Experimental Lung Research, 39, 441–452, 2013 Copyright © 2013 Informa Healthcare USA, Inc. ISSN: 0190-2148 print / 1521-0499 online DOI: 10.3109/01902148.2013.845626
ORIGINAL ARTICLE
Participation of autophagy in acute lung injury induced by seawater Qiu-ping Liu,1 Dang-xia Zhou,2,3 Pu Lin,4 Xiao-li Gao,2 Lei Pan,5 and Fa-guang Jin5 Exp Lung Res Downloaded from informahealthcare.com by Biblioteka Uniwersytetu Warszawskiego on 01/09/15 For personal use only.
1 2 3 4 5
Third Ward of VIP, 323 Hospital of PLA, Xi’an, China Pathology department, Medical School, Xi’an Jiaotong University, Xi’an, China Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, China Electric Power Science Research Institute of Shaanxi Province, Xi’an, China Department of Respiratory Medicine, Tangdu Hospital, Fourth Military Medical University, Xi’an, China A B STRA CT Seawater drowning can lead to acute lung injury (ALI). However, the molecular and cellular mechanisms underlying this phenomenon remain elusive. The overall aim of this study is to clarify the role of autophagy in seawater-induced ALI, by which we can further understand the molecular mechanism and develop new methods for prevention and treatment of seawater-induced ALI. In this study, electron microscopy, western blot analysis, and RT-PCR were used to detect autophagy in lung tissues. Moreover, arterial blood gas analysis, lung weight coefficient, TNF-α, IL-8 in bronchoalveolar fluid (BALF), histopathology were used to detect the lung injury of seawater exposure. An inhibitor of autophagy (3-Methyladenine, 3-MA) was injected intraperitoneally before seawater exposure to further explore the role of autophagy in ALI. Electron microscopy revealed increasing autophagosomes in alveolar epithelial cell in seawater group compared with the control. The transcription and expression levels (mRNA and protein levels) of the LC3 II significantly increased in lung tissue of seawater group compared with those in control group. Furthermore, the alterations of autophage were basically consistent with the changes in arterial blood gas, lung weight coefficient, TNF-α, IL-8 in BALF and morphologic findings. In addition, inhibition of autophagy by 3-MA partly ameliorated seawater-induced ALI, as indicated by reduced lung weight coefficient and TNF-α in BALF, as well as increased PaO2 . In conclusion, seawater aspiration triggered autophagy, and autophagy may be a scathing factor responsible for ALI induced by seawater. KEYWORDS 3-methyladenine, acute lung injury, alveolar epithelial cell, autophagy, seawater
INTRODUCTION Drowning is one of important causes of accidental deaths. It has been showed that drowning accounts for approximately 450,000 deaths each year in the world [1]. Similar to stress situations such as trauma, burns, and sepsis, seawater aspiration can also lead to ALI (acute pulmonary injury)/ARDS (acute respiratory distress syndrome) [2]. The sites of drowning are diverse. As seawater is high osmolarity, ALI including lung edema, hypoxemia and inflammatory reaction are more serious in seawater drowning comReceived 22 June 2013; accepted 13 September 2013. Address correspondence to Dang-xia Zhou, Pathology department, Medical School, Xi’an Jiaotong University, Xi’an 710061, China. E-mail: [email protected]
pared to those in freshwater drowning [3]. However, the mechanism of seawater-induced ALI is still not clearly understood and thus currently there are no effective strategies to treat it. Therefore, it is urgent to understand how seawater aspiration influences lung tissue. The damage of alveolar epithelial cells and the activation of inflammatory response are hallmark of ALI/ARDS [4, 5]. Studies have showed that seawater drowning results in increasing alveolar epithelial cells degeneration, death and loss [2, 6]. Previous studies demonstrate that apoptosis of alveolar epithelial cells is an important contributor to the pathogenesis of ALI [6–8]. However, apoptosis is not the sole means of programmed cell death. More recently, autophagy, a process in which de novo-formed membrane-enclosed vesicles engulf and consume 441
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cellular components, has been shown to engage in programmed cell death process [9]. Autophagy, which can occur in almost all cells that have mitochondria, is a conserved cellular process for the disposal of damaged organelles or denatured proteins through a lysosomal degradation pathway [10]. Autophagy occurs at low basal levels in cells to execute homeostatic function. It is upregulated rapidly when intracellular nutrition and energy are deficient, as in hypoxia, starvation, and growth factor depletion [11]. Autophagy can also induce typeII programmed cell death when it is inappropriately activated. Increasing evidence suggests that autophagy, and its regulatory proteins, may critically influence vital cellular processes such as programmed cell death, cell proliferation, inflammation, and innate immune functions and thereby may play a critical role in the pathogenesis of human disease, including caner, infection, and neurodegenerative diseases, etc. [12]. Recently, it is increasingly clear that autophagy is relevant to many pulmonary diseases including ALI, COPD, α1-antitrypsin deficiency, pulmonary hypertension, cystic fibrosis and respiratory infection, etc. [13–15]. However, as far as what is known, few studies have examined the regulation and functional significance of autophagy in ALI induced by seawater. Therefore, the purpose of this study was to investigate the roles of autophagy in seawater-ALI, by which we can further understand the molecular mechanism of ALI and might identify new targets for prevention and treatment of ALI.
MATERIALS AND METHODS Animals and Treatment Healthy adult male Sprague-Dawley rats weighing 180–200 g from Experimental Animal Center of Xi’an Jiaotong University were obtained and housed in solid-bottomed polycarbonate cages in SPF animal laboratory with a temperature 21–25◦ C and a relative humidity of 40–60%. Rats were acclimatized at a 12 hours light/12 hours dark cycle and fed a standard diet and tap water ad libitum before the experiments. Experiments were performed in accordance with the Animal Experimentation Committee Regulation. According to the methods of previous study [6], seawater (osmolality 1300 mmol/L, SW 1.05, pH 8.2, NaCl 26.518 g/L, MgSO4 3.305 g/L, MgCl2 2.447 g/L, CaCl2 1.141 g/L, KCl 0.725 g/L, NaHCO3 0.202 g/L, and NaBr 0.083 g/L) was prepared. The rats were divided at random into five groups: a control group, 0.5 hours seawater group, 1 hour
seawater group, 2 hours seawater group, and 4 hours seawater group, each comprising 10 individuals. The rats in the experimental groups were anesthetized with 20% urethane (5 mL/kg) intraperitoneally and maintained in the supine position during experiments. A tube was inserted into trachea through a tracheostomy, and then 3 mL/kg of seawater was aspirated into trachea within 5 minutes.
Arterial Blood Gas Analysis The rats were sacrificed by aortic puncture at the indicated time points. The blood samples were immediately taken for arterial blood gas analysis. Hydrogen ion concentration (pH), arterial oxygen tension (PaO2 ), and arterial carbon dioxide tension (PaCO2) was measured with a blood gas analyzer.
Lung Weight Coefficient The lung weight coefficient was determined as an index of pulmonary edema. The lung tissue were immediately removed and weighed after the surface blood was aspirated. The lung weight coefficient was calculated by dividing the lung weight by body weight in each rat.
Measurement of TNF-α, IL-8 in BALF The BALF was taken by a method as described by Han et al. [6]. Briefly, the left lungs were excised integrally from rats in each group. The bronchoalveolar lavage (BAL) was made with intratracheal injections of 2 mL of physiological saline at 37◦ C for three times. The BALF was retrieved and centrifuged, and TNFα, IL-8 were then determined by the ELISA method (R&D Systems Inc. Minneapolis, MN, USA).
Histopathological Examination Lung tissue for histological study was fixed in fresh 4% formaldehyde solution for 24 hours and then dehydrated and embedded in paraffin, finally 4 μm sections were got and stained with hematoxylin & eosin (HE). The tissue sections were observed under a light microscope for the lung histopathology. Lung injury was evaluated according to the degree of alveolar edema, interstitial edema, neutrophil infiltration, and hemorrhage.
Transmission Electron Microscopy For ultrastructural examination, lung tissue from each group were fixed with 2.5% glutaraldehyde, then postfixed with 2% OsO4 and embedded in Araldite. Ultrathin sections were stained with uranyl acetate Experimental Lung Research
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and lead citrate and inspected using a transmission electron microscope (H-7650, Hitachi, Japan).
