTAB) buffer (0.5% HTAB in 50 mM phosphate buffer, pH 6.0) with a Polytron homogenizer. The homogenate was sonicated on ice for 15 s, frozen at –70 °C, thawed three times, and then centrifuged at 40,000 x g for 15 min. The supernatant was assayed for MPO activity spectrophotometrically. Then, 20 µl of supernatant was combined with 12 µl of 25 mM H2O2, 30 µl of 40 mM o-dianisidine hydrochloride, and 1.938 ml of 50 mM phosphate buffer (pH 6.0). The change in absorbance was measured at 460 nm on a Beckman spectrometer (model 25; Beckman, Fullerton, CA, USA). One unit of MPO activity is defined as the activity degrading 1 µmol of H2O2/min at 25 °C.

2.3.5 Percent lung tissue water
Lung samples were taken at the end time points in three groups, weighed, incubated at 105 °C for 24 h, and then weighed again. The percent tissue water was calculated as ((wet weight – dry weight)/wet weight) x 100.

2.3.6 Tissue preparation for light microscopy
Either at the end of the 150 min reperfusion procedure in SB group and CPB group or after 210 min observation in S group, samples of right upper lobes were fixed in 10% formalin, dehydrated, embedded in paraffin, cut into 5 µm sections, and mounted. After deparaffinization, tissues were stained with hematoxylin and eosin for histologic study.

2.4 Statistical analysis
Data were expressed as mean ± SD. Statistical analysis of the results was performed by analysis of variance (ANOVA). Between groups, variance was determined using the Student–Newman–Keuls post hoc test. We considered p < 0.05 to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Physiological data over the study period
Formula in the CPB and SB groups decreased significantly than that observed in the S groups 60 min, 120 min after CPB, but Formula in the SB group remained higher than that in the S group 120 min after CPB (p < 0.05).

Rats underwent CPB had higher peak airway pressure (P-AwP) than the S group after CPB (p < 0.01). Although mean P-AwP in the CBP group was slightly higher than that observed in the SB groups 60 min, 120 min after CPB, the difference among the two groups was not statistically significant (p = 0.67 and p = 0.054, respectively) (Table 1 ).


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  		Table 1. Physiological data over the study period
 
3.2 Effects of SB203580 on pulmonary p38 MAPK and JNK activity
The total p38 MAPK protein content was similar between three groups. Representative blots are shown in Fig. 1A. Phosphorylated p38 MAP kinase was evidently increased in CPB group compared with the S group (4.69 ± 0.41-fold increase, p < 0.01, 3.01 ± 0.38-fold increase, p < 0.01, 1.39 ± 0.105-fold increase, p < 0.05) at three time points, respectively. Levels of activated p38 reach peak at 10 min after pulmonary artery declamped. Treatment with SB203580 resulted in a decrease in lung-phosphorylated p38 MAP kinase (1.83 ± 0.48 vs 4.69 ± 0.41-fold decrease, SB vs CPB, p < 0.01, 3.01 ± 0.38 vs 1.28 ± 0.15-fold decrease, CPB vs SB, p < 0.01, and 1.39 ± 0.105 vs 1.06 ± 0.29-fold decrease, SB vs CPB, p < 0.05) at 60 min and 150 min after pulmonary artery declamping, respectively, no significant differences were observed in activated p38 levels in the S group at any time point. (Fig. 1C)


Figure 1
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  		Fig. 1. (A) Representative Western blots of P38 MAPK and activated P38 MAPK in three groups. (B) Representative Western blots of JNK in three groups. SB203580 had no significant effect on the CPB-mediated increase in JNK activity. (C) Levels of activated P38 MAPK in lung tissue samples were significantly increased in the CPB group compared with the S group at three time points (** p < 0.01, * p < 0.05 CPB vs S). Treatment with SB203580 showed a 78%, 87%, and 80% reduction of activated P38 MAPK increase in three time points, respectively ( p < 0.01, p < 0.05 SB vs CPB). The values were expressed as fold change over the S group in each comparison. Results were given as mean ± SD of six animals, ANOVA.

