j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e7
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Hydrogen water alleviates lung injury induced by one-lung ventilation Qifei Wu, PhD,a Jingyao Zhang, PhD,b Yong Wan, MM,b Sidong Song, MM,b Yong Zhang, PhD,a Guangjian Zhang, PhD,a Chang Liu, PhD,b and Junke Fu, PhDa,* a
Department of Thoracic Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China b Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China
article info
abstract
Article history:
Background: With the development of thoracic surgeries, one-lung ventilation (OLV) has
Received 24 February 2015
been routinely used to facilitate surgical exposure. However, OLV can cause lung injury
Received in revised form
during the surgical process and becomes an important factor affecting the outcomes. To
18 May 2015
date, effective treatments for the prevention of lung injury caused by OLV are lacking.
Accepted 9 June 2015
Hydrogen has been demonstrated to have effective protection against tissue injuries
Available online xxx
caused by oxidative stress, inflammation, and apoptosis. This study investigated the efficacy of hydrogen water consumption on the prevention of lung injury induced by OLV
Keywords:
in rats.
Hydrogen
Materials and Methods: Male SpragueeDawley rats (n ¼ 32, 240e260 g) were divided
Lung injury
randomly into the following four groups: sham group, sham þ H2 group, OLV group,
Oxidative stress
OLV þ H2 group. The rats drank hydrogen water or degassed hydrogen water for 4 wk
Inflammation
before the operation and received OLV for 60 min and two-lung ventilation for 60 min. Lung
Nuclear factor kappa B
tissues were assayed for wet-to-dry ratio, oxidative stress variables, proinflammatory cytokines, and hematoxylineeosin staining. Results: Hydrogen water consumption reduced wet-to-dry weight ratio, malondialdehyde and myeloperoxidase activity and decreased the concentration of TNF-a, IL-1b, and IL-6 in the lung tissues compared with sham group and sham þ H2 group. Hydrogen water consumption further attenuated NF-kB activation and caused histopathologic alterations. Conclusions: Our data demonstrated that hydrogen water consumption ameliorated OLVinduced lung injury, and it may exert its protective role by its anti-inflammation, antioxidation and reducing NF-kB activity in the lung tissues. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
In recent years, with the development of complicated and minimally invasive thoracic surgeries, one-lung ventilation
(OLV) has been routinely used to facilitate surgical exposure. However, OLV can cause lung injury during the surgical process and becomes an important factor affecting the outcomes [1,2]. There is no single mechanism that can fully explain
* Corresponding author. Department of Thoracic Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, No.277 Yan Ta West Road, Xi’an, Shaanxi, 710061, China. Tel.: þ86 29-8532-3860; fax: þ86 29-8526-3190. E-mail address: [email protected] (J. Fu). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.06.017
2
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e7
OLV-induced lung injury, and its etiology is most likely multifactorial, including ischemia and/or reperfusion, mechanical ventilation, and hypoxemia [3]. In general, generation of reactive oxygen species (ROS) and release of proinflammatory cytokines play an important role in the lung injury [4]. To date, effective treatments for the prevention of lung injury caused by OLV are lacking. Hydrogen, a gaseous molecule without known toxicity, has been recently discovered to be a novel therapeutic medical gas in a variety of biomedical fields and possed the potent antioxidant and anti-inflammatory efficacies by selectively reducing cytotoxic oxygen radicals in several animal models [5]. Hydrogen can be administrated in many different ways, including inhalation, intravenous injection of hydrogen saline, intraperitoneal injection of hydrogen saline, and drinking hydrogen-containing water [6,7]. Drinking hydrogencontaining water is a promising way for hydrogen usage because of its convenience and safety. Previous study has demonstrated that hydrogen consumption through inhalation or intravenous injection could reduce lung injury induced by mechanical ventilation and high concentrations of oxygen [4,8,9]. However, the potential effect of hydrogen water consumption by drinking on lung injury caused by OLV has not been examined. Therefore, in the present study, we have investigated the effect of hydrogen water consumption by drinking on the lung injury of rats caused by OLV.
anesthesia; (2) Sham þ H2 group: the rats that drank hydrogen water for 4 wk before the operation and received only anesthesia; (3) OLV group: the rats that drank degassed hydrogen water for 4 wk before the operation and received OLV for 60 min and then two-lung ventilation for 60 min; (4) OLV þ H2 group: the rats that drank hydrogen water for 4 wk before the operation and received OLV for 60 min and then two-lung ventilation for 60 min.
2.4.
Rats were anesthetized with intraperitoneal injection of 10% chloral hydrate (0.4 mL/100 g body weight). Then tracheotomy and endotracheal intubation were performed. At the same time, external jugular vein was exposed, and heparin 100 U/kg was injected. After these manipulations, mechanical ventilation (tidal volume 20 mL/kg without an end expiratory pressure at a respiratory rate of 80 breaths/min) was performed (Small Animal Ventilator, Model DH-150, Medical Instrument Limited Company of Zhejiang University, China). Thoracotomy was performed at the fourth intercostal space at the left edge of the sternum. Then, the left main bronchus was separated from the pulmonary artery, and vein, and occluded with a micro bulldog clamp. OLV was sustained for 60 min, then the microclamp was released, and the left lung was ventilated for 60 min. Then the rats were killed for further experiments.
2.5.
2.
Materials and methods
2.1.
Animals
Adult male SpragueeDawley rats weighing 240e260 g were used in all experiments. The animals were housed in individual cages in a temperature-controlled room with a 12-h light and/or dark cycle and free access to food and water. All the protocols were approved by the Xi’an Jiaotong University, China, in accordance with the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health (publication no. 96-01).