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RT-PCR Total RNA was extracted from lung tissue using the Trizol reagent and reverse-transcribed into cDNA using commercial kits (Fermentas, Lithuania). PCR was carried out in 20 μL reaction volumes, containing 7 μL of PCR Mix (10× Taq Buffer with (NH4 )2 SO4 , 0.2 mmol/L dNTP, 1.5 mmol/L MgCl2 ), 1 μL of each primer, 9 μL ddH2 O, and 2 μL of cDNA. PCR cycling was performed using the MyCycler Thermal Cycler (Bio-Rad) with conditions: 95◦ C, 5 minutes, followed by 35 cycles of 30 seconds at 95◦ C, 30 seconds at 60◦ C and 45 seconds at 72◦ C, after cycles at 72◦ C, 10 minutes. The primer sequences and the expected sizes of PCR products were as follows: LC3 (sense): 5 CCATGCCGTCCGAGAAGACCTTC-3 and (antisense) 5 -GACCAGCTTCCGCTGGTAACGTC3 (452bp). GAPDH: (sense) 5 -GCAAGTTCA ACGGCACAG-3 and (antisense) 5 -GCCAGTA GACTCCACGACAT −3 (140 bp).
Western Blotting In brief, lung tissue in each group was homogenized in ice-cold RIPA lysate buffer (Sigma) and centrifuged at 15,000 × g for 15 minutes at 4◦ C. The total protein concentration was determined with a UV 3000 ultraviolet spectrophotometer (Nano Drop, Wilmington, DE). The samples (100 ug protein per lane) were separated with 15% gradient SDS-PAGE gel and then transferred onto a PVDF membrane. Nonspecific binding to the membrane was blocked with 5% nonfat milk in Tris buffered saline-Tween 20 (TBST, pH 7.4) for 2 hours at room temperature. After that, the membranes were incubated overnight with rabbit polyclonal antibody against LC3 (1:1000 dilution, Cell signaling technology) and β-actin (1:1000 dilution, Sigma) at 4◦ C. After washed with TBST for three times, the membrane were incubated in goat anti-rabbit IgG (1:5000 dilution, Beijing ZhongShan Biotechnology CO., Ltd., Beijing, China) for 2 hours at room temperature, and bands visualized by using the ECL kit (Proteintech, USA). The ratio of LC3 level II/LC3 I level was used as an indicator of autophagic level.
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before seawater aspiration. The dose of 3-MA was according to the Zhang et al.’s study in rats [11]. The control rats received vehicle alone in the same time. Lung injury was evaluated by detecting arterial blood gas, lung weight coefficient, TNF-α, IL-8 in BALF in both 3-MA and vehicle-treated rats 1 hour after seawater aspiration.
Statistical Analysis All statistical analyses were carried out using SPSS statistical software version 13.0 (SPSS, Chicago, USA). Data were summarized as mean + SD. Oneway ANOVA and post-hoc comparisons were used to determine the differences among multiple groups. P < .05 was regarded as statistically significant.
RESULTS Effects of Seawater Aspiration on Arterial Blood Gas Compared with control group, seawater aspiration caused significant changes in the arterial blood gas parameters, which was manifested by an obvious decrease in PaO2 and pH value, as well as an significant increase in PaCO2 (Figure 1). As shown in Figure 1, the most serious respiratory failure occurred at the time point of 30 minutes to 1 hour after seawater aspiration, and then slowly reversed with time.
Effects of Seawater Aspiration on Lung Weight Coefficient Lung weight coefficient significantly increased in the seawater group when compared with that in the control group. The obvious change occurred at 30 minutes and 1 hour after seawater aspiration, and then gradually decreased (Figure 2).
Effects of Seawater Aspiration on TNF-α, IL-8 in BALF TNF-α and IL-8 in BALF significantly increased in the seawater group when compared with that in the control group. They were elevated from the beginning of the experiment, and then gradually increased with time (Table 1).
Suppression of Autophagy in vivo To further explore the role of autophagy, the rats were injected intraperitoneally with a pharmacological inhibitor of autophagy, 3-Methyladenine (3-MA, Sigma, 30 mg/kg dissolved in 1 mL saline) 20 minutes C
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Effect of Seawater Aspiration on Lung Histopathological Changes Normal lung tissue structure and clear alveoli were observed in rats of control group (Figure 3A).
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FIGURE 1. Effects of seawater aspiration on arterial blood gas parameters of rats. (A) PaO2 ; (B)
PaCO2 ; (C) pH. Data are expressed as Mean ± S.D. ∗ P < .05 versus control group.
Compared with the control, diffuse alveolar damage was observed in seawater groups (Figure 3B–D). Some alveoli were collapsed, others were distended. In addition, the lung tissue demonstrated capillary congestion, interstitial and intraalveolar edema and hemorrhage, thickened alveolar wall and increasing netrophils within the vascular space, the interstitium, and the alveoli.
Effects of Seawater Aspiration on Electron Microscopic Changes of Lung Tissues As shown in Figure 4, the evacuation of substance from lamellar bodies in the type II alveolar epithelial cell in the seawater groups was observed. In addition, autophagosome was more frequently observed in the alveolar epithelial cells of rats in seawater aspiration groups compared with the control group. Experimental Lung Research
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FIGURE 2. Effects of seawater aspiration on lung weight coefficient of rats. Data are expressed as Mean ± SD ∗ P < .05, compared with control group.
Effect of Seawater Aspiration on LC3 mRNA level by RT-PCR
Effect of Seawater Aspiration on LC3 Conversion by Western Blotting
Compared with the control group, a notable upregulation of LC3 mRNA level was observed in a rat lung tissue of the seawater groups (Figure 5).
The conversion of LC3-I to LC3-II is a necessary step during the process of autophagy. The level of LC3-II is known to correlate well with the number of
FIGURE 3. Light micrographs of lung tissues stained with hematoxylin-eosin (HE (×400). (A) Lung
tissue in a rat of the control group. (B) Lung tissue in a rat of the seawater groups showing some collapsed alveoli, distended alveoli and thickened alveolar wall. (C) Lung tissue in a rat after seawater aspiration showing capillary congestion, interstitial and intraalveolar edema and hemorrhage. (D) Lung tissue in a rat after seawater aspiration demonstrating increasing netrophils within the vascular space, the interstitium, and the alveoli. C
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FIGURE 4. High resolution electron micrographs of lung tissues (×50,000). (A) Control group. (B) Seawater group. The evacuation of
substance from lamellar bodies in the type II alveolar epithelial cell. The arrows indicated the double membrane-surrounded autophagosomes in the type II alveolar epithelial cell. (C) Seawater group. The arrows indicated the double membrane-surrounded autophagosomes in the type I alveolar epithelial cells.
autophagosomes. Our studies showed that the LC3II protein were hardly detectable in normal lung tissue by using western blotting analysis. However, a marked upregulation of the ratios of LC3-II/LC3-I was observed, in rats of seawater groups (Figure 6).
Inhibition of Autophagy by 3-MA Attenuates Acute Lung Injury Further experiments were performed to explore the role of autophagy in seawater-induced ALI by using an inhibitor of autophagy, 3-MA. At first, In order to confirm the inhibitory effect of 3-MA on autophagy, we detected LC3-II conversion in rats by western blotting. As shown in Figure 7, 30 mg/kg 3-MA could significantly reduce LC3-II conversion in lung tissues compared with vehicle-treated rats. In addition, As shown in Figure 8, lung injury indicators including PaO2 , lung weight coefficient, and TNF-α in BALF were obviously ameliorated by 3MA treatment when compared with vehicle-treated rats (Figure 8).
TABLE 1. Effects of Seawater Aspiration on TNF-α and IL-8 in Bronchoalveolar Fluid of Rats Group
TNF-α (pg/mL)
IL-8 (pg/mL)
Control 0.5 hours 1 hour 2 hours 4 hours
21.67 ± 6.86 40.67 ± 11.78∗ 51.66 ± 14.05∗ 52.83 ± 16.74∗ 54.50 ± 15.61∗
30.61 ± 5.28 43.17 ± 7.19∗ 50.01 ± 11.03∗ 55.50 ± 8.96∗ 52.18 ± 11.36∗
∗
P < .05, compared with control group.
