Inhibition of hydrogen sulfide generation contributes to lung injury after experimental orthotopic lung transplantation

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Inhibition of hydrogen sulfide generation contributes to lung injury after experimental orthotopic lung transplantation Jingxiang Wu, MD,a,1 Jionglin Wei, MMed,a,b,1 Xingji You, PhD,c Xu Chen, MMed,a Hongwei Zhu, MMed,a Xiaoyan Zhu, PhD,c Yujian Liu, PhD,d,* and Meiying Xu, MDa,** a

Department of Anesthesiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai, China Department of Anesthesiology, Shanghai Huadong Hospital, Shanghai Fudan University, Shanghai, China c Department of Physiology, Second Military Medical University, Shanghai, China d Department of Pathophysiology, Second Military Medical University, Shanghai, China b

article info

abstract

Article history:

Background: Lung injury induced by ischemia or reperfusion significantly accounts for the

Received 19 July 2012

risk of early mortality of lung transplantation (LT). Recent studies have demonstrated

Received in revised form

that hydrogen sulfide (H2S) and its endogenous synthase cystathionine-g-lyase (CSE)

17 September 2012

confer protection against injury induced by ischemia or reperfusion in various organs.

Accepted 19 September 2012

This prompted us to define the role of CSE/H2S pathway in transplantation-induced lung

Available online 4 October 2012

injury. Methods: We performed single left LT in male SpragueeDawley rats after 3 h of cold

Keywords:

ischemia time. H2S donor NaHS (14 mmol/kg, intraperitoneally) or CSE inhibitor prop-

Lung transplantation

argylglycine (37.5 mg/kg, intraperitoneally) was administered 15 min before the start of the

Ischemiaereperfusion injury

LT. CSE protein expression, H2S generation, and the severity of pulmonary graft injuries

Hydrogen sulfide

were estimated at 24 h after reperfusion.

Cystathionine-g-lyase

Results: Both CSE protein expression and H2S generation were markedly decreased in transplanted rat lungs compared with those in sham-operated lungs. In the lungtransplanted rats, NaHS administration significantly improved pulmonary function and decreased lipid peroxidation and myeloperoxidase activity. In addition, NaHS inhibited the production of interleukin 1b but increased interleukin 10 levels in graft lung tissues. In contrast, propargylglycine further exacerbated pulmonary function and lung injuries after experimental orthotopic LT. Conclusions: To our knowledge, this study for the first time has demonstrated that the suppression of CSE expression and H2S production is associated with transplantationinduced lung injury. Both exogenous and endogenous H2S seem to have protective effects against acute LT injury by their multiple functions including antioxidation and antiinflammation, suggesting that modulation of H2S levels may be considered a potential therapeutic approach in LT. ª 2013 Elsevier Inc. All rights reserved.

* Corresponding author. Department of Pathophysiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China. Tel./fax: þ86 21 35090031. ** Corresponding author. Department of Anesthesiology, Shanghai Chest Hospital, Shanghai Jiaotong University, 241 west Huaihai Road, Shanghai 200030, China. Tel./fax: þ86 21 32260806. E-mail addresses: [email protected] (Y. Liu), [email protected] (M. Xu). 1 These authors contributed equally to this work. 0022-4804/$ e see front matter ª 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2012.09.028

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Introduction

Lung transplantation (LT) has become an accepted treatment effective for end-stage lung diseases. However, despite multiple significant therapeutic advances in anesthesia, surgical techniques, lung preservation, and intensive care medicine, lung injury induced by ischemia or reperfusion (I/R) still remains a major unsolved problem and significantly accounts for the risk of early mortality of LT [1,2]. During the variable period of cold ischemia and transplantation-related reperfusion of the donor lung, the generation of multiple proinflammatory mediators and reactive oxygen species (ROS) can overwhelm the lung’s anti-inflammatory and antioxidant defenses and finally leads to a primary graft dysfunction [3,4]. Thus, agents exhibiting anti-inflammatory and antioxidative activities may contribute to ameliorating I/R-induced lung injury after transplantation. Hydrogen sulfide (H2S) has been well known to be toxic at high concentrations because of its ability to interfere with the mitochondrial electron transport chain. However, recent studies show that H2S is naturally synthesized in the mammalian body from L-cysteine mainly by the activity of two enzymes, cystathionine-g-lyase (CSE) and cystathionine-b-synthetase, with a physiological level varying from 30 to 300 mM [5,6]. In lung tissue, H2S was generated predominantly by CSE [7]. Endogenous H2S has been implicated to play critical roles in the pathogenesis of various lung diseases, including chronic obstructive pulmonary diseases [8], inflammatory lung injury [9], and bleomycin-induced lung fibrosis [10]. More recently, George et al. [11,12] show that the exogenous H2S administration exhibits significant antioxidant effects and improves pulmonary function in an ex vivo cold I/R model of rabbit lung. However, whether the endogenous CSE/H2S pathway is involved in the pathogenesis of lung injury after orthotopic LT still remains to be elucidated. In the present study, we aimed to define the role of CSE/H2S pathway in orthotopic transplantation-induced lung injury, and whether H2S treatment may be recognized as a potential therapeutic strategy for LT.