2.2.
Hydrogen water administration
Establishment of animal models
Tissue collection
After the rats were killed, the lungs were removed en bloc and drained of blood. The left lung was isolated and dissected into three portions with a sharp blade. One portion for wet-to-dry (W/D) ratio measurement, one portion for histologic examination, and the other portion was immediately snap frozen in liquid nitrogen and stored at 80 C for assays.
2.6.
Lung tissue W/D ratio measurement
The portion of left lung for W/D ratio measurement was weighed immediately after collection and placed into a 60 C oven to dry for 2 d. The dried tissue was weighed to determine the W/D ratio.
The hydrogen water was provided by Naturally Plus Japan International Co Ltd (Tokyo, Japan). The main technology of this product was dissolving the hydrogen in water under high pressure to the supersaturated level using hydrogen watereproducing apparatus. The saturated hydrogen water was stored under atmospheric pressure at 4 C in an aluminum bag with no dead volume. Each day, hydrogen water from the aluminum bag was placed in a closed vessel to confirm the content of hydrogen. Hydrogen water degassed by gentle stirring was used for control.
2.7. Measurement of malondialdehyde and myeloperoxidase in lung tissues
2.3.
Lung tissues were thawed and then homogenized immediately on ice in 1-mL normal saline. The homogenates were centrifuged at 3000 g for 15 min at 4 C. Levels of TNF-a, IL-1b, and IL-6 were measured with commercial enzyme-linked immunosorbent assay kits from Dakewe Biotech Co
Experimental protocol
Animals were randomly assigned to the following four groups with eight rats in each group: (1) Sham group: the rats that drank degassed hydrogen water for 4 wk and received only
Pulmonary malondialdehyde (MDA) and myeloperoxidase (MPO) contents were determined using a commercial assay kits as the manufacturer’s instructions (Nanjing Jiancheng Biochemistry Co, Nanjing, China) [4,10].
2.8. Determination of TNF-a, IL-1b, and IL-6 levels in lung tissues
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e7
3
(Shenzhen, China) after the instructions. The absorbance was read on a microplate reader, and the concentrations were calculated according to the standard curve. Protein content in the sample was determined by Coomassie blue assay, and the results were corrected per microgram of protein.
2.9. Determination of NF-kB transcription factor activity in lung tissue To analyze the specific binding activity of nuclear p65 NF-kB, nuclear protein extracts of lung tissue were isolated. Protein concentrations were quantified using Pierce 660 nm Protein Assay Kit (Thermo Scientific Pierce, Rockford, IL) for equal protein loading. NF-kB transcription factor activity was measured using the TransAM NF-kB p65 Transcription Factor Assay Kit (Active Motif, Carlsbad, CA), according to the manufacturer’s recommendations. Results were read on a BioTek ELx800 Plate Reader (Biotek, Winooski, VT). Data were expressed as the percentage of NF-kB transcription factor activity relative to the sham group. The assay was conducted three times for each sample.
2.10.
Histologic investigation
The portions of the left lung were harvested and flushed with normal saline, fixed with 10% formalin for 24 h and embedded in paraffin; sections of 4 mm were stained with hematoxylin and eosin for light microscope observation and evaluated blindly by an independent consultant histopathologist. Damage to the lung tissue was graded by the pathologist on a scale of 1 (no injury) e 4 (worst), a method described previously [11].
2.11.
Statistical analysis
Results were expressed as mean standard deviation. Differences of all the measured parameters among the groups were assessed with one-way analysis of variance followed by StudenteNewmaneKeuls test for multiple comparisons if analysis of variance indicated a significant overall effect. A value of P < 0.05 was considered to be statistically significant. All data were analyzed using the SPSS version 13 statistical software package (SPSS Inc, Chicago, IL).
3.
Results
3.1.
Lung tissue W/D ratio measurement
To evaluate the extent of OLV-induced lung damage, the W/D ratios were measured in different groups as shown in Figure 1A. The W/D ratio was significantly increased in OLV group compared with other three groups (P < 0.01). Although hydrogen water consumption in rats significantly reduced the W/D ratios compared with OLV group, significant differences were still observed between sham þ H2 group and OLV þ H2 group (P < 0.01). There was no significant difference in W/D ratios between sham and sham þ H2 groups (P ¼ 0.52).
Fig. 1 e Effects of hydrogen water consumption on the lung W/D ratio, MDA levels, and MPO activity in lung tissues of mice after the OLV. The lung W/D ratio (A), MDA levels and MPO activity (B) in lung tissues were determined after 60 min of OLV followed by 60 min of two-lung ventilation. Data are presented as the mean ± standard deviation (n [ 8 in each group). *P < 0.01 versus sham group and sham D H2 group, #P < 0.01 versus OLV group.
3.2.
Levels of MDA and MPO in lung tissues
To ascertain whether hydrogen water consumption in rats protects the lung injury from OLV, the activities of MDA and MPO in different groups were measured. We found that hydrogen water consumption in rats significantly decreased the MDA level and MPO activity compared with those of OLV group (P < 0.01; Fig. 1B, C). However, compared with sham þ H2 group, pulmonary MDA level and MPO activity increased in OLV þ H2 group (P < 0.01; Fig. 1B, C). There was no significant
4
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e7
difference in pulmonary MDA level and MPO activity between sham and sham þ H2 groups (P ¼ 0.33 in MDA level; P ¼ 0.22 in MPO activity; Fig. 1B, C).