DISCUSSION Although seawater drowning is a common cause of acute lung injury (ALI), the molecular and cellular mechanisms underlying this phenomenon remain elusive. The pathophysiology of ALI and its most severe form, ARDS, is characterized by increased vascular and epithelial permeability, hypercoagulation, hypofibrinolysis, and diffuse inflammation reactions [16]. The damage of alveolar epithelial cells and the activation of inflammatory responses are hallmark of ALI/ARDS [4, 5]. Previous studies on mechanisms related to alveolar epithelial damage mostly focused on apoptosis, which has been considered as an underlying mechanism in ALI/ARDS and multiorgan dysfunction syndrome [6–8]. However, Apoptosis is not the sole means by which the cell can undergo a genetically programmed death. Autophagy has also been linked to the actual death process [17]. Thus, this study was designed to explore possible role of autophagy in ALI induced by seawater. Our research, for the first time, found that seawater aspiration triggered autophagy in alveolar epithelial cells with increased pulmonary epithelial permeability in an in vivo model. The alterations of autophage were basically consistent with the changes in arterial blood gas, lung weight coefficient, TNF-α, IL-8 in BALF, histopathologic and transmission electron microscopic findings. In addition, inhibition of autophagy by 3-MA partly ameliorated seawaterinduced ALI, as indicated by reduced lung weight coefficient and TNF-αin BALF, as well as increased PaO2 . The results demonstrated that autophagy was involved in the ALI pathophysiological process, and Experimental Lung Research
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FIGURE 5. Effects of seawater aspiration on LC3 mRNA level in rat lung tissue. (A)
Electrophorogram picture for LC3 mRNA in lung tissues after seawater aspiration. (B) The ratios of LC3 mRNA/GADPH mRNA were presented in bar chart. ∗ P < .05, compared with the control group. M: marker; lanes 1, 3, 5, 7, and 9 represented the LC3 mRNA (452 bp) in control, 0.5, 1, 2, and 4 hours seawater aspiration groups respectively; lanes 2, 4, 6, 8, and 10 represented the GADPH mRNA (140 bp) in control, 0.5, 1, 2, and 4 hours seawater aspiration groups respectively.
it may be a scathing factor in ALI after seawater aspiration. Autophagy is an evolutionarily conserved physiological process that provides a membrane-dependent mechanism for the sequestration, transport, and lysosomal turnover of subcellular components, including proteins and organelles [12]. Autophagic pathway consists of several distinct steps: (1) the formation of an isolation membrane; (2) the formation of an autophagosome with encapsulated cargo; (3) the fusion of the autophagosome to the lysosome; and (4) the degradative phase with the digestion of lysosomal contents [10]. It is an endogenous tightly regulated process responsible for the degradation of damaged and dysfunctional cellular organelles and protein aggregates [18]. Autophagy principally serves an adaptive role that promotes cell survival C
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[19]. Recently, autophagy is also considered a form of nonapoptotic programmed cell death called type II programmed cell death or autophagic cell death [20]. It is not yet understood what factors determine whether autophagy is cytoprotective or cytotoxic. Possible factors that might control the cellular “decision” between the two responses include potentially variable thresholds for each process, molecular links that coordinately regulate apoptosis and autophagy, and mutual inhibition or activation of each pathway by the other [21]. Recently, evidence of autophagy in the pathogenesis of ALI has gradually come to light [22]. For example, Li et al. reported that nanoparticles triggers autophagic cell death and the autophagy inhibitor 3-methyladenine might rescue cell death and ameliorate ALI caused by nanoparticles in mice
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FIGURE 6. Effects of seawater aspiration on LC3 conversion in rat lung tissue. (A) LC3 expression in lung tissues after seawater aspiration. (B) The ratios of LC3-II/LC3-I were presented in bar chart. ∗ P < .05, compared with the control group.
[23]. Sun et al. concluded that the autophagic cell death of alveolar epithelial cells likely plays a crucial role in the high mortality rate of H5 N1 infection and autophagy-blocking agents might be useful as prophylactics and therapeutics against infection of humans by the H5 N1 virus [24]. Our present study further verified that autophagy plays an important role in ALI induced by seawater. Our data showed that the autophagy was elevated after seawater aspiration, inhibition of autophagy by 3-MA partly ameliorated seawater-induced ALI. All above results indicated that autophagy might be a damaging factor in lung injury. However, some studies concluded an opposite conclusion in different organs or diseases. For instance, Jiang et al. reported that autophagy was not obvious during the ischemia period, but was significantly enhanced during reperfusion. Inhibition of autophagy by 3-methyladenine and chloroquine worsened renal ischemia/reperfusion injury. They concluded that autophagy was a protective mechanism during in vitro hypoxia and in vivo renal I/R
injury [25]. Takahashi et al. found that autophagyrelated processes are properly activated in the liver in a mouse model of sepsis; Inhibition of autophagy process by chloroquine administration resulted in elevated serum transaminase levels and a significant increase in mortality. They argued that autophagy appears to play a protective role in liver of septic animals [26]. These results have been controversial as to whether high levels autophagy lessen or aggravate cell injury. We speculated that these inconsistent results indicated that the role of autophagy might be model-dependent and/or tissue-dependent. In our study, various methods including arterial blood gas analysis, lung weight coefficient measuring, TNF-α, IL-8 measuring in BALF, histopathology examination were used to detect the lung injury of seawater exposure. We found that seawater aspiration could cause lung injury, which was characterized by decreased PaO2 and increased PaCO2, lung weight coefficient, TNF-α, IL-8 in BALF. The results were consistent with histopathologic findings including Experimental Lung Research
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FIGURE 7. Effects of 3-MA treatment on LC3 conversion in lung tissue of seawater aspiration rats. (A) LC3 expression in lung tissues. (B) The ratios of LC3-II/LC3-I were presented in bar chart. ∗ P < .05, compared with the vehicle group.
pulmonary edema, inflammation reaction in lung tissue. All these data suggested that the intratracheal aspiration of seawater in the rat model induced topical ALI, which was also in accordance with the findings of other authors [2, 6, 27]. Static measures of autophagy included quantifying the number of autophagosomes within cells by electron microscopy and measuring steady-state levels of pathway constituents such as LC3II by western blot analysis. The measurements were considered as sufficient indications of autophagic activity [12]. LC3 is a mammalian homologus protein of yeast Atg8 protein. In our study, ultrastructural electron microscopy analysis of lung tissue from rats in seawater groups revealed increasing autophagosomes relative to the control group. In addition, the transcription and expression levels (mRNA and protein levels) of the LC3 II, an indicator of autophagosome formation, increased in lung tissues of seawater groups compared with those in the control group. Moreover, the alterations of autophage were basically consistent with the changes in arterial blood gas, lung weight coefficient, TNF-α, IL-8 in BALF and morphologic findings. In addition, we used a widely used pharmacological inhibitor of autophagy, 3-MA to further explore the role of autophagy in ALI. 3-MA potently blocks the initial autophagic sequestration and autopagosome forma C
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tion at the early stage and often used to understand the role of autophagy [11, 23, 25]. We found that the autophagy inhibitor 3-MA could partly ameliorated seawater-induced ALI, as indicated by reduced lung weight coefficient and TNF-α in BALF, as well as increased PaO2 . arterial blood gas, lung weight coefficient, TNF-α and IL-8 in BALF are widely accepted indicators for quantitating lung injury. Therefore, all these results further confirmed that autophagy exists in the lung, and may be a damaging factor responsible for seawater-induced ALI in rats. It was reported that autophagy could be activated under various stress conditions such as hypoxia, ischemia, oxidative stress and endoplasmic reticulum stress, etc. [17, 18, 28]. Autophagy is a process that can be regulated by hypoxia in cells and tissues and may be important for hypoxia-induced cell injury [12]. In this study, seawater aspiration caused a significant decrease in PaO2 and an increase PaCO2 . These results clearly demonstrated that hypoxia occurs in rat model. We speculated that the airway is blocked and respiration is simultaneously inhibited, which is the main cause of hypoxia after drowning. Studies showed that hypoxia can trigger a complex cascade of events, such as burst of reactive oxygen species (ROS) and Ca2+ overload in mitochondria [11]. These events are effective triggers for autophagy. The
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FIGURE 8. Effects of 3-MA treatment on PaO2 , PaCO2 , lung weight coefficient, TNF-α,
and IL-8 in BALF. (A) PaO2 and PaCO2 ; (B) Lung weight coefficient; (C) TNF-α and IL-8 in BALF. ∗ P < .05, compared with the vehicle group.
