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Address correspondence to Jianguo Xu, M.D, Department of Anesthesiology, Jinling Hospital, 305 East Zhongshan Road, Nanjing 210002, P. R. China; tel 86 25 8480 6839; fax 86 25 8480 6839; e-mail: nulvran{at}yahoo.com.cn.
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Abstract |
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, interleukin (IL)-1ß, and IL-6), mRNA expressions (cytokine-induced neutrophil chemoattractant (CINC), P-selectin, CD11b/CD18 (Mac-1), and intercellular adhesion molecule-1 (ICAM-1)), and activities of nuclear factor (NF)-
B and activator protein (AP)-1 were determined in each group. HES, in a dose-related manner, significantly reduced pulmonary capillary permeability in the CLP model of sepsis. HES also down-regulated pulmonary proinflammatory cytokines (TNF-, IL-1ß, and IL-6) and mRNA expressions (CINC and P-selectin), and inhibited pulmonary activities of NF-B and AP-1. The results suggest that during sepsis HES reduces pulmonary capillary permeability and this beneficial effect of HES may act through down-regulation of inflammatory mediators and suppression of NF-B and AP-1 activation. (received 6 November 2004; accepted 22 December 2004)
Keywords: hydroxyethyl starch, sepsis, inflammatory mediators, NF-B, AP-1, capillary permeability
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementReferences |
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, interleukin (IL)-1ß, and IL-6), chemokines (eg, cytokine-induced neutrophil chemoattractant (CINC)) and adhesion molecules (eg, P-selectin, CD11b/CD18 (Mac-1), and intercellular adhesion molecule-1 (ICAM-1)). Hydroxyethyl starch (HES) is a colloidal, synthetically modified polymer of amylopectin, a waxy starch derived from maize or sorghum. Clinically, HES is frequently used for volume replacement to maintain or improve tissue perfusion in patients with sepsis, trauma, shock, or surgical stress [2,3]. In addition to its effect on maintenance of stability of hemodynamic parameters, recent studies have shown that HES may reduce capillary leakage in many pathological conditions that increase capillary permeability [4,5]. Previous studies [6,7] in our laboratory demonstrated that during endotoxemia HES may induce down-regulation of inflammatory mediators in lung and therefore ameliorate pulmonary microvascular permeability. To extend application of HES to the ARDS/ALI caused by sepsis, it is necessary to examine the effects of HES on other animal models of sepsis.
Compared to endotoxin administration, the cecal ligation and puncture (CLP) model of sepsis produces delayed and prolonged inflammatory mediator expression. An additional important difference is that a specific focus of inflammation does not exist in the endotoxin model, which may not be completely representative of a typical episode of clinical human sepsis. In the CLP model, however, the subsequent response to the cecal products, which leak into the abdomen, resembles that described for septic patients [8].
Given that events that result in inflammatory mediator expression all involve the activation of transcription factors, such as nuclear factor (NF)-B and activator protein (AP)-1 [9,10], we used the rat CLP model in the present study to determine whether or not HES reduces pulmonary capillary permeability through down-regulation of inflammatory mediators (TNF-, IL-1ß, IL-6, CINC, P-selectin, Mac-1, and ICAM-1) and inhibits activation of transcription factors (NF-B, AP-1).
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Materials and Methods |
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Experimental protocol. The rats were randomly assigned to 6 groups (6 rats/group): (a) saline controls; (b) CLP alone; (c, d, and e) CLP plus HES (7.5, 15, or 30 ml/kg); and (f) HES alone (30 ml/ kg). Immediately after the baseline parameters were monitored (0 min), sepsis was induced by cecal ligation with a single, 18-gauge puncture, as previously described [11]. In brief, after a 2-cm midline incision, the cecum was exposed, ligated just distal to the ileocecal valve to avoid intestinal obstruction, and punctured once with an 18-gauge needle. The punctured cecum was squeezed to expel a small amount of feces, and the abdominal incision was closed in two layers. The rats in the saline control and the HES-alone groups underwent the same surgical procedure except that the cecum was neither ligated nor punctured. These surgical procedures were all finished within 10 min.
To test the influence of different solutions on CLP-induced changes, rats in the CLP and HES (7.5, 15, or 30 ml/kg) and CLP-alone groups were treated at 3 hr after CLP with HES (7.5, 15, or 30 ml/kg) (hydroxyethyl starch, medium molecular weight, low degree of substitution; HAES-steril 200/ 0.5, 6%, Fresenius Kabi, Germany) and 0.9% NaCl solution (30 ml/kg) via the right jugular vein, respectively. The rate of infusion was 0.2 ml/min. Similarly, HES (30 ml/kg) and saline (30 ml/kg) were infused, respectively, in the HES-alone and saline control groups.
