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Eur J Cardiothorac Surg 2004;25:530-536
© 2004 Elsevier Science NL
ligand, on lung ischemia-reperfusion injury in rats
a Department of Cardiovascular and Thoracic Surgery, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan
b Department of Internal Medicine, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan
Received 18 June 2003; received in revised form 29 November 2003; accepted 14 December 2003.
* Corresponding author. Tel.: +81-75-251-5752; fax: +81-75-257-5910
e-mail: kazuitoh{at}koto.kpu-m.ac.jp
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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(PPAR-) belongs to the nuclear receptor superfamily and has an anti-inflammatory effect by preventing the activation of transcription factors such as nuclear factor-
B (NF-B). NF-B regulates the expression of many genes of early response products in the development of acute inflammation. We examined the effects of pioglitazone, a synthetic ligand of PPAR-, against lung ischemia-reperfusion injury in rats. Methods: The left lungs of male Wistar rats were rendered ischemic for 90 min and then reperfused for 2 h. Treated animals received pioglitazone (10 mg/kg) 2 h before induction of ischemia. Lung injury was quantified in terms of lung microvascular permeability (Evans blue dye extravasation), tissue lipid peroxidation (thiobarbituric acid reactive substances), and tissue polymorphonuclear leukocyte accumulation (myeloperoxidase activity). The tissue concentrations of tumor necrosis factor-
(TNF-) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were also measured. Statistical analyses were performed by one-way analysis of variance, followed by Sheffe's multiple comparison test. Results: The lung vascular permeability in pioglitazone-treated animals was reduced by 55% of the increase of Evans blue dye extravasation relative to control animals (P=0.003). The protective effects of pioglitazone treatment were correlated with the reduction by 79% of the increase of thiobarbituric acid reactive substances (P=0.045) and the reduction by 58% of myeloperoxidase activity increase (P<0.001). The production of TNF- was reduced by 63% of the increase (P<0.001) and the reduction of CINC-1 was 45% (P<0.001). Pioglitazone did not affect the lung in the sham animals. Conclusions: Pioglitazone treatment before ischemia attenuated lung ischemia-reperfusion injury in rats. The mechanism of these protective effects involves inhibition of the production of proinflammatory cytokines, polymorphonuclear leukocyte accumulation, and tissue lipid peroxidation, resulting in reduced lung edema.
Key Words: Ischemia-reperfusion injury • Lung • Pioglitazone • Peroxisome proliferator-activated receptor-
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Thiazolidinediones, such as pioglitazone, troglitazone, and rosiglitazone, are oral anti-diabetic drugs which exert their insulin sensitizing action by stimulating nuclear transcriptional factor peroxisome proliferator-activated receptor- (PPAR-). PPAR- is a member of the nuclear hormone receptor superfamily of ligand-activated transcriptional factors that are related to retinoid, steroid, and thyroid hormone receptors [2]. PPAR- is also involved in the control of inflammation, in particular, in modulating the production of inflammatory mediators by preventing the activation of transcription factors, such as nuclear factor-B (NF-B), signal transducer and activators of transcription (STAT), and activation protein-1 (AP-1) [3,4]. Recent studies have demonstrated that the transcription factor NF-B regulates the expression of many genes of early response products in the development of acute inflammation [5]. The inhibition of NF-B activation attenuates I/R injury in the heart [6], liver [7], and lung [8].
