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Ischemia postconditioning preventing lung ischemia–reperfusion injury
Gene 554 (2015) 120–124
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Ischemia postconditioning preventing lung ischemia–reperfusion injury Qi-Feng Cao ⁎, Mei-Jun Qu, Wei-Qin Yang, Dan-Ping Wang, Ming-Hui Zhang, Song-Bo Di 3rd Department of Internal Medicine, Integrated Chinese and Western Medicine Hospital of Taizhou, Zhejiang Province 317523, China
a r t i c l e
i n f o
Article history: Received 15 May 2014 Accepted 5 October 2014 Available online 7 October 2014 Keywords: Ischemic postconditioning Lung ischemia reperfusion Antioxidant
a b s t r a c t Objective: This study evaluates the inhibitory effect of IPO against ischemia reperfusion (I/R) induced lung injury in rats. Methods: Anesthetized and mechanically ventilated adult Sprague–Dawley rats were randomly assigned to one of the following groups (n = 12 each): the sham operated control group, the ischemia–reperfusion (IR) group (30 min of left-lung ischemia and 24 h of reperfusion), the IPO group (three successive cycles of 30-s reperfusion per 30-s occlusion before restoring full perfusion), and the dexamethasone plus IPO group (rats were injected with dexamethasone (3 mg/kg·day−1) 10 min prior to the experiment and the rest of the procedures were the same as the IPO group). Lung injury was assessed by wet-to-dry lung weight ratio and tissue apoptosis and biochemical changes. Results: Lung ischemia–reperfusion increased lung MDA production, serum proinflammatory cytokine count, and MPO activity and reduced antioxidant enzyme activities (all p b 0.05 I/R versus sham), accompanied with a compensatory increase in caspase-3, bax, Fas, FasL proteins and a decrease in Bcl-2 protein. Plasma levels of TNF-α, IL6, and IL-1β were increased in the I/R group (all p b 0.05 versus sham). IPO attenuated or prevented all the above changes. Treatment with dexamethasone enhanced all the protective effects of postconditioning. Conclusion: Postconditioning obviously inhibits I/R induced lung injury by its antioxidant, anti-inflammatory and anti-apoptosis activities. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Lung ischemia–reperfusion injury is clinically common in lung transplantation and extracorporeal circulation operation (Melotte et al., 2010; Mukherjee et al., 2012; Sun et al., 2011). Lung I/R injury can lead to lung dysfunction and is the leading cause of death after lung transplantation (Chen et al., 2012). Lung I/R injury still causes significant morbidity and mortality and is characterized by neutrophil extravasation, interstitial edema, disruption of epithelial integrity, and leakage of protein into the alveolar space, all associated with severe alterations in gas exchange (Tang et al., 2008). Reperfusion injury after pulmonary transplantation can contribute significantly to postoperative pulmonary dysfunction. To date, the mechanisms underlying lung I/R injury remain unclear, and effective treatments for its prevention are lacking. Murry et al. (1986) first introduced the definition of ischemic preconditioning (IPC), in which repetitive brief periods of ischemia protected the myocardium from a subsequent greater period of
Abbreviations: I/R, ischemia reperfusion; IPC, ischemic preconditioning; IPO, ischemic postconditioning. ⁎ Corresponding author. E-mail address: [email protected] (Q.-F. Cao).
http://dx.doi.org/10.1016/j.gene.2014.10.009 0378-1119/© 2014 Elsevier B.V. All rights reserved.
ischemic insult. Ischemic preconditioning (IPC) in which repetitive brief periods of intestinal ischemia protect the intestine from a subsequent longer ischemic insult has been demonstrated to attenuate acute lung injury induced by I/R (Tamion et al., 2002). Although IPC has been shown to be beneficial in the human heart (Tomai et al., 2001), prospectively controlled studies in humans involving IPC of the intestine are lacking. The major problem is the inability to predict the onset of ischemia in some clinical settings related to I/R. Recently, some studies showed that ischemic postconditioning (IPO), which consists of one or more short cycles of reperfusion followed by one or more short cycles of ischemia, immediately after an ischemic phase and before the permanent reperfusion occurs, was as efficient as the IPC in preventing myocardial reperfusion injury (Zhao & Vinten-Johansen, 2006). It has also been reproduced in other organs such as the spinal cord (Chen & Liu, 2007), renal (Liu et al., 2007a), and cerebral (Rehni & Singh, 2007) tissues. Recent studies have demonstrated that brief intermittent cycles of ischemia alternating with reperfusion applied after the prolonged ischemic event, a novel approach termed “ischemic postconditioning (IPO)”, attenuated IRI in a wide range of organs, including the heart, brain, spinal cord, liver, and kidney (Zhao, 2010). This study aimed to observe the influence of IPO on lung I/R rat lung tissue oxidative injury and the mechanism. We established the rat lung I/R model, used IPO, and observe its prevention effect of lung I/R injury in rats.
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2. Material and methods
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absorbance of 1 per minute) was expressed as units per gram of tissue. MPO product was measured spectrophotometrically at 650 nm.
2.1. Animals 2.6. Analysis of antioxidant enzyme activities Three months old male albino Wistar rats, weighting between 150 and 250 g, were used. Animals were raised in the Animal House of the Faculty of Science, Taizhou University. They were maintained in a temperature room (22 ± 2 °C) on a 12 h light–dark natural cycle. Rats were fed with standard diet and water ad libitum. These studies were conducted with the approval of the China National Ethical Committee. 2.2. Surgical procedure and experimental protocol The rats were randomly assigned to one of four groups (n = 12 in each group). Under aseptic conditions, an in situ unilateral lung warm ischemia model was used. In brief, a left anterolateral thoracotomy in the fifth intercostal space was created. The left lung was mobilized, the pulmonary hilum was dissected, and the perivascular and peribronchial tissues were removed. Next, all the rats received 500 U/kg of heparin intravenously in saline (total volume 500 mL). In group 1 (sham), the rats underwent sham thoracotomy and hilar dissection, but the lungs were not rendered ischemic. In group 2 (I/R), 5 min after heparin administration, the left pulmonary artery, bronchus, and pulmonary vein were occluded with a noncrushing microvascular clamp, maintaining the lung in a partially inflated state. The lungs were kept moist with periodic applications of warm, sterile saline, and the incision was covered to minimize evaporative losses. The ischemic period was held constant at 30 min, after which the clamp was removed and the lung reperfused for 24 h. In group 3 (IPO), postconditioning was performed by three successive cycles of 30 s of reperfusion for each 30 s of occlusion, starting immediately after the release of the index ischemia. In group 4 (dexamethasone + IPO) rats were injected with dexamethasone (3 mg/kg·day−1) 10 min prior to the experiment and the rest of the procedures were the same as the IPO group. 2.3. Lung wet/dry weight ratio At the end of the experiments, the left lower lobe of the lung was dissected and dried at a constant temperature of 80 °C for 24 h to obtain a dehydrate consistency. The lung wet/dry (W/D) ratio was calculated as an indicator of edema. 2.4. In situ detection of apoptotic cells Serial sections of 4-μm thickness were prepared. TUNEL staining was performed with the use of an in situ apoptosis detection kit according to the manufacturer's instruction (Boehringer Mannheim, Germany) and examined by light microscopy. The apoptotic index (AI) was calculated as the percentage of stained cells, namely: AI = number of apoptotic cells × 100 / total number of nucleated cells.