The animals were allowed to awaken and were given free access to water, but denied food. At timed intervals, the rats were reanesthetized with 2% sodium pentobarbital in saline (40 mg/kg, ip; Sigma, USA). The lungs were collected either at 5 hr after CLP for determination of cytokine levels (TNF-, IL-1ß, and IL-6), mRNA expressions (CINC, P-selectin, Mac-1, and ICAM-1), and transcription factor activities (NF-B and AP-1), or at 12 hr after CLP for determination of the lung wet/dry weight ratio and pulmonary capillary permeability.
Lung wet/dry weight ratio and pulmonary capillary permeability. The lung wet/dry weight ratio was assessed as described by Tian et al [7]. Pulmonary capillary permeability was determined with the Evans blue dye extravasation method. Rats were injected with Evans blue (20 mg/kg; Sigma) via the right jugular vein at 15 min before killing. The lung tissues were excised and weighed. The dye was then extracted from the tissue by incubation with 4 ml of formamide for 24 hr at 37°C. The absorbance of the fluid with dye was measured at 620 nm. The total amount of dye was calculated by means of a standard calibration curve. Results were expressed as µg/g of wet tissue.
Enzyme-linked immunosorbent assay (ELISA).
The pulmonary levels of proinflammatory cytokines were quantified using ELISA kits specific for rat cytokines according to the manufacturer’s instructions (TNF- from Diaclone Research, France; IL-1ß and IL-6 from Biosource Europe SA, Belgium). Values were expressed as pg/mg protein.
RNA isolation and cDNA synthesis. The tissue samples were homogenized in TRIzol reagent (Roche Molecular Biochemicals, USA). Total RNA was extracted from the tissue according to the manufacturer’s protocol. Total RNA concentration was determined from spectrophotometric absorbance measurements (260 and 280 nm). For each sample tested, the ratio between the spectro-photometric readings at 260 nm and 280 nm (A260/ A280) was used to estimate the purity of the nucleic acid, and the ratio in all samples ranged between 1.7 and 2.0.
Reverse transcriptase reactions were then carried out using the Reverse Transcription System Kit (Promega, USA). Each reaction tube contained 1 µg of total RNA in a volume of 20 µl containing 5 mmol/L MgCl2, 1x Reverse Transcription Buffer, 1mmol/L of each dNTP, 1 U/µl of RNase inhibitor, 15 U/µg of AMV Reverse Transcriptase, 0.025 µg/ µl of Oligo(dT)15 Primer, and DEPC-treated water to volume. Reverse transcriptase reactions were carried out in a DNA Thermal Cycler (MiniCycler PTC 150, MJ Research, USA) at 42°C for 60 min and 95°C for 5 min. The cDNA was then stored at -20°C.
Semiquantitative PCR.
PCR was performed using 0.5 U of Taq-polymerase (Promega), 0.005 µmol dNTP, and 50 pmol of each primer (Sangon Co., Shanghai, China) in a total volume of 25 µl in a DNA Thermal Cycler. The primers, cycle numbers, and amounts of cDNA used are listed in Table 1
. Each PCR cycle consisted of 30 sec at 95°C, 30 sec at 58°C, and 60 sec at 72°C. The PCR products were electrophoresed on a 2% agarose gel stained with ethidium bromide. The gel was captured as a digital image and analyzed using Scion Image software (Scion, USA). The relative levels of cytokine mRNAs were normalized to ß-actin transcript from the same reaction.
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B and 5'-CGCTTGATGAGTCAGCCGGAA-3' for AP-1) were labeled with [
-32P] ATP (Free Biotech, Beijing, China) with T4 polynucleotide kinase. Equal amounts of nuclear extract (80 µg) were added to 9 µl of gel shift binding buffer (Tris-HCl 10 mmol/L (pH 7.5), NaCl 50 mmol/L, EDTA 0.5 mmol/L, MgCl2 1 mmol/L, DTT 0.5 mmol/L, 4% glycerol, 0.05 g/L Poly-dIdC) (15 min, room temperature). The mixture was incubated for 30 min with 1 µl of the 32P-labelled oligonucleotide probe. Then, 1 µl of loading buffer was added to stop the reaction and the sample was electrophoresed in a 4% polyacrylamide gel. The dried gel was exposed to X-ray film (Fuji Hyperfilm) at -70°C. The intensity of the NF-B or AP-1 complex was quantified by densitometry. Survival study. In additional groups of anaesthetized animals, HES (7.5, 15, or 30 ml/kg) or saline (30 ml/kg) were infused intravenously in 15 rats at 3 hr after CLP operation, respectively. Then the animals were monitored for 72 hr to record times of death.