 
Fig. 1B shows that, phosphorylated JNK was evidently increased in CPB group compared with the S group (2.69 ± 0.21-fold increase, p < 0.01, 1.81 ± 0.29-fold increase, p < 0.01, 1.99 ± 0.26-fold increase, p < 0.01) at three time points, respectively. Whereas SB203580 had no significant effect on the CPB-mediated increase in JNK activity (2.69 ± 0.21 vs 2.51 ± 0.31, SB vs CPB, p > 0.05, 1.81 ± 0.29 vs 1.76 ± 0.25, CPB vs SB, p > 0.05, and 1.99 ± 0.26 vs 1.82 ± 0.29, SB vs CPB, p > 0.05 at three time points, respectively).

3.3 Activities of NF-{kappa}B and AP-1 in lung tissue
Relative to the S group, there was a significant rise in NF-{kappa}B activation in CPB group, as determined by assay of NF-{kappa}B translocation to the nuclear fraction. Pretreatment with SB203580, however, resulted in a decrease of NF-{kappa}B activation (4.52 ± 0.58 vs 2.96 ± 0.47-fold increase, CPB vs SB, p < 0.01, 4.05 ± 0.61 vs 2.27 ± 0.32, CPB vs SB, p < 0.01, and 2.24 ± 0.51 vs 1.57 ± 0.34-fold increase, CPB vs SB, p < 0.05) at three time points after pulmonary artery declamped, respectively, no significant differences were observed in NF-{kappa}B activation levels in the S group at any time point (Fig. 2A and C).


Figure 2
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  		Fig. 2. Representative EMSA of NF-{kappa}B activation (A) and Western blots of phosphorylated I{kappa}B{alpha} (B) in three groups. (C) Levels of NF-{kappa}B activation in lung tissue samples in three groups (** p < 0.01, CPB vs S). Treatment with SB203580 showed a 46%, 58%, and 54% reduction of NF-{kappa}B activation increase, respectively ( p < 0.01, p < 0.05 SB vs CPB). (D) Levels of phosphorylated I{kappa}B{alpha} in lung tissue samples in three groups (** p < 0.01, CPB vs S). Treatment with SB203580 reduced phosphorylated I{kappa}B{alpha} increase as shown (** p < 0.01, SB vs CPB). The values were expressed as fold change from the S group in each comparison. Results were given as mean ± SD of six animals, ANOVA.

 
Phosphorylation of I{kappa}B{alpha}, which required for NF-{kappa}B activation, was evidently increased in CPB group compared with the S group. Treatment with SB203580 resulted in a decrease in lung-phosphorylated I{kappa}B{alpha} (4.02 ± 0.48 vs 1.86 ± 0.24-fold increase, CPB vs SB, p < 0.01; 2.86 ± 0.56 vs 2.02 ± 0.51, CPB vs SB, p < 0.01; and 1.65 ± 0.295 vs 1.28 ± 0.22, CPB vs SB, p = 0.072) at three time points, respectively. There were no significant differences being observed in phospho-I{kappa}B{alpha} levels in the S group at any time point (Fig. 2B and D).

Compared with the S group, there was also a significant rise of AP-1 activation in CPB group, as determined by assay of AP-1 translocation to the nuclear fraction. Pretreatment with SB203580, however, resulted in a decrease of AP-1 activation (1.698 ± 0.278 vs 1.176 ± 0. 26, CPB vs SB, p < 0.05; 2.69 ± 0.33 vs 1.12 ± 0.29, CPB vs SB, p < 0.01, and 1.90 ± 0.46 vs 1.24 ± 0.18, CPB vs SB, p < 0.01) at three time points, respectively. There were no significant differences being observed in AP-1 activation levels between S group and SB group (Fig. 3A and B).


Figure 3
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  		Fig. 3. Representative EMSA of AP-1 activation in three groups (A). (B) Levels of AP-1 activation in lung tissue samples were significantly increased in the CPB group compared with the S group (** p < 0.01, CPB vs S). Treatment with SB203580 showed a 46%, 58%, and 54% reduction of AP-1 activation increase at three time points, respectively ( p < 0.01 SB vs CPB). The values were expressed as fold change from the S group in each comparison. Results were given as mean ± SD of six animals, ANOVA.

 
3.4 TNF-{alpha} and IL-1ß expression and production
Taqman quantitative real-time PCR analysis was performed to examine the expressions of TNF-{alpha} and IL-1ß mRNA in lung of rats at the end of the time points of each group (each n = 6). TNF-{alpha} and IL-1ß mRNA levels were up-regulated in CPB group comparing with the S group, both of which were significantly decreased by administration with SB203580 (Fig. 4 ).