Lipid peroxidation level in the tissue samples was expressed in MDA. Measurement was based on the method of Ohkawa et al. (1979). The reaction mixture contained 0.1 mL of sample, 0.2 mL of 8.1% sodium dodecyl sulfate, 1.5 mL of 20% acetic acid, and 1.5 mL of 0.8% aqueous solution of TBA. The mixture pH was adjusted to 3.5 and the volume was finally made up to 4.0 mL with distilled water and 5.0 mL of the mixture of n-butanol and pyridine (15:1, vol/vol) was added. The mixture was shaken vigorously. After centrifugation at 4000 rpm for 10 min, the absorbance of the organic layer was measured at 532 nm. MDA was expressed as nmol/mg protein. GPx activity was measured by the method described by Beutler (1975). The reaction mixture contained 1 M Tris buffer (pH 8.0), 0.1 M GSH, 2 mM nicotinamide adenine dinucleotide phosphate (NADPH), and 10 U/mL glutathione reductase. Decrease in absorbance (OD) was followed at 340 nm and enzyme activity in samples was calculated as U/g protein. SOD activity was determined according to the method Sun et al. (1988) with a slight modification (Durak et al., 1993). The principle of the method is based on the inhibition of NBT reduction by the xanthine–xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the sample after 1.0 mL ethanol/ chloroform mixture (5/3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. Catalase activity was evaluated by measuring the decrease in H2O2 concentration at 240 nm (Aebi, 1984). Working solution (phosphate buffer 100 mM; H2O2 10 mM) and sample were mixed in a cuvette. The change in absorbance per minute at 240 nm (DA240) was calculated. Enzyme activity was expressed in units of CAT activity per milligram of protein. One unit of CAT activity is defined as the amount of enzyme needed to reduce 1 μmol H2O2/min. 2.7. Analysis of serum TNF-α, IL-1β and IL-6 levels The levels of sera TNF-α, IL-1β and IL-6 were measured using commercially available kits. The TNF-α, IL-1β and IL-6 assay kits were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). These experiments were performed according to the manufacturer's instructions. 2.8. Analysis of PaO2, NO and NOS levels Arterial blood gas analysis is used to measure the partial pressure of venous oxygen (PaO2). NO and NOS levels were measured with commercially available kits. These experiments were performed according to the manufacturer's instructions.
2.5. Myeloperoxidase activity 2.9. Western blot The activity of MPO was used as a marker of neutrophil infiltration (Bradley et al., 1982; Day et al., 2005; Sener et al., 2005). Lung MPO activity was determined as described previously (Serafin et al., 2002). Lung tissue was homogenized in 2 mL of 10 mmol/L phosphate buffer (pH 7.4). After centrifugation at 10,000 g for 20 min, the pellet was resuspended in 1 mL of 10 mmol/L phosphate buffer (pH 7.0) containing 0.5% hexadecyltrimethylammonium and sonicated for 10 s. After heating at 60 °C for 2 h, the samples were centrifuged at 8000 g for 10 min. The assay mixture consisted of 20 μL of the supernatant, 10 μL of 3,3′5,5′-tetramethylbenzidine (final concentration, 1.6 mmol/L) dissolved in dimethyl sulfoxide, and 70 μL of H2O2 (final concentration, 3 mmol/L) diluted in 80 mmol/L of phosphate buffer (pH 5.4), and incubated at 37 °C for 5 min. MPO activity (1 unit defined as change in
Lung tissues were homogenized and sonicated in a tissue lysis buffer containing Tris–HCl (20 mM, pH 7.4), NaCl (150 mM), ethylenediaminetetraacetate (l mM), ethylene glycol tetraacetic acid (l mM), βglycerolphosphate (1 mM), sodium pyrophosphate (2.5 mM), Triton X-100 (1%), phenylmethylsulfonyl fluoride (1 mM), dithiothreitol (1 mM), leupeptin (1 mg/mL), aprotinin (1 mg/mL), and pepstatin (1 mg/mL). The homogenate was centrifuged at 1000 g for 10 min at 4 °C to collect the supernatant as a total protein preparation. Equal amounts of protein were combined with 5 × sodium dodecyl sulfate loading buffer and boiled for 5 min and then separated using 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and subsequently transferred to polyvinyldine diflouride membrane for immunoblot
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Table 1 Effects of IPO on myocardium antioxidant status and lipid peroxidation. Group
MDA
I II III IV
4.19 8.66 7.28 5.72
b c d
± ± ± ±
SOD 0.45 0.92b 0.85c 0.63d
207.8 95.29 152.58 184.11
MPO ± ± ± ±
22.17 8.69b 16.38d 19.05d
0.27 0.84 0.61 0.38
CAT ± ± ± ±
0.03 0.09b 0.07d 0.04d
24.98 11.39 17.05 22.63
GSH-Px ± ± ± ±
3.15 1.31b 1.84d 2.66d
62.95 27.47 40.71 58.03
± ± ± ±
7.31 3.03b 4.25d 6.09d
p b 0.01, group II vs group I. p b 0.05. p b 0.01, groups III and IV vs group II.
analysis. The membranes were blocked in 5% nonfat milk for 2 h at room temperature and then incubated overnight at 4 °C with primary antibodies against caspase-3 (1:1000, Cell Signaling Technology, Beverly, MA), Fas (1:1000, Cell Signaling Technology), FasL (1:500, Santa Cruz Biotechnology), Bax (1:1000, Santa Cruz Biotechnology), and Bcl-2 (1:1000, Santa Cruz Biotechnology). After being washed with Tris-buffered saline-Tween 20, the membranes were incubated with proper secondary horseradish peroxidase-conjugated antibodies (1:5000–1:10,000, Cell Signaling Technology) and developed with enhanced chemiluminescence reagent (GE Healthcare, Rahway, NJ). The membranes were subsequently reblotted for β-actin (1:2000; Cell Signaling Technology), and the results were normalized to β-actin to correct for loading. 2.10. Statistical analysis Data are expressed as mean ± standard deviation (SD). Group differences were analyzed using a oneway ANOVA with the Student– Newman–Keuls or Dunnett's test. p b 0.05 was considered statistically significant. Data were analyzed using SPSS 16.0 (SPSS, Chicago, IL, USA). 3. Results The effects of IPO on myocardium antioxidant status and lipid peroxidation were investigated. The I/R caused an increase in myocardium antioxidant enzyme activities (SOD, CAT, GSH-Px) with a concomitant decrease in MDA and MPO values as shown in Table 1. Both IPO group (3) and dexamethasone + IPO group (4) showed an improvement in myocardium antioxidant status as assessed by MDA and antioxidant enzyme activities which were significantly decreased and increased as compared with the I/R group (p b 0.05). However, the TRAP value in two groups was less than that of the RD group (p b 0.05). There was also considerable variation in the serum TNF-α, IL-1β and IL-6 contents in rat groups. As shown in Table 2, serum TNF-α, IL-1β and IL-6 contents in the I/R group were significantly higher than those in the sham group. Treatment of IPO and dexamethasone + IPO significantly decreased serum TNF-α, IL-1β and IL-6 contents when compared to the I/R group (p b 0.01). There were significant differences in the contents of sera TNF-α, IL-1β and IL-6 between IPO and dexamethasone + IPO groups (p b 0.01). Serum TNF-α, IL-1β and IL-6 concentration of the dexamethasone + IPO group was markedly lower when compared with the IPO group (p b 0.01). There was also considerable variation in the PaO2, NO and NOS values in rat groups. As shown in Table 3, PaO2, NO and NOS values in Table 2 Effects of IPO on serum TNF-α, IL-1β and IL-6 contents.