Statistical analysis. All data were expressed as mean ± SD. The groups were compared using the Kruskal-Wallis test, followed by the Tukey test for multiple comparisons when appropriate. Comparisons between groups were performed using the Mann-Whitney Rank Sum test. Significant differences within the respective groups between the different time points to baseline were tested by the paired Student t test. In addition, the Kaplan-Meier method was used to compare survival rates. Differences were considered to be statistically significant if p was <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementReferences |
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). The single 18-gauge puncture model did not induce significant changes in HR and MAP over time. Accordingly, there were no significant differences between the experimental groups at the time points studied (Table 2).
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) and the pulmonary microvascular permeability (Fig. 2) were markedly increased at 12 hr after CLP. Treatment with HES in CLP rats significantly diminished these increased values. At 15 ml/kg, HES caused more reduction of capillary permeability than at the other two dosages.
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(Fig. 3A), IL-1ß (Fig. 3B), and IL-6 (Fig. 3C) at 5 hr after the CLP operation. The CLP-induced increases in these cytokines were significantly blunted in rats treated with HES. The effect of HES on down-regulation of proinflammatory cytokines was dose-dependent. The levels of TNF-, IL-1ß, and IL-6 did not differ between the saline control and HES-alone groups.
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), P-selectin (Fig. 4B), Mac-1 (Fig. 4C), and ICAM-1 (Fig. 4D) were markedly increased at 5 hr after CLP. Infusion of HES in septic rats significantly reduced the increases of CINC and P-selectin mRNA expression, but not affect the changes in Mac-1 and ICAM-1 mRNA expression. The effects of HES in CLP rats on inhibition of CINC and P-selectin mRNA expression were dose-related, and the maximal inhibition was observed at 15 ml/kg.
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B and AP-1.
Compared with saline control and HES-alone, the CLP model activated NF-B and AP-1 in the rat lung significantly at 5 hr after the operation (Fig. 5). Activities of these 2 transcription factors in CLP rats infused with HES decreased markedly. However, the effect of HES on inhibition of NF-B activation was dose-dependent and the effect on inhibition of the AP-1 activation was dose-related.
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B or AP-1) oligonucleotide (specific competitor), or unlabeled SP1 oligonucleotide (nonspecific competitor), was added to the binding reaction 10 min before the addition of radiolabeled probe using Hela nuclear extract (Fig. 6).
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Discussion |
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B and AP-1 activations. Early pathologic changes in ALI/ARDS include pulmonary neutrophil sequestration with subsequent injury to the alveolar-capillary barrier, leading to increased pulmonary vascular permeability, progressive lung inflammation, and edema [13,14]. Normally, small amounts of plasma proteins reach the alveolar epithelial surface by a size-selective process that restricts the passage of very large molecules. However, the size-selectivity of plasma proteins has been found to be compromised in the lungs of ARDS patients [15]. In this study, we used the Evans blue dye extravasation method to study lung protein permeability. At 12 hr after the CLP, the protein permeability in the lung increased markedly. In contrast, treatment with HES reduced the increased pulmonary capillary permeability. In addition, the action of HES on the lung wet/dry weight ratio (another index of tissue microvascular permeability) further underlined the beneficial effect of HES on reducing pulmonary capillary permeability.
These findings provide confirmation of our previous study [7], which showed that HES reduces LPS-induced increases in pulmonary capillary permeability. Moreover, several other studies have demonstrated such beneficial effects of HES during ischemia. For example, Kaplan [4], using the asphyxial model, confirmed that HES attenuates ischemia-induced increase in vascular permeability.
Neutrophil sequestration within the area of inflammation is a multiple process that begins with neutrophil activation and attraction by chemo-attractant factors (eg, CINC) as well as adhesion to the endothelium through adhesion molecules (eg, P-selectin, Mac-1, and ICAM-1). Finally, activated cells migrate across the endothelial barrier and accumulate in surrounding tissue [9]. Thus, chemokines and adhesion molecules play an important role in the development of ALI/ARDS.