3.3. Levels of TNF-a, IL-1b, and IL-6 levels in lung tissues The levels of TNF-a, IL-1b, and IL-6 in lung tissue are shown in Figure 2. We found that hydrogen water consumption in rats significantly decreased TNF-a, IL-1b, and IL-6 levels compared with those of OLV group (P < 0.01). However, compared with sham þ H2 group, pulmonary TNF-a, IL-1b and IL-6 levels increased in OLV þ H2 group (P < 0.01). There was no significant difference of TNF-a, IL-1b, and IL-6 levels between sham and sham þ H2 groups (P ¼ 0.84 in TNF-a level; P ¼ 0.81 in IL-1b level; and P ¼ 0.81 in IL-6 level).
3.4.
NF-kB transcription factor activity in lung tissue
The levels of NF-kB transcription factor activity in lung tissue are shown in Figure 2. We found that hydrogen water consumption in rats significantly decreased NF-kB transcription factor activity compared with those of OLV group (P < 0.01). However, compared with sham þ H2 group, NF-kB transcription factor activity was increased in OLV þ H2 group (P < 0.01). There was no significant difference of NF-kB transcription factor activity between sham and sham þ H2 groups (P ¼ 0.52).
3.5.
Histologic investigation
The representative lung injuries in different groups are shown in Figure 3. Lung microscopic examination revealed that the
lung tissues of rats were severely damaged in OLV group with alveolar, perivascular and interstitial edema, atelectasis, disruption of alveolar and bronchiolar epithelial cells, and hemorrhage. Hydrogen water consumption obviously suppressed these morphological changes, and the injury degree of lung was minor in OLV þ H2 group than OLV group (P < 0.01). Compared with the sham þ H2 group, the injury degree of lung still increased in OLV þ H2 group (P < 0.01). There was no significant difference in the lung injury score between sham and sham þ H2 groups (P ¼ 0.60).
4.
Discussion
Anesthesia for thoracic surgery routinely involves OLV to facilitate surgical exposure and to isolate and protect the lungs during the procedure. However, there is an increasing concern about the effects of lung injury as a consequence of OLV [12e14], so developing approaches to minimize this kind of lung injury will advance OLV and could have substantial clinical impact. Recently, mounting evidence has shown that hydrogen can behave as an antioxidant in biological systems because of its ability to reduce ROS and potential anti-inflammatory effects [9,15,16]. To develop a new strategy to alleviate lung injury during OLV, we designed the present study to evaluate the effects of hydrogen water consumption on OLV-induced lung injury. The results demonstrated that hydrogen water consumption before OLV can alleviate OLV-induced lung injury. This protective effect is supported by reduced lung injury as measured by lower level of W/D ratio, MDA, MPO activity, proinflammatory cytokines, and marked preservation of lung
Fig. 2 e Effects of hydrogen water consumption on the TNF-a, IL-1b and IL-6 levels and NF-kB activity in the lung tissues of mice after the OLV. TNF-a (A), IL-1b (B), and IL-6 (C) levels, and NF-kB activity were determined (D) in the lung tissues after 60 min of OLV followed by 60 min of two-lung ventilation. Data are presented as the mean ± standard deviation (n [ 8 in each group). *P < 0.01 versus sham group and sham D H2 group, #P < 0.01 versus OLV group.
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e7
5
Fig. 3 e Effect of hydrogen on histopathologic changes in lung tissues of mice after the OLV) ( 3 200). Photomicrographs of left lung sections from the sham group (A), sham D H2 group (B) and OLV group (C) and OLV D H2 group (D) after 60 min of OLV followed by 60 min of two-lung ventilation. The lung in the OLV group showed a thickened alveolar wall, edema, hemorrhage, and less alveolar space. Hydrogen water consumption significantly prevented the histopathologic changes caused by OLV. (E) The severity of lung injury was scored, and the data are presented as the mean ± standard deviation (n [ 8 in each group). *P < 0.01 versus sham group and sham D H2 group, #P < 0.01 versus OLV group. (Color version of the figure is available online.)
tissue structure microscopically. In addition, hydrogen water consumption has been shown to ameliorate NF-kB expression in the lung tissues. The mechanism of lung injury response to OLV is very complicated, which at least includes ischemia and/or reperfusion, mechanical ventilation, and hypoxemia [3,17].