the I/R group were significantly lower than those in the sham group. Treatment of IPO and dexamethasone + IPO significantly increased PaO2, NO and NOS values when compared to the I/R group (p b 0.01). There were significant differences in the PaO2, NO and NOS values between IPO and dexamethasone + IPO groups (p b 0.01). PaO2, NO and NOS values of the dexamethasone + IPO group were markedly higher when compared with the IPO group (p b 0.01). The effect of IPO treatment on W/D is presented in Table 4. Rats in the I/R group had the highest W/D, while the HF + MD group had the lowest weight gain. Both the IPO group (3) and dexamethasone + IPO group (4) showed less W/D than the I/R group (p b 0.01). However, there were no significant differences found in W/D of the IPO group (3) when compared to the dexamethasone + IPO group (4). Table 5 shows that the AI was significantly higher in the I/R group than in the sham group (p b 0.05). The treatment of IPO and dexamethasone + IPO markedly reduced AI in the I/R rats. AI in dexamethasone + IPO group was significantly lower than that in IPO group. Fig. 1 shows that the myocardium caspase-3, Fas and FasL protein expression levels were significantly higher in the I/R group than in the sham group (p b 0.01). The treatment of IPO and dexamethasone + IPO markedly reduced myocardium caspase-3, Fas and FasL proteins expression levels in the I/R rats. Myocardium caspase-3, Fas and FasL protein expression levels in the dexamethasone + IPO group were significantly lower than those in the IPO group. In the I/R group, the level of myocardium Bcl-2 protein expression was significantly lower, whereas the level of Bax protein expression was higher compared to those in the sham group (p b 0.01) (Fig. 2). Treatment of IPO and dexamethasone + IPO significantly increased the level of myocardium Bcl-2 protein expression and significantly decreased the myocardium Bax protein expression (p b 0.01). 4. Discussion ROS, which play an important role in the IR injury, are produced due to the reduction of molecular oxygen by the intracellular oxidative enzymes. Major ROS are superoxide (O2−), hydrogen peroxide (H2O2), and hydroxyl anions (OH−) (Slater, 1984; Valko et al., 2006). O2 − is considered to be the ‘primary’ ROS and produced by the enzyme xanthine oxidase in the early stages of ischemia. After O2 − is produced, the enzyme superoxide dismutase catalyzes the dismutation of O2− to O2 and the less reactive H2O2, and H2O2 is converted to H2O and O2 by the enzymes, catalase, and glutathione peroxidase (Li et al., 2014). Thus, superoxide dismutase and catalase are naturally occurring intracellular enzymes, which play a critical role in the defence of cells against Table 3 Effects of IPO on PaO2, NO and NOS values.
Group
TNF-α (ng/mL)
IL-1β (ng/mL)
IL-6 (ng/L)
Group
PaO2 (mm Hg)
NO (μmol/l)
NOS (U/mg)
I II III IV
267.49 351.96 310.52 288.48
170.63 241.63 219.07 185.49
427.42 659.26 581.32 495.36
I II III IV
436.71 235.47 359.33 418.25
0.91 0.53 0.68 0.79
1.94 1.52 1.72 1.83
b c d
± ± ± ±
30.51 33.61b 30.28d 30.53d
p b 0.01, group II vs group I. p b 0.05. p b 0.01, groups III and IV vs group II.
± ± ± ±
19.08 22.39b 22.51c 20.54d
± ± ± ±
44.29 68.97b 60.11c 52.53d
b c d
± ± ± ±
44.72 25.07b 38.11d 38.69d
p b 0.01, group II vs group I. p b 0.05. p b 0.01, groups III and IV vs group II.
± ± ± ±
0.09 0.06b 0.07c 0.08d
± ± ± ±
0.17 0.16b 0.19c 0.16d
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Table 4 Effects of IPO on W/D. Group
W/D
I II III IV
5.04 6.78 6.16 5.49
b c
± ± ± ±
p b 0.01, group II vs group I. p b 0.05, groups III and IV vs group II.
G
E
G
G
G
AI (%)
I II III IV
2.15 27.81 16.85 10.62
,
,,
,,,
0.22 2.91b 2.22d 1.19d
,9
Fig. 1. Effects of IPO on caspase-3, Fas and FasL protein expression. bp b 0.01, group II vs group I; dp b 0.01, groups III and IV vs group II.
Bcl-2-binding protein that shares significant sequence homology with Bcl-2. Bcl-2 is a membrane-bound protein that is present in the membranes of the mitochondrion, nucleus, and endoplasmic reticulum (Hockenbery et al., 1990). Thus, Bcl-2 may have an antioxidant role, preventing lipid peroxidation and associated cellular toxicity. Recent data highlight the pivotal role of caspase-3 in the execution of ischemia-induced apoptosis (Zheng et al., 2003). Caspase-3 inhibitors can prevent delayed cell death after ischemia (Chang & Karin, 2001). Thus, caspase-3 is a key step in the execution process of apoptosis, and its inhibition can block apoptotic cell death. Fas activates caspase3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active-site thiol (Mannick et al., 1999). FasL initiates apoptosis by binding to the Fas receptor, which could ultimately contribute to Fas receptormediated programmed cell death. An alternative possibility is that IPO may directly decrease Fas and FasL protein expression. On the other hand, Fas/FasL triggers the activation of caspase 8. Activated caspase 8 cleaves pro-caspase 3, which then undergoes autocatalysis to form active caspase 3, a principle effector caspase of apoptosis (Lee et al., 2008). We further demonstrated that the I/R-induced protein levels of activated caspase-3 and Bax were significantly inhibited by IPO treatment, indicating that the I/R-induced lung injury may be partly inhibited through Fas/FasL and Bcl-2/Bax system-mediated apoptosis pathway. The present study revealed that IPO treatment significantly inhibited the expression of caspase-3, Fas, FasL, Bax and lung cell apoptosis resulted from ischemia/reperfusion. The antiapoptotic activity observed with IPO can be attributed to the inhibition of caspase-3, Fas, FasL, Bax expression and induction of Bcl-2 protein. Also, free radical scavenging
%FO£DFWLQ
%D[£DFWLQ
E
G
G
G
E
± ± ± ±
G
G
Group
p b 0.01, group II vs group I. p b 0.01, groups III and IV vs group II.
)DV/£DFWLQ
E
0.42 0.47b 0.53 0.58c
Table 5 Effects of IPO on AI.
d
)DV£DFWLQ
E
the toxic effects of ROS products by their scavenging capacity (Maxwell & Lip, 1997). Increased levels of superoxide dismutase and catalase have also been reported in lung and kidney injuries induced by IR (Baltalarlı et al., 2006; Mun et al., 2003). However, we think that the level of antioxidant enzymes during I/R is determined by several factors, including the duration of IR periods, organ subjected to IR injury, and the magnitude of IR injury. In the present study, we found that I/R increased the lung MDA level and myocardium antioxidant enzyme activities in rats. IPO treatment could significantly alleviate I/R induced lung oxidative injury. We for the first time also found that dexamethasone treatment after ischemic insult enhanced the well-documented protective effect of IPO. Several previous investigations demonstrated elevated MPO activities in lung tissue after I/R injury clearly exhibiting the inflammatory response and the active role of MPO in this cascade (Ai et al., 2013; Liu et al., 2007b). Our findings were in parallel with the others: lung tissue MPO activities of the rats subjected to I/R were significantly higher. In IPO and dexamethasone + IPO treated groups, lung tissue MPO levels showed a significant decrease. Our results strengthen the fact that IPO may act as a potent anti-inflammatory agent by preventing the migration of neutrophils into the tissue, inhibiting NADPH oxidase and/or reversing superoxide anion radical production induced by reperfusion sequence. Excessive NO production has been attributed to inducible NO synthase that is not present under normal conditions but can be induced in response to systemic inflammatory states such as I/R injury (Liu et al., 2007b). In the present study, I/R caused an increase in NO levels both in the lung tissue which was significantly reversed by IPO administration, confirming the evidence related to the role of nitrosative stress in response to I/R injury and preventive effects of IPO. The observed antioxidant effect of IPO may be related to its indirect anticytotoxic and antinitrosative effects and the ability of protecting thiol-dependent antioxidant proteins from oxidative damage. The signaling molecules IL-6, IL-1β, and TNF-α, discharged from activated macrophages and neutrophils, exert a considerable amplifying effect on the systemic inflammatory response. The severity of lung injury has been shown to correlate with IL-6, IL-1β, and TNF-α activities (Amaral et al., 2007; Douzinas et al., 2004). In the present study, I/R significantly decreased the sera IL-6, IL-1β, and TNF-α, which suggests that the preventive effect of IPO on lung injury could be mediated by the depression of IL-6, IL-1β, and TNF-α. Apoptosis is an important form of cell death that is modulated by a number of gene products, in particular the members of the Bcl-2 family. Bcl-2 and Bax are recognized as modulators of apoptotic events, and their relative levels determine the fate of cells. Bcl-2 has been shown to inhibit apoptosis and to prolong cell survival, while Bax, a pro-apoptotic antagonist of Bcl-2, has been characterized as a
b
123
G
,
,,
,,,
,9
Fig. 2. Effects of IPO on Bcl-2 and Bax protein expression. bp b 0.01, group II vs group I; d p b 0.01, groups III and IV vs group II.