Pioglitazone, (±)-5-[4-[2-(5-ethyl-2-pyridyl) ethoxy] benzyl] thiazolidine-2,4-dione monohydrochloride (C19H20N2O3S·HCl), is a synthetic ligand of PPAR- and has been clinically used to treat patients with type 2 diabetes. In addition to its insulin-sensitizing effect, pioglitazone is reported to ameliorate I/R-induced intestinal damage [9], gastric mucosal damage [10], and reduce myocardial infarction size [11] when administered before ischemia. However, the effects of pioglitazone associated with I/R-induced lung injury have not yet been investigated. We hypothesized that PPAR- activation inhibits proinflammatory cytokine expression by antagonizing the activities of NF-B, which results in reduced I/R-induced lung injury. In the present study, lung injury was induced by I/R in rats to evaluate the effects of the PPAR- ligand pioglitazone by measuring the changes in pulmonary microvascular permeability, lipid peroxidation, tissue-associated polymorphonuclear leukocyte accumulation, and proinflammatory cytokine production.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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2.1. Experimental protocol
Animals were anesthetized with 50 mg/kg of intraperitoneally administered sodium pentobarbital (Nembutal®, Dainippon Pharmaceutical Co., Osaka, Japan). A 14-gauge angiocatheter was inserted into the trachea through a midline neck incision and secured with a 2–0 braided suture. Animals were then placed on a ventilator (model SN480-7, Shinano, Tokyo, Japan) with an inspired oxygen content of 40%, at a rate of 80 breaths/min with a positive end-expiratory pressure of 2 cmH2O. The tidal volume was adjusted to maintain the maximal peak pressure below 10 mmHg (range 2.5–3.0 ml). A 22-gauge cannula inserted into the right carotid artery was used to monitor the arterial pressure. A 20-gauge cannula was inserted into the right jugular vein. Pentobarbital was diluted with saline (15 mg/ml) and was infused intravenously at 0.3 ml/h to maintain anesthesia and hydration during the experiment. Surgery was conducted under an operating microscope, and a warming blanket was placed underneath each animal to keep the esophageal temperature between 36.5 and 37.5 °C throughout the experiment.
A left parasternal incision and anterioaxillar thoracotomy in the eighth intercostal space was performed. The mediastinal component was gently mobilized toward the right, the left lung hilus was stripped, and the inferior pulmonary ligament was divided without touching the left lung. Animals then received 100 units of intravenous heparin. Five minutes after heparin administration, the left pulmonary artery, vein, and bronchus were clamped at the end of inflation. Ischemia was maintained for a period of 90 min. At the end of the ischemic period, the left hilus was declamped, and the lung was allowed to ventilate and reperfuse for 2 h. During the experiment, the incision was covered with Saran WrapTM (a thin polyvinylidene chloride sheet) to minimize evaporative losses. At the end of the reperfusion period, a midline laparotomy was made and blood samples were obtained from the inferior vena cava just before the animal was killed by administration of 150 mg/kg pentobarbital through the arterial cannula. An 18-gauge cannula was inserted into the main pulmonary artery and secured with a 2–0 braided suture. The left atrial appendage and left ventricle were incised to allow free flow of effluent blood from the lung. The pulmonary vasculature was cleared with 50 ml of phosphate-buffered saline (PBS; pH 7.4) from a height of 20 cm. The left lungs were then resected and stored in liquid nitrogen.
Animals were randomly divided into four experimental groups: the sham (n=5), the pioglitazone-treated sham (n=5), the I/R (n=8), and the pioglitazone-treated I/R (n=8) groups (Fig. 1) . A total of 52 rats were used: 26 for measurement of lung microvascular permeability and 26 for the biochemical assays (lipid peroxidation, proinflammatory cytokines, and myeloperoxidase (MPO) activity). Treated animals received 10 mg/kg of pioglitazone intraperitoneally 2 h before the beginning of ischemia. The dose of pioglitazone was determined based on the results of our previous study in which preischemic treatment with this agent inhibited the I/R-induced gastric and intestinal injury in a dose-dependent manner (1–30 mg/kg) [9,10]. Pioglitazone was dissolved in 0.5% dimethylsulfoxide (DMSO) in PBS. Untreated animals received only a vehicle (0.5% DMSO in PBS) at the same time point. Time-matched sham groups underwent the same procedure without left lung hilus clamping.
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2.3. Biochemical assays
Another series of animals underwent the same surgical procedure and the lung tissue was stored in liquid nitrogen. The lung tissue was homogenized with 1.0 ml of cold 10 mM potassium phosphate buffer containing 30 mM KCl (pH 7.8) in a glass chamber on ice. The aliquots of 10 µl of the homogenates were used to determine the protein concentration in the homogenates according to the method of Lowry et al. [14].