Generation of ROS and release of proinflammatory cytokines were widely recognized, taking important roles in the OLVinduced lung injury [18,19]. ROS are oxidants derived from the metabolism of oxygen, which encompasses a broad range of biochemical interactions and chemical properties. They include superoxide, H2O2, hydroxyl radical, peroxyl radical,
6
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e7
alkoxyl radical, ozone, and singlet oxygen. In cellular process, oxidative phosphorylation in mitochondria is the major endogenous source of ROS. Under normal physiological conditions, ROS can be safely reduced to water by superoxide dismutase, catalase, and glutathione peroxidase. However, in the pathologic conditions such as ischemiaereperfusion injury, organ transplantation, and sepsis, the balance between the generation of ROS and the mechanisms to detoxify them can be upset, resulting in the accumulation of ROS in the tissues, where they quickly react with lipids, proteins, and nucleic acids, causing severe cellular damage, unwanted cell death, and consequently vascular barrier disruption and pulmonary edema, even respiratory failure [20]. Our findings were in agreement with these reports, such that we also found that OLV increased the level of MDA, a reliable marker of oxidative stress-mediated lipid peroxidation, in the lung tissue, and drinking hydrogen water significantly reduced the MDA level. In addition, OLV increased the activity of MPO, an enzyme present in neutrophils, in the lung tissue, and drinking hydrogen water significantly reduced the MPO activity, which means drinking hydrogen water decreases the neutrophil recruitment in the lung tissue. The mechanism for this protection of lung tissue may be that hydrogen can modulate the gene transcription of antioxidation genes [21]. Another possibility is that hydrogen permeates mitochondria and directly reduces the production of superoxide [22]. However, the accurate mechanism for hydrogen protection to tissues is still unclear. Previous studies have shown that inflammatory mediators, including TNF-a, IL-1b, and IL-6, are the major generators of lung injury induction, which play important roles in the initiation and amplification of inflammatory responses [23e26]. In the present study, we investigated the levels of TNF-a, IL-1b, and IL-6 in the lung tissues, and our results show that the concentrations of TNF-a, IL-1b, and IL-6 in the lung tissues significantly increased after OLV, and hydrogen water pretreatment reduced the concentrations of cytokines in the lung tissues. The release of proinflammatory cytokines, including TNFa, IL-1b, and IL-6, is promoted by the transcription factor NFkB, which plays a central role in intracellular signaling for the production of proinflammatory cytokines. In previous studies, scientists have proved that hydrogen had the ability of inhibiting the activation of NF-kB [15,16,27,28]. So, in this study we also investigated the activities of NF-kB in the lung tissues of different groups, and we found that hydrogen water consumption decreased the activity of NF-kB. This may be an explanation for the increase of proinflammatory cytokines in OLV group. This study also has some limitations. First, we did not measure the serum and/or lung tissue hydrogen concentrations in this study, as we have little experience in this aspect. However, it has been reported to detect these changes using a novel method, and these results can better validate the cause and effect of hydrogen water consumption and its protective effect [29]. Next, we did not detect the changes of hemodynamic parameters, peak airway pressures, and arterial blood gases among different groups. These parameters may give us more clues for the accurate mechanism study related to the lung protective effect of hydrogen water consumption. In
addition, we used external clamping of the bronchus to block the ventilation of left lung. This method is not the same as the clinical situation (with bronchial blocker or double lumen tube) and may disrupt the bronchial arterial supply. In conclusion, the results of this study demonstrate that hydrogen water consumption ameliorated OLV-induced acute lung injury by reducing oxidative stress and the production of proinflammatory cytokines in lung tissue. However, the intensive mechanism involved in the protective role of hydrogen water consumption to OLV-induced lung injury still requires further study.
Acknowledgment This project was supported by China Postdoctoral Science Foundation funded project (2013M532058) and Key Science and Technology Project of Shaanxi Province (2013K12-08-01). Authors’ contributions: J.Z., Y.W., S.S., Y.Z., G.Z. collected the data and drafted the article. Q.W. conceived and created the framework for the paper, evaluated the data, and drafted and edited the article. C.L. and J.F. conceived and created the framework for the paper, evaluated the data, and edited the article. All the authors have read and approved the final article.
Disclosure The authors have no conflicts of interest to declare.
references
[1] Amato MB, Barbas CS, Medeiros DM, et al. Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome. N Engl J Med 1998;338:347. [2] Jeon K, Yoon JW, Suh GY, et al. Risk factors for postpneumonectomy acute lung injury/acute respiratory distress syndrome in primary lung cancer patients. Anaesth Intensive Care 2009;37:14. [3] Baudouin SV. Lung injury after thoracotomy. Br J Anaesth 2003;91:132. [4] Sun Q, Cai J, Liu S, et al. Hydrogen-rich saline provides protection against hyperoxic lung injury. J Surg Res 2011; 165:e43. [5] Zhang J-Y, Liu C, Zhou L, et al. A review of hydrogen as a new medical therapy. Hepatogastroenterology 2012;59:1026. [6] Ohsawa I, Nishimaki K, Yamagata K, Ishikawa M, Ohta S. Consumption of hydrogen water prevents atherosclerosis in apolipoprotein E knockout mice. Biochem Biophys Res Commun 2008;377:1195. [7] Buchholz BM, Kaczorowski DJ, Sugimoto R, et al. Hydrogen inhalation ameliorates oxidative stress in transplantation induced intestinal graft injury. Am J Transplant 2008;8:2015. [8] Huang CS, Kawamura T, Lee S, et al. Hydrogen inhalation ameliorates ventilator-induced lung injury. Crit Care 2010;14: R234. [9] Li H, Zhou R, Liu J, et al. Hydrogen-rich saline attenuates lung ischemia-reperfusion injury in rabbits. J Surg Res 2012; 174:e11. [10] Liu C, Wu Q, Li Q, et al. Mesenteric lymphatic ducts ligation decreases the degree of gut-induced lung injury in a portal
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vein occlusion and reperfusion canine model. J Surg Res 2009;154:45. Kostopanagiotou G, Avgerinos E, Costopanagiotou C, et al. Acute lung injury in a rat model of intestinal ischemiareperfusion: the potential time depended role of phospholipases A(2). J Surg Res 2008;147:108. Cheng YD, Gao Y, Zhang H, Duan CJ, Zhang CF. Effects of OLV preconditioning and postconditioning on lung injury in thoracotomy. Asian J Surg 2014;37:80. Eguchi T, Yoshida K, Kondo R, et al. Sivelestat prevents cytoskeletal rearrangements in neutrophils resulting from lung re-expansion following one-lung ventilation during thoracic surgery. Inflammation 2013;36:1479. Bastin AJ, Sato H, Davidson SJ, Quinlan GJ, Griffiths MJ. Biomarkers of lung injury after one-lung ventilation for lung resection. Respirology 2011;16:138. Huang CS, Kawamura T, Peng X, et al. Hydrogen inhalation reduced epithelial apoptosis in ventilatorinduced lung injury via a mechanism involving nuclear factor-kappa B activation. Biochem Biophys Res Commun 2011;408:253. Wang C, Li J, Liu Q, et al. Hydrogen-rich saline reduces oxidative stress and inflammation by inhibit of JNK and NF-kappaB activation in a rat model of amyloid-betainduced Alzheimer’s disease. Neurosci Lett 2011; 491:127. Olivant Fisher A, Husain K, Wolfson MR, et al. Hyperoxia during one lung ventilation: inflammatory and oxidative responses. Pediatr Pulmonol 2012;47:979. Leite CF, Calixto MC, Toro IF, Antunes E, Mussi RK. Characterization of pulmonary and systemic inflammatory responses produced by lung re-expansion after one-lung ventilation. J Cardiothorac Vasc Anesth 2012;26:427.