Figure 4
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  		Fig. 4. Taqman real-time PCR analysis of TNF-{alpha} and IL-1ß mRNA expression in lung of rats at the end time point of each group. The mRNA expression of TNF-{alpha} and IL-1ß in CPB group increased significantly in CPB group in comparison with the S group whereas the values in animals receiving SB203580 were markedly lower. Results are expressed as fold change from the S group in each comparison. ** p < 0.01, CPB compared with S, and p < 0.01, SB compared with CPB. Results were given as mean ± SD of six animals, ANOVA.

 
The concentration of TNF-{alpha} in the lung tissue at the end of the time points of each group (each n = 6) increased significantly (p < 0.01) in the CPB group (17.616 ± 5.887 pg/mg protein) as compared with the S group (5.975 ± 1.835 pg/mg protein). This increase was significantly reduced in the SB group (9.129 ± 2.058 pg/mg protein, SB vs CPB, p < 0:01; Fig. 5 ). The concentration of IL-1ß in the lung tissue at the end time point of each group (each n = 6) increased significantly (p < 0.05) in the CPB group (5.966 ± 2.192 pg/mg protein) as compared with the S group (3.613 ± 1.175 pg/mg protein). This increase was significantly reduced in the SB group (4.120 ± 0.480 pg/mg protein (SB vs CPB, p < 0:05, Fig. 5).


Figure 5
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  		Fig. 5. TNF-{alpha} and IL-1ß concentrations in lung tissue at the end time point of each group. Rats in SB group showed a 73% reduction of TNF-{alpha} increase (** p < 0.01, CPB vs S; p < 0.01, SB vs CPB). Rats in SB group showed a 78.5% reduction of IL-1ß increase (* p < 0.05, CPB vs S group; p < 0.05, SB vs CPB). Results were given as mean ± SD of six animals, ANOVA.

 
3.5 MPO activity
At the end of the time points of each group, lung MPO activity of the CPB group was higher than the S group (0.62 ± 0.04 U/100 mg vs 0.32 ± 0.04 U/100 mg, p < 0.01). Lung MPO activity of the SB group was lower than the CPB group and not different from the S group (0.37 ± 0.05 U/100 mg vs 0.32 ± 0.04 U/100 mg, SB vs S, p > 0.05).

3.6 Percent lung tissue water
The percent tissue water in lung samples was significantly increased in the CPB group compared with the S group (85.3252 ± 1.37449 vs 82.0842 ± 1.68336, CPB vs S, p ≤ 0.01), whereas the values in SB group were significantly lower and similar to the S group (83.3018 ± 1.30791 vs 82.0842 ± 1.68336, SB vs S, p > 0.05; vs CPB 85.3252 ± 1.37449, p < 0.05).

3.7 Morphologic changes
Qualitative light microscopic examination of lungs after 150 min reperfusion in SB group and CPB group and after 210 min observation in S group. In CPB group polymorphonuclear leukocytes were observed in peribronchovascular capillaries, the alveolar walls were thickened and infiltrated with numerous inflammatory cells, and severe intra-alveolar hemorrhage was often observed. While treatment with SB203580 showed a well-preserved pulmonary structure, the alveolar walls were thin, with only mild peribronchovascular edema and intra-alveolar inflammatory cell infiltration (Fig. 6 ).