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and anti-inflammatory activities with reduced TNF-α, IL-1β and IL-6 production may contribute to the antiapoptotic effect of IPO. References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Ai, Songtao, Fan, Xindong, Fan, Linfeng, Sun, Qi, Liu, Yu, Tao, Xiaofeng, Dai, Kerong, 2013. Extraction and chemical characterization of Angelica sinensis polysaccharides and its antioxidant activity. Carbohydr. Polym. 94 (2), 731–736 (15 May). Amaral, F.A., Fagundes, C.T., Guabiraba, R., Vieira, A.T., Souza, A.L., Russo, R.C., Soares, M.P., Teixeira, M.M., Souza, D.G., 2007. The role of macrophage migration inhibitory factor in the cascade of events leading to reperfusion-induced inflammatory injury and lethality. Am. J. Pathol. 171, 1887–1893. Baltalarlı, A., Ozcan, V., Bir, F., et al., 2006. Ascorbic acid (vitamin C) and ilioprost attenuate the lung injury caused by ischemia/reperfusion of the lower extremities of rats. Ann. Vasc. Surg. 20, 49. Beutler, E., 1975. Red Cell Metabolism, 2nd ed. Grune and Stratton Co., New York, pp. 261–265. Bradley, P.P., Priebat, D.A., Christensen, R.D., Rothstein, G., 1982. Measurement of cutaneous inflammation estimation of neutrophil content with an enzyme marker. J. Invest. Dermatol. 78, 206–209. Chang, L., Karin, M., 2001. Mammalian MAP kinase signaling cascade. Nature 410, 37–40. Chen, Z., Liu, J., 2007. Effect of ischemic post-conditioning on spinal cord ischemic–reperfusion injury in rabbits. Can. J. Anaesth. 54, 42. Chen, J., Shen, B.Y., Deng, X.X., Zhan, Q., Peng, C.H., 2012. SKP1-CULLIN1-F-box (SCF)mediated DRG2 degradation facilitated chemotherapeutic drugs induced apoptosis in hepatocellular carcinoma cells. Biochem. Biophys. Res. Commun. 420 (3), 651–655. Day, A.L., Wick, E., Jordan, T.H., et al., 2005. Neutral endopeptidase determines the severity of pancreatitis-associated lung injury. J. Surg. Res. 128, 21–27. Douzinas, E.E., Kollias, S., Tiniakos, D., Evangelou, E., Papalois, A., Rapidis, A.D., Tsoukalas, G.D., Patsouris, E., Roussos, C., 2004. Hypoxemic reperfusion after 120 mins of intestinal ischemia attenuates the histopathologic and inflammatory response. Crit. Care Med. 32, 2279–2283. Durak, I., Yurtarslanl, Z., Canbolat, O., Akyol, O., 1993. A methodological approach to superoxide dismutase (SOD) activity assay based on inhibition of nitroblue tetrazolium (NBT) reduction. Clin. Chim. Acta 214, 103–104. Hockenbery, D., Nunez, G., Milliman, C., Schreiber, R.D., Korsmeyer, S.J., 1990. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348, 334–336. Lee, S.D., Kuo, W.W., Ho, Y.J., Lin, A.C., Tsai, C.H., Wang, H.F., Kuo, C.H., Yang, A.L., Huang, C.Y., Hwang, J.M., 2008. Cardiac Fas-dependent and mitochondria-dependent apoptosis in ovariectomized rats. Maturitas 61, 268–277. Li, Yan-Wei, Zhang, Yan, Zhang, Ling, Li, Xu, Yu, Jian-Bo, Zhang, Hong-Tao, Tan, Bin-Bin, Jiang, Lian-Hao, Wang, Ya-Xin, Liang, Yu, Zhang, Xiu-Shan, Wang, Wen-Sheng, Liu, Hai-Gen, 2014. Protective effect of tea polyphenols on renal ischemia/reperfusion injury via suppressing the activation of TLR4/NF-κB p65 signal pathway. Gene 542 (1), 46–51 (25 May). Liu, X., Chen, H., Zhan, B., et al., 2007a. Attenuation of reperfusion injury by renal ischemic postconditioning: the role of NO. Biochem. Biophys. Res. Commun. 359, 628.
Liu, K.X., Wu, W.K., He, W., et al., 2007b. Ginkgo biloba extract (EGb 761) attenuates lung injury induced by intestinal ischemia/reperfusion in rats: roles of oxidative stress and nitric oxide. World J. Gastroenterol. 13, 299. Mannick, J.B., Hausladen, A., Liu, L., Hess, D.T., Zeng, M., Miao, Q.X., Kane, L.S., Gow, A.J., Stamler, J.S., 1999. Fas-induced caspase denitrosylation. Science 284, 651–654. Maxwell, S.R.J., Lip, G.Y.H., 1997. Reperfusion injury: a review of the pathophysiology, clinical manifestations and therapeutic options. Int. J. Cardiol. 58, 95. Melotte, V., Qu, X., Ongenaert, M., van Criekinge, W., de Bruïne, A.P., Baldwin, H.S., et al., 2010. The N-myc downstream regulated gene (NDRG) family: diverse functions, multiple applications. FASEB J. 24 (11), 4153–4166. Mukherjee, S., Kim, S., Gibbons, L.E., Nho, K., Risacher, S.L., Glymour, M.M., et al., 2012. Genetic architecture of resilience of executive functioning. Brain Imaging Behav. 6 (4), 621–633. Mun, K.C., Lee, H.G., Lee, T.H., et al., 2003. Effect of modified polyhemoglobin on the ischemia/reperfusion injury in kidney. Transplant. Proc. 35, 99. Murry, C.E., Jennings, R.B., Reimer, K.A., 1986. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74, 1124–1136. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351. Rehni, A.K., Singh, N., 2007. Role of phosphoinositide 3-kinase in ischemic postconditioning-induced attenuation of cerebral ischemia-evoked behavioral deficits in mice. Pharmacol. Rep. 59, 192. Sener, G., Sehirli, O., Ercan, F., Sirvanci, S., Gedik, N., Kacmaz, A., 2005. Protective effect of MESNA (2-mercaptoethane sulfonate) against hepatic ischemia/reperfusion injury in rats. Surg. Today 35, 575–580. Serafin, A., Rosello-Catafau, J., Prats, N., Xaus, C., Gelpi, E., Peralta, C., 2002. Ischemic preconditioning increases the tolerance of fatty liver to hepatic ischemia–reperfusion injury in the rat. Am. J. Pathol. 161, 587–601. Slater, T.F., 1984. Free radical mechanisms in tissue injury. Biochem. J. 222, 1. Sun, Y., Oberley, L.W., Li, Y., 1988. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 34, 497–500. Sun, Z., Shen, L., Sun, X., Han, T., Yang, G., Zhang, J., et al., 2011. Variation of NDRG2 and c-Myc expression in rat heart during the acute stage of ischemia/reperfusion injury. Histochem. Cell Biol. 135 (1), 27–35. Tamion, F., Richard, V., Lacoume, Y., et al., 2002. Intestinal preconditioning prevents systemic inflammatory response in hemorrhagic shock. Role of HO-1. Am. J. Physiol. Gastrointest. Liver Physiol. 283, G408. Tang, P.S., Mura, M., Seth, R., et al., 2008. Acute lung injury and cell death: how many ways can cells die? Am. J. Physiol. Lung Cell. Mol. Physiol. 294, L632. Tomai, F., Crea, F., Chiariello, L., et al., 2001. Ischemic preconditioning in humans: models, mediators, and clinical relevance. Circulation 100, 559. Valko, M., Rhodes, C.J., Moncol, J., et al., 2006. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 160, 1. Zhao, Z.Q., 2010. Postconditioning in reperfusion injury: A status report. Cardiovasc. Drugs Ther. 24, 265. Zhao, Z.Q., Vinten-Johansen, J., 2006. Postconditioning: reduction of reperfusion-induced injury. Cardiovasc. Res. 70, 200. Zheng, Z., Zhao, H., Steinberg, G.K., Yenari, M.A., 2003. Cellular and molecular events underlying ischemia-induced neuronal apoptosis. Drug News Perspect. 16, 497–503.
