Interact CardioVasc Thorac Surg 2009;9:159-162. doi:10.1510/icvts.2008.196089
© 2009 European Association of Cardio-Thoracic Surgery
Institutional report - Pulmonary |
Role of heme oxygenase-1/carbon monoxide system in pulmonary ischemia-reperfusion injury
Wantie Wanga,
Fangyan Wanga,
Lu Shia,
Xuguang Jiaa and
Lina Linb,*
a Department of Pathophysiology, Wenzhou Medical College, Wenzhou 325035, Zhejiang, China
b Department of Anesthesiology, First Affiliated Hospital of Wenzhou Medical College, Wenzhou 325003, Zhejiang, China
Received 9 October 2008;
received in revised form 1 May 2009;
accepted 5 May 2009
 This research project was supported by The Scientific Research of Zhejiang Provincial Department of Education (Item number: 20000670) and Emphasis Project of Wenzhou City Scientific Plan (Item number: Y2005A080).
*Corresponding author. Tel.: +86-577-88069459; fax: +86-577-88383956.
E-mail address: wzlinlina{at}tom.com (L. Lin).
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Abstract
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The aim of this study is to investigate the effect of heme oxygenase-1 (HO-1)/carbon monoxide (CO) system in pulmonary ischemia-reperfusion injury (PIRI) in rabbits. The rabbits were randomly assigned to three groups (n=10, in each): control group (C), PIR group (I-R), PIR+Hemin group (H) and PIR+zinc protoporphyrin IX (ZnPP) group (Z). There were changes to several parameters which included plasma carboxyhemoglobin (COHb), wet to dry ratio of lung tissue weight (W/D), the injured alveoli rate (IAR) and the HO-1 enzymatic activity. Immunohistochemistry and in situ hybridization for HO-1 was detected in lung. The electron microscopic observation for lung tissue injury was done after PIRI. The plasma content of COHb increased by reperfusion was strengthened by hemin but weakened by ZnPP. The HO-1 activity in lung tissue was upregulated by PIRI, further enhanced by hemin and abolished by ZnPP. Except for the C group, HO-1 was upregulated in all other groups in the pulmonary endothelial cells, some pulmonary vascular smooth muscle cells, extima of vessels and epithelial cells of airway. The injury parameters were highest in the Z group, the second was in the IR group, then the H group and the C group. HO-1/CO system was activated and may be one of the protective signal pathway during PIRI in rabbits.
Key Words: Ischemia-reperfusion injury; Lung; Heme oxygenase-1; Carbon monoxide
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1. Introduction
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Some previous studies [1–3] indicated that induction of HO-1 can protect myocardium, liver and kidney from ischemia-reperfusion injury. But, there are few reports about the function of heme oxygenase-1 (HO-1)/carbon monoxide (CO) pathway in pulmonary ischemia-reperfusion injury (PIRI). This study will investigate the role of HO-1/CO pathway in PIRI.
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2. Materials and methods
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2.1. Animals, main equipments and main reagents
Forty healthy Japanese big ear rabbits (either gender), body weight of 1.7–2.7 kg, were offered by the Experimental Animal Center of Wenzhou Medical College. All animals received humane care in compliance with the European Convention on Animal Care. The following experimental protocol was approved by the Wenzhou Medical College Animal Care and Use Committee.
A H-600-transmission electron microscope (Japan Hitachi) and a CMIA-color medical image analysis system were employed. Hemin (H5533, Lot 072K1221), dihydronicotinamide adenine dinucleotide phosphate (NADPH, N1630, Lot 081K7059) and ZnPP IX (ALX-430-049-M100, L09617
[GenBank]
) were provided by Canada Alexis Company. The primary antibody against HO-1, hybridization in situ kit for HO-1 (Wuhan Boster Biological Technology Co, Ltd.) was employed.
Hemin and ZnPP IX were melted by 0.1 mol/l NaOH, adjusted temporarily pH to 7.4 by 0.1 mol/l HCl, when used, and diluted to the content needed by normal saline. Operation and storage was absent from light. NADPH was solved by 0.01 mol/l NaOH.
2.2. Animal model and grouping
After some conventional operations, dissociate and pass ligatures around the hilum of the left lung, as described by Sekido [4], was carried at to duplicate the rabbit PIRI model.