cellular responses triggered by oxidative stress include the altered regulation of signaling pathways that culminate in the regulation of cell survival or cell death pathways. Recent studies suggest that autophagy may represent a general cellular and tissue response to oxidative stress [29]. In addition, increased Ca2+ overload in mitochondria can upregulate autophagy by activating calmondulin-dependent kinase or stim-
ulating calpains, which also contribute to autophagy [30]. The damage of alveolar epithelial cells and the activation of inflammatory responses are hallmark of ALI/ARDS [4]. In this study, we have also observed increased morphological and biochemical markers of autophagy in lung tissues from the ALI-rats induced by seawater aspiration. We hypothesize that increased Experimental Lung Research
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autophagy contributes to ALI pathogenesis by promoting epithelial cell death. Alveolar epithelial cells death not only increases pulmonary permeability, but also destroys the alveolar epithelial barrier function. Lung weight coefficient significantly increased in rats after seawater aspiration, further suggesting the hypothesis. In addition, studies showed that autophagy exerts a critical influence on inflammatory responses. Autophagy plays a potentially pivotal role in the regulation of inflammatory responses. In particular, autophagy regulates endogenous inflammasome activators, as well as inflammasome components and pro-IL-1β. As a result, autophagy acts a key modulator of IL-1β and IL-18, as well as IL-1α, release etc. [31]. The alterations of autophage and changes of TNF-α, IL-8 levels in BALF further supported previous findings. In conclusion, this study discloses that autophagy might play important roles in the disruption of epithelial cells in ALI induced by seawater aspiration. Our data demonstrate autophage increases after seawater aspiration and the alterations are consistent with the severity of lung injury. Inhibition of autophagy by 3MA partly ameliorated ALI induced by seawater. On the basis of these findings, it is tempting to speculate that the selective targeting of autophagic proteins using pharmacological or genetic strategies may serve as the basis for developing novel therapies for ALI. However, the signaling mechanisms involved in the regulation of alveolar epithelial cells autophagy, as well as the relationship between autophagy and apoptosis are still unclear. Therefore, further investigations concerning the discrete molecular signaling mechanisms and correlation between autophagy and apoptosis are necessary.
[3]
[4]
[5]
[6]
[7]
[8] [9]
[10] [11]
[12] [13]
[14]
[15]
[16]
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This research was financially supported by National Natural Science Fundings of China (No. 81273018), Science Technical Development Project Funding of Lanzhou Military District (NO, CLZ11JA17; CLZ12JB26), and Science Funding of Health Department, Shaanxi Province (2012D58).
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[18]
[19] [20] [21]
REFERENCES [22] [1]
[2]
C
Soar J, Deakin CD, Nolan JP, Abbas G, Alfonzo A, Handley AJ, Lockey D, Perkins GD, Thies K: European Resuscitation Council. European Resuscitation Council guidelines for resuscitation 2005. Section 7. Cardiac arrest in special circumstances. Resuscitation. 2005;67(Suppl 1):S135–S170. Li J, Xu M, Fan Q, Xie X, Zhang Y, Mu D, Zhao P, Zhang B, Cao F, Wang Y, Jin F, Li Z: Tanshinone IIA ameliorates
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[23]
451
seawater exposure-induced lung injury by inhibiting aquaporins (AQP) 1 and AQP5 expression in lung. Respir Physiol Neurobiol. 2011;176(1–2):39–49. Salomez F, Vincent JL: Drowning: a review of epidemiology, pathophysiology, treatment and prevention. Resuscitation. 2004;63(3):261–268. Xu YL, Liu YL, Wang Q, Li G, Lu¨ XD, Kong B: Intravenous transplantation of mesenchymal stem cells attenuates oleic acid induced acute lung injury in rats. Chin Med J (Engl). 2012;125(11):2012–2018. You K, Xu X, Fu J, Xu S, Yue X, Yu Z, Xue X: Hyperoxia disrupts pulmonary epithelial barrier in newborn rats via the deterioration of occludin and ZO-1. Respir Res. 2012;13:36. doi: 10.1186/1465-9921-13-36. Han F, Luo Y, Li Y, Liu Z, Xu D, Jin F, Li Z: Seawater induces apoptosis in alveolar epithelial cells via the Fas/FasL-mediated pathway. Respir Physiol Neurobiol. 2012;182(2–3):71–80. Galani V, Tatsaki E, Bai M, Kitsoulis P, Lekka M, Nakos G, Kanavaros P: The role of apoptosis in the pathophysiology of Acute Respiratory Distress Syndrome (ARDS): an up-to-date cell-specific review. Pathol Res Pract. 2010;206(3):145–150. Schmidt EP, Tuder RM: Role of apoptosis in amplifying inflammatory responses in lung diseases. J Cell Death. 2010; 3:41–53. Eisenberg-Lerner A, Bialik S, Simon HU, Kimchi A: Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ. 2009;16(7):966–975. Ryter SW, Nakahira K, Haspel JA, Choi AM: Autophagy in pulmonary diseases. Annu Rev Physiol. 2012;74:377–401. Zhang J, Wang JS, Zheng ZK, Tang J, Fan K, Guo H, Wang JJ: Participation of autophagy in lung ischemia-reperfusion injury in vivo. J Surg Res. 2012a; pii: S0022–S4804(12)01860–01864. Ryter SW, Choi AM: Autophagy in the lung. Proc Am Thorac Soc. 2010;7(1):13–21. Patel AS, Morse D, Choi AM: Regulation and functional significance of autophagy in respiratory cell biology and disease. Am J Respir Cell Mol Biol. 2013;48(1):1–9. Lahm T, Petrache I: LC3 as a potential therapeutic target in hypoxia-induced pulmonary hypertension. Autophagy. 2012;8(7):1146–1147. Mizumura K, Cloonan SM, Haspel JA, Choi AM: The emerging importance of autophagy in pulmonary diseases. Chest. 2012;142(5):1289–1299. McVey M, Tabuchi A, Kuebler WM: Microparticles and acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2012;303(5):L 364–L381. Zhang M, Jiang M, Bi Y, Zhu H, Zhou Z, Sha J: Autophagy and apoptosis act as parterns to induce germ cell death after heat stress in mice. Plos One. 2012b;7(7):e41412. Balduini W, Carloni S, Buonocore G: Autophagy in hypoxiaischemia induced brain injury. J Matern Fetal Neonatal Med. 2012; 25(Suppl 1):30–34. Levine B, Kroemer G: Autophagy in the pathogenesis of disease. Cell. 2008;132(1):27–42. Choi AM, Ryter SW, Levine B: Autophagy in human health and disease. J Engl J Med. 2013;368(7):651–662. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G: Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol. 2007;8:741–752. Tang PS, Mura M, Seth R, Liu M: Acute lung injury and cell death: how many ways can cells die? Am J Physiol Lung Cell Mol Physiol. 2008;294(4):L632–L641. Li C, Liu H, Sun Y, Wang H, Guo F, Rao S, Deng J, Zhang Y, Miao Y, Guo C, Meng J, Chen X, Li L, Li D, Xu H, Wang H, Li B, Jiang C: PAMAM nanoparticles promote acute lung injury by inducing autophagic cell death through the Akt-TSC2-mTOR signaling pathway. J Mol Cell Biol. 2009;1(1):37–45.
Exp Lung Res Downloaded from informahealthcare.com by Biblioteka Uniwersytetu Warszawskiego on 01/09/15 For personal use only.
452
Q.-p. Liu et al.
[24] Sun Y, Li C, Shu Y, Ju X, Zou Z, Wang H, Rao S, Guo F, Liu H, Nan W, Zhao Y, Yan Y, Tang J, Zhao C, Yang P, Liu K, Wang S, Lu H, Li X, Tan L, Gao R, Song J, Gao X, Tian X, Qin Y, Xu KF, Li D, Jin N, Jiang C: Inhibition of autophagy ameliorates acute lung injury caused by avian influenza A H5N1 infection. Sci Signal. 2012;5(212):ra16. [25] Jiang M, Liu K, Luo J, Dong Z: Autophagy is a renoprotective mechanism during in virtro hypoxia and in vivo ischemiareperfusion injury. Am J Pathol. 2010;176(3):1181–1192. [26] Takahashi W, Watanabe E, Fujimura L, Watanabe-Takano H, Yoshidome H, Swanson PE, Tokuhisa T, Oda S, Hatano M: Kinetics and protective role of autophagy in a mouse cecal ligation and puncture-induced sepsis. Crit Care. 2013;17(4):R160. [27] Fan Q, Zhao P, Li J, Xie X, Xu M, Zhang Y, Mu D, Li W, Sun R, Liu W, Nan Y, Zhang B, Jin F, Li Z: 17β-Estradiol
[28] [29]
[30]
[31]
administration attenuates seawater aspiration-induced acute lung injury in rats. Pulm Pharmacol Ther. 2011;24(6):673– 681. Blagosklonny MV: Hypoxia, MTOR and autophagy: converging on senescence or quiescence. Autophagy. 2013;9(2): 260–262. Ryter SW, Choi AM: Regulation of autophagy in oxygendependent cellular stress. Curr Pharm Des. 2013;19(15): 2747–2756. Høyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T, Bianchi K, Fehrenbacher N, Elling F, Rizzuto R, Mathiasen IS, J¨aa¨ ttel¨a M: Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell. 2007;25(2):193–205. Harris J: Autophagy and IL-1 Family cytokines. Front Immunol. 2013;4:83. doi: 10.3389/fimmu.2013.00083.