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Short communication
Ischemia postconditioning preventing lung ischemia–reperfusion injury Qi-Feng Cao ⁎, Mei-Jun Qu, Wei-Qin Yang, Dan-Ping Wang, Ming-Hui Zhang, Song-Bo Di 3rd Department of Internal Medicine, Integrated Chinese and Western Medicine Hospital of Taizhou, Zhejiang Province 317523, China
a r t i c l e
i n f o
Article history: Received 15 May 2014 Accepted 5 October 2014 Available online 7 October 2014 Keywords: Ischemic postconditioning Lung ischemia reperfusion Antioxidant
a b s t r a c t Objective: This study evaluates the inhibitory effect of IPO against ischemia reperfusion (I/R) induced lung injury in rats. Methods: Anesthetized and mechanically ventilated adult Sprague–Dawley rats were randomly assigned to one of the following groups (n = 12 each): the sham operated control group, the ischemia–reperfusion (IR) group (30 min of left-lung ischemia and 24 h of reperfusion), the IPO group (three successive cycles of 30-s reperfusion per 30-s occlusion before restoring full perfusion), and the dexamethasone plus IPO group (rats were injected with dexamethasone (3 mg/kg·day−1) 10 min prior to the experiment and the rest of the procedures were the same as the IPO group). Lung injury was assessed by wet-to-dry lung weight ratio and tissue apoptosis and biochemical changes. Results: Lung ischemia–reperfusion increased lung MDA production, serum proinflammatory cytokine count, and MPO activity and reduced antioxidant enzyme activities (all p b 0.05 I/R versus sham), accompanied with a compensatory increase in caspase-3, bax, Fas, FasL proteins and a decrease in Bcl-2 protein. Plasma levels of TNF-α, IL6, and IL-1β were increased in the I/R group (all p b 0.05 versus sham). IPO attenuated or prevented all the above changes. Treatment with dexamethasone enhanced all the protective effects of postconditioning. Conclusion: Postconditioning obviously inhibits I/R induced lung injury by its antioxidant, anti-inflammatory and anti-apoptosis activities. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Lung ischemia–reperfusion injury is clinically common in lung transplantation and extracorporeal circulation operation (Melotte et al., 2010; Mukherjee et al., 2012; Sun et al., 2011). Lung I/R injury can lead to lung dysfunction and is the leading cause of death after lung transplantation (Chen et al., 2012). Lung I/R injury still causes significant morbidity and mortality and is characterized by neutrophil extravasation, interstitial edema, disruption of epithelial integrity, and leakage of protein into the alveolar space, all associated with severe alterations in gas exchange (Tang et al., 2008). Reperfusion injury after pulmonary transplantation can contribute significantly to postoperative pulmonary dysfunction. To date, the mechanisms underlying lung I/R injury remain unclear, and effective treatments for its prevention are lacking. Murry et al. (1986) first introduced the definition of ischemic preconditioning (IPC), in which repetitive brief periods of ischemia protected the myocardium from a subsequent greater period of
Abbreviations: I/R, ischemia reperfusion; IPC, ischemic preconditioning; IPO, ischemic postconditioning. ⁎ Corresponding author. E-mail address: [email protected] (Q.-F. Cao).
http://dx.doi.org/10.1016/j.gene.2014.10.009 0378-1119/© 2014 Elsevier B.V. All rights reserved.
ischemic insult. Ischemic preconditioning (IPC) in which repetitive brief periods of intestinal ischemia protect the intestine from a subsequent longer ischemic insult has been demonstrated to attenuate acute lung injury induced by I/R (Tamion et al., 2002). Although IPC has been shown to be beneficial in the human heart (Tomai et al., 2001), prospectively controlled studies in humans involving IPC of the intestine are lacking. The major problem is the inability to predict the onset of ischemia in some clinical settings related to I/R. Recently, some studies showed that ischemic postconditioning (IPO), which consists of one or more short cycles of reperfusion followed by one or more short cycles of ischemia, immediately after an ischemic phase and before the permanent reperfusion occurs, was as efficient as the IPC in preventing myocardial reperfusion injury (Zhao & Vinten-Johansen, 2006). It has also been reproduced in other organs such as the spinal cord (Chen & Liu, 2007), renal (Liu et al., 2007a), and cerebral (Rehni & Singh, 2007) tissues. Recent studies have demonstrated that brief intermittent cycles of ischemia alternating with reperfusion applied after the prolonged ischemic event, a novel approach termed “ischemic postconditioning (IPO)”, attenuated IRI in a wide range of organs, including the heart, brain, spinal cord, liver, and kidney (Zhao, 2010). This study aimed to observe the influence of IPO on lung I/R rat lung tissue oxidative injury and the mechanism. We established the rat lung I/R model, used IPO, and observe its prevention effect of lung I/R injury in rats.
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2. Material and methods
121
absorbance of 1 per minute) was expressed as units per gram of tissue. MPO product was measured spectrophotometrically at 650 nm.
2.1. Animals 2.6. Analysis of antioxidant enzyme activities Three months old male albino Wistar rats, weighting between 150 and 250 g, were used. Animals were raised in the Animal House of the Faculty of Science, Taizhou University. They were maintained in a temperature room (22 ± 2 °C) on a 12 h light–dark natural cycle. Rats were fed with standard diet and water ad libitum. These studies were conducted with the approval of the China National Ethical Committee. 2.2. Surgical procedure and experimental protocol The rats were randomly assigned to one of four groups (n = 12 in each group). Under aseptic conditions, an in situ unilateral lung warm ischemia model was used. In brief, a left anterolateral thoracotomy in the fifth intercostal space was created. The left lung was mobilized, the pulmonary hilum was dissected, and the perivascular and peribronchial tissues were removed. Next, all the rats received 500 U/kg of heparin intravenously in saline (total volume 500 mL). In group 1 (sham), the rats underwent sham thoracotomy and hilar dissection, but the lungs were not rendered ischemic. In group 2 (I/R), 5 min after heparin administration, the left pulmonary artery, bronchus, and pulmonary vein were occluded with a noncrushing microvascular clamp, maintaining the lung in a partially inflated state. The lungs were kept moist with periodic applications of warm, sterile saline, and the incision was covered to minimize evaporative losses. The ischemic period was held constant at 30 min, after which the clamp was removed and the lung reperfused for 24 h. In group 3 (IPO), postconditioning was performed by three successive cycles of 30 s of reperfusion for each 30 s of occlusion, starting immediately after the release of the index ischemia. In group 4 (dexamethasone + IPO) rats were injected with dexamethasone (3 mg/kg·day−1) 10 min prior to the experiment and the rest of the procedures were the same as the IPO group. 2.3. Lung wet/dry weight ratio At the end of the experiments, the left lower lobe of the lung was dissected and dried at a constant temperature of 80 °C for 24 h to obtain a dehydrate consistency. The lung wet/dry (W/D) ratio was calculated as an indicator of edema. 2.4. In situ detection of apoptotic cells Serial sections of 4-μm thickness were prepared. TUNEL staining was performed with the use of an in situ apoptosis detection kit according to the manufacturer's instruction (Boehringer Mannheim, Germany) and examined by light microscopy. The apoptotic index (AI) was calculated as the percentage of stained cells, namely: AI = number of apoptotic cells × 100 / total number of nucleated cells.