2.3.1. Lipid peroxidation
The concentration of thiobarbituric acid-reactive substances (TBA-RS) in the lung homogenates was measured as an index of lipid peroxidation by the method of Ohkawa et al. [15]. To samples of 0.2 ml of tissue homogenate were added 0.2 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of aqueous solution of 0.8% TBA with 1.5 ml of 20% acetate buffer (pH 3.5), and 0.04 ml of 1.0% butylated hydroxytoluene dissolved with absolute ethanol. The mixture was finally made up to 4.0 ml with distilled water, and heated at 95 °C for 60 min in an oil bath. After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of a mixture of n-butanol and pyridine (15:1, v/v) were added and the mixture was shaken vigorously. After centrifugation at 3000 rev./min for 10 min, the absorbance of the upper layer was measured at 535 nm. The concentration of TBA-RS is expressed as nanomoles of malondialdehyde per milligram of tissue protein using 1,1,3,3-tetramethoxypropane as the standard.
2.3.2. Proinflammatory cytokines
The residual volume of homogenates was centrifuged at 40 000xg for 15 min at 4 °C. The supernatant was used for analysis of tissue cytokine concentration. The content of tumor necrosis factor- (TNF-) in the lung tissue was determined by using a rat TNF- ELISA kit (Bio Source International, Inc., CA). The content of cytokine-induced neutrophil chemoattractant-1 (CINC-1) was determined by using a GRO/CINC-1 ELISA kit (Immuno-Biological Laboratories Co., Ltd, Shizuoka, Japan). These assays were performed according to the manufacturer's instructions.
2.3.3. Myeloperoxidase activity
The pellet was used to determine pulmonary tissue MPO activity. Tissue MPO activity was measured to quantify polymorphonuclear leukocyte accumulation in the lungs by the method of Krawisz et al. [16]. The pellet was suspended in 0.3 ml of 0.5% of hexadecyltrimethylammonium bromide buffer (pH 6.0) and sonicated on ice three times for 5 s each time. The mixture was centrifuged at 40 000xg for 15 min at 4 °C and the supernatant was assayed for MPO activity. Assay buffer was composed of 0.167 mg/ml o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. Aliquots of 0.05 ml of each sample were mixed with 0.95 ml of assay buffer, and the change in absorbance at 460 nm over 1 min was recorded. One unit of MPO activity is defined as that degrading 1 µmol of peroxide per minute at 25 °C.
2.4. Statistical analysis
All data are presented as means±SD. The sample size in each of the experimental groups was greater than or equal to 5. Comparisons between multiple groups were performed by one-way analysis of variance, followed by Sheffe's multiple comparison test. These analyses were performed using StatView J-5.0 (SAS Institute Inc., NC). P values of less than 0.05 were considered statistically significant.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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3.1. Lung microvascular permeability
The permeability index increased significantly (P<0.001) in the I/R group (0.142±0.012 mg-1 tissue) as compared with the sham group (0.100±0.006 mg-1 tissue). This increase was significantly (P=0.003) reduced in the pioglitazone-treated I/R group (0.119±0.012 mg-1 tissue). The permeability index in the pioglitazone-treated sham group (0.098±0.006 mg-1 tissue) did not differ from that in the untreated sham group (P=0.987; Fig. 2)
. The permeability index in the pioglitazone-treated I/R group is significantly higher than that in the sham group (P=0.014).
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in the lung tissue increased significantly (P<0.001) in the I/R group (13.345±2.898 pg/mg protein) as compared with the sham group (4.568±0.632 pg/mg protein). This increase was significantly (P<0.001) reduced in the pioglitazone-treated I/R group (7.816±2.030 pg/mg protein). The tissue concentration of TNF- in the pioglitazone-treated sham group (4.640±0.862 pg/mg protein) did not differ from that in the untreated sham group (P>0.999; Fig. 5)
. The concentration of TNF- in the pioglitazone-treated I/R group did not differ from that in the sham group (P=0.079).
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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, significantly attenuated I/R-induced lung injury in rats. We also ascertained that pioglitazone did not affect normal lungs comparing the sham and the pioglitazone-treated sham groups. In the present study, we assessed I/R-induced lung injury by various methods, including lung microvascular permeability, lipid peroxidation, tissue-associated polymorphonuclear leukocyte infiltration, and proinflammatory cytokine production. Each assessment indicated that preischemic treatment with pioglitazone significantly reduced I/R-induced lung injury. These findings can be explained at least in part as follows: (1) activation of PPAR- inhibits gene expression and inflammatory cytokine production in part by antagonizing the activities of the transcription factors such as NF-B [3]; (2) preventing NF-B activation inhibits proinflammatory genes and their products (TNF-, cyclooxygenase-2, CINC, and intercellular adhesion molecule-1) [17,18], (3) TNF- and CINC-1 increase the neutrophil population in the lung [19,20], and (4) TNF- causes lung endothelial injury resulting in lung edema [21]. Thus, preischemic treatment with pioglitazone, a synthetic ligand of PPAR-, decreased proinflammatory cytokine production and reduced neutrophil accumulation in rat lung exposed to I/R, which inhibited increases in pulmonary microvascular permeability. Another mechanism of protective effects of PPAR- agonist, troglitazone, against I/R lung injury is reported by suppression of transcription factor early growth response gene-1 and its inflammatory gene targets, such as interleukin-1ß, monocyte chemotactic protein-1, and macrophage inflammatory protein-2 [22].