Experimental Lung Research
ORIGINAL ARTICLE
Participation of autophagy in acute lung injury induced by seawater Qiu-ping Liu,1 Dang-xia Zhou,2,3 Pu Lin,4 Xiao-li Gao,2 Lei Pan,5 and Fa-guang Jin5 Exp Lung Res Downloaded from informahealthcare.com by Biblioteka Uniwersytetu Warszawskiego on 01/09/15 For personal use only.
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Third Ward of VIP, 323 Hospital of PLA, Xi’an, China Pathology department, Medical School, Xi’an Jiaotong University, Xi’an, China Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, China Electric Power Science Research Institute of Shaanxi Province, Xi’an, China Department of Respiratory Medicine, Tangdu Hospital, Fourth Military Medical University, Xi’an, China A B STRA CT Seawater drowning can lead to acute lung injury (ALI). However, the molecular and cellular mechanisms underlying this phenomenon remain elusive. The overall aim of this study is to clarify the role of autophagy in seawater-induced ALI, by which we can further understand the molecular mechanism and develop new methods for prevention and treatment of seawater-induced ALI. In this study, electron microscopy, western blot analysis, and RT-PCR were used to detect autophagy in lung tissues. Moreover, arterial blood gas analysis, lung weight coefficient, TNF-α, IL-8 in bronchoalveolar fluid (BALF), histopathology were used to detect the lung injury of seawater exposure. An inhibitor of autophagy (3-Methyladenine, 3-MA) was injected intraperitoneally before seawater exposure to further explore the role of autophagy in ALI. Electron microscopy revealed increasing autophagosomes in alveolar epithelial cell in seawater group compared with the control. The transcription and expression levels (mRNA and protein levels) of the LC3 II significantly increased in lung tissue of seawater group compared with those in control group. Furthermore, the alterations of autophage were basically consistent with the changes in arterial blood gas, lung weight coefficient, TNF-α, IL-8 in BALF and morphologic findings. In addition, inhibition of autophagy by 3-MA partly ameliorated seawater-induced ALI, as indicated by reduced lung weight coefficient and TNF-α in BALF, as well as increased PaO2 . In conclusion, seawater aspiration triggered autophagy, and autophagy may be a scathing factor responsible for ALI induced by seawater. KEYWORDS 3-methyladenine, acute lung injury, alveolar epithelial cell, autophagy, seawater
INTRODUCTION Drowning is one of important causes of accidental deaths. It has been showed that drowning accounts for approximately 450,000 deaths each year in the world [1]. Similar to stress situations such as trauma, burns, and sepsis, seawater aspiration can also lead to ALI (acute pulmonary injury)/ARDS (acute respiratory distress syndrome) [2]. The sites of drowning are diverse. As seawater is high osmolarity, ALI including lung edema, hypoxemia and inflammatory reaction are more serious in seawater drowning comReceived 22 June 2013; accepted 13 September 2013. Address correspondence to Dang-xia Zhou, Pathology department, Medical School, Xi’an Jiaotong University, Xi’an 710061, China. E-mail: [email protected]
pared to those in freshwater drowning [3]. However, the mechanism of seawater-induced ALI is still not clearly understood and thus currently there are no effective strategies to treat it. Therefore, it is urgent to understand how seawater aspiration influences lung tissue. The damage of alveolar epithelial cells and the activation of inflammatory response are hallmark of ALI/ARDS [4, 5]. Studies have showed that seawater drowning results in increasing alveolar epithelial cells degeneration, death and loss [2, 6]. Previous studies demonstrate that apoptosis of alveolar epithelial cells is an important contributor to the pathogenesis of ALI [6–8]. However, apoptosis is not the sole means of programmed cell death. More recently, autophagy, a process in which de novo-formed membrane-enclosed vesicles engulf and consume 441
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cellular components, has been shown to engage in programmed cell death process [9]. Autophagy, which can occur in almost all cells that have mitochondria, is a conserved cellular process for the disposal of damaged organelles or denatured proteins through a lysosomal degradation pathway [10]. Autophagy occurs at low basal levels in cells to execute homeostatic function. It is upregulated rapidly when intracellular nutrition and energy are deficient, as in hypoxia, starvation, and growth factor depletion [11]. Autophagy can also induce typeII programmed cell death when it is inappropriately activated. Increasing evidence suggests that autophagy, and its regulatory proteins, may critically influence vital cellular processes such as programmed cell death, cell proliferation, inflammation, and innate immune functions and thereby may play a critical role in the pathogenesis of human disease, including caner, infection, and neurodegenerative diseases, etc. [12]. Recently, it is increasingly clear that autophagy is relevant to many pulmonary diseases including ALI, COPD, α1-antitrypsin deficiency, pulmonary hypertension, cystic fibrosis and respiratory infection, etc. [13–15]. However, as far as what is known, few studies have examined the regulation and functional significance of autophagy in ALI induced by seawater. Therefore, the purpose of this study was to investigate the roles of autophagy in seawater-ALI, by which we can further understand the molecular mechanism of ALI and might identify new targets for prevention and treatment of ALI.
MATERIALS AND METHODS Animals and Treatment Healthy adult male Sprague-Dawley rats weighing 180–200 g from Experimental Animal Center of Xi’an Jiaotong University were obtained and housed in solid-bottomed polycarbonate cages in SPF animal laboratory with a temperature 21–25◦ C and a relative humidity of 40–60%. Rats were acclimatized at a 12 hours light/12 hours dark cycle and fed a standard diet and tap water ad libitum before the experiments. Experiments were performed in accordance with the Animal Experimentation Committee Regulation. According to the methods of previous study [6], seawater (osmolality 1300 mmol/L, SW 1.05, pH 8.2, NaCl 26.518 g/L, MgSO4 3.305 g/L, MgCl2 2.447 g/L, CaCl2 1.141 g/L, KCl 0.725 g/L, NaHCO3 0.202 g/L, and NaBr 0.083 g/L) was prepared. The rats were divided at random into five groups: a control group, 0.5 hours seawater group, 1 hour
seawater group, 2 hours seawater group, and 4 hours seawater group, each comprising 10 individuals. The rats in the experimental groups were anesthetized with 20% urethane (5 mL/kg) intraperitoneally and maintained in the supine position during experiments. A tube was inserted into trachea through a tracheostomy, and then 3 mL/kg of seawater was aspirated into trachea within 5 minutes.
Arterial Blood Gas Analysis The rats were sacrificed by aortic puncture at the indicated time points. The blood samples were immediately taken for arterial blood gas analysis. Hydrogen ion concentration (pH), arterial oxygen tension (PaO2 ), and arterial carbon dioxide tension (PaCO2) was measured with a blood gas analyzer.
Lung Weight Coefficient The lung weight coefficient was determined as an index of pulmonary edema. The lung tissue were immediately removed and weighed after the surface blood was aspirated. The lung weight coefficient was calculated by dividing the lung weight by body weight in each rat.
Measurement of TNF-α, IL-8 in BALF The BALF was taken by a method as described by Han et al. [6]. Briefly, the left lungs were excised integrally from rats in each group. The bronchoalveolar lavage (BAL) was made with intratracheal injections of 2 mL of physiological saline at 37◦ C for three times. The BALF was retrieved and centrifuged, and TNFα, IL-8 were then determined by the ELISA method (R&D Systems Inc. Minneapolis, MN, USA).
Histopathological Examination Lung tissue for histological study was fixed in fresh 4% formaldehyde solution for 24 hours and then dehydrated and embedded in paraffin, finally 4 μm sections were got and stained with hematoxylin & eosin (HE). The tissue sections were observed under a light microscope for the lung histopathology. Lung injury was evaluated according to the degree of alveolar edema, interstitial edema, neutrophil infiltration, and hemorrhage.
Transmission Electron Microscopy For ultrastructural examination, lung tissue from each group were fixed with 2.5% glutaraldehyde, then postfixed with 2% OsO4 and embedded in Araldite. Ultrathin sections were stained with uranyl acetate Experimental Lung Research
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and lead citrate and inspected using a transmission electron microscope (H-7650, Hitachi, Japan).
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RT-PCR Total RNA was extracted from lung tissue using the Trizol reagent and reverse-transcribed into cDNA using commercial kits (Fermentas, Lithuania). PCR was carried out in 20 μL reaction volumes, containing 7 μL of PCR Mix (10× Taq Buffer with (NH4 )2 SO4 , 0.2 mmol/L dNTP, 1.5 mmol/L MgCl2 ), 1 μL of each primer, 9 μL ddH2 O, and 2 μL of cDNA. PCR cycling was performed using the MyCycler Thermal Cycler (Bio-Rad) with conditions: 95◦ C, 5 minutes, followed by 35 cycles of 30 seconds at 95◦ C, 30 seconds at 60◦ C and 45 seconds at 72◦ C, after cycles at 72◦ C, 10 minutes. The primer sequences and the expected sizes of PCR products were as follows: LC3 (sense): 5 CCATGCCGTCCGAGAAGACCTTC-3 and (antisense) 5 -GACCAGCTTCCGCTGGTAACGTC3 (452bp). GAPDH: (sense) 5 -GCAAGTTCA ACGGCACAG-3 and (antisense) 5 -GCCAGTA GACTCCACGACAT −3 (140 bp).
Western Blotting In brief, lung tissue in each group was homogenized in ice-cold RIPA lysate buffer (Sigma) and centrifuged at 15,000 × g for 15 minutes at 4◦ C. The total protein concentration was determined with a UV 3000 ultraviolet spectrophotometer (Nano Drop, Wilmington, DE). The samples (100 ug protein per lane) were separated with 15% gradient SDS-PAGE gel and then transferred onto a PVDF membrane. Nonspecific binding to the membrane was blocked with 5% nonfat milk in Tris buffered saline-Tween 20 (TBST, pH 7.4) for 2 hours at room temperature. After that, the membranes were incubated overnight with rabbit polyclonal antibody against LC3 (1:1000 dilution, Cell signaling technology) and β-actin (1:1000 dilution, Sigma) at 4◦ C. After washed with TBST for three times, the membrane were incubated in goat anti-rabbit IgG (1:5000 dilution, Beijing ZhongShan Biotechnology CO., Ltd., Beijing, China) for 2 hours at room temperature, and bands visualized by using the ECL kit (Proteintech, USA). The ratio of LC3 level II/LC3 I level was used as an indicator of autophagic level.
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before seawater aspiration. The dose of 3-MA was according to the Zhang et al.’s study in rats [11]. The control rats received vehicle alone in the same time. Lung injury was evaluated by detecting arterial blood gas, lung weight coefficient, TNF-α, IL-8 in BALF in both 3-MA and vehicle-treated rats 1 hour after seawater aspiration.
Statistical Analysis All statistical analyses were carried out using SPSS statistical software version 13.0 (SPSS, Chicago, USA). Data were summarized as mean + SD. Oneway ANOVA and post-hoc comparisons were used to determine the differences among multiple groups. P < .05 was regarded as statistically significant.
RESULTS Effects of Seawater Aspiration on Arterial Blood Gas Compared with control group, seawater aspiration caused significant changes in the arterial blood gas parameters, which was manifested by an obvious decrease in PaO2 and pH value, as well as an significant increase in PaCO2 (Figure 1). As shown in Figure 1, the most serious respiratory failure occurred at the time point of 30 minutes to 1 hour after seawater aspiration, and then slowly reversed with time.
Effects of Seawater Aspiration on Lung Weight Coefficient Lung weight coefficient significantly increased in the seawater group when compared with that in the control group. The obvious change occurred at 30 minutes and 1 hour after seawater aspiration, and then gradually decreased (Figure 2).
Effects of Seawater Aspiration on TNF-α, IL-8 in BALF TNF-α and IL-8 in BALF significantly increased in the seawater group when compared with that in the control group. They were elevated from the beginning of the experiment, and then gradually increased with time (Table 1).
Suppression of Autophagy in vivo To further explore the role of autophagy, the rats were injected intraperitoneally with a pharmacological inhibitor of autophagy, 3-Methyladenine (3-MA, Sigma, 30 mg/kg dissolved in 1 mL saline) 20 minutes C
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Effect of Seawater Aspiration on Lung Histopathological Changes Normal lung tissue structure and clear alveoli were observed in rats of control group (Figure 3A).
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FIGURE 1. Effects of seawater aspiration on arterial blood gas parameters of rats. (A) PaO2 ; (B)
PaCO2 ; (C) pH. Data are expressed as Mean ± S.D. ∗ P < .05 versus control group.
Compared with the control, diffuse alveolar damage was observed in seawater groups (Figure 3B–D). Some alveoli were collapsed, others were distended. In addition, the lung tissue demonstrated capillary congestion, interstitial and intraalveolar edema and hemorrhage, thickened alveolar wall and increasing netrophils within the vascular space, the interstitium, and the alveoli.
Effects of Seawater Aspiration on Electron Microscopic Changes of Lung Tissues As shown in Figure 4, the evacuation of substance from lamellar bodies in the type II alveolar epithelial cell in the seawater groups was observed. In addition, autophagosome was more frequently observed in the alveolar epithelial cells of rats in seawater aspiration groups compared with the control group. Experimental Lung Research
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FIGURE 2. Effects of seawater aspiration on lung weight coefficient of rats. Data are expressed as Mean ± SD ∗ P < .05, compared with control group.
Effect of Seawater Aspiration on LC3 mRNA level by RT-PCR
Effect of Seawater Aspiration on LC3 Conversion by Western Blotting
Compared with the control group, a notable upregulation of LC3 mRNA level was observed in a rat lung tissue of the seawater groups (Figure 5).
The conversion of LC3-I to LC3-II is a necessary step during the process of autophagy. The level of LC3-II is known to correlate well with the number of
FIGURE 3. Light micrographs of lung tissues stained with hematoxylin-eosin (HE (×400). (A) Lung
tissue in a rat of the control group. (B) Lung tissue in a rat of the seawater groups showing some collapsed alveoli, distended alveoli and thickened alveolar wall. (C) Lung tissue in a rat after seawater aspiration showing capillary congestion, interstitial and intraalveolar edema and hemorrhage. (D) Lung tissue in a rat after seawater aspiration demonstrating increasing netrophils within the vascular space, the interstitium, and the alveoli. C
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FIGURE 4. High resolution electron micrographs of lung tissues (×50,000). (A) Control group. (B) Seawater group. The evacuation of
substance from lamellar bodies in the type II alveolar epithelial cell. The arrows indicated the double membrane-surrounded autophagosomes in the type II alveolar epithelial cell. (C) Seawater group. The arrows indicated the double membrane-surrounded autophagosomes in the type I alveolar epithelial cells.
autophagosomes. Our studies showed that the LC3II protein were hardly detectable in normal lung tissue by using western blotting analysis. However, a marked upregulation of the ratios of LC3-II/LC3-I was observed, in rats of seawater groups (Figure 6).
Inhibition of Autophagy by 3-MA Attenuates Acute Lung Injury Further experiments were performed to explore the role of autophagy in seawater-induced ALI by using an inhibitor of autophagy, 3-MA. At first, In order to confirm the inhibitory effect of 3-MA on autophagy, we detected LC3-II conversion in rats by western blotting. As shown in Figure 7, 30 mg/kg 3-MA could significantly reduce LC3-II conversion in lung tissues compared with vehicle-treated rats. In addition, As shown in Figure 8, lung injury indicators including PaO2 , lung weight coefficient, and TNF-α in BALF were obviously ameliorated by 3MA treatment when compared with vehicle-treated rats (Figure 8).
TABLE 1. Effects of Seawater Aspiration on TNF-α and IL-8 in Bronchoalveolar Fluid of Rats Group
TNF-α (pg/mL)
IL-8 (pg/mL)
Control 0.5 hours 1 hour 2 hours 4 hours
21.67 ± 6.86 40.67 ± 11.78∗ 51.66 ± 14.05∗ 52.83 ± 16.74∗ 54.50 ± 15.61∗
30.61 ± 5.28 43.17 ± 7.19∗ 50.01 ± 11.03∗ 55.50 ± 8.96∗ 52.18 ± 11.36∗
∗
P < .05, compared with control group.
DISCUSSION Although seawater drowning is a common cause of acute lung injury (ALI), the molecular and cellular mechanisms underlying this phenomenon remain elusive. The pathophysiology of ALI and its most severe form, ARDS, is characterized by increased vascular and epithelial permeability, hypercoagulation, hypofibrinolysis, and diffuse inflammation reactions [16]. The damage of alveolar epithelial cells and the activation of inflammatory responses are hallmark of ALI/ARDS [4, 5]. Previous studies on mechanisms related to alveolar epithelial damage mostly focused on apoptosis, which has been considered as an underlying mechanism in ALI/ARDS and multiorgan dysfunction syndrome [6–8]. However, Apoptosis is not the sole means by which the cell can undergo a genetically programmed death. Autophagy has also been linked to the actual death process [17]. Thus, this study was designed to explore possible role of autophagy in ALI induced by seawater. Our research, for the first time, found that seawater aspiration triggered autophagy in alveolar epithelial cells with increased pulmonary epithelial permeability in an in vivo model. The alterations of autophage were basically consistent with the changes in arterial blood gas, lung weight coefficient, TNF-α, IL-8 in BALF, histopathologic and transmission electron microscopic findings. In addition, inhibition of autophagy by 3-MA partly ameliorated seawaterinduced ALI, as indicated by reduced lung weight coefficient and TNF-αin BALF, as well as increased PaO2 . The results demonstrated that autophagy was involved in the ALI pathophysiological process, and Experimental Lung Research
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FIGURE 5. Effects of seawater aspiration on LC3 mRNA level in rat lung tissue. (A)
Electrophorogram picture for LC3 mRNA in lung tissues after seawater aspiration. (B) The ratios of LC3 mRNA/GADPH mRNA were presented in bar chart. ∗ P < .05, compared with the control group. M: marker; lanes 1, 3, 5, 7, and 9 represented the LC3 mRNA (452 bp) in control, 0.5, 1, 2, and 4 hours seawater aspiration groups respectively; lanes 2, 4, 6, 8, and 10 represented the GADPH mRNA (140 bp) in control, 0.5, 1, 2, and 4 hours seawater aspiration groups respectively.
it may be a scathing factor in ALI after seawater aspiration. Autophagy is an evolutionarily conserved physiological process that provides a membrane-dependent mechanism for the sequestration, transport, and lysosomal turnover of subcellular components, including proteins and organelles [12]. Autophagic pathway consists of several distinct steps: (1) the formation of an isolation membrane; (2) the formation of an autophagosome with encapsulated cargo; (3) the fusion of the autophagosome to the lysosome; and (4) the degradative phase with the digestion of lysosomal contents [10]. It is an endogenous tightly regulated process responsible for the degradation of damaged and dysfunctional cellular organelles and protein aggregates [18]. Autophagy principally serves an adaptive role that promotes cell survival C
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[19]. Recently, autophagy is also considered a form of nonapoptotic programmed cell death called type II programmed cell death or autophagic cell death [20]. It is not yet understood what factors determine whether autophagy is cytoprotective or cytotoxic. Possible factors that might control the cellular “decision” between the two responses include potentially variable thresholds for each process, molecular links that coordinately regulate apoptosis and autophagy, and mutual inhibition or activation of each pathway by the other [21]. Recently, evidence of autophagy in the pathogenesis of ALI has gradually come to light [22]. For example, Li et al. reported that nanoparticles triggers autophagic cell death and the autophagy inhibitor 3-methyladenine might rescue cell death and ameliorate ALI caused by nanoparticles in mice
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FIGURE 6. Effects of seawater aspiration on LC3 conversion in rat lung tissue. (A) LC3 expression in lung tissues after seawater aspiration. (B) The ratios of LC3-II/LC3-I were presented in bar chart. ∗ P < .05, compared with the control group.
[23]. Sun et al. concluded that the autophagic cell death of alveolar epithelial cells likely plays a crucial role in the high mortality rate of H5 N1 infection and autophagy-blocking agents might be useful as prophylactics and therapeutics against infection of humans by the H5 N1 virus [24]. Our present study further verified that autophagy plays an important role in ALI induced by seawater. Our data showed that the autophagy was elevated after seawater aspiration, inhibition of autophagy by 3-MA partly ameliorated seawater-induced ALI. All above results indicated that autophagy might be a damaging factor in lung injury. However, some studies concluded an opposite conclusion in different organs or diseases. For instance, Jiang et al. reported that autophagy was not obvious during the ischemia period, but was significantly enhanced during reperfusion. Inhibition of autophagy by 3-methyladenine and chloroquine worsened renal ischemia/reperfusion injury. They concluded that autophagy was a protective mechanism during in vitro hypoxia and in vivo renal I/R
injury [25]. Takahashi et al. found that autophagyrelated processes are properly activated in the liver in a mouse model of sepsis; Inhibition of autophagy process by chloroquine administration resulted in elevated serum transaminase levels and a significant increase in mortality. They argued that autophagy appears to play a protective role in liver of septic animals [26]. These results have been controversial as to whether high levels autophagy lessen or aggravate cell injury. We speculated that these inconsistent results indicated that the role of autophagy might be model-dependent and/or tissue-dependent. In our study, various methods including arterial blood gas analysis, lung weight coefficient measuring, TNF-α, IL-8 measuring in BALF, histopathology examination were used to detect the lung injury of seawater exposure. We found that seawater aspiration could cause lung injury, which was characterized by decreased PaO2 and increased PaCO2, lung weight coefficient, TNF-α, IL-8 in BALF. The results were consistent with histopathologic findings including Experimental Lung Research
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FIGURE 7. Effects of 3-MA treatment on LC3 conversion in lung tissue of seawater aspiration rats. (A) LC3 expression in lung tissues. (B) The ratios of LC3-II/LC3-I were presented in bar chart. ∗ P < .05, compared with the vehicle group.
pulmonary edema, inflammation reaction in lung tissue. All these data suggested that the intratracheal aspiration of seawater in the rat model induced topical ALI, which was also in accordance with the findings of other authors [2, 6, 27]. Static measures of autophagy included quantifying the number of autophagosomes within cells by electron microscopy and measuring steady-state levels of pathway constituents such as LC3II by western blot analysis. The measurements were considered as sufficient indications of autophagic activity [12]. LC3 is a mammalian homologus protein of yeast Atg8 protein. In our study, ultrastructural electron microscopy analysis of lung tissue from rats in seawater groups revealed increasing autophagosomes relative to the control group. In addition, the transcription and expression levels (mRNA and protein levels) of the LC3 II, an indicator of autophagosome formation, increased in lung tissues of seawater groups compared with those in the control group. Moreover, the alterations of autophage were basically consistent with the changes in arterial blood gas, lung weight coefficient, TNF-α, IL-8 in BALF and morphologic findings. In addition, we used a widely used pharmacological inhibitor of autophagy, 3-MA to further explore the role of autophagy in ALI. 3-MA potently blocks the initial autophagic sequestration and autopagosome forma C
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tion at the early stage and often used to understand the role of autophagy [11, 23, 25]. We found that the autophagy inhibitor 3-MA could partly ameliorated seawater-induced ALI, as indicated by reduced lung weight coefficient and TNF-α in BALF, as well as increased PaO2 . arterial blood gas, lung weight coefficient, TNF-α and IL-8 in BALF are widely accepted indicators for quantitating lung injury. Therefore, all these results further confirmed that autophagy exists in the lung, and may be a damaging factor responsible for seawater-induced ALI in rats. It was reported that autophagy could be activated under various stress conditions such as hypoxia, ischemia, oxidative stress and endoplasmic reticulum stress, etc. [17, 18, 28]. Autophagy is a process that can be regulated by hypoxia in cells and tissues and may be important for hypoxia-induced cell injury [12]. In this study, seawater aspiration caused a significant decrease in PaO2 and an increase PaCO2 . These results clearly demonstrated that hypoxia occurs in rat model. We speculated that the airway is blocked and respiration is simultaneously inhibited, which is the main cause of hypoxia after drowning. Studies showed that hypoxia can trigger a complex cascade of events, such as burst of reactive oxygen species (ROS) and Ca2+ overload in mitochondria [11]. These events are effective triggers for autophagy. The
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FIGURE 8. Effects of 3-MA treatment on PaO2 , PaCO2 , lung weight coefficient, TNF-α,
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cellular responses triggered by oxidative stress include the altered regulation of signaling pathways that culminate in the regulation of cell survival or cell death pathways. Recent studies suggest that autophagy may represent a general cellular and tissue response to oxidative stress [29]. In addition, increased Ca2+ overload in mitochondria can upregulate autophagy by activating calmondulin-dependent kinase or stim-
ulating calpains, which also contribute to autophagy [30]. The damage of alveolar epithelial cells and the activation of inflammatory responses are hallmark of ALI/ARDS [4]. In this study, we have also observed increased morphological and biochemical markers of autophagy in lung tissues from the ALI-rats induced by seawater aspiration. We hypothesize that increased Experimental Lung Research
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Seawater Induces Autophagy in Lung
autophagy contributes to ALI pathogenesis by promoting epithelial cell death. Alveolar epithelial cells death not only increases pulmonary permeability, but also destroys the alveolar epithelial barrier function. Lung weight coefficient significantly increased in rats after seawater aspiration, further suggesting the hypothesis. In addition, studies showed that autophagy exerts a critical influence on inflammatory responses. Autophagy plays a potentially pivotal role in the regulation of inflammatory responses. In particular, autophagy regulates endogenous inflammasome activators, as well as inflammasome components and pro-IL-1β. As a result, autophagy acts a key modulator of IL-1β and IL-18, as well as IL-1α, release etc. [31]. The alterations of autophage and changes of TNF-α, IL-8 levels in BALF further supported previous findings. In conclusion, this study discloses that autophagy might play important roles in the disruption of epithelial cells in ALI induced by seawater aspiration. Our data demonstrate autophage increases after seawater aspiration and the alterations are consistent with the severity of lung injury. Inhibition of autophagy by 3MA partly ameliorated ALI induced by seawater. On the basis of these findings, it is tempting to speculate that the selective targeting of autophagic proteins using pharmacological or genetic strategies may serve as the basis for developing novel therapies for ALI. However, the signaling mechanisms involved in the regulation of alveolar epithelial cells autophagy, as well as the relationship between autophagy and apoptosis are still unclear. Therefore, further investigations concerning the discrete molecular signaling mechanisms and correlation between autophagy and apoptosis are necessary.
[3]
[4]
[5]
[6]
[7]
[8] [9]
[10] [11]
[12] [13]
[14]
[15]
[16]
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This research was financially supported by National Natural Science Fundings of China (No. 81273018), Science Technical Development Project Funding of Lanzhou Military District (NO, CLZ11JA17; CLZ12JB26), and Science Funding of Health Department, Shaanxi Province (2012D58).
[17]
[18]
[19] [20] [21]
REFERENCES [22] [1]
[2]
C
Soar J, Deakin CD, Nolan JP, Abbas G, Alfonzo A, Handley AJ, Lockey D, Perkins GD, Thies K: European Resuscitation Council. European Resuscitation Council guidelines for resuscitation 2005. Section 7. Cardiac arrest in special circumstances. Resuscitation. 2005;67(Suppl 1):S135–S170. Li J, Xu M, Fan Q, Xie X, Zhang Y, Mu D, Zhao P, Zhang B, Cao F, Wang Y, Jin F, Li Z: Tanshinone IIA ameliorates
2013 Informa Healthcare USA, Inc.
[23]
451
seawater exposure-induced lung injury by inhibiting aquaporins (AQP) 1 and AQP5 expression in lung. Respir Physiol Neurobiol. 2011;176(1–2):39–49. Salomez F, Vincent JL: Drowning: a review of epidemiology, pathophysiology, treatment and prevention. Resuscitation. 2004;63(3):261–268. Xu YL, Liu YL, Wang Q, Li G, Lu¨ XD, Kong B: Intravenous transplantation of mesenchymal stem cells attenuates oleic acid induced acute lung injury in rats. Chin Med J (Engl). 2012;125(11):2012–2018. You K, Xu X, Fu J, Xu S, Yue X, Yu Z, Xue X: Hyperoxia disrupts pulmonary epithelial barrier in newborn rats via the deterioration of occludin and ZO-1. Respir Res. 2012;13:36. doi: 10.1186/1465-9921-13-36. Han F, Luo Y, Li Y, Liu Z, Xu D, Jin F, Li Z: Seawater induces apoptosis in alveolar epithelial cells via the Fas/FasL-mediated pathway. Respir Physiol Neurobiol. 2012;182(2–3):71–80. Galani V, Tatsaki E, Bai M, Kitsoulis P, Lekka M, Nakos G, Kanavaros P: The role of apoptosis in the pathophysiology of Acute Respiratory Distress Syndrome (ARDS): an up-to-date cell-specific review. Pathol Res Pract. 2010;206(3):145–150. Schmidt EP, Tuder RM: Role of apoptosis in amplifying inflammatory responses in lung diseases. J Cell Death. 2010; 3:41–53. Eisenberg-Lerner A, Bialik S, Simon HU, Kimchi A: Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ. 2009;16(7):966–975. Ryter SW, Nakahira K, Haspel JA, Choi AM: Autophagy in pulmonary diseases. Annu Rev Physiol. 2012;74:377–401. Zhang J, Wang JS, Zheng ZK, Tang J, Fan K, Guo H, Wang JJ: Participation of autophagy in lung ischemia-reperfusion injury in vivo. J Surg Res. 2012a; pii: S0022–S4804(12)01860–01864. Ryter SW, Choi AM: Autophagy in the lung. Proc Am Thorac Soc. 2010;7(1):13–21. Patel AS, Morse D, Choi AM: Regulation and functional significance of autophagy in respiratory cell biology and disease. Am J Respir Cell Mol Biol. 2013;48(1):1–9. Lahm T, Petrache I: LC3 as a potential therapeutic target in hypoxia-induced pulmonary hypertension. Autophagy. 2012;8(7):1146–1147. Mizumura K, Cloonan SM, Haspel JA, Choi AM: The emerging importance of autophagy in pulmonary diseases. Chest. 2012;142(5):1289–1299. McVey M, Tabuchi A, Kuebler WM: Microparticles and acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2012;303(5):L 364–L381. Zhang M, Jiang M, Bi Y, Zhu H, Zhou Z, Sha J: Autophagy and apoptosis act as parterns to induce germ cell death after heat stress in mice. Plos One. 2012b;7(7):e41412. Balduini W, Carloni S, Buonocore G: Autophagy in hypoxiaischemia induced brain injury. J Matern Fetal Neonatal Med. 2012; 25(Suppl 1):30–34. Levine B, Kroemer G: Autophagy in the pathogenesis of disease. Cell. 2008;132(1):27–42. Choi AM, Ryter SW, Levine B: Autophagy in human health and disease. J Engl J Med. 2013;368(7):651–662. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G: Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol. 2007;8:741–752. Tang PS, Mura M, Seth R, Liu M: Acute lung injury and cell death: how many ways can cells die? Am J Physiol Lung Cell Mol Physiol. 2008;294(4):L632–L641. Li C, Liu H, Sun Y, Wang H, Guo F, Rao S, Deng J, Zhang Y, Miao Y, Guo C, Meng J, Chen X, Li L, Li D, Xu H, Wang H, Li B, Jiang C: PAMAM nanoparticles promote acute lung injury by inducing autophagic cell death through the Akt-TSC2-mTOR signaling pathway. J Mol Cell Biol. 2009;1(1):37–45.
Exp Lung Res Downloaded from informahealthcare.com by Biblioteka Uniwersytetu Warszawskiego on 01/09/15 For personal use only.
452
Q.-p. Liu et al.
[24] Sun Y, Li C, Shu Y, Ju X, Zou Z, Wang H, Rao S, Guo F, Liu H, Nan W, Zhao Y, Yan Y, Tang J, Zhao C, Yang P, Liu K, Wang S, Lu H, Li X, Tan L, Gao R, Song J, Gao X, Tian X, Qin Y, Xu KF, Li D, Jin N, Jiang C: Inhibition of autophagy ameliorates acute lung injury caused by avian influenza A H5N1 infection. Sci Signal. 2012;5(212):ra16. [25] Jiang M, Liu K, Luo J, Dong Z: Autophagy is a renoprotective mechanism during in virtro hypoxia and in vivo ischemiareperfusion injury. Am J Pathol. 2010;176(3):1181–1192. [26] Takahashi W, Watanabe E, Fujimura L, Watanabe-Takano H, Yoshidome H, Swanson PE, Tokuhisa T, Oda S, Hatano M: Kinetics and protective role of autophagy in a mouse cecal ligation and puncture-induced sepsis. Crit Care. 2013;17(4):R160. [27] Fan Q, Zhao P, Li J, Xie X, Xu M, Zhang Y, Mu D, Li W, Sun R, Liu W, Nan Y, Zhang B, Jin F, Li Z: 17β-Estradiol
[28] [29]
[30]
[31]
administration attenuates seawater aspiration-induced acute lung injury in rats. Pulm Pharmacol Ther. 2011;24(6):673– 681. Blagosklonny MV: Hypoxia, MTOR and autophagy: converging on senescence or quiescence. Autophagy. 2013;9(2): 260–262. Ryter SW, Choi AM: Regulation of autophagy in oxygendependent cellular stress. Curr Pharm Des. 2013;19(15): 2747–2756. Høyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T, Bianchi K, Fehrenbacher N, Elling F, Rizzuto R, Mathiasen IS, J¨aa¨ ttel¨a M: Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell. 2007;25(2):193–205. Harris J: Autophagy and IL-1 Family cytokines. Front Immunol. 2013;4:83. doi: 10.3389/fimmu.2013.00083.
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