Free Radical Biology and Medicine 71 (2014) 321–331

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Original Contribution

Stanniocalcin-1 ameliorates lipopolysaccharide-induced pulmonary oxidative stress, inflammation, and apoptosis in mice Shih-En Tang a,b, Chin-Pyng Wu c, Shu-Yu Wu d, Chung-Kan Peng b, Wann-Cherng Perng b, Bor-Hwang Kang d, Shi-Jye Chu e,n, Kun-Lun Huang b,d,n,n a

Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei 114, Taiwan Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan c Department of Critical Care Medicine, Landseed Hospital, Taoyuan, Taiwan d Institute of Aerospace and Undersea Medicine, National Defense Medical Center, Taipei 114, Taiwan e Division of Rheumatology, Immunology, and Allergy, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan b

art ic l e i nf o

a b s t r a c t

Article history: Received 6 December 2013 Received in revised form 20 March 2014 Accepted 22 March 2014 Available online 28 March 2014

Stanniocalcin-1 (STC1) is an endogenous glycoprotein whose anti-inflammatory effects occur through induction of uncoupling proteins to reduce oxidative stress. In this study, we tested the hypothesis that exogenous recombinant human STC1 (rhSTC1) protects against lipopolysaccharide (LPS)-induced acute lung injury in mice. Anesthetized C57BL/6 mice underwent intratracheal spraying of LPS (20 mg/10 g body wt), and lung injury was assessed 24 h later by analyzing pulmonary edema, bronchoalveolar lavage fluid, and lung histopathology. Lung inflammation, oxidative stress, and expression of STC1 and its downstream uncoupling protein 2 (UCP2) were analyzed at specific time points. Expression of UCP2 was suppressed initially but was subsequently upregulated after STC1 elevation in response to intratracheal administration of LPS. Intratracheal rhSTC1 treatment 1 h before or after LPS spraying significantly attenuated pulmonary inflammation, oxidative stress, cell apoptosis, and acute lung injury. Pretreatment with STC1 short interfering RNA 48 h before LPS spraying inhibited the expression of STC1 and UCP2 and significantly increased the extent of lung injury. These findings suggest that STC1 is an endogenous stress protein that may counteract LPS-induced lung injury by inhibiting the inflammatory cascade and inducing antioxidant and antiapoptotic mechanisms. However, the potential clinical application of STC1 and the direct linkage between UCP2 and LPS-induced lung injury remain to be further investigated. & 2014 Elsevier Inc. All rights reserved.

Keywords: Acute lung injury Free radicals Lipopolysaccharide Oxidative stress Stanniocalcin-1 Stress proteins Uncoupling protein 2

Pneumonia and sepsis are the most common causes of acute lung injury/acute respiratory distress syndrome (ALI/ARDS), which is a consequence of fulminant inflammation of the lungs. The pathogenesis involves apoptosis of the pulmonary endothelium and epithelium,

Abbreviations: ALI, acute lung injury; ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; Bcl-2, B-cell CLL/lymphoma 2; CXCL2, chemokine (C-X-C motif) ligand 2; DNPH, 2,4-dinitrophenylhydrazine; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; IκB, inhibitor of NF-κB; PARP, poly(ADP-ribose) polymerase; ROS, reactive oxygen species; STC1, stanniocalcin-1; siRNA, short interfering RNA; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; UCP2, uncoupling protein 2; VDAC1, voltage-dependent anion channel 1 n Corresponding author. nn Corresponding author at: Institute of Aerospace and Undersea Medicine, National Defense Medical Center, 161 Ming-Chuan E. Road, Sec. 6, Room 8117, Taipei 114, Taiwan E-mail addresses: [email protected] (S.-J. Chu), [email protected] (K.-L. Huang). http://dx.doi.org/10.1016/j.freeradbiomed.2014.03.034 0891-5849/& 2014 Elsevier Inc. All rights reserved.

increase in permeability of the alveolar–capillary barrier, influx of protein-rich edema fluid into the air spaces, and oxidative damage of the lung parenchyma [1,2]. Numerous synthetic or natural chemicals have been investigated widely [2] with the aim of reducing the intensity of inflammatory [3] and proapoptotic responses [4] during the course of sepsis-induced ALI. However, the translational application of these results to clinical treatment has been very limited, and increasingly interest has been diverted to endogenous antiinflammatory or antiapoptotic regulators, including surfactants [5], keratinocyte growth factor [6], and hepatocyte growth factor [7]. Because endogenous anti-inflammatory factors are important stresscoping mechanisms, the exogenous application of these factors could be useful in treatment. Understanding the functional mechanisms of these endogenous proteins may facilitate the design of a novel therapeutic intervention for ALI. Stanniocalcin-1 (STC1) is a highly evolutionarily conserved glycoprotein that is expressed widely in a variety of tissues, including the lungs [8], and has been shown to be involved in