Lipid peroxidation level in the tissue samples was expressed in MDA. Measurement was based on the method of Ohkawa et al. (1979). The reaction mixture contained 0.1 mL of sample, 0.2 mL of 8.1% sodium dodecyl sulfate, 1.5 mL of 20% acetic acid, and 1.5 mL of 0.8% aqueous solution of TBA. The mixture pH was adjusted to 3.5 and the volume was finally made up to 4.0 mL with distilled water and 5.0 mL of the mixture of n-butanol and pyridine (15:1, vol/vol) was added. The mixture was shaken vigorously. After centrifugation at 4000 rpm for 10 min, the absorbance of the organic layer was measured at 532 nm. MDA was expressed as nmol/mg protein. GPx activity was measured by the method described by Beutler (1975). The reaction mixture contained 1 M Tris buffer (pH 8.0), 0.1 M GSH, 2 mM nicotinamide adenine dinucleotide phosphate (NADPH), and 10 U/mL glutathione reductase. Decrease in absorbance (OD) was followed at 340 nm and enzyme activity in samples was calculated as U/g protein. SOD activity was determined according to the method Sun et al. (1988) with a slight modification (Durak et al., 1993). The principle of the method is based on the inhibition of NBT reduction by the xanthine–xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the sample after 1.0 mL ethanol/ chloroform mixture (5/3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. Catalase activity was evaluated by measuring the decrease in H2O2 concentration at 240 nm (Aebi, 1984). Working solution (phosphate buffer 100 mM; H2O2 10 mM) and sample were mixed in a cuvette. The change in absorbance per minute at 240 nm (DA240) was calculated. Enzyme activity was expressed in units of CAT activity per milligram of protein. One unit of CAT activity is defined as the amount of enzyme needed to reduce 1 μmol H2O2/min. 2.7. Analysis of serum TNF-α, IL-1β and IL-6 levels The levels of sera TNF-α, IL-1β and IL-6 were measured using commercially available kits. The TNF-α, IL-1β and IL-6 assay kits were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). These experiments were performed according to the manufacturer's instructions. 2.8. Analysis of PaO2, NO and NOS levels Arterial blood gas analysis is used to measure the partial pressure of venous oxygen (PaO2). NO and NOS levels were measured with commercially available kits. These experiments were performed according to the manufacturer's instructions.
2.5. Myeloperoxidase activity 2.9. Western blot The activity of MPO was used as a marker of neutrophil infiltration (Bradley et al., 1982; Day et al., 2005; Sener et al., 2005). Lung MPO activity was determined as described previously (Serafin et al., 2002). Lung tissue was homogenized in 2 mL of 10 mmol/L phosphate buffer (pH 7.4). After centrifugation at 10,000 g for 20 min, the pellet was resuspended in 1 mL of 10 mmol/L phosphate buffer (pH 7.0) containing 0.5% hexadecyltrimethylammonium and sonicated for 10 s. After heating at 60 °C for 2 h, the samples were centrifuged at 8000 g for 10 min. The assay mixture consisted of 20 μL of the supernatant, 10 μL of 3,3′5,5′-tetramethylbenzidine (final concentration, 1.6 mmol/L) dissolved in dimethyl sulfoxide, and 70 μL of H2O2 (final concentration, 3 mmol/L) diluted in 80 mmol/L of phosphate buffer (pH 5.4), and incubated at 37 °C for 5 min. MPO activity (1 unit defined as change in
Lung tissues were homogenized and sonicated in a tissue lysis buffer containing Tris–HCl (20 mM, pH 7.4), NaCl (150 mM), ethylenediaminetetraacetate (l mM), ethylene glycol tetraacetic acid (l mM), βglycerolphosphate (1 mM), sodium pyrophosphate (2.5 mM), Triton X-100 (1%), phenylmethylsulfonyl fluoride (1 mM), dithiothreitol (1 mM), leupeptin (1 mg/mL), aprotinin (1 mg/mL), and pepstatin (1 mg/mL). The homogenate was centrifuged at 1000 g for 10 min at 4 °C to collect the supernatant as a total protein preparation. Equal amounts of protein were combined with 5 × sodium dodecyl sulfate loading buffer and boiled for 5 min and then separated using 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and subsequently transferred to polyvinyldine diflouride membrane for immunoblot
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Table 1 Effects of IPO on myocardium antioxidant status and lipid peroxidation. Group
MDA
I II III IV
4.19 8.66 7.28 5.72
b c d
± ± ± ±
SOD 0.45 0.92b 0.85c 0.63d
207.8 95.29 152.58 184.11
MPO ± ± ± ±
22.17 8.69b 16.38d 19.05d
0.27 0.84 0.61 0.38
CAT ± ± ± ±
0.03 0.09b 0.07d 0.04d
24.98 11.39 17.05 22.63
GSH-Px ± ± ± ±
3.15 1.31b 1.84d 2.66d
62.95 27.47 40.71 58.03
± ± ± ±
7.31 3.03b 4.25d 6.09d
p b 0.01, group II vs group I. p b 0.05. p b 0.01, groups III and IV vs group II.
analysis. The membranes were blocked in 5% nonfat milk for 2 h at room temperature and then incubated overnight at 4 °C with primary antibodies against caspase-3 (1:1000, Cell Signaling Technology, Beverly, MA), Fas (1:1000, Cell Signaling Technology), FasL (1:500, Santa Cruz Biotechnology), Bax (1:1000, Santa Cruz Biotechnology), and Bcl-2 (1:1000, Santa Cruz Biotechnology). After being washed with Tris-buffered saline-Tween 20, the membranes were incubated with proper secondary horseradish peroxidase-conjugated antibodies (1:5000–1:10,000, Cell Signaling Technology) and developed with enhanced chemiluminescence reagent (GE Healthcare, Rahway, NJ). The membranes were subsequently reblotted for β-actin (1:2000; Cell Signaling Technology), and the results were normalized to β-actin to correct for loading. 2.10. Statistical analysis Data are expressed as mean ± standard deviation (SD). Group differences were analyzed using a oneway ANOVA with the Student– Newman–Keuls or Dunnett's test. p b 0.05 was considered statistically significant. Data were analyzed using SPSS 16.0 (SPSS, Chicago, IL, USA). 3. Results The effects of IPO on myocardium antioxidant status and lipid peroxidation were investigated. The I/R caused an increase in myocardium antioxidant enzyme activities (SOD, CAT, GSH-Px) with a concomitant decrease in MDA and MPO values as shown in Table 1. Both IPO group (3) and dexamethasone + IPO group (4) showed an improvement in myocardium antioxidant status as assessed by MDA and antioxidant enzyme activities which were significantly decreased and increased as compared with the I/R group (p b 0.05). However, the TRAP value in two groups was less than that of the RD group (p b 0.05). There was also considerable variation in the serum TNF-α, IL-1β and IL-6 contents in rat groups. As shown in Table 2, serum TNF-α, IL-1β and IL-6 contents in the I/R group were significantly higher than those in the sham group. Treatment of IPO and dexamethasone + IPO significantly decreased serum TNF-α, IL-1β and IL-6 contents when compared to the I/R group (p b 0.01). There were significant differences in the contents of sera TNF-α, IL-1β and IL-6 between IPO and dexamethasone + IPO groups (p b 0.01). Serum TNF-α, IL-1β and IL-6 concentration of the dexamethasone + IPO group was markedly lower when compared with the IPO group (p b 0.01). There was also considerable variation in the PaO2, NO and NOS values in rat groups. As shown in Table 3, PaO2, NO and NOS values in Table 2 Effects of IPO on serum TNF-α, IL-1β and IL-6 contents.