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[19] Gothard J. Lung injury after thoracic surgery and one-lung ventilation. Curr Opin Anaesthesiol 2006;19:5. [20] Davidovich N, DiPaolo BC, Lawrence GG, Chhour P, Yehya N, Margulies SS. Cyclic stretch-induced oxidative stress increases pulmonary alveolar epithelial permeability. Am J Respir Cell Mol Biol 2013;49:156. [21] Ohsawa I, Ishikawa M, Takahashi K, et al. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med 2007;13:688. [22] Sato Y, Kajiyama S, Amano A, et al. Hydrogen-rich pure water prevents superoxide formation in brain slices of vitamin C-depleted SMP30/GNL knockout mice. Biochem Biophys Res Commun 2008;375:346. [23] Goodman RB, Pugin J, Lee JS, Matthay MA. Cytokinemediated inflammation in acute lung injury. Cytokine Growth Factor Rev 2003;14:523. [24] Qiu X, Li H, Tang H, et al. Hydrogen inhalation ameliorates lipopolysaccharide-induced acute lung injury in mice. Int Immunopharmacol 2011;11:2130. [25] Slutsky AS, Ranieri VM. Ventilator-induced lung injury. N Engl J Med 2013;369:2126. [26] Lai CC, Liu WL, Chen CM. Glutamine attenuates acute lung injury caused by acid aspiration. Nutrients 2014;6:3101. [27] Zhang J, Wu Q, Song S, et al. Effect of hydrogen-rich water on acute peritonitis of rat models. Int Immunopharmacol 2014; 21:94. [28] Chen H, Sun YP, Li Y, et al. Hydrogen-rich saline ameliorates the severity of l-arginine-induced acute pancreatitis in rats. Biochem Biophys Res Commun 2010;393:308. [29] Liu C, Kurokawa R, Fujino M, Hirano S, Sato B, Li XK. Estimation of the hydrogen concentration in rat tissue using an airtight tube following the administration of hydrogen via various routes. Sci Rep 2014;4:5485.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Hydrogen water alleviates lung injury induced by one-lung ventilation Qifei Wu, PhD,a Jingyao Zhang, PhD,b Yong Wan, MM,b Sidong Song, MM,b Yong Zhang, PhD,a Guangjian Zhang, PhD,a Chang Liu, PhD,b and Junke Fu, PhDa,* a
Department of Thoracic Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China b Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China
article info
abstract
Article history:
Background: With the development of thoracic surgeries, one-lung ventilation (OLV) has
Received 24 February 2015
been routinely used to facilitate surgical exposure. However, OLV can cause lung injury
Received in revised form
during the surgical process and becomes an important factor affecting the outcomes. To
18 May 2015
date, effective treatments for the prevention of lung injury caused by OLV are lacking.
Accepted 9 June 2015
Hydrogen has been demonstrated to have effective protection against tissue injuries
Available online xxx
caused by oxidative stress, inflammation, and apoptosis. This study investigated the efficacy of hydrogen water consumption on the prevention of lung injury induced by OLV
Keywords:
in rats.
Hydrogen
Materials and Methods: Male SpragueeDawley rats (n ¼ 32, 240e260 g) were divided
Lung injury
randomly into the following four groups: sham group, sham þ H2 group, OLV group,
Oxidative stress
OLV þ H2 group. The rats drank hydrogen water or degassed hydrogen water for 4 wk
Inflammation
before the operation and received OLV for 60 min and two-lung ventilation for 60 min. Lung
Nuclear factor kappa B
tissues were assayed for wet-to-dry ratio, oxidative stress variables, proinflammatory cytokines, and hematoxylineeosin staining. Results: Hydrogen water consumption reduced wet-to-dry weight ratio, malondialdehyde and myeloperoxidase activity and decreased the concentration of TNF-a, IL-1b, and IL-6 in the lung tissues compared with sham group and sham þ H2 group. Hydrogen water consumption further attenuated NF-kB activation and caused histopathologic alterations. Conclusions: Our data demonstrated that hydrogen water consumption ameliorated OLVinduced lung injury, and it may exert its protective role by its anti-inflammation, antioxidation and reducing NF-kB activity in the lung tissues. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
In recent years, with the development of complicated and minimally invasive thoracic surgeries, one-lung ventilation
(OLV) has been routinely used to facilitate surgical exposure. However, OLV can cause lung injury during the surgical process and becomes an important factor affecting the outcomes [1,2]. There is no single mechanism that can fully explain
* Corresponding author. Department of Thoracic Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, No.277 Yan Ta West Road, Xi’an, Shaanxi, 710061, China. Tel.: þ86 29-8532-3860; fax: þ86 29-8526-3190. E-mail address: [email protected] (J. Fu). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.06.017
2
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e7
OLV-induced lung injury, and its etiology is most likely multifactorial, including ischemia and/or reperfusion, mechanical ventilation, and hypoxemia [3]. In general, generation of reactive oxygen species (ROS) and release of proinflammatory cytokines play an important role in the lung injury [4]. To date, effective treatments for the prevention of lung injury caused by OLV are lacking. Hydrogen, a gaseous molecule without known toxicity, has been recently discovered to be a novel therapeutic medical gas in a variety of biomedical fields and possed the potent antioxidant and anti-inflammatory efficacies by selectively reducing cytotoxic oxygen radicals in several animal models [5]. Hydrogen can be administrated in many different ways, including inhalation, intravenous injection of hydrogen saline, intraperitoneal injection of hydrogen saline, and drinking hydrogen-containing water [6,7]. Drinking hydrogencontaining water is a promising way for hydrogen usage because of its convenience and safety. Previous study has demonstrated that hydrogen consumption through inhalation or intravenous injection could reduce lung injury induced by mechanical ventilation and high concentrations of oxygen [4,8,9]. However, the potential effect of hydrogen water consumption by drinking on lung injury caused by OLV has not been examined. Therefore, in the present study, we have investigated the effect of hydrogen water consumption by drinking on the lung injury of rats caused by OLV.