2.

Materials and methods

2.1.

Rat orthotopic left single LT model of cold I/R injury

This study was performed in accordance with the Guidelines for Animal Experiments of the Shanghai Jiaotong University, China. Male SpragueeDawley rats (250e300 g) were housed with free access to food and water under a natural day/night cycle. All rats were acclimated for a week before any experimental procedures. The orthotopic left LT was performed by means of the modified cuff technique [13,14]. Donor rats were anesthetized by intraperitoneal injection of ketamine chloride (100 mg/kg) and intubated through a tracheotomy. Rats were mechanically ventilated with 100% oxygen, tidal volume of 10 mL/kg, and respiratory rate of 70 breaths/min. After heparinization (1000 U/kg intravenously), a median sternotomy was

performed, and the lungs were flushed through the main pulmonary artery with 20 mL of cold low-potassium dextran solution (Perfadex, Vitrolife, Gothenburg, Sweden) delivered from a height of 25 cm. The pulmonary artery, vein, and main stem bronchus were ligated and then tied off, and the inflated left lung was placed into low-potassium dextran solution solution at 4 C for 3 h. Recipient rats were anesthetized, intubated, and mechanically ventilated as same as the donor rats. Animals were then maintained under general anesthesia with a mixture of isoflurane and oxygen. A left thoracotomy was performed through the fourth intercostal space. The hilum of the left lung was dissected, and the pulmonary artery, pulmonary vein, and the main bronchus were clamped with microvascular clamps. After incising the pulmonary artery and vein, the donor lung was anastomosed with the corresponding recipient structures using multiple interrupted sutures for each anastomosis. A chest tube was placed, and the thorax was closed. The mechanical ventilation was maintained until animals recovered consciousness. In separate experiments, the recipient rats received saline (LT group), 14 mmol/kg H2S donor NaHS (LT-NaHS group), or 37.5 mg/kg CSE inhibitor propargylglycine (PAG; LT-PAG group) 15 min before the start of LT. The sham-operated controls were subjected to the same perioperative protocols as the recipient rats but not to LT. All the rats were killed by the overdose of chloralhydrate 24 h after the lung reperfusion. The left lower lung tissues were fixed in 4% paraformaldehyde for histopathologic analysis. Other parts of lung tissues were removed and then stored at 80 C until use.

2.2.

Histopathologic analysis

For histopathologic analysis, the left lower lung tissues were fixed as described previously. The samples were dehydrated and embedded in paraffin. Sections (4-mm thickness) were cut and stained with hematoxylin and eosin (n ¼ 6 for each group and n ¼ 6 for each animal). The total surface of the slides was scored by two blinded pathologists with expertise in lung pathology. Briefly, the criteria for scoring lung inflammation was set up as previously described with the modifications [15]: 0, normal tissue; 1, minimal inflammatory change; 2, no obvious damage to the lung architecture with small amount of leukocyte infiltration in the interstitium; 3, thickening of the alveolar septae with edema and leukocyte infiltration in the interstitium and alveoli; 4, formation of nodules or areas of pneumonitis that distorted the normal architecture; and 5, total obliteration of the field.

2.3.

Assessment of lung function

Transplant graft function was assessed by measuring the partial arterial pressure of oxygen (PaO2)-to-fraction of inspired oxygen (FiO2) ratio and the amount of blood shunted (Qs) or the total blood flow (Qt) levels in blood taken from the pulmonary vein at the completion of the 24-h reperfusion period (n ¼ 6 for each group). Wet-to-dry lung tissue weight ratios were calculated at the end of the 24-h reperfusion period as a measurement of lung edema (n ¼ 6 for each

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group). The lung tissue was weighed and placed in an oven at 80 C for 48 h. Then, the dried lung portion was reweighed, and the ratio of the lung weight before and after drying was calculated.

2.4.