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various biological processes such as body fluid homeostasis [9], angiogenesis [10], and wound healing [11]. STC1 was also identified recently as an endogenous anti-inflammatory and antiapoptotic regulator. Huang et al. [12] demonstrated an anti-inflammatory effects of STC1 in a murine model of anti-glomerular basement membrane glomerulonephritis. An antiapoptotic effect of exogenous STC1 has also been demonstrated in cell death induced by hypoxia [13] and by oxidants [14] in vitro in pneumocytes. STC1 may produce its effects by inducing mitochondrial uncoupling proteins (UCPs) [15], which in turn may reduce the formation of reactive oxygen species (ROS) [16] and suppress transendothelial leukocyte migration [17,18]. Exogenous STC1 can be internalized rapidly by the mitochondria of inflammatory cells [16], suggesting a potential therapeutic strategy such as intratracheal instillation directly into the inflammatory lung tissues. The purpose of this study was to use a model of lipopolysaccharide (LPS)-induced lung injury in mice to test the hypothesis that intratracheal administration of recombinant human STC1 (rhSTC1) acts by reducing the generation of ROS, suppressing the activation of nuclear factor-κB (NF-κB), and blocking the apoptotic pathway to reduce oxidative stress, inflammation, and apoptosis.

serum or bronchoalveolar lavage fluid (BALF) were measured using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA). Pulmonary edema The total lung weight was measured as an indicator of pulmonary edema. The lungs were removed at the end of each experiment and weighed to calculate the lung weight to body weight ratio. BALF analysis BALF was collected at the end of the experiment by irrigating the right lung with two separate 0.7-ml aliquots of phosphatebuffered saline (PBS), of which 1.2 ml was recovered consistently. One BALF aliquot was used immediately to measure the total and differential cell counts, as described below. The remaining fluid was centrifuged at 200 g for 10 min, and the concentration of protein in the supernatant was determined using bicinchoninic acid (BCA) protein assay reagents (Pierce, Rockford, IL, USA). Total and differential cell counts

Materials and methods Animals Adult male C57BL/6 mice (8–10 weeks; 25 72.5 g) were obtained from Charles River Technology (Taipei, Taiwan). The animals used in this study were cared for according to the National Institutes of Health guidelines (National Academy Press, Washington, DC, 1996). Approval for the project protocol was provided by the National Science Council and the Institutional Animal Care and Use Committee at the National Defense Medical Center (Taipei, Taiwan). During the procedures, the mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). The solutions were delivered into the lungs using a MicroSprayer aerosolizer (IA-1C; Penn-Century, Philadelphia, PA, USA), which was inserted into the trachea. LPS solution (Escherichia coli serotype 0111: B4; Sigma, St. Louis, MO, USA) was prepared in saline at a concentration of 1 mg/ml. RhSTC1 (Cat. No. RD172095100) was obtained as a Flag-tagged fusion protein from BioVendor Laboratory Medicine (Modrice, Czech Republic). Distilled water was used to dilute the rhSTC1 protein to produce a final concentration of 5 mg/ml. Although the rhSTC1 used in this study is a human-derived recombinant protein, its efficacy has been demonstrated in rodents in previous studies [19,20]. Experimental protocols The mice were divided into different experimental groups as follows (nZ8 in each group). The control group received an intratracheal distilled-water spray (50 ml) followed 1 h later by a saline spray. The STC1 group received an intratracheal rhSTC1 (250 ng/mouse) spray followed 1 h later by a saline spray. The LPS group received an intratracheal distilled-water spray followed 1 h later by an LPS spray (2 mg/kg). The STC1þLPS group received an intratracheal rhSTC1 spray followed 1 h later by an LPS spray. The rhSTC1 dose used in this study was based on our preliminary data and the dose–response curves of UCP2 expression (data not shown). Measurements of STC1, cytokine, and chemokine levels STC1 levels in the serum were measured using a mouse STC1 enzyme-linked immunosorbent assay (ELISA) kit (MyBioSource, San Diego, CA, USA). Tumor necrosis factor-α and CXCL2 levels in