Figure 6
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  		Fig. 6. Histopathologic appearance of the pulmonary tissue. S group (A), interstitial and alveolar edema along with numerous inflammatory cell infiltration was observed in the CPB group (C), whereas only slight interstitial and alveolar edema along with inflammatory cells infiltration was observed in the SB group (B) (original magnification 200x).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
CPB-induced pulmonary inflammation after major cardiac surgery is thought to be associated with systemic inflammatory response (SIR) and pulmonary ischemia/reperfusion (I/R) injury. Proinflammatory cytokines secreted by monocytes and other cell types are known to be the key mediators of both pathologic mechanisms. The release of cytokines can be stimulated by a number of factors including ischemia–reperfusion, CPB, complement activation, endotoxin release, and the effect of other cytokines [12,13]. Recently, attention has focused on the p38 MAPK due to their consistent activation by proinflammatory cytokines and to their role in nuclear signaling. The p38 subfamily of MAPKs consists of four isoforms ({alpha}, ß, {gamma} and {delta}), {alpha} and ß are 75% homologous, and {gamma} and {delta}, which are more distant relatives, only p38{alpha} and p38ß have been detected in the lung tissue [14]. The p38 family has been particularly intense following the discovery that p38 inhibitors are anti-inflammatory and anti-ischemia/reperfusion injury in vivo models [15,16]. However, the role and the contribution of p38 kinase in CPB-induced lung inflammation are less clear. The present study uses a rat CPB model to ascertain that P38 MAP kinase activation is one important aspect of the signaling event that may mediate the activation of AP-1 and NF-{kappa}B, subsequent expression and production cytokines in postpump lung.

The lung is an important source of inflammatory cytokines as well as the target organ for the effects of these mediators. CPB and I/R injury induced excess proinflammatory cytokine release in conjunction with lung inflammatory response [17,18]. Moreover, some authors have reported that proinflammatory cytokines were secreted in the lung in cases of I/R injury to other organs [19]. CPB-induced lung inflammatory response is thought to result from an intense local and systemic inflammatory response involving cellular (neutrophil, endothelial cell, and monocyte) activation and elaboration of inflammatory mediators including cytokines, chemokines, and adhesion molecules. These inflammatory mediators in turn are capable of further activating multiple cell types and thus propagating an inflammatory response. P38 MAP kinase inhibition has recently gained much attention as a new target for pulmonary protection from ischemia–reperfusion injury and inflammatory response [8,20]. Both in vivo and in vitro studies have documented that SB203580 is a specific p38 MAP kinase inhibitor which has no effect on the other members of MAP kinase family such as ERK1/2, JNK, and ERK5 [14,21], although recently SB203580 was shown to inhibit JNK in rat neonatal ventricular myocytes [22]. However, we chose to examine the effects of SB203580 on JNK activity in our model of CPB. The CPB produced significant JNK activation during and after CPB, whereas this activity was not inhibited by in vivo administration of SB203580 (10 mg/kg), confirming the selectivity of this inhibitor for p38 MAPK. Our results indicate that in murine lung subjected to CPB, P38 MAPK and inflammatory transcription factors become activated, resulting in elaboration of multiple inflammatory mediators and inflammatory cell infiltration in pulmonary tissue. The in vivo administration of SB203580 prevented up-regulation of lung-phosphorylated p38 MAP kinase, decreased pulmonary tissue levels of proinflammatory cytokines, MPO, water content and inflammatory cell infiltration, improved lung histologic changes. These findings suggest that there is a direct correlation between activation of p38 MAP kinase and CPB-induced lung inflammation.

Because TNF-{alpha} and IL-1ß mediate the postpump lung inflammatory reaction [23], it was reasonable to expect that inhibiting one aspect of the signal transduction pathway that regulates TNF-{alpha} and IL-1ß transcription and translation would provide a measure of postpump lung protection. It is well accepted that the production of proinflammatory cytokines, such as TNF-{alpha}, IL-1ß, was regulated through activation of p38 MAP kinase. In this study, we found that the p38 MAP kinase specific inhibitor SB203580 treatment resulted in decreasing the levels of TNF-{alpha} and IL-1ß expressions and productions in pulmonary tissue, concomitant with the decrease of lung inflammation. These results suggest that the p38 MAP kinase mediates the production of TNF-{alpha} and IL-1ß in the lung and contributes to postpump lung inflammatory reaction.

P38MAP kinase is activated by dual phosphorylation on tyrosine and threonine in response to extracellular stimuli [14]. Once activated, p38 is translocated to the nucleus, where it phosphorylates and activates different transcription factors and transactivates target genes. A key component of the proinflammatory cytokines transcriptional machinery involves the transcription factor AP-1. The gene of proinflammatory cytokines such as TNF-{alpha} and IL-1ß contains a number of AP-1-like binding sites within its promoter region [24]. AP-1 is a two-subunit DNA-binding protein composed of heterodimers or homodimers of the c-Fos and c-Jun proto-oncogene families; c-Jun transcription can be initiated by MEF2C or by the dimmer ATF-2-c-Jun, whereas Elk-1 initiates transcription of c-Fos [14]. MEF2C, ATF-2, and Elk-1 are all substrates of p38 MAP kinase, suggesting that p38 MAP kinase play an important role in the activation of AP-1. Our results that AP-1 was activated in the lung after CPB and this activation was abolished by administration with SB203580, the p38 MAP kinase selective inhibitor, confirmed the role for p38 MAP kinase in the activation of AP-1.