Myeloperoxidase determination

Activity of myeloperoxidase (MPO), an enzyme present in neutrophils, was used as a marker of neutrophil infiltration. MPO activity was determined in lung as described previously [15]. Lung tissues (100 mg of tissue) were homogenized in 2 mL of 20 mM potassium phosphate buffer (pH 7.4). After centrifugation at 15,000g for 20 min, the pellet was resuspended in 2 mL of 50 mM potassium phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide and sonicated for 30 s. After being heated at 60 C for 2 h, the samples were centrifuged at 10,000g for 10 min. The supernatant (25 mL) was added to 725 mL of 50 mM phosphate buffer (pH 6.0) containing 0.167 mg/mL of o-dianisidine and 5  104% hydrogen peroxide. The MPO activity was measured spectrophotometrically as the change in absorbance at 460 nm at 37 C, using a Spectramax microplate reader (Molecular Devices, Sunnyvale, CA). The results are expressed as units of MPO activity per gram wet tissue (1 unit is defined as the change in absorbance of 1/min) (n ¼ 6 for each group).

2.5.

Malondialdehyde level determination

Levels of malondialdehyde (MDA), as an index of membrane lipid peroxidation, were determined as previously described [15]. Lung tissues were homogenized (100 mg/mL) in 10 volumes of 1.15% KCl solution containing 0.85% NaCl and then centrifuged at 1500g for 15 min. A total of 200 mL of the homogenates were then added to a reaction mixture consisting of 1.5 mL of 0.8% thiobarbituric acid, 200 mL of 8.1% sodium dodecyl sulfate, 1.5 mL of 20% acetic acid (adjusted to pH 3.5 with NaOH), and 600 mL distilled H2O. The mixture was then heated at 95 C for 40 min. After cooling to room temperature, the samples were cleared by centrifugation (10,000  g, 10 min) and their absorbance measured at 532 nm, using 1,1,3,3-tetramethoxypropane as an external standard. The level of lipid peroxides was expressed as nanomoles of MDA per milligram of protein (Bradford assay) (n ¼ 6 for each group).

2.6.

Measurement of cytokine levels in lung tissues

Lung tissues (100 mg) were homogenized and sonicated in 1 mL of phosphate-buffered saline (PBS) containing protease inhibitors (2 mM phenylmethylsulfonyl fluoride and 1 mg/mL each antipain, leupeptin, and pepstatin A). Lung homogenates were then centrifuged at 1000g for 10 min. The supernatants were filtered through 0.45 mm pore-size sterile filters and frozen at 80 C until use for the measurement of the levels of cytokines in lung tissues. The levels of interleukin (IL) 1b and IL-10 were measured by enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN), following the manufacturer’s instruction (n ¼ 6 for each group).

2.7.

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Western blot analysis

Rat lung tissues were homogenized in cold T-Per lysis buffer (Pierce Biotechnology, Thermo Fischer, Rockford, IL) (n ¼ 6 for each group). Then, the lysates were quickly sonified in ice bath, boiled for 5 min at 95 C, and stored at 80 C until used. Sodium dodecyl sulfateepolyacrylamide gel electrophoresis and immunoblotting were performed as previously described [16]. Antibody against b-actin was obtained from SigmaAldrich (Shanghai, China), and the antibodies against CSE were purchased from Abnova (Taipei, Taiwan).

2.8.

Real-time H2S production measurement

The real-time kinetics of H2S production by rat lung tissue were measured as previously described [16,17]. Briefly, approximately 100 mg of rat lung tissue was placed in PBS (approximately five times the volume of the tissue) containing protease inhibitor (2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 10 mg/mL of aprotinin) on ice, then the tissues were diced with double scissors to small particles, washed three times with ice-cold PBS, and homogenized for 15 s. After centrifugation at 5000 rpm for 5min, the supernatants were placed into a temperaturecontrolled micro-respiration chamber (Unisense, Aarhus, Denmark) and were used for real-time H2S production measurements by using a miniaturized H2S microrespiration sensor (Model H2S-MRCh; Unisense) coupled to Unisense PA2000 amplifier (n ¼ 5 for each group). The supernatant protein concentration was determined using a modified Bradford assay against a standard curve constructed with bovine serum albumin.

2.9.

Statistical analysis

Data were expressed as means  standard deviation. The statistical significance was estimated by one-way analysis of variance followed by the StudenteNewmaneeKeuls test. A P value <0.05 was considered statistically significant.

3.