One BALF aliquot was analyzed immediately. The erythrocytes were lysed using erythrocyte lysis buffer (Sigma), and the BALF was centrifuged at 400 g for 5 min. The supernatant was discarded, and the pelleted cells were resuspended in 1.0 ml of PBS (pH 7.4) for further analysis. The total leukocyte number was counted using a hemocytometer. For differential cell counts, BALF cells were centrifuged at 600 g for 5 min using a Cytospin 3 (Shandon, Cheshire, UK) and stained with Liu's stain (ASK Tonyar Biotech., Taipei, Taiwan) according to the manufacturer's instructions. The differential counts were determined by counting at least 200 cells using standard hematology criteria to classify the cells. Lactate dehydrogenase (LDH) activity assay in BALF Twenty microliters of supernatant was incubated with 500 ml of 0.24 mM nicotinamide adenine dinucleotide in Tris/NaCl buffer (pH 7.2). The reaction was initiated with the addition of 100 ml of 9.8 mM pyruvate and was monitored spectrophotometrically at 340 nm at 30 1C for 2 min. Measurement of myeloperoxidase activity in the lungs To determine the extent of neutrophil infiltration, myeloperoxidase activity was assessed as described previously [21]. After bronchoalveolar lavage, the right lung was removed from the thorax, blotted with gauze to remove blood, and frozen at  80 1C until assayed. Immunoblotting Immunoblotting was performed as described previously [22]. In brief, cytoplasmic, nuclear, and mitochondrial proteins were extracted from frozen lung tissue using a Nuclear/Cytosol Extraction Kit and a Mitochondria/Cytosol Fractionation Kit (BioVision, Mountain View, CA, USA) according to the manufacturer's instructions. The protein concentrations were measured using a BCA protein assay kit (Pierce). Equal amounts of lung homogenates (30 mg/lane) were fractionated using 8–15% sodium dodecyl sulfate–polyacrylamide electrophoresis (SDS–PAGE), transferred to polyvinylidene fluoride membranes, and immunoblotted with antibodies against STC1, UCP2 (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), IκB-α, NF-κB p65, cleaved caspase-3,

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PARP, Bcl-2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), β-actin (for cytoplasmic proteins, diluted 1:10,000; Sigma), VDAC1 (for mitochondrial proteins, diluted 1:1000; Abcam), or TATA (for nuclear proteins, diluted 1:2000; Abcam). Determination of protein carbonyl levels Changes in the protein carbonylation levels in lung homogenates were detected using a commercial kit (OxyBlot Protein Oxidation Detection; Millipore, Billerica, MA, USA) according to the manufacturer's instructions. The carbonyl groups in the protein side chains were derivatized to 2,4-dinitrophenylhydrazone by reaction with 2,4-dinitrophenylhydrazine (DNPH). The DNPH-derivatized proteins were separated using 12% SDS–PAGE. The separated proteins were probed with a rabbit polyclonal antibody to the dinitrophenyl moiety (1:150 dilution; Millipore) and an anti-α-tubulin antibody (diluted 1:1000; Millipore) before measurement of protein carbonyls by immunoblotting. Real-time quantitative polymerase chain reaction (PCR) The RNA was extracted and cDNA was synthesized using the AxyPrep Multisource Total RNA Miniprep Kit (Axygen Biosciences, Union City, CA, USA) and the MMLV Reverse Transcription Kit (Protech, Taipei, Taiwan), respectively, according to the manufacturers’ instructions. The primers for mouse Stc1, Ucp2, and Actb were used to perform PCR amplification with the TaqMan Gene Expression Assay (Applied Biosystems assay ID for primers: Stc1, Mm01322191; Ucp2, Mm627599; Actb, Mm607939). Thereafter, real-time PCR was performed using the Line-Gene 9660 qPCR System (Bioer, Hangzhou, China) and 2  Smart Quant Probe Master Mix (Protech). The real-time PCR mixture contained 1 ml of 20  TaqMan Gene Expression Assay, 10 ml of 2  Smart Quant Probe Master Mix, and 1 ml of cDNA template in a total volume of 20 ml. The PCR program was an initial incubation at 95 1C for 10 min, followed by 40 cycles of denaturation at 95 1C for 15 s and annealing and extension at 60 1C for 1 min.

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Downingtown, PA, USA). For immunohistochemistry staining of lung tissue, formalin-fixed paraffin sections (4 mm) were deparaffinized before antigen retrieval and endogenous peroxidase was blocked using 3% H2O2 in methanol for 15 min. The slides were then incubated for 60 min with a polyclonal antibody against 3-nitrotyrosine (1:200 dilution; Millipore). After being washed, the slides were sequentially incubated with mouse tissue-specific horseradish peroxidase–polymer anti-rabbit antibody (Nichirei Corp., Tokyo, Japan) for 30 min. The horseradish peroxidase was then reacted with diaminobenzidine substrate for 3 min, and the sections were then counterstained with hematoxylin. DNA nick-end labeling of lung tissue sections Terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL) was performed to detect DNA strand breaks. The TUNEL assay was performed in 5-mm-thick sections of paraffin-embedded lung tissue using an ApopTag Plus Peroxidase in situ Apoptosis Detection Kit (Chemicon, Temecula, CA, USA), according to the manufacturer's instructions. Intratracheal delivery of STC1 short interfering RNA (siRNA) For the in vivo study, chemically synthesized Accell (Dharmacon/ Thermo Fisher, Lafayette, CO, USA) siRNA against STC1 (si-STC1; 50 CCAAAUUGAGUGAUAGGGA-30 ) or the nontargeting control siRNA (si-NTC) was administered through intratracheal delivery without the need for viral vectors or transfection reagents.The knockdown effect of the si-STC1 has been validated in vitro in a murine pulmonary epithelial cell line (LA-4; data not shown). The mice received STC1 siRNA (3 mg/kg) or equivalent doses of si-NTC in a volume of 50 ml through intratracheal spray 2 days before LPS exposure (2 mg/kg). For confirming the protective effect of STC1, an intratracheal rhSTC1 spray (250 ng/mouse) was given 1 h before the LPS spray in the STC1 siRNA-pretreated mice. Treatment with rhSTC1 after LPS administration (therapeutic protocols)