In the present study, we found that HES inhibited the expression of CINC and P-selectin mRNAs, although no effects on Mac-1 and ICAM-1 mRNAs were observed. This suggests that the effect of HES on reducing pulmonary capillary permeability may be through inhibition of endothelial cell activation and neutrophil adhesion. Our results are consistent with an in vitro study [16], which demonstrated that HES can inhibit the rapid expression of P-selectin, but not affect E-selectin expression on LPS-stimulated endothelial cells or Mac-1 expression on neutrophils. P-selectin, which is expressed on the endo-thelium, can interact with counter-receptors on neutrophils, namely, P-selectin glycoprotein-1. This interaction leads to low-affinity binding between neutrophils and endothelial cells, which has been characterized as "rolling" of neutrophils. Other adhesion molecules, such as Mac-1 and ICAM-1, represent high affinity binding interactions that result in cessation of lateral neutrophil movement [9]. The different effects of HES on adhesion molecules in this study indicate the selectivity of HES in inhibiting pulmonary inflammatory mediators.
Although neutrophils have received much attention as a key component of the common pathway underlying ARDS, proinflammatory cytokines play a role in the inflammatory response through neutrophil influx and activation. In this study, we evaluated the effects of HES on reducing inflammatory cytokines by assessing the pulmonary levels of TNF-, IL-1ß, and IL-6. The results showed that in CLP-induced sepsis HES down-regulated pulmonary proinflammatory cytokines, which may contribute to the beneficial effect of HES on reducing pulmonary capillary permeability.
In sepsis, NF-B appears to be a particularly important transcription factor. After being activated extracellularly, it binds to the promoter region of inflammatory genes to increase their rates of transcription [17,18]. Several studies have also implicated the NF-B pathway in the pathogenesis of ALI/ARDS [19]. Based on these observations, we postulated that, in the CLP model of sepsis, HES might inhibit the inflammatory mediators and reduce pulmonary capillary permeability through the NF-B signaling pathway. Our findings that HES reduced pulmonary NF-B activity and deceased pulmonary inflammatory mediator levels or expressions have confirmed that hypothesis.
To increase the clinical relevance of the experimental protocol, we chose 3 doses of HES (7.5, 15, and 30 ml/kg). Interestingly, we found that the effects of HES on down-regulating proinflam-matory cytokines (TNF-, IL-1ß and IL-6) are dose-dependent and the effects of HES on reducing pulmonary capillary permeability and inhibiting expression of CINC and P-selectin mRNAs are dose-related. HES at 30 ml/kg caused less inhibition of expression of CINC and P-selectin mRNAs than 15 ml/kg HES, although the higher dose produced greater inhibition of NF-B activation.
The dissociation between inhibition of NF-B activation and CINC and P-selectin mRNAs expression may be explained by involvement of other transcription factors, such as AP-1. Compared with HES (15 ml/kg), HES (30 ml/kg) induced less inhibition of AP-1 activation. Thus, it is likely that HES (30 ml/kg), in contrast to HES (15 ml/kg), inhibited the NF-B-mediated CINC and P-selectin mRNAs expression but augmented AP-1-mediated transcription of the CINC and P-selectin genes; the inhibitory effect of HES (30 ml/kg) mediated through NF-B inhibition was consequently offset by the AP-1-mediated stimulatory effect. In addition to these 2 transcription factors, other proinflam-matory signaling pathways should be studied.
To exclude influences of macrohemodynamics in this study, we chose a single 18-gauge puncture model, which is known to produce a moderate insult without the development of shock [11]. The data showed that mortality induced by this CLP model before 72 hr was infrequent and there were no significant differences in macrohemodynamic parameters between the experimental groups at the different time points studied. This implies that HES may exert its anti-inflammatory effect on reducing pulmonary capillary permeability in sepsis independent of its actions on macrohemodynamics.
Although fluid therapy increases hydrostatic pressures in the lungs and promotes fluid filtration and edema formation, patients with sepsis/septic shock, especially associated with hypovolemia, still need fluid resuscitation. Currently, HES is often used for volume support during septic disorders. Our results illustrate that in rats with sepsis secondary to CLP, HES may play a beneficial role in reducing pulmonary capillary permeability in parallel with down-regulating inflammatory mediators. This anti-inflammatory action of HES may have important clinical implications, particularly as an adjunct to fluid resuscitation and may shed light on the clinical applications of HES in sepsis with ALI/ARDS.
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Acknowledgement |
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