Results

3.1. CSE protein levels and H2S generation are decreased after rat LT We examined CSE protein levels and H2S generation in the transplanted lung tissues at 24 h after rat orthotopic LT. As illustrated in Figure 1A, Western blot analysis demonstrated markedly decreased CSE protein expression in the transplanted lungs compared with that in the sham-operated lungs. In the meantime, the initial real-time H2S production rate was also significantly decreased in the transplanted lung tissues compared with that obtained from the sham-operated group (Fig. 1B).

3.2.

Histology and grading of transplanted lungs

Sections of lung tissue were stained with hematoxylin and eosin and scored by a histopathologic analysis. As shown in

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Fig. 1 e CSE protein levels and H2S generation are decreased after rat LT. (A) Western blot analysis of CSE protein in shamand LT-operated lung tissues. (B) Semiquantitation of Western blot signals of CSE in sham- (n [ 6) and LT-operated (n [ 6) graft lung tissues. (C) Representative traces of H2S production in the homogenate of lung tissues. H2S production was initiated by the addition of L-cysteine and pyridoxal 50 -phosphate (PLP). (D) Cumulative data of H2S production rate in sham(n [ 5) and LT-operated (n [ 5) graft lung tissues. Data were presented as means ± standard error of the mean. **P < 0.01 versus sham group. L-Cys [ L-cysteine.

Figure 2, the histologic examination of lung sections from transplanted rats demonstrated a clear evidence of interstitial edema, alveolar thickening, and extensive leukocyte infiltration in the interstitium and alveoli, with a histopathologic damage score of 3.33  0.51 (Fig. 2B and E). Administration of NaHS resulted in a significant attenuation of these findings and reduced the lung injury score to 1.83  0.75 (Fig. 2C and E). In contrast, administration of PAG, an irreversible CSE inhibitor, significantly aggravated the lung injury after transplantation, as evidenced by an increased lung injury score of 4.33  0.82 (Fig. 2D and E).

3.3.

Pulmonary function

There were no significant changes in pulmonary function in the sham-operated rats. The transplanted rats developed significant pulmonary dysfunction as evidenced by decreased PaO2-to-FiO2 ratio, increased intrapulmonary shunt (Qs/Qt), and lung edema (wet-to-dry weight ratio) (Fig. 3). In addition, we found that NaHS administration significantly improved the pulmonary function in the transplanted lung. In contrast, administration of PAG resulted in a lower PaO2-to-FiO2 ratio and a higher Qs/Qt value in rats with orthotopic LT, indicating

that inhibition of H2S generation may worsen pulmonary function.

3.4. NaHS decreased, whereas CSE inhibitor enhanced neutrophils in transplanted lung The activity of MPO, an enzyme principally present in neutrophils, was used as a marker of neutrophil infiltration. We measured the MPO activity in whole-lung extracts and found that the MPO activity in the LT group was significantly higher than that in the sham group (P < 0.01) and administration of NaHS significantly decreased (P < 0.01), whereas PAG further increased (P < 0.05) the transplantation-induced MPO activity in lung tissues (Fig. 4).

3.5. NaHS decreased, whereas CSE inhibitor enhanced membrane lipid peroxidation in the transplanted lung We measured MDA as an index of membrane lipid peroxidation activity in whole-lung extracts. As shown in Figure 5, it was found that MDA level in the LT group was significantly higher than that in the sham group (P < 0.01) and administration of NaHS significantly decreased (P < 0.05), whereas

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Fig. 2 e Effect of H2S donor or CSE inhibitor on morphologic changes in lung sections from transplanted rats. Twenty-four hours after lung reperfusion, the left lower lung tissues were fixed in 4% paraformaldehyde for hematoxylin and eosin staining and histopathologic analysis. Original magnification, 3200. Representative images from six animals per group were shown. (A) Sham-operated group, (B) LT group, (C) H2S donor NaHS (14 mmol/kg) was administrated 15 min before the start of the LT (LTeNaHS group), (D) CSE inhibitor PAG (37.5 mg/kg) was administrated 15 min before the start of the LT (LTePAG group); (E) the severity of lung injury was scored as described in Materials and Methods section. Data are means ± standard error of the mean of six rats for each group. **P < 0.01 versus sham group; #P < 0.05, ##P < 0.01 versus LT group.

PAG further increased (P < 0.01) the transplantation-induced MDA levels in lung tissues.

3.6.

inflammation in transplanted lung as evidenced with elevated IL-1b (P < 0.01) and decreased IL-10 levels (P < 0.01).