Besides the p38 MAP kinase, NF-{kappa}B system is another major signaling pathway responsible for the proinflammatory cytokine expressions. NF-{kappa}B activation is involved in CPB-induced lung inflammation. However, these previous results may not be directly extrapolated to the relation between MAPKs and NF-{kappa}B in CPB-induced acute lung injury, and therefore the roles of P38 MAPK remain to be elucidated in these clinically relevant pathophysiologic conditions. In this study, we found that the prospho-I{kappa}B{alpha} was markedly increased and NF-{kappa}B was significantly activated in the lungs of rats after CPB, concomitant with the increase of proinflammatory cytokine production and lung inflammatory reaction. The role of NF-{kappa}B system in the postpump proinflammatory cytokine release and CPB-induced acute injury in lung remains to be further studied. However, there are few reports regarding the correlation of p38 MAP kinase with the activation of NF-{kappa}B. In the present study, we examined the effect of SB203580 on the I{kappa}B{alpha} phosphorylation and the NF-{kappa}B DNA binding so as to characterize whether p38 MAP kinase was required for NF-{kappa}B activation in lung during and after CPB. Our results showed that both the increase of prospho-I{kappa}B{alpha} and NF-{kappa}B DNA binding were affected by in vivo administration with SB203580. These results provided a preliminary evidence that p38 MAP kinase influence NF-{kappa}B activation in the lung during and after CPB. The precise inter-relationship between the activation of p38 MAP kinase and activation of the transcription factor NF-{kappa}B activation after CPB in lung is warranted to be further assessed.