the I/R group were significantly lower than those in the sham group. Treatment of IPO and dexamethasone + IPO significantly increased PaO2, NO and NOS values when compared to the I/R group (p b 0.01). There were significant differences in the PaO2, NO and NOS values between IPO and dexamethasone + IPO groups (p b 0.01). PaO2, NO and NOS values of the dexamethasone + IPO group were markedly higher when compared with the IPO group (p b 0.01). The effect of IPO treatment on W/D is presented in Table 4. Rats in the I/R group had the highest W/D, while the HF + MD group had the lowest weight gain. Both the IPO group (3) and dexamethasone + IPO group (4) showed less W/D than the I/R group (p b 0.01). However, there were no significant differences found in W/D of the IPO group (3) when compared to the dexamethasone + IPO group (4). Table 5 shows that the AI was significantly higher in the I/R group than in the sham group (p b 0.05). The treatment of IPO and dexamethasone + IPO markedly reduced AI in the I/R rats. AI in dexamethasone + IPO group was significantly lower than that in IPO group. Fig. 1 shows that the myocardium caspase-3, Fas and FasL protein expression levels were significantly higher in the I/R group than in the sham group (p b 0.01). The treatment of IPO and dexamethasone + IPO markedly reduced myocardium caspase-3, Fas and FasL proteins expression levels in the I/R rats. Myocardium caspase-3, Fas and FasL protein expression levels in the dexamethasone + IPO group were significantly lower than those in the IPO group. In the I/R group, the level of myocardium Bcl-2 protein expression was significantly lower, whereas the level of Bax protein expression was higher compared to those in the sham group (p b 0.01) (Fig. 2). Treatment of IPO and dexamethasone + IPO significantly increased the level of myocardium Bcl-2 protein expression and significantly decreased the myocardium Bax protein expression (p b 0.01). 4. Discussion ROS, which play an important role in the IR injury, are produced due to the reduction of molecular oxygen by the intracellular oxidative enzymes. Major ROS are superoxide (O2−), hydrogen peroxide (H2O2), and hydroxyl anions (OH−) (Slater, 1984; Valko et al., 2006). O2 − is considered to be the ‘primary’ ROS and produced by the enzyme xanthine oxidase in the early stages of ischemia. After O2 − is produced, the enzyme superoxide dismutase catalyzes the dismutation of O2− to O2 and the less reactive H2O2, and H2O2 is converted to H2O and O2 by the enzymes, catalase, and glutathione peroxidase (Li et al., 2014). Thus, superoxide dismutase and catalase are naturally occurring intracellular enzymes, which play a critical role in the defence of cells against Table 3 Effects of IPO on PaO2, NO and NOS values.
Group
TNF-α (ng/mL)
IL-1β (ng/mL)
IL-6 (ng/L)
Group
PaO2 (mm Hg)
NO (μmol/l)
NOS (U/mg)
I II III IV
267.49 351.96 310.52 288.48
170.63 241.63 219.07 185.49
427.42 659.26 581.32 495.36
I II III IV
436.71 235.47 359.33 418.25
0.91 0.53 0.68 0.79
1.94 1.52 1.72 1.83
b c d
± ± ± ±
30.51 33.61b 30.28d 30.53d
p b 0.01, group II vs group I. p b 0.05. p b 0.01, groups III and IV vs group II.
± ± ± ±
19.08 22.39b 22.51c 20.54d
± ± ± ±
44.29 68.97b 60.11c 52.53d
b c d
± ± ± ±
44.72 25.07b 38.11d 38.69d
p b 0.01, group II vs group I. p b 0.05. p b 0.01, groups III and IV vs group II.
± ± ± ±
0.09 0.06b 0.07c 0.08d
± ± ± ±
0.17 0.16b 0.19c 0.16d
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Table 4 Effects of IPO on W/D. Group
W/D
I II III IV
5.04 6.78 6.16 5.49
b c
± ± ± ±
p b 0.01, group II vs group I. p b 0.05, groups III and IV vs group II.
G
E
G
G
G
AI (%)
I II III IV
2.15 27.81 16.85 10.62
,
,,
,,,
0.22 2.91b 2.22d 1.19d
,9
Fig. 1. Effects of IPO on caspase-3, Fas and FasL protein expression. bp b 0.01, group II vs group I; dp b 0.01, groups III and IV vs group II.
Bcl-2-binding protein that shares significant sequence homology with Bcl-2. Bcl-2 is a membrane-bound protein that is present in the membranes of the mitochondrion, nucleus, and endoplasmic reticulum (Hockenbery et al., 1990). Thus, Bcl-2 may have an antioxidant role, preventing lipid peroxidation and associated cellular toxicity. Recent data highlight the pivotal role of caspase-3 in the execution of ischemia-induced apoptosis (Zheng et al., 2003). Caspase-3 inhibitors can prevent delayed cell death after ischemia (Chang & Karin, 2001). Thus, caspase-3 is a key step in the execution process of apoptosis, and its inhibition can block apoptotic cell death. Fas activates caspase3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active-site thiol (Mannick et al., 1999). FasL initiates apoptosis by binding to the Fas receptor, which could ultimately contribute to Fas receptormediated programmed cell death. An alternative possibility is that IPO may directly decrease Fas and FasL protein expression. On the other hand, Fas/FasL triggers the activation of caspase 8. Activated caspase 8 cleaves pro-caspase 3, which then undergoes autocatalysis to form active caspase 3, a principle effector caspase of apoptosis (Lee et al., 2008). We further demonstrated that the I/R-induced protein levels of activated caspase-3 and Bax were significantly inhibited by IPO treatment, indicating that the I/R-induced lung injury may be partly inhibited through Fas/FasL and Bcl-2/Bax system-mediated apoptosis pathway. The present study revealed that IPO treatment significantly inhibited the expression of caspase-3, Fas, FasL, Bax and lung cell apoptosis resulted from ischemia/reperfusion. The antiapoptotic activity observed with IPO can be attributed to the inhibition of caspase-3, Fas, FasL, Bax expression and induction of Bcl-2 protein. Also, free radical scavenging
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%D[£DFWLQ
E
G
G
G
E
± ± ± ±
G
G
Group
p b 0.01, group II vs group I. p b 0.01, groups III and IV vs group II.
)DV/£DFWLQ
E
0.42 0.47b 0.53 0.58c
Table 5 Effects of IPO on AI.
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the toxic effects of ROS products by their scavenging capacity (Maxwell & Lip, 1997). Increased levels of superoxide dismutase and catalase have also been reported in lung and kidney injuries induced by IR (Baltalarlı et al., 2006; Mun et al., 2003). However, we think that the level of antioxidant enzymes during I/R is determined by several factors, including the duration of IR periods, organ subjected to IR injury, and the magnitude of IR injury. In the present study, we found that I/R increased the lung MDA level and myocardium antioxidant enzyme activities in rats. IPO treatment could significantly alleviate I/R induced lung oxidative injury. We for the first time also found that dexamethasone treatment after ischemic insult enhanced the well-documented protective effect of IPO. Several previous investigations demonstrated elevated MPO activities in lung tissue after I/R injury clearly exhibiting the inflammatory response and the active role of MPO in this cascade (Ai et al., 2013; Liu et al., 2007b). Our findings were in parallel with the others: lung tissue MPO activities of the rats subjected to I/R were significantly higher. In IPO and dexamethasone + IPO treated groups, lung tissue MPO levels showed a significant decrease. Our results strengthen the fact that IPO may act as a potent anti-inflammatory agent by preventing the migration of neutrophils into the tissue, inhibiting NADPH oxidase and/or reversing superoxide anion radical production induced by reperfusion sequence. Excessive NO production has been attributed to inducible NO synthase that is not present under normal conditions but can be induced in response to systemic inflammatory states such as I/R injury (Liu et al., 2007b). In the present study, I/R caused an increase in NO levels both in the lung tissue which was significantly reversed by IPO administration, confirming the evidence related to the role of nitrosative stress in response to I/R injury and preventive effects of IPO. The observed antioxidant effect of IPO may be related to its indirect anticytotoxic and antinitrosative effects and the ability of protecting thiol-dependent antioxidant proteins from oxidative damage. The signaling molecules IL-6, IL-1β, and TNF-α, discharged from activated macrophages and neutrophils, exert a considerable amplifying effect on the systemic inflammatory response. The severity of lung injury has been shown to correlate with IL-6, IL-1β, and TNF-α activities (Amaral et al., 2007; Douzinas et al., 2004). In the present study, I/R significantly decreased the sera IL-6, IL-1β, and TNF-α, which suggests that the preventive effect of IPO on lung injury could be mediated by the depression of IL-6, IL-1β, and TNF-α. Apoptosis is an important form of cell death that is modulated by a number of gene products, in particular the members of the Bcl-2 family. Bcl-2 and Bax are recognized as modulators of apoptotic events, and their relative levels determine the fate of cells. Bcl-2 has been shown to inhibit apoptosis and to prolong cell survival, while Bax, a pro-apoptotic antagonist of Bcl-2, has been characterized as a
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Fig. 2. Effects of IPO on Bcl-2 and Bax protein expression. bp b 0.01, group II vs group I; d p b 0.01, groups III and IV vs group II.