Cytokines in the transplanted lung tissues

4. We measured the proinflammatory cytokine IL-1b and antiinflammatory cytokine IL-10 levels as an index of inflammation in whole-lung extracts. As shown in Figure 6, it was found that IL-1b level in LT group was higher and IL-10 level was lower than that in the sham group (both P < 0.01) and administration of NaHS in rats with LT significantly decreased the IL-1b levels (P < 0.01), whereas increased IL-10 levels in the lung (P < 0.01). In contrast, CSE inhibitor aggravated the

Discussion

The present study is the first to show that CSE, the main synthase of H2S in lung tissue, is markedly downregulated after rat orthotopic LT. Moreover, CSE inhibitor PAG significantly aggravates, whereas exogenous H2S donor NaHS attenuates lung injury and dysfunction caused by LT. These results suggest that inhibition of H2S generation contributes to lung injury after experimental orthotopic LT.

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Fig. 4 e Effect of H2S donor or CSE inhibitor on lung MPO activity of transplanted rats. H2S donor NaHS (14 mmol/kg) or CSE inhibitor PAG (37.5 mg/kg) was administrated 15 min before the start of the experimental orthotopic LT. The neutrophil infiltration was assessed by measuring MPO activity at the completion of the 24-h reperfusion period. Data are means ± standard error of the mean of six rats for each group. **P < 0.01 versus sham group; #P < 0.05, ##P < 0.01 versus LT group.

Fig. 3 e Effect of H2S donor or CSE inhibitor on lung functions of transplanted rats. H2S donor NaHS (14 mmol/ kg) or CSE inhibitor PAG (37.5 mg/kg) was administrated 15 min before the start of the experimental orthotopic LT. Transplant graft function was assessed by measuring (A) PaO2-to-FiO2, (B) Qs-to-Qt, and (C) wet-to-dry ratio levels at the completion of the 24-h reperfusion period. Data are means ± standard error of the mean of six rats for each group. **P < 0.01 versus sham group; ##P < 0.01 versus LT group.

LT has been well recognized as a therapeutic option for the treatment of end-stage lung disease [1,2]. To decrease the metabolic energy requirement until implantation in the

recipient, donor lungs are generally stored in hypothermic preservation solution. However, although hypothermia is essential during donor lung storage and significantly extends organ viability, it is associated with sodium pump inactivation, oxidative stress, and the release of proinflammatory mediators that are ultimately deleterious to the donor lung at the time of reperfusion [3,18]. Thus, the obligatory cold I/R injury remains the major causes of recipient morbidity and mortality during LT. H2S has been increasingly recognized as a protective mediator against I/R injury in a number of mammalian tissues, such as heart [19e21], skin [22], liver [23,24], and kidney [25]. As for lung, Fu et al. [7] first demonstrates the protective effects of H2S donor in an isolated rat lung I/R model. More recently, George et al. [11,12] investigated the therapeutic potential of H2S donor in an ex vivo cold I/R model of rabbit lung. They found that even after 18-h cold storage, the infusion of H2S during reperfusion was associated with a significant decrease of ROS in lung tissues. In agreement with these publications, the present study provides several lines of evidence that H2S exerts the capacity to prevent lung injury after experimental orthotopic LT. A novel finding of this study is the demonstration that CSE expression, as well as H2S generation, is markedly downregulated in lung tissues after 3 h of cold ischemia and 24-h reperfusion. Downregulation of CSE/H2S pathway has been found in mouse kidney I/R injury model [26] and renal transplant tolerance model [27]. In contrast, Fu et al. [7] demonstrate the increase of H2S content and CSE activity in the isolated rat lung I/R model. Several protocol differences may account for the discrepancy between our findings and those of Fu et al. First of all, an ex vivo warm I/R model of rat-isolated lungs was used in the study by Fu et al., whereas the present

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Fig. 5 e Effect of H2S donor or CSE inhibitor on lung MDA level of transplanted rats. H2S donor NaHS (14 mmol/kg) or CSE inhibitor PAG (37.5 mg/kg) was administrated 15 min before the start of the experimental orthotopic LT. The membrane lipid peroxidation was assessed by measuring MDA levels at the completion of the 24-h reperfusion period. Data are means ± standard error of the mean of six rats for each group. **P < 0.01 versus sham group; #P < 0.05, ##P < 0.01 versus LT group.

study used an orthotopic LT model which resulted in cold I/Rinduced lung injury. In addition, the lungs were subjected to 45-min ischemia followed by 45-min reperfusion in the study by Fu et al., whereas they were subjected to 3-h ischemia followed by 24-h reperfusion in the present study. Taken together, we believe that the different types of I/R injury and the different I/R durations may have affected the responsiveness of pulmonary CSE and H2S pathway to I/R stress. Upregulation of CSE expression is also reported in hepatic I/R injury models [24]. Despite these inconsistent data on the responsiveness of CSE/H2S pathway to different types of I/R