anesthesia; (2) Sham þ H2 group: the rats that drank hydrogen water for 4 wk before the operation and received only anesthesia; (3) OLV group: the rats that drank degassed hydrogen water for 4 wk before the operation and received OLV for 60 min and then two-lung ventilation for 60 min; (4) OLV þ H2 group: the rats that drank hydrogen water for 4 wk before the operation and received OLV for 60 min and then two-lung ventilation for 60 min.
2.4.
Rats were anesthetized with intraperitoneal injection of 10% chloral hydrate (0.4 mL/100 g body weight). Then tracheotomy and endotracheal intubation were performed. At the same time, external jugular vein was exposed, and heparin 100 U/kg was injected. After these manipulations, mechanical ventilation (tidal volume 20 mL/kg without an end expiratory pressure at a respiratory rate of 80 breaths/min) was performed (Small Animal Ventilator, Model DH-150, Medical Instrument Limited Company of Zhejiang University, China). Thoracotomy was performed at the fourth intercostal space at the left edge of the sternum. Then, the left main bronchus was separated from the pulmonary artery, and vein, and occluded with a micro bulldog clamp. OLV was sustained for 60 min, then the microclamp was released, and the left lung was ventilated for 60 min. Then the rats were killed for further experiments.
2.5.
2.
Materials and methods
2.1.
Animals
Adult male SpragueeDawley rats weighing 240e260 g were used in all experiments. The animals were housed in individual cages in a temperature-controlled room with a 12-h light and/or dark cycle and free access to food and water. All the protocols were approved by the Xi’an Jiaotong University, China, in accordance with the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health (publication no. 96-01).
2.2.
Hydrogen water administration
Establishment of animal models
Tissue collection
After the rats were killed, the lungs were removed en bloc and drained of blood. The left lung was isolated and dissected into three portions with a sharp blade. One portion for wet-to-dry (W/D) ratio measurement, one portion for histologic examination, and the other portion was immediately snap frozen in liquid nitrogen and stored at 80 C for assays.
2.6.
Lung tissue W/D ratio measurement
The portion of left lung for W/D ratio measurement was weighed immediately after collection and placed into a 60 C oven to dry for 2 d. The dried tissue was weighed to determine the W/D ratio.
The hydrogen water was provided by Naturally Plus Japan International Co Ltd (Tokyo, Japan). The main technology of this product was dissolving the hydrogen in water under high pressure to the supersaturated level using hydrogen watereproducing apparatus. The saturated hydrogen water was stored under atmospheric pressure at 4 C in an aluminum bag with no dead volume. Each day, hydrogen water from the aluminum bag was placed in a closed vessel to confirm the content of hydrogen. Hydrogen water degassed by gentle stirring was used for control.
2.7. Measurement of malondialdehyde and myeloperoxidase in lung tissues
2.3.
Lung tissues were thawed and then homogenized immediately on ice in 1-mL normal saline. The homogenates were centrifuged at 3000 g for 15 min at 4 C. Levels of TNF-a, IL-1b, and IL-6 were measured with commercial enzyme-linked immunosorbent assay kits from Dakewe Biotech Co
Experimental protocol
Animals were randomly assigned to the following four groups with eight rats in each group: (1) Sham group: the rats that drank degassed hydrogen water for 4 wk and received only
Pulmonary malondialdehyde (MDA) and myeloperoxidase (MPO) contents were determined using a commercial assay kits as the manufacturer’s instructions (Nanjing Jiancheng Biochemistry Co, Nanjing, China) [4,10].
2.8. Determination of TNF-a, IL-1b, and IL-6 levels in lung tissues
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(Shenzhen, China) after the instructions. The absorbance was read on a microplate reader, and the concentrations were calculated according to the standard curve. Protein content in the sample was determined by Coomassie blue assay, and the results were corrected per microgram of protein.
2.9. Determination of NF-kB transcription factor activity in lung tissue To analyze the specific binding activity of nuclear p65 NF-kB, nuclear protein extracts of lung tissue were isolated. Protein concentrations were quantified using Pierce 660 nm Protein Assay Kit (Thermo Scientific Pierce, Rockford, IL) for equal protein loading. NF-kB transcription factor activity was measured using the TransAM NF-kB p65 Transcription Factor Assay Kit (Active Motif, Carlsbad, CA), according to the manufacturer’s recommendations. Results were read on a BioTek ELx800 Plate Reader (Biotek, Winooski, VT). Data were expressed as the percentage of NF-kB transcription factor activity relative to the sham group. The assay was conducted three times for each sample.