The rabbits were divided into four groups (n=10, each). (1) Control group (C), ligatures passed hilum of left lung but did not ligate. (2) Ischemia-reperfusion group (I-R), clamp hilum, block 1 h, then immediately unclamped the hilum to perform reperfusion for 3 h at the end of the experiment to obtain the lung specimen. (3) PIR+Hemin group (H), before the operation 48 h, hemin (40 µmol/kg, 2 times/d) [5] was administered by intraperitoneal injection in order to induce the expression of HO-1. (4) PIR+zinc protoporphyrin IX (ZnPP) group (Z), before undoing the ligatures 15 min, ZnPP IX (10 µmol/kg) was injected intravenously [6]. Blood was drawn at the time points T0 (pre-ischemia before ligation of hilum), T1 (ischemia 1 h), T2 (reperfusion 1 h), T3 (reperfusion 2 h), T4 (reperfusion 3 h).
2.3. Assay of plasmatic (COHb) and detection of the activity of HO-1 in lung tissue
Assay of plasmatic COHb was determined as previously reported [7].
Detection of the activity of HO-1 in lung tissue followed Zhou et al. [8]. Determination of protein level by staining was done with Coomassie Blue.
2.4. Immunohistochemistry (IHC) analysis of the expression of HO-1 in lung tissue
The streptavidin-biotin peroxidase method was used for IHC. Computerized image analysis (the software was developed by East China University of Science and Technology) was performed to quantitate the immuno-stained HO-1.
2.5. Hybridization in situ analysis of the expression of HO-1 in lung tissue
The sequence of HO-1 oligonucleotide probe:
- 5'-AGAAT GCTGA GTTCA TGAGG AACTT TCAGA-3'
- 5'-GCTGC TGGTG GCCCA CGCCT ACACC CGCTA-3'
- 5'-TTCCT GCTCA ACATC CAGCT CTTTG AGGAG-3'
Computerized image analysis (the software was developed by East China University of Science and Technology) was performed to quantitate the expression of HO-1.
2.6. Wet to dry ratio weight (W/D) of lung tissue
Harvest specimen of lung near the side of hilum and rinse with normal sodium, remove the superfluous water and weigh as wet weight. Dry the specimen in an electric airblast drier at 70 °C for 24 h, and determine dry weight. Thus we can determine the wet to dry weight ratio of lung tissue.
2.7. The injured alveoli rate (IAR)
Lung tissue was stained by HE conventionally. We observed the changes of histological structure under microscope. At 200 magnification field of vision, we continuously observed 200 pulmonary alveoli, counted the quantity of those injury alveoli which there were >2 erythrocytes and/or leucocytes inside of, and calculated the percentage of the injury alveoli as a quantitative assessment index of pulmonary alveoli impairment [9].
2.8. Observation of lung tissue with electron microscope
Harvest 2 or 3 pieces of lung tissue near the left hilum, the size of each piece is approximately 0.1x0.1x0.1 (cm3). The specimen were conventionally stained with acetic acid and lead nitrate, and finally observed with a transmission electron microscope.
2.9. Statistical analysis
Our data was analyzed by statistical software SPSS 11.5 and presented as means±S.D. (standard deviation). Differences between groups were analyzed by using an analysis of variance (ANOVA), and means at different stages in groups were compared by repeated measure, followed by the SNK test as a post-hoc test or Dunnett's T3 test. A P<0.05 was considered statistically significant. Correlation analysis was completed by Pearson correlation in the bivariate, we obtained the coefficient by utilizing linear regression.
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3. Results
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3.1. Change of plasma COHb level in different time points during PIR among four groups (Table 1, Fig. 1)
Compared with the C group, the serum content of COHb in the IR, the H, and the Z group increased in a time-dependent manner after IR. But the increment of the H group was higher than that of the IR group, while that of the Z group is lower.
3.2. Change of HO-1 activity in lung tissue, average optical density value (AV) of IHC in situ hybridization (ISH) (Table 2 and Fig. 2)
The contents of the HO-1 activity in lung tissue were highest in the H group, followed by the IR group, the Z group, and the C group. IHC and ISH demonstrated that, except for the C group, HO-1 was upregulated in all other groups in the pulmonary endothelial cells, part of pulmonary vascular smooth muscle cells, extima of vessels and epithelial cells of airway. The H group had the highest average optical density value compared to the IR group, the Z group and the C group.
3.3. Change of W/D and IAR values in lung tissue among four groups in rabbits (Table 3)
The value of W/T and IAR was highest in the Z group, the second was in the IR group, then the H group and the C group.