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stress in different tissues, a decrease of endogenous H2S formation by CSE inhibitors has been shown to aggravate I/Rinduced tissue injury in various organs, including heart [27e29], liver [24], kidney [30], and lung [7], which is in agreement with the present study. Nevertheless, our findings provide evidence that a decrease of endogenous H2S generation occurs in the lung tissues after experimental orthotopic LT and even contributes to the transplantation-induced lung injury. It is well accepted that the lung tissue is particularly sensitive to oxidative stress because it possesses the largest endothelial surface area of any organ in the body [31]. On this basis, the use of antioxidants and/or radical scavengers has been proposed to reduce cold I/R-induced injury during LT. The protective effects of H2S against oxidative stresseinduced injury have been highlighted in various in vitro and in vivo models [32,33]. H2S exerts potent antioxidant activities not only via directly scavenging ROS and peroxynitrite [34] but also indirectly by enhancing the activity of antioxidant enzymes, such as catalase and superoxide dismutase [35]. In the present study, we demonstrated that the therapeutic treatment with H2S during reperfusion significantly protected rats against transplantation-induced neutrophil infiltration and lung tissue injury. In addition, H2S treatment significantly alleviated membrane lipid peroxidation levels in the lungs after transplantation in rats. These findings were also supported by the observation that infusion of H2S during reperfusion resulted in a significant decrease of ROS in rabbit lung tissues after cold I/R. In addition, the present study also found that inhibition of endogenous H2S generation further increased membrane lipid peroxidation levels in rat lungs after transplantation. These data suggest that the protective effects of both exogenous and endogenous H2S against transplantation-associated lung injury may be partly because of their potent antioxidant properties. Inflammatory events also have an important role in the development and full manifestation of transplantationinduced lung injury [36]. Extensive recruitment of leukocytes into the tissues accounts for the production of ROS and release

Fig. 6 e Effect of H2S donor or CSE inhibitor on IL-1b and IL-10 protein levels in lung tissues of transplanted rats. H2S donor NaHS (14 mmol/kg) or CSE inhibitor PAG (37.5 mg/kg) was administrated 15 min before the start of the experimental orthotopic LT. (A) IL-1b and (B) IL-10 concentration in the lung tissues were determined as described in the Materials and Methods section at the completion of the 24-h reperfusion period. Data are means ± standard error of the mean of six rats for each group. **P < 0.01 versus sham group; ##P < 0.01 versus LT group.

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of granule enzymes and cytokines. In addition, clinical and basic science studies have indicated that the detrimental effects of ROS are partly mediated by proinflammatory signal cascades [37,38], which subsequently leads to the activation of immune responses, including the generation of proinflammatory mediators that further amplify the inflammatory response [39]. There are conflicting data regarding the roles of H2S in the regulation of inflammation in lung tissue. For example, Li et al. [40] describes that H2S per se provokes an inflammatory reaction in lung tissue, meanwhile the inhibition of CSE activity displays distinct anti-inflammatory effects as evidenced by the attenuation of endotoxin-induced lung injury. On the other hand, recent studies show that H2S exerts anti-inflammatory effects in several acute lung injury rodent models, as evidenced by reducing neutrophil accumulation, inhibiting the release of proinflammatory cytokines, and increasing antiinflammatory IL-10 [9]. In the present study, we found that administration of H2S donor in the lung-transplanted rats significantly decreased IL-1b and MPO levels, whereas reduced IL-10 levels in the lung tissues. In contrast, inhibition of endogenous H2S production further aggravated inflammation in lung tissues after transplantation. Thus, our findings suggest that the anti-inflammatory effects of both exogenous and endogenous H2S may partly account for their protective effects against transplantation-induced lung injury. In conclusion, we have for the first time demonstrated that the suppression of CSE expression and H2S production is associated with transplantation-induced lung injury. Both exogenous and endogenous H2S seem to have protective effects against acute LT injury, by their multiple functions including antioxidation and anti-inflammation. These results indicate that the modulation of H2S levels may be considered as a potential therapeutic approach in the LT.