2.10.
Histologic investigation
The portions of the left lung were harvested and flushed with normal saline, fixed with 10% formalin for 24 h and embedded in paraffin; sections of 4 mm were stained with hematoxylin and eosin for light microscope observation and evaluated blindly by an independent consultant histopathologist. Damage to the lung tissue was graded by the pathologist on a scale of 1 (no injury) e 4 (worst), a method described previously [11].
2.11.
Statistical analysis
Results were expressed as mean standard deviation. Differences of all the measured parameters among the groups were assessed with one-way analysis of variance followed by StudenteNewmaneKeuls test for multiple comparisons if analysis of variance indicated a significant overall effect. A value of P < 0.05 was considered to be statistically significant. All data were analyzed using the SPSS version 13 statistical software package (SPSS Inc, Chicago, IL).
3.
Results
3.1.
Lung tissue W/D ratio measurement
To evaluate the extent of OLV-induced lung damage, the W/D ratios were measured in different groups as shown in Figure 1A. The W/D ratio was significantly increased in OLV group compared with other three groups (P < 0.01). Although hydrogen water consumption in rats significantly reduced the W/D ratios compared with OLV group, significant differences were still observed between sham þ H2 group and OLV þ H2 group (P < 0.01). There was no significant difference in W/D ratios between sham and sham þ H2 groups (P ¼ 0.52).
Fig. 1 e Effects of hydrogen water consumption on the lung W/D ratio, MDA levels, and MPO activity in lung tissues of mice after the OLV. The lung W/D ratio (A), MDA levels and MPO activity (B) in lung tissues were determined after 60 min of OLV followed by 60 min of two-lung ventilation. Data are presented as the mean ± standard deviation (n [ 8 in each group). *P < 0.01 versus sham group and sham D H2 group, #P < 0.01 versus OLV group.
3.2.
Levels of MDA and MPO in lung tissues
To ascertain whether hydrogen water consumption in rats protects the lung injury from OLV, the activities of MDA and MPO in different groups were measured. We found that hydrogen water consumption in rats significantly decreased the MDA level and MPO activity compared with those of OLV group (P < 0.01; Fig. 1B, C). However, compared with sham þ H2 group, pulmonary MDA level and MPO activity increased in OLV þ H2 group (P < 0.01; Fig. 1B, C). There was no significant
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difference in pulmonary MDA level and MPO activity between sham and sham þ H2 groups (P ¼ 0.33 in MDA level; P ¼ 0.22 in MPO activity; Fig. 1B, C).
3.3. Levels of TNF-a, IL-1b, and IL-6 levels in lung tissues The levels of TNF-a, IL-1b, and IL-6 in lung tissue are shown in Figure 2. We found that hydrogen water consumption in rats significantly decreased TNF-a, IL-1b, and IL-6 levels compared with those of OLV group (P < 0.01). However, compared with sham þ H2 group, pulmonary TNF-a, IL-1b and IL-6 levels increased in OLV þ H2 group (P < 0.01). There was no significant difference of TNF-a, IL-1b, and IL-6 levels between sham and sham þ H2 groups (P ¼ 0.84 in TNF-a level; P ¼ 0.81 in IL-1b level; and P ¼ 0.81 in IL-6 level).
3.4.
NF-kB transcription factor activity in lung tissue
The levels of NF-kB transcription factor activity in lung tissue are shown in Figure 2. We found that hydrogen water consumption in rats significantly decreased NF-kB transcription factor activity compared with those of OLV group (P < 0.01). However, compared with sham þ H2 group, NF-kB transcription factor activity was increased in OLV þ H2 group (P < 0.01). There was no significant difference of NF-kB transcription factor activity between sham and sham þ H2 groups (P ¼ 0.52).
3.5.
Histologic investigation
The representative lung injuries in different groups are shown in Figure 3. Lung microscopic examination revealed that the
lung tissues of rats were severely damaged in OLV group with alveolar, perivascular and interstitial edema, atelectasis, disruption of alveolar and bronchiolar epithelial cells, and hemorrhage. Hydrogen water consumption obviously suppressed these morphological changes, and the injury degree of lung was minor in OLV þ H2 group than OLV group (P < 0.01). Compared with the sham þ H2 group, the injury degree of lung still increased in OLV þ H2 group (P < 0.01). There was no significant difference in the lung injury score between sham and sham þ H2 groups (P ¼ 0.60).
4.
Discussion
Anesthesia for thoracic surgery routinely involves OLV to facilitate surgical exposure and to isolate and protect the lungs during the procedure. However, there is an increasing concern about the effects of lung injury as a consequence of OLV [12e14], so developing approaches to minimize this kind of lung injury will advance OLV and could have substantial clinical impact. Recently, mounting evidence has shown that hydrogen can behave as an antioxidant in biological systems because of its ability to reduce ROS and potential anti-inflammatory effects [9,15,16]. To develop a new strategy to alleviate lung injury during OLV, we designed the present study to evaluate the effects of hydrogen water consumption on OLV-induced lung injury. The results demonstrated that hydrogen water consumption before OLV can alleviate OLV-induced lung injury. This protective effect is supported by reduced lung injury as measured by lower level of W/D ratio, MDA, MPO activity, proinflammatory cytokines, and marked preservation of lung
Fig. 2 e Effects of hydrogen water consumption on the TNF-a, IL-1b and IL-6 levels and NF-kB activity in the lung tissues of mice after the OLV. TNF-a (A), IL-1b (B), and IL-6 (C) levels, and NF-kB activity were determined (D) in the lung tissues after 60 min of OLV followed by 60 min of two-lung ventilation. Data are presented as the mean ± standard deviation (n [ 8 in each group). *P < 0.01 versus sham group and sham D H2 group, #P < 0.01 versus OLV group.