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and anti-inflammatory activities with reduced TNF-α, IL-1β and IL-6 production may contribute to the antiapoptotic effect of IPO. References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Ai, Songtao, Fan, Xindong, Fan, Linfeng, Sun, Qi, Liu, Yu, Tao, Xiaofeng, Dai, Kerong, 2013. Extraction and chemical characterization of Angelica sinensis polysaccharides and its antioxidant activity. Carbohydr. Polym. 94 (2), 731–736 (15 May). Amaral, F.A., Fagundes, C.T., Guabiraba, R., Vieira, A.T., Souza, A.L., Russo, R.C., Soares, M.P., Teixeira, M.M., Souza, D.G., 2007. The role of macrophage migration inhibitory factor in the cascade of events leading to reperfusion-induced inflammatory injury and lethality. Am. J. Pathol. 171, 1887–1893. Baltalarlı, A., Ozcan, V., Bir, F., et al., 2006. Ascorbic acid (vitamin C) and ilioprost attenuate the lung injury caused by ischemia/reperfusion of the lower extremities of rats. Ann. Vasc. Surg. 20, 49. Beutler, E., 1975. Red Cell Metabolism, 2nd ed. Grune and Stratton Co., New York, pp. 261–265. Bradley, P.P., Priebat, D.A., Christensen, R.D., Rothstein, G., 1982. Measurement of cutaneous inflammation estimation of neutrophil content with an enzyme marker. J. Invest. Dermatol. 78, 206–209. Chang, L., Karin, M., 2001. Mammalian MAP kinase signaling cascade. Nature 410, 37–40. Chen, Z., Liu, J., 2007. Effect of ischemic post-conditioning on spinal cord ischemic–reperfusion injury in rabbits. Can. J. Anaesth. 54, 42. Chen, J., Shen, B.Y., Deng, X.X., Zhan, Q., Peng, C.H., 2012. SKP1-CULLIN1-F-box (SCF)mediated DRG2 degradation facilitated chemotherapeutic drugs induced apoptosis in hepatocellular carcinoma cells. Biochem. Biophys. Res. Commun. 420 (3), 651–655. Day, A.L., Wick, E., Jordan, T.H., et al., 2005. Neutral endopeptidase determines the severity of pancreatitis-associated lung injury. J. Surg. Res. 128, 21–27. Douzinas, E.E., Kollias, S., Tiniakos, D., Evangelou, E., Papalois, A., Rapidis, A.D., Tsoukalas, G.D., Patsouris, E., Roussos, C., 2004. Hypoxemic reperfusion after 120 mins of intestinal ischemia attenuates the histopathologic and inflammatory response. Crit. Care Med. 32, 2279–2283. Durak, I., Yurtarslanl, Z., Canbolat, O., Akyol, O., 1993. A methodological approach to superoxide dismutase (SOD) activity assay based on inhibition of nitroblue tetrazolium (NBT) reduction. Clin. Chim. Acta 214, 103–104. Hockenbery, D., Nunez, G., Milliman, C., Schreiber, R.D., Korsmeyer, S.J., 1990. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348, 334–336. Lee, S.D., Kuo, W.W., Ho, Y.J., Lin, A.C., Tsai, C.H., Wang, H.F., Kuo, C.H., Yang, A.L., Huang, C.Y., Hwang, J.M., 2008. Cardiac Fas-dependent and mitochondria-dependent apoptosis in ovariectomized rats. Maturitas 61, 268–277. Li, Yan-Wei, Zhang, Yan, Zhang, Ling, Li, Xu, Yu, Jian-Bo, Zhang, Hong-Tao, Tan, Bin-Bin, Jiang, Lian-Hao, Wang, Ya-Xin, Liang, Yu, Zhang, Xiu-Shan, Wang, Wen-Sheng, Liu, Hai-Gen, 2014. Protective effect of tea polyphenols on renal ischemia/reperfusion injury via suppressing the activation of TLR4/NF-κB p65 signal pathway. Gene 542 (1), 46–51 (25 May). Liu, X., Chen, H., Zhan, B., et al., 2007a. Attenuation of reperfusion injury by renal ischemic postconditioning: the role of NO. Biochem. Biophys. Res. Commun. 359, 628.
Liu, K.X., Wu, W.K., He, W., et al., 2007b. Ginkgo biloba extract (EGb 761) attenuates lung injury induced by intestinal ischemia/reperfusion in rats: roles of oxidative stress and nitric oxide. World J. Gastroenterol. 13, 299. Mannick, J.B., Hausladen, A., Liu, L., Hess, D.T., Zeng, M., Miao, Q.X., Kane, L.S., Gow, A.J., Stamler, J.S., 1999. Fas-induced caspase denitrosylation. Science 284, 651–654. Maxwell, S.R.J., Lip, G.Y.H., 1997. Reperfusion injury: a review of the pathophysiology, clinical manifestations and therapeutic options. Int. J. Cardiol. 58, 95. Melotte, V., Qu, X., Ongenaert, M., van Criekinge, W., de Bruïne, A.P., Baldwin, H.S., et al., 2010. The N-myc downstream regulated gene (NDRG) family: diverse functions, multiple applications. FASEB J. 24 (11), 4153–4166. Mukherjee, S., Kim, S., Gibbons, L.E., Nho, K., Risacher, S.L., Glymour, M.M., et al., 2012. Genetic architecture of resilience of executive functioning. Brain Imaging Behav. 6 (4), 621–633. Mun, K.C., Lee, H.G., Lee, T.H., et al., 2003. Effect of modified polyhemoglobin on the ischemia/reperfusion injury in kidney. Transplant. Proc. 35, 99. Murry, C.E., Jennings, R.B., Reimer, K.A., 1986. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74, 1124–1136. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351. Rehni, A.K., Singh, N., 2007. Role of phosphoinositide 3-kinase in ischemic postconditioning-induced attenuation of cerebral ischemia-evoked behavioral deficits in mice. Pharmacol. Rep. 59, 192. Sener, G., Sehirli, O., Ercan, F., Sirvanci, S., Gedik, N., Kacmaz, A., 2005. Protective effect of MESNA (2-mercaptoethane sulfonate) against hepatic ischemia/reperfusion injury in rats. Surg. Today 35, 575–580. Serafin, A., Rosello-Catafau, J., Prats, N., Xaus, C., Gelpi, E., Peralta, C., 2002. Ischemic preconditioning increases the tolerance of fatty liver to hepatic ischemia–reperfusion injury in the rat. Am. J. Pathol. 161, 587–601. Slater, T.F., 1984. Free radical mechanisms in tissue injury. Biochem. J. 222, 1. Sun, Y., Oberley, L.W., Li, Y., 1988. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 34, 497–500. Sun, Z., Shen, L., Sun, X., Han, T., Yang, G., Zhang, J., et al., 2011. Variation of NDRG2 and c-Myc expression in rat heart during the acute stage of ischemia/reperfusion injury. Histochem. Cell Biol. 135 (1), 27–35. Tamion, F., Richard, V., Lacoume, Y., et al., 2002. Intestinal preconditioning prevents systemic inflammatory response in hemorrhagic shock. Role of HO-1. Am. J. Physiol. Gastrointest. Liver Physiol. 283, G408. Tang, P.S., Mura, M., Seth, R., et al., 2008. Acute lung injury and cell death: how many ways can cells die? Am. J. Physiol. Lung Cell. Mol. Physiol. 294, L632. Tomai, F., Crea, F., Chiariello, L., et al., 2001. Ischemic preconditioning in humans: models, mediators, and clinical relevance. Circulation 100, 559. Valko, M., Rhodes, C.J., Moncol, J., et al., 2006. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 160, 1. Zhao, Z.Q., 2010. Postconditioning in reperfusion injury: A status report. Cardiovasc. Drugs Ther. 24, 265. Zhao, Z.Q., Vinten-Johansen, J., 2006. Postconditioning: reduction of reperfusion-induced injury. Cardiovasc. Res. 70, 200. Zheng, Z., Zhao, H., Steinberg, G.K., Yenari, M.A., 2003. Cellular and molecular events underlying ischemia-induced neuronal apoptosis. Drug News Perspect. 16, 497–503.