Lung histopathology The lungs were fixed in 10% neutral-buffered formalin for 24 h. The paraffin-embedded lungs were sectioned at a thickness of 5 mm and stained with hematoxylin and eosin. Immunofluorescence staining and immunohistochemistry staining for 3-nitrotyrosine Immunofluorescence staining of paraffin-embedded lung tissue was performed as described previously, with minor modifications [23]. The sections were deparaffinized, rehydrated, and fixed in acetone for 15 min and incubated at room temperature with 5% normal goat serum for 30 min. The sections were then incubated for 1 h at room temperature with a mouse-on-mouse Ig blocking reagent (Santa Cruz Biotechnology) to reduce nonspecific binding. Next, the sections were incubated at 4 1C overnight in a humid box with a mouse monoclonal anti-3-nitrotyrosine antibody (diluted 1:50; Abcam, Cambridge, MA, USA). The labeled antibodies were visualized after incubation for 30 min at room temperature with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (diluted 1:100; Santa Cruz Biotechnology). The nuclei were counterstained with DAPI. The slides were mounted with Prolong Gold antifade solution (Invitrogen, Eugene, OR, USA). The images were captured using a fluorescence microscope (Leica DM 2500; Leica Microsystems GmbH, Wetzlar, Germany) equipped with an EMCCD camera (LucaEM R DL-604M, Andor Technology, Belfast, UK), using MetaMorph digital analysis software (Universal Imaging,

Other groups of mice were used in the therapeutic protocols. The mice were divided into an LPS group and an LPS þ STC1 group. The mice received an intratracheal LPS spray (50 ml, 2 mg/kg). After 1 h, the mice received an intratracheal spray of rhSTC1 (50 ml, 250 ng/mouse) or sterile water (50 ml). Statistical analyses All data are expressed as the mean 7SD and were compared using Student's t test, one-way analysis of variance, or two-way analysis of variance followed by Bonferroni's post hoc test where appropriate. A p value of o0.05 was considered significant. All statistical calculations were performed using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA).

Results STC1 expression in lung tissue induced by LPS We examined whether LPS would induce STC1 expression in the lungs. STC1 messenger RNA (mRNA) expression in the lungs increased significantly 2 h after intratracheal instillation of LPS (Fig. 1A). Significant elevation of STC1 expression was observed 4 h after LPS spraying in both the serum (Fig. 1B) and the lung tissue (Fig. 1C). LPS suppressed UCP2 expression in the first 4 h after LPS spraying and then induced the expression of UCP2 at 8 to 24 h

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Fig. 1. The effects of intratracheal administration of lipopolysaccharide (LPS) on endogenous stanniocalcin-1 (STC1) expression. C57BL/6 mice were challenged with LPS (2 mg/kg intratracheally). Control animals were treated with sterile saline solution. (A) STC1 mRNA expression in lung tissues 2 h after LPS was determined by real-time PCR analysis using TaqMan probes directed at STC1 and β-actin. The data are expressed as the fold change, corrected for β-actin, mean 7 SD (n¼6 per group). nSignificantly different from control (p o0.05). (B) STC1 protein concentrations in the serum at the indicated time points after LPS were quantified by enzyme-linked immunosorbent assay. The data are expressed as the mean 7SD (n ¼6 per group). nSignificantly different from 0 h (p o 0.05). (C) STC1 and UCP2 protein levels in lung tissues at the indicated time points after LPS were determined by Western blot analysis. β-Actin was used as the loading control. The data are expressed as the mean 7 SD (n ¼3 per group). n Significantly different from 0 h (p o0.05).

with a 4-h lag after the expression of STC1 (Fig. 1C). These results indicate that the expression of UCP2 was suppressed initially and subsequently upregulated after the elevation of its upstream STC1 in response to LPS-induced lung injury. Attenuation of inflammatory lung injury by rhSTC1 pretreatment To determine whether exogenous STC1 plays an antiinflammatory role in the lungs, rhSTC1 was sprayed into the lungs as a pretreatment 1 h before LPS. Intratracheal instillation of LPS caused pulmonary edema, microvascular protein leakage, and cell damage, which were reflected, respectively, in an increased ratio of lung weight to body weight (Fig. 2A), BALF protein concentration (Fig. 2B), and BALF LDH activity (Fig. 2B). Pretreatment with rhSTC1 significantly reduced the extent of pulmonary edema, microvascular protein leakage, and cell damage in the lungs of the LPS-treated mice (Fig. 2A and B). The extent of lung inflammation was evaluated based on histological inflammatory cell infiltration, myeloperoxidase activity in lung tissues, and neutrophil counts in BALF. Pretreatment with