3.4. Ultramicrostructure change of lung tissue (Fig. 3)
For the C group, the structure of capillary endothelial cells were normal, connected closely. Basement membrane was normal. Type I alveolar epithelial cells showed no change similar to the structure of type II alveolar epithelial cells. Mitochondria in endothelial cells were normal, too. The number of lamellar body was unchanged, microvilli did not reduce.
For the IR group, capillary endothelial cells swelled, nuclear chromatin was seen to concentrate at the side of nuclear membrane, nucleus tended to condense, and the gaps enlarged. There were a few pinocytosis bulbs in type I alveolar epithelial cells. Microvilli reduced on the surface of type II alveolar epithelial cells, mitochondrium swelled, lamellar body was rare, some vacuole appeared. Interalveolar septum caused edema. Inflammatory cells, mainly neutrophil, affiliated to interalveolar septum and capillary, lumen.
For the H group, the structure of capillary and alveolar epithelial cell was improved, inflammatory cells in the capillary reduced, chromatin scattered normally. Pinocytosis bulbs were found in type I alveolar epithelial cells. Microvilli of type II alveolar epithelial cells were normal and lamellar body increased. Interalveolar septum formed slight edema.
For the Z group, capillary and alveolar epithelial cells were obviously damaged. Vascular endothelial cellular swelling was significant, protruding towards the intracavitary like a column, margin between cells were vague, some cells appeared karyopyknotic, endoelastic plank was deformed severely, the basement of endothelial cells detached from endoelastic plank, like an arch bridge. Type I alveolar epithelial cells had a few pinocytosis bullule. The surface of type II alveolar epithelial cells was flat, microvilli were rare, lamellar body was scare and out of kytoplasm with vacuolus. Interalveolar septum formed edema and some inflammatory cells recruited there.
3.5. Correlation analysis
The content of COHb in blood plasma at the end of reperfusion and the activity of HO-1 of lung presented a positive correlation relationship (r=0.967, P<0.01); W/D of lung tissue was found to be correlated with IAR (r=0.845, P<0.01); the activity of HO-1 showed negative correlation with W/D. IAR coefficient was –0.799 and –0.862, respectively, each P<0.01; AV of IHC and ISH appeared to be positively correlated with IAR (r=0.801, 0.780, respectively, both P<0.01).
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4. Discussion
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Heme oxygenase has three isozymes belonging to distinct gene products: HO-1, HO-2 and HO-3. HO catalyzes the metabolism of heme to biliverdin (which is rapidly converted to bilirubin by biliverdin reductase), free iron (which leads to the induction of ferritin and combines with it), and CO. Biliverdin, CO and iron-binding protein induced by iron has the ability of anti-oxidation, is an anti-inflammatory, anti-proliferative candidate of HO-1 and is also the focus of present research.
Endogenous CO occurs from at least two ways [10]: (1) the predominant source is that the oxidizing reaction of protoheme is catalyzed by HO; (2) the minor sources include the oxidation of organic molecules. This includes anti-oxidation of phenols, lipid peroxidation of membrane lipids and photo-oxidation of organic compounds. The CO thus formed diffuses into the blood, is carried via hemoglobin, and is excreted in the lungs. Therefore, HO activity can be assessed by measuring the rate of total body CO excretion, blood COHb concentration.
This study found that COHb concentration in the IR group kept increasing with the persistence of reperfusion, and at each time point, compared to either pre-ischemia and post-ischemia or the same time point in the C group, showed significant deviation. The activity of lung HO in and IR group was higher than that in the C group. In addition, it indicated I-R can induce the expression of lung HO-1, and that IHC and ISH showed generally positive expression of HO-1 in the IR group. This result is in accordance with Zhang et al. [11]. Furthermore, W/D and IAR of the IR group was much higher than those of the C group, the abnormal ultramicrostructure indicated that I-R induced the damage of lung, too. However, the induction of HO-1 was to mediate PIRI or a compensatory protective mechanism for PIRI. Therefore, we administered an agonist and inhibitor of HO-1 to further study its function. In the H group, we observed evidence of obvious up-regulation of HO-1, such as activity of blood COHb, and lung tissue HO-1 was higher than those in the I-R group, expression of HO-1 was strongly positive, IAR improved more in contrast to the I-R group. In the Z group the inhibitor of HO-1 was used before reperfusion, and HO-1 activity was suppressed, PIRI aggravated. This result indicated that the upregulation of HO-1 can be one of the results to reduce the damage of lung ischemia-reperfusion.
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Abstract
1. Introduction
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