Acknowledgment The authors thank Dr Wei-gang Guo, Dr Qiang Tan, and Yi-qingYang for their advice and helpful suggestions. This work was supported by National Natural Science Foundation of China (31171120 and 31271270), Shanghai Natural Science Foundation (12ZR1428700), and Shanghai Joint development project for Municipal hospitals (SHDC12010222).

references

[1] Stehlik J, Edwards LB, Kucheryavaya AY, Aurora P, Christie JD, et al. The Registry of the International Society for Heart and Lung Transplantation: twenty-seventh official adult heart transplant reportd2010. J Heart Lung Transplant 2010;29:1089. [2] Christie JD, Edwards LB, Kucheryavaya AY, Aurora P, Dobbels F, et al. The Registry of the International Society for Heart and Lung Transplantation: twenty-seventh official adult lung and heart-lung transplant reportd2010. J Heart Lung Transplant 2010;29:1104.

[3] de Perrot M, Liu M, Waddell TK, Keshavjee S. Ischemiareperfusion-induced lung injury. Am J Respir Crit Care Med 2003;167:490. [4] den Hengst WA, Gielis JF, Lin JY, Van Schil PE, De Windt LJ, et al. Lung ischemia-reperfusion injury: a molecular and clinical view on a complex pathophysiological process. Am J Physiol Heart Circ Physiol 2010;299:H1283. [5] Li L, Rose P, Moore PK. Hydrogen sulfide and cell signaling. Annu Rev Pharmacol Toxicol 2011;51:169. [6] Calvert JW, Coetzee WA, Lefer DJ. Novel insights into hydrogen sulfidedmediated cytoprotection. Antioxid Redox Signal 2010;12:1203. [7] Fu Z, Liu X, Geng B, Fang L, Tang C. Hydrogen sulfide protects rat lung from ischemia-reperfusion injury. Life Sci 2008;82:1196. [8] Chen Y, Wang R. The message in the air: hydrogen sulfide metabolism in chronic respiratory diseases. Respir Physiol Neurobiol; 2012. [Epub ahead of print]. [9] Li T, Zhao B, Wang C, Wang H, Liu Z, et al. Regulatory effects of hydrogen sulfide on IL-6, IL-8 and IL-10 levels in the plasma and pulmonary tissue of rats with acute lung injury. Exp Biol Med (Maywood) 2008;233:1081. [10] Fang L, Li H, Tang C, Geng B, Qi Y, et al. Hydrogen sulfide attenuates the pathogenesis of pulmonary fibrosis induced by bleomycin in rats. Can J Physiol Pharmacol 2009; 87:531. [11] George TJ, Arnaoutakis GJ, Beaty CA, Jandu SK, Santhanam L, et al. Hydrogen sulfide decreases reactive oxygen in a model of lung transplantation. J Surg Res 2012;178:494. [12] George TJ, Arnaoutakis GJ, Beaty CA, Jandu SK, Santhanam L, et al. Inhaled hydrogen sulfide improves graft function in an experimental model of lung transplantation. J Surg Res; 2012. [Epub ahead of print]. [13] Mizobuchi T, Sekine Y, Yasufuku K, Fujisawa T, Wilkes DS. Comparison of surgical procedures for vascular and airway anastomoses that utilize a modified non-suture external cuff technique for experimental lung transplantation in rats. J Heart Lung Transplant 2004;23:889. [14] Belperio JA, Keane MP, Burdick MD, Gomperts BN, Xue YY, et al. CXCR2/CXCR2 ligand biology during lung transplant ischemia-reperfusion injury. J Immunol 2005;175:6931. [15] Yang T, Mao YF, Liu SQ, Hou J, Cai ZY, et al. Protective effects of the free radical scavenger edaravone on acute pancreatitisassociated lung injury. Eur J Pharmacol 2010;630:152. [16] Zhu XY, Liu SJ, Liu YJ, Wang S, Ni X. Glucocorticoids suppress cystathionine gamma-lyase expression and H2S production in lipopolysaccharide-treated macrophages. Cellular and molecular life sciences: CMLS 2010;67:1119. [17] You XJ, Xu C, Lu JQ, Zhu XY, Gao L, et al. Expression of cystathionine b-synthase and cystathionine gamma-lyase in human pregnant myometrium and their roles in the control of uterine contractility. PLoS One 2011;6:e23788. [18] Zaouali MA, Ben Abdennebi H, Padrissa-Altes S, MahfoudhBoussaid A, Rosello-Catafau J. Pharmacological strategies against cold ischemia reperfusion injury. Expert Opin Pharmacother 2010;11:537. [19] Johansen D, Ytrehus K, Baxter GF. Exogenous hydrogen sulfide (H2S) protects against regional myocardial ischemiareperfusion injurydevidence for a role of K ATP channels. Basic Res Cardiol 2006;101:53. [20] Sivarajah A, Collino M, Yasin M, Benetti E, Gallicchio M, et al. Anti-apoptotic and anti-inflammatory effects of hydrogen sulfide in a rat model of regional myocardial I/R. Shock 2009; 31:267. [21] Osipov RM, Robich MP, Feng J, Liu Y, Clements RT, et al. Effect of hydrogen sulfide in a porcine model of myocardial ischemia-reperfusion: comparison of different administration regimens and characterization of the