rhSTC1 significantly reduced LPS-induced neutrophil sequestration in the lungs (Fig. 2C and D) and migration into the alveolar spaces (Fig. 2C). The activation of the NF-κB pathway and the production of cytokines and chemokines were assessed to investigate the mechanism underlying the anti-inflammatory actions of rhSTC1. rhSTC1 pretreatment significantly inhibited IκB-α degradation (Fig. 3A) and NF-κB p65 nuclear translocation (Fig. 3B) induced by LPS. The production of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) in serum was suppressed significantly by rhSTC1 (Fig. 3C). Pretreatment with rhSTC1 also significantly inhibited the secretion of TNF-α (Fig. 3D) and of the chemokine CXCL2 (Fig. 3E). These results demonstrate that early supplement of STC1 suppressed LPS-induced pulmonary inflammation and attenuated ALI. Antioxidant and antiapoptotic effects of rhSTC1 To examine the reduction in oxidative stress after STC1 pretreatment, the production of protein carbonyl and nitrotyrosine

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Fig. 2. The effects of intratracheal administration of recombinant human stanniocalcin-1 (rhSTC1) on lipopolysaccharide (LPS)-induced pulmonary edema, inflammatory cell infiltration, and inflammatory lung injury. C57BL/6 mice were challenged with LPS (2 mg/kg intratracheally) with or without concurrent rhSTC1 (250 ng intratracheally, 1 h before LPS administration). Control animals were treated with distilled water and sterile saline solution. (A) The lung weight to body weight ratio (LW/BW) was recorded 24 h after treatments. (B) Total protein concentrations and lactate dehydrogenase (LDH) activity were determined in bronchoalveolar lavage fluid (BALF) collected 24 h after treatments. (C) Myeloperoxidase (MPO) activity in lung tissues post-bronchoalveolar lavage was measured in lung tissue homogenates, and neutrophil count was determined in BALF. The data are expressed as the mean7SD (n¼ 6 per group). nSignificantly different from the control group (po0.05); #significantly different from the LPS group (po0.05). (D) Representative images of hematoxylin and eosin-stained lung sections from one of the three mice per experimental group are presented. Original magnification 40  .

was measured by immunoblotting and immunofluorescence/ immunohistochemistry in lung tissue, respectively. LPS elicited significant increases in protein carbonyl formation (Fig. 4A and B) and nitrotyrosine production in lung tissues (Fig. 4C and D). STC1 significantly suppressed the production of both protein carbonyl and nitrotyrosine in the lungs of LPS-treated mice (Fig. 4A–D). The expression of UCP2 was suppressed in the first 4 h after LPS stimulation. Pretreatment with rhSTC1 induced UCP2 mRNA at 2 h (Fig. 5A) and protein expression at 2 and 4 h (Fig. 5B). These results suggest that exogenous STC1 may produce its antioxidative effects by increasing UCP2 expression. TUNEL was used to detect LPS-induced apoptotic DNA strand breaks in lung tissue sections. The number of TUNEL-positive cells increased significantly in LPS-treated mice. The extent of apoptosis decreased significantly in the lungs after rhSTC1 pretreatment (Fig. 6A). The expression of Bcl-2, an antiapoptotic protein that preserves mitochondrial integrity, was suppressed 24 h after LPS stimulation (Fig. 6B). By contrast, the protein levels of cleaved caspase-3 (Fig. 6C) and cleaved PARP (Fig. 6D), one of the main cleavage targets of active caspase-3, increased significantly. STC1 pretreatment restored Bcl-2 expression and reduced the magnitude of the increases in cleaved caspase-3 and cleaved PARP (Fig. 6B–D). These data suggest that exogenous STC1 protected against LPSinduced lung injury partially through its antiapoptotic effects. Increased LPS-induced lung injury by in vivo silencing of STC1 To investigate the role of endogenous STC1 protein, STC1 expression in the lungs was reduced by intratracheal instillation of STC1 siRNA. The mice received siRNA (3 mg/kg) 2 days before intratracheal instillation of LPS, and pulmonary edema and