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Fig. 3 e Effect of hydrogen on histopathologic changes in lung tissues of mice after the OLV) ( 3 200). Photomicrographs of left lung sections from the sham group (A), sham D H2 group (B) and OLV group (C) and OLV D H2 group (D) after 60 min of OLV followed by 60 min of two-lung ventilation. The lung in the OLV group showed a thickened alveolar wall, edema, hemorrhage, and less alveolar space. Hydrogen water consumption significantly prevented the histopathologic changes caused by OLV. (E) The severity of lung injury was scored, and the data are presented as the mean ± standard deviation (n [ 8 in each group). *P < 0.01 versus sham group and sham D H2 group, #P < 0.01 versus OLV group. (Color version of the figure is available online.)
tissue structure microscopically. In addition, hydrogen water consumption has been shown to ameliorate NF-kB expression in the lung tissues. The mechanism of lung injury response to OLV is very complicated, which at least includes ischemia and/or reperfusion, mechanical ventilation, and hypoxemia [3,17].
Generation of ROS and release of proinflammatory cytokines were widely recognized, taking important roles in the OLVinduced lung injury [18,19]. ROS are oxidants derived from the metabolism of oxygen, which encompasses a broad range of biochemical interactions and chemical properties. They include superoxide, H2O2, hydroxyl radical, peroxyl radical,
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alkoxyl radical, ozone, and singlet oxygen. In cellular process, oxidative phosphorylation in mitochondria is the major endogenous source of ROS. Under normal physiological conditions, ROS can be safely reduced to water by superoxide dismutase, catalase, and glutathione peroxidase. However, in the pathologic conditions such as ischemiaereperfusion injury, organ transplantation, and sepsis, the balance between the generation of ROS and the mechanisms to detoxify them can be upset, resulting in the accumulation of ROS in the tissues, where they quickly react with lipids, proteins, and nucleic acids, causing severe cellular damage, unwanted cell death, and consequently vascular barrier disruption and pulmonary edema, even respiratory failure [20]. Our findings were in agreement with these reports, such that we also found that OLV increased the level of MDA, a reliable marker of oxidative stress-mediated lipid peroxidation, in the lung tissue, and drinking hydrogen water significantly reduced the MDA level. In addition, OLV increased the activity of MPO, an enzyme present in neutrophils, in the lung tissue, and drinking hydrogen water significantly reduced the MPO activity, which means drinking hydrogen water decreases the neutrophil recruitment in the lung tissue. The mechanism for this protection of lung tissue may be that hydrogen can modulate the gene transcription of antioxidation genes [21]. Another possibility is that hydrogen permeates mitochondria and directly reduces the production of superoxide [22]. However, the accurate mechanism for hydrogen protection to tissues is still unclear. Previous studies have shown that inflammatory mediators, including TNF-a, IL-1b, and IL-6, are the major generators of lung injury induction, which play important roles in the initiation and amplification of inflammatory responses [23e26]. In the present study, we investigated the levels of TNF-a, IL-1b, and IL-6 in the lung tissues, and our results show that the concentrations of TNF-a, IL-1b, and IL-6 in the lung tissues significantly increased after OLV, and hydrogen water pretreatment reduced the concentrations of cytokines in the lung tissues. The release of proinflammatory cytokines, including TNFa, IL-1b, and IL-6, is promoted by the transcription factor NFkB, which plays a central role in intracellular signaling for the production of proinflammatory cytokines. In previous studies, scientists have proved that hydrogen had the ability of inhibiting the activation of NF-kB [15,16,27,28]. So, in this study we also investigated the activities of NF-kB in the lung tissues of different groups, and we found that hydrogen water consumption decreased the activity of NF-kB. This may be an explanation for the increase of proinflammatory cytokines in OLV group. This study also has some limitations. First, we did not measure the serum and/or lung tissue hydrogen concentrations in this study, as we have little experience in this aspect. However, it has been reported to detect these changes using a novel method, and these results can better validate the cause and effect of hydrogen water consumption and its protective effect [29]. Next, we did not detect the changes of hemodynamic parameters, peak airway pressures, and arterial blood gases among different groups. These parameters may give us more clues for the accurate mechanism study related to the lung protective effect of hydrogen water consumption. In
addition, we used external clamping of the bronchus to block the ventilation of left lung. This method is not the same as the clinical situation (with bronchial blocker or double lumen tube) and may disrupt the bronchial arterial supply. In conclusion, the results of this study demonstrate that hydrogen water consumption ameliorated OLV-induced acute lung injury by reducing oxidative stress and the production of proinflammatory cytokines in lung tissue. However, the intensive mechanism involved in the protective role of hydrogen water consumption to OLV-induced lung injury still requires further study.
Acknowledgment This project was supported by China Postdoctoral Science Foundation funded project (2013M532058) and Key Science and Technology Project of Shaanxi Province (2013K12-08-01). Authors’ contributions: J.Z., Y.W., S.S., Y.Z., G.Z. collected the data and drafted the article. Q.W. conceived and created the framework for the paper, evaluated the data, and drafted and edited the article. C.L. and J.F. conceived and created the framework for the paper, evaluated the data, and edited the article. All the authors have read and approved the final article.
Disclosure The authors have no conflicts of interest to declare.
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