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[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

cellular mechanisms of protection. J Cardiovasc Pharmacol 2009;54:287. Henderson PW, Singh SP, Belkin D, Nagineni V, Weinstein AL, et al. Hydrogen sulfide protects against ischemia-reperfusion injury in an in vitro model of cutaneous tissue transplantation. J Surg Res 2010; 159:451. Jha S, Calvert JW, Duranski MR, Ramachandran A, Lefer DJ. Hydrogen sulfide attenuates hepatic ischemia-reperfusion injury: role of antioxidant and antiapoptotic signaling. Am J Physiol Heart Circ Physiol 2008;295:H801. Kang K, Zhao M, Jiang H, Tan G, Pan S, et al. Role of hydrogen sulfide in hepatic ischemia-reperfusion-induced injury in rats. Liver Transpl 2009;15:1306. Bos EM, Leuvenink HG, Snijder PM, Kloosterhuis NJ, Hillebrands JL, et al. Hydrogen sulfide-induced hypometabolism prevents renal ischemia/reperfusion injury. J Am Soc Nephrol 2009;20:1901. Kim JI, Choi SH, Jung KJ, Lee E, Kim HY, et al. Protective role of methionine sulfoxide reductase A against ischemia/ reperfusion injury in mouse kidney and its involvement in the regulation of trans-sulfuration pathway. Antioxid Redox Signal; 2012. [Epub ahead of print]. Vuillefroy de Silly R, Coulon F, Poirier N, Jovanovic V, Brouard S, et al. Transplant tolerance is associated with reduced expression of cystathionine-gamma-lyase that controls IL-12 production by dendritic cells and TH-1 immune responses. Blood 2012;119:2633. Salloum FN, Das A, Samidurai A, Hoke NN, Chau VQ, et al. Cinaciguat, a novel activator of soluble guanylate cyclase, protects against ischemia/reperfusion injury: role of hydrogen sulfide. Am J Physiol Heart Circ Physiol 2012; 302:H1347. Elsey DJ, Fowkes RC, Baxter GF. L-cysteine stimulates hydrogen sulfide synthesis in myocardium associated with attenuation of ischemia-reperfusion injury. Journal

[30]

[31]

[32]

[33] [34]

[35]

[36] [37]

[38] [39]

[40]

e33

of cardiovascular pharmacology and therapeutics 2010; 15:53. Tripatara P, Patel NS, Collino M, Gallicchio M, Kieswich J, et al. Generation of endogenous hydrogen sulfide by cystathionine gamma-lyase limits renal ischemia/ reperfusion injury and dysfunction. Lab Invest 2008;88:1038. Rahman I, MacNee W. Lung glutathione and oxidative stress: implications in cigarette smoke-induced airway disease. Am J Physiol 1999;277:L1067. Nakao A, Sugimoto R, Billiar TR, McCurry KR. Therapeutic antioxidant medical gas. Journal of clinical biochemistry and nutrition 2009;44:1. Moody BF, Calvert JW. Emergent role of gasotransmitters in ischemia-reperfusion injury. Medical gas research 2011;1:3. Whiteman M, Armstrong JS, Chu SH, Jia-Ling S, Wong BS, et al. The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite ‘scavenger’? J Neurochem 2004; 90:765. Chang L, Geng B, Yu F, Zhao J, Jiang H, et al. Hydrogen sulfide inhibits myocardial injury induced by homocysteine in rats. Amino acids 2008;34:573. Laubach VE, Kron IL. Pulmonary inflammation after lung transplantation. Surgery 2009;146:1. Naik E, Dixit VM. Mitochondrial reactive oxygen species drive proinflammatory cytokine production. J Exp Med 2011; 208:417. Bartosz G. Reactive oxygen species: destroyers or messengers? Biochem Pharmacol 2009;77:1303. Sabroe I, Dower SK, Whyte MK. The role of Toll-like receptors in the regulation of neutrophil migration, activation, and apoptosis. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 2005;41(Suppl 7):S421. Li L, Bhatia M, Zhu YZ, Zhu YC, Ramnath RD, et al. Hydrogen sulfide is a novel mediator of lipopolysaccharide-induced inflammation in the mouse. FASEB J 2005;19:1196.