lung injury were assessed 24 h after intratracheal instillation of LPS. Intrapulmonary spraying of LPS induced consistent UCP2 responses in both the LPS group (Fig. 5B) and the si-NTCpretreated animals (Fig. 7A). Intratracheal administration of STC1 siRNA significantly suppressed the expression of STC1 (Supplementary Fig. S1) and UCP2 in the lungs (Fig. 7A) and caused greater increases in the lung weight to body weight ratio (LW/BW) (Fig. 7B), BALF protein concentration, LDH activity (Fig. 7C), CXCL2 level, and inflammatory cell count (Fig. 7D) compared with the si-NTC þ LPS group. After LPS spraying, on a gross level, the lungs of the STC1 siRNA-treated mice appeared to be more congested and swollen than those treated with the si-NTC. The extent of lung injury (Fig. 7A–D) and the gross appearance of congestion and swelling (Supplementary Fig. S2) were ameliorated by rhSTC1 replacement in STC1 siRNA-treated mice. These data are consistent with those suggesting that STC1 is an endogenous stress protein that can counteract LPS-induced lung injury. Therapeutic effect of rhSTC1 on LPS-induced lung injury To test the therapeutic effects of rhSTC1 on LPS-induced lung injury, rhSTC1 was sprayed into the lungs 1 h after intratracheal administration of LPS. Treatment with rhSTC1 significantly attenuated the increases in LW/BW (Fig. 8A), BALF protein concentration, LDH activity (Fig. 8B), CXCL2 level, and inflammatory cell count (Fig. 8C) induced by intratracheal administration of LPS. The gross appearance of congestion and swelling was ameliorated by treatment with rhSTC1 given 1 h after LPS (Supplementary Fig. S3). These results suggest that early treatment with STC1 attenuated LPS-induced ALI.

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Fig. 3. The effects of intratracheal administration of recombinant human stanniocalcin-1 (rhSTC1) on lipopolysaccharide (LPS)-induced NF-κB activation and induction of TNF-α and CXCL2. C57BL/6 mice were challenged with LPS (2 mg/kg intratracheally) with or without concurrent rhSTC1 (250 ng intratracheally, 1 h before LPS administration). Control animals were treated with distilled water and sterile saline solution. (A) The cytosolic levels of IκB-α at the indicated time points and (B) nuclear levels of NF-κB p65 were determined in lung tissues 4 h after LPS 7 rhSTC1 administration by Western blot analysis with specific antibodies. β-Actin and TATA were used as the loading controls for cytoplasmic and nuclear proteins, respectively. The data are expressed as the mean 7 SD (n¼ 3 per group). nSignificantly different from the control group (po 0.05); #significantly different from the LPS group (p o 0.05). (C) TNF-α in the serum and (D) BALF at the indicated time points and (E) CXCL2 in BALF 24 h after LPS 7 rhSTC1 administration were determined by enzyme-linked immunosorbent assay. The data are expressed as the mean 7SD (n ¼6 per group). nSignificantly different from the control group (po 0.05); #significantly different from the LPS group (p o 0.05).

Discussion In this study, we demonstrate for the first time the protective effect of exogenous rhSTC1 in a murine model of LPS-induced ALI. Intrapulmonary administration of LPS elicited leukocyte infiltration and cytokine production, activated the NF-κB pathway,

triggered the apoptosis cascade, and elevated alveolar–capillary protein permeability, suggesting an inflammatory lung injury. Pretreatment with intratracheal administration of rhSTC1 significantly inhibited NF-κB activation, suppressed inflammatory cell infiltration, and restored the alveolar–capillary integrity. We also found that rhSTC1 reduced the extent of caspase-3 activation,

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PARP cleavage, and DNA fragmentation in the LPS-treated lungs, suggesting an antiapoptotic effect of STC1. These findings are consistent with those of other studies in the kidney that have

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demonstrated a renal protective effect of STC1 transgenic overexpression in models of anti-glomerular basement membrane glomerulonephritis [12] and renal ischemia–reperfusion injury [24]. Zhang et al. [25] also demonstrated in an STC1 cDNA transfection model that STC1 overexpression protects neurons from hypoxia- or hypercalcemia-induced cell apoptosis. Therefore, our results suggest that exogenous STC1 attenuates LPS-induced lung injury by suppressing the inflammatory response and reducing cell apoptosis in the lungs. Oxidative stress resulting from the activation of cellular ROS production is part of the innate immune response to foreign pathogens, such as LPS, on the cell wall of gram-negative bacteria. Production of ROS serves as an important mechanism to eliminate invading bacteria; however, uncontrolled oxidative stress can cause severe tissue damage [26,27], such as ALI/ARDS [28–30]. In our study, pretreatment with rhSTC1 significantly suppressed the elevation of protein carbonyl and nitrotyrosine levels in the lungs after LPS spraying (Fig. 4), suggesting an antioxidative effect of STC1. Increasing evidence suggests that STC1 exerts this effect indirectly by inducing mitochondrial UCPs [17]. Mitochondrial UCPs are members of the family of mitochondrial anion carrier proteins [31]. Many different roles of UCP2 have been suggested, including the attenuation of ROS production [32–34], regulation of glucose-stimulated insulin secretion [35], and regulation of mitochondrial Ca2 þ levels [36]. Certain species of ROS, particularly H2O2, have been appreciated as important factors in cellular signal transduction processes [37]. The mild uncoupling effect of UCP2 may provide a redox signal, as ROS have a role as a signal derived from mitochondria [38–40]. Moreover, UCP2 has been suggested to attenuate the production of ROS in mitochondria directly or indirectly [40–43]. Absence of UCP2 causes global oxidative stress in various organs, including the lungs, as shown in a previous study using different UCP2-knockout mouse models [44]. A recent study showed that UCP2, functional owing to fatty acids released by redox-activated mitochondrial calcium-independent phospholipase A2 in lung and spleen, suppresses mitochondrial superoxide production by its uncoupling action [45]. STC1 can upregulate mitochondrial UCP2 expression in macrophages [18], the kidney [24], and the retina [19]. In agreement with these previous studies, our study found that exogenous STC1 increased UCP2 mRNA transcription and protein expression in LPS-treated lungs (Fig. 5A and B). UCP2 expression was associated with a significant reduction in tissue oxidative stress, as evidenced by lower levels of protein carbonyls and nitrotyrosine in the lungs (Fig. 4). By contrast, the expression of UCP2 in the lungs was suppressed significantly by administration of STC1 siRNA before or after LPS treatment (Fig. 7A). These findings suggest that UCP2 induction may play a role in mediating the lung-protective effects of STC1.