Tissue lipid peroxidation induced by oxygen-free radicals is an important cause of I/R-induced lung injury [23,24]. In the present study, we showed that pioglitazone reduced the concentration of TBA-RS in I/R-induced lung tissue. However, neither pioglitazone nor other PPAR- ligands have been reported to have anti-oxidant activities in vitro, particularly the inhibition of lipid peroxidation. The inhibitory effect of pioglitazone on I/R-induced lipid peroxidation in the rat lung may result from its anti-inflammatory activity rather than from its direct anti-oxidant activity.
The left lung I/R injury in rats was estimated by 90 min ischemia followed by 4 h reperfusion [25], however, in this study, we set the period of ischemia 90 min and the reperfusion 2 h, based on the results of our preliminary time-course experiments, in which the pulmonary microvascular permeability at various time points of reperfusion after 90 min ischemia was gradually increased during the first 2 h of reperfusion and became a plateau up to 4 h (data not shown). Since each assessment showed significant lung injury in the I/R group compared with the sham group, the period of 90 min ischemia and 2 h reperfusion will be acceptable.
I/R lung injury was established by clamping whole hilar components of the vasculatures (pulmonary artery, vein and bronchial artery) and the airways, since this study was assumed to be suitable for the condition of the graft in the clinical lung transplantation, in which the graft is preserved inflated and circulatory arrest. Our animal model will also be applicable to the pulmonary re-expansion injury model by clamping only the bronchus and the pulmonary embolism model by clamping only the pulmonary artery.
This investigation suggests that pioglitazone has potential as a new therapeutic agent for I/R lung injury to apply to the clinical lung transplantation, although we must solve some problems, such as the toxicity, the proper dose, and the way of administration. The important side effects of pioglitazone in clinical use are hypoglycemia, liver dysfunction and edema. The acute toxicity when administered once is not reported in animals (LD50>1814 mg/kg in rats). We administered 10 mg/kg of pioglitazone intraperitoneally owing to our previous experiments, in which I/R induced intestinal injury was reduced by 60% by 10 mg/kg of pioglitazone [10]. Since the dose of pioglitazone for the treatment of diabetes is 15–45 mg/body per day, the dose of 10 mg/kg used as the preischemic treatment in this study is much larger. However, smaller dose of preischemic treatment of pioglitazone demonstrated 30% reduction in rat intestinal I/R injury (3 mg/kg) [10], and 20% reduction in rat myocardial infarct size (0.3 mg/kg) [11]. The most proper dose of pioglitazone for human lung must be examined. The most effective way to administer pioglitazone is to add in the preservation solution, as pioglitazone is soluble in DMSO/PBS solution. This will alternatively be administered by an oral route to the donors before ischemia. The results of the present study in which the drug was administered just before ischemia suggest that preoperative treatment with pioglitazone reduces reperfusion lung injury. We speculate that pioglitazone may be useful in the treatment of other conditions associated with I/R of the lung, such as pulmonary resection with arterioplasty, pulmonary thromboendoarteriectomy, and operations with cardiopulmonary bypass.
In conclusion, preischemic treatment with pioglitazone at a dose of 10 mg/kg reduced I/R-induced lung injury in rats. Pioglitazone inhibited the production of proinflammatory cytokines (TNF-, CINC-1) and infiltration of polymorphonuclear leukocyte into the lung interstitium, resulting in inhibition of pulmonary edema. Pioglitazone has potential as a new therapeutic agent for lung reperfusion injury, however, further examinations are necessary to determine the proper dose and the way of administration in human.
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Footnotes |
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References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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