In summary, we can conclude that (1) p38 MAP kinase activation is one important aspect of the signaling event that may mediate the release of TNF-{alpha} and IL-1ß and contributes to CPB-induced lung inflammation, (2) SB203580 selectively inhibiting p38 MAP kinase activation efficaciously reduces lung inflammation after CPB, and (3) p38 MAP kinase influences the activation of NF-{kappa}B in the lung during and after CPB.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Edmunds Jr. LH. Inflammatory response to cardiopulmonary bypass. Ann Thorac Surg 1998;66(5 Suppl.):S12[discussion S25. 3. Lee JC, Young PR (1996)].[Abstract/Free Full Text]
  2. Lee JC, Young PR. Role of CSBP/p38/RK stress response kinase in LPS and cytokine signaling mechanisms. J Leukoc Biol 1996;59:152-157.[Abstract]
  3. Matsumoto K, Hashimoto S, Gon Y, Nakayama T, Horie T. Proinfammatory cytokine-induced and chemical mediator-induced IL-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase-dependent pathway. J Allergy Clin Immunol 1998;101:825-831.[CrossRef][Medline]
  4. Khan TA, Bianchi C, Araujo EG, Ruel M, Voisine P, Sellke FW. Activation of pulmonary mitogen-activated protein kinases during cardiopulmonary bypass. J Surg Res 2003;115(1):56-62.[Medline]
  5. Khan TA, Bianchi C, Ruel M, Voisine P, Li J, Liddicoat JR, Sellke FW. Mitogen-activated protein kinase inhibition and cardioplegia-cardiopulmonary bypass reduce coronary myogenic tone. Circulation 2003;108(Suppl. 1):II348-II353.
  6. Yan W, Zhao K, Jiang Y, Huang Q, Wang J, Kan W, Wang S. Role of p38 MAPK in ICAM-1expression of vascular endothelial cells induced by lipopolysaccharide. Shock 2002;17:433-438.[CrossRef][Medline]
  7. Parhar K, Ray A, Steinbrecher U, Nelson C, Salh B. The p38 mitogen-activated protein kinase regulates interleukin-1 beta-induced IL-8 expression via an effect on the IL-8 promoter in intestinal epithelial cells. Immunology 2003;108:502-512.[CrossRef][Medline]
  8. Madrid LV, Mayo MW, Reuther JY, Baldwin Jr. AS. Akt stimulates the transactivation potential of the RelA/p65 subunit of NF-kappa B through utilization of the Ikappa B kinase and activation of the mitogen-activated protein kinase p38. J Biol Chem 2001;276:18934-18940.[Abstract/Free Full Text]
  9. Chen XL, Xia ZF, Yu YX, Wei D, Wang CR, Ben DF. p38 mitogen-activated protein kinase inhibition attenuates burn-induced liver injury in rats. Burns 2005;31(3):320-330[Epub 2005 January 21].[Medline]
  10. Gabriel C, Justicia C, Camins A, Planas AM. Activation of nuclear factor kappa B in the rat brain after transient focal ischemia. Brain Res Mol Brain Res 1999;65:61-69.[Medline]
  11. Mullane KM, Kraemer R, Smith B. Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration in ischemic myocardium. J Pharmacol Methods 1985;14:157-167.[CrossRef][Medline]
  12. Wan S, LeClerc JL, Vincent JL. Cytokine responses to cardiopulmonary bypass: lessons learned from cardiac transplantation. Ann Thorac Surg 1997;63:269-276.[Abstract/Free Full Text]
  13. Fong Y, Moldawer LL, Marano M, Wei H, Tatter SB, Clarick RH, Santhanam U, Sherris D, May LT, Sehgal PB. Endotoxemia elicits increased circulating beta 2-IFN/IL-6 in man. J Immunol 1989;142:2321-2324.[Abstract]
  14. Ono K, Han J. The p38 signal transduction pathway: activation and function. Cell Signal 2000;12:1-13.[CrossRef][Medline]
  15. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 1994;372:739-746.[CrossRef][Medline]
  16. Kobayashi M, Takeyoshi I, Yoshinari D, Matsumoto K, Morishita Y. P38 mitogen-activated protein kinase inhibition attenuates ischemia-reperfusion injury of the rat liver. Surgery 2002;131(3):344-349.[CrossRef][Medline]
  17. Serrick C, La Franchesca S, Giaid A, Shennib H. Cytokineinterleukin-2, tumor necrosis factor-alpha, and interferongamma release after ischemia/reperfusion injury in a novel lung autograft animal model. Am J Respir Crit Care Med 1995;152:277-282.[Abstract]
  18. Kamoshita N, Takeyoshi I, Ohwada S, Iino Y, Morishita Y. The effect of FR167653 on pulmonary ischemia-reperfusion injury in dogs. J Heart Lung Transplant 1997;16:1062-1072.[Medline]
  19. Carrick JB, Martins Jr. O, Snider CC, Means ND, Enderson DL, Frame SB, Morris SA, Karlstad MD. The effect of LPS on cytokine synthesis and lung neutrophil in flux after hepatic ischemia/reperfusion injury in the rat. J Surg Res 1997;68:16-23.[CrossRef][Medline]
  20. Nick JA, Young SK, Brown KK, Avdi NJ, Arndt PG, Suratt BT, Janes MS, Henson PM, Worthen GS. Role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation. J Immunol 2000;164(4):2151-2159.[Abstract/Free Full Text]
  21. Ballard-Croft C, White DJ, Maass DL, Hybki DP, Horton JW. Role of p38 mitogen-activated protein kinase in cardiac myocyte secretion of the inflammatory cytokine TNF-alpha. Am J Physiol Heart Circ Physiol 2001;280:H1970-H1981.[Abstract/Free Full Text]
  22. Clerk A, Sugden PH. The p38 MAPK inhibitor, SB203580, inhibits cardiac stress-activated protein kinases/C-jun N-terminal kinases (SAPKs/JNKs). FEBS Lett 1998;426:93-96.[CrossRef][Medline]
  23. Nieman G, Searles B, Carney D, McCann U, Schiller H, Lutz C, Finck C, Gatto LA, Hodell M, Picone A. Systemic inflammation induced by cardiopulmonary bypass: a review of pathogenesis and treatment. J Extra Corpor Technol 1999;31(4):202-210.[Medline]
  24. Eder J. Tumour necrosis factor alpha and interleukin-1 signalling: do MAPKK kinases connect it all?. Trends Pharmacol Sci 1997;18:312-319.[Medline]



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