Fig. 4. The effects of intratracheal administration of recombinant human stanniocalcin-1 (rhSTC1) on oxidative stress. C57BL/6 mice were challenged with LPS (2 mg/kg intratracheally) with or without concurrent rhSTC1 (250 ng intratracheally, 1 h before LPS administration). Control animals were treated with distilled water and sterile saline solution. (A) Protein carbonyl levels in lung tissues 4 h after LPS7rhSTC1 administration were measured. Carbonyl groups were derivatized to 2,4-dinitrophenylhydrazone (DNP) and detected by Western blot analysis with an anti-DNP antibody. Representative Western blot analysis of bands detected with the anti-DNP antibody (top) and with the anti-α-tubulin (bottom) in samples from the different animals. (B) Densitometric analysis of carbonyl and α-tubulin bands. The data are expressed as the fold change, corrected for α-tubulin, mean7SD (n¼ 3 per group). nSignificantly different from the control (po0.05); #significantly different from the LPS group (po0.05). (C) Immunofluorescence images of 3-nitrotyrosine (NT, green) in representative lung sections from mice exposed to LPS7rhSTC1. Nuclei were counterstained with DAPI (blue). Original magnification 20  . A representative image from one of the three mice per experimental group is presented. (D) Immunohistochemistry images of NT (brown) in representative lung sections from mice exposed to LPS7rhSTC1. Original magnification 40  . A representative image from one of the three mice per experimental group is presented.

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Fig. 5. The effects of intratracheal administration of recombinant human stanniocalcin-1 (rhSTC1) on UCP2 expression. (A) C57BL/6 mice were instilled intratracheally with rhSTC1 (250 ng). Control animals were treated with distilled water. UCP2 mRNA expression in lung tissues 4 h after rhSTC1 administration was determined by real-time PCR analysis using TaqMan probes directed at UCP2 and β-actin. The data are expressed as the fold change, corrected for β-actin, mean 7 SD (n¼ 3 per group). nSignificantly different from the control (p o0.05). (B) Then mice were challenged with LPS (2 mg/kg intratracheally) with or without concurrent rhSTC1 (250 ng intratracheally, 1 h before LPS administration). Control animals were treated with distilled water and sterile saline solution. UCP2 protein expression in lung tissues at the indicated time points after LPS 7 rhSTC1 administration was determined by Western blot analysis. The data are expressed as the fold change, corrected for β-actin, mean7 SD (n ¼3 per group). n Significantly different from the control (Con) (p o 0.05); #significantly different from the LPS group (p o 0.05).

Fig. 6. The effects of intratracheal administration of recombinant human stanniocalcin-1 (rhSTC1) on the expression of apoptosis-related proteins and DNA fragmentation. C57BL/6 mice were challenged with LPS (2 mg/kg intratracheally) with or without concurrent rhSTC1 (250 ng intratracheally, 1 h before LPS administration). Control animals were treated with distilled water and sterile saline solution. (A) DNA fragmentation in lung sections at 24 h was detected by using TUNEL assay and presented as the percentage of TUNEL-positive cells. Quantitative apoptosis measurements were performed by counting TUNEL-positive and -negative pneumocytes. The cells were counted in 10–15 random fields (400  ) until reaching a total number of 1000 pneumocytes and expressed as the percentage of TUNEL-positive cells among the total pneumocytes. The data are expressed as the mean 7 SD (n¼ 3 per group). nSignificantly different from the control group (p o 0.05); #significantly different from the LPS group (p o 0.05). (B) Bcl-2 mitochondrial levels in lung tissues 24 h after LPS 7rhSTC1 administration was determined by Western blot analysis. VDAC1 was used as the loading control. (C) The cytosolic level of cleaved caspase-3 and (D) the nuclear level of cleaved PARP in lung tissues 24 h after LPS 7 rhSTC1 administration were determined by Western blot analysis. β-Actin and TATA were used as the loading controls for cytosolic and nuclear proteins, respectively. The data are expressed as the fold change, corrected for VDAC1, β-actin, or TATA, mean 7 SD (n¼ 3 per group). nSignificantly different from the control group (po 0.05); #significantly different from the LPS group (p o 0.05).

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Fig. 7. The effects of intratracheal instillation of stanniocalcin-1 (STC1) siRNA on LPS-induced lung injury. C57BL/6 mice were treated with STC1 siRNA (3 mg/kg) or equivalent doses of nontarget control (NTC) siRNA in a volume of 50 μl through an intratracheal instillation 2 days before LPS exposure (si-NTC þ LPS group and si-STC1þ LPS group). Then the mice were challenged with LPS (2 mg/kg intratracheally). For confirming the protective effect of STC1, an intratracheal rhSTC1 spray (250 ng/mouse) was given 1 h before the LPS spray in the STC1 siRNA-pretreated mice (si-STC1þ STC1þLPS group). (A) UCP2 in lung tissues 4 h after LPS was determined by Western blot analysis. β-Actin was used as the loading control (n¼ 3 per group). nSignificantly different from si-NTC (p o 0.05). #Significantly different between the indicated groups (po 0.05). (B) The lung weight to body weight ratio (LW/BW) was recorded and (C) total protein concentration, lactate dehydrogenase (LDH) activity, (D) total cell count (TCC), and enzyme-linked immunosorbent assay analysis of CXCL2 were determined in the BALF 24 h after LPS. The data are expressed as the mean 7SD (n¼ 5–8 per group). n Significantly different from si-NTCþ LPS (p o0.05). #Significantly different from si-STC1þ LPS (po 0.05).

However, the direct linkage between UCP2 and LPS-induced lung injury remains to be further investigated. Interestingly, we found that intrapulmonary spraying of LPS by itself induced STC1 expression in the mouse lung. The promoter of the STC1 gene contains hypoxia-inducible factor-1 and NF-κB binding sites [46], but the detailed mechanism of the upregulation of STC1 in LPS-induced lung injury needs further investigation. This result raises the question of whether endogenous STC1 mediates the pathogenesis of LPS-induced lung injury. In our study, the extent of lung injury in LPS-treated mice was severely exacerbated by intratracheal pretreatment with STC1 siRNA to knock down endogenous STC1 expression in the lung (Fig. 7). This suggests that STC1 plays a protective role in the lung by counteracting the LPSinduced lung injury and that the increase in STC1 expression after LPS administration may act as a compensatory mechanism in response to LPS-induced oxidative and inflammatory stress. This finding is consistent with previous data showing that STC1 is a stress response protein responsible for tissue protection against several models of injuries, such as renal ischemia–reperfusion [24], myocardial infarction [47,48], and cerebral infarction [25]. The stress response is a self-protection mechanism that occurs when prior sublethal stresses increase the ability of tissues to withstand subsequent insults. This response can be induced by various types

of stresses, such as heat, ischemia, hypoxia, hypoglycemia, drugs, and inflammation, and may produce a “cross-tolerance” to other types of insult [49,50]. In rats, recovery from LPS-induced shock makes the animal more resistant to ischemia–reperfusion injury to the heart [51], suggesting that LPS is an inducer of stress proteins. Westberg et al. [48] demonstrated recently that STC1 mediates the hypoxic preconditioning-induced tolerance to the myocardial injury after ischemia and reperfusion. In our study, intrapulmonary administration of LPS induced significant STC1 expression in the lungs, which attenuated the consequent lung injury. Taken together, the above-mentioned evidence suggests that STC1 is a stress response protein with cytoprotective functions. Stress proteins produce maximally protective effects when highly expressed before the insult. Our results showed that STC1 in the lung reached a significantly high level 4 h after LPS spraying, a time point long after the initiation of inflammatory response by LPS (Fig. 1). To increase the tissue level of STC1 before the initiation of the inflammatory vicious cycle, in this study, exogenous rhSTC1 was administered simultaneous with LPS as a prevention or 1 h later as a treatment. In addition to its prevention effects, intratracheal administration of rhSTC1 1 h after LPS produced substantial anti-inflammatory and antiapoptotic effects and significantly attenuated the LPS-induced lung injury (Fig. 8). However, the treatment effects decreased when

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of ALI. However, the therapeutic time window and the best treatment protocol for rhSTC1 require further investigation.

Acknowledgments This work was supported in part by Grant NSC 100-2314-B706-001-MY3 from the National Science Council and Grants TSGHC100-041 and TSGH-C101-087 from Tri-Service General Hospital, National Defense Medical Center, Taiwan.

Appendix A.

Supplementary Information

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.freeradbiomed. 2014.03.034. References

Fig. 8. The treatment effects of recombinant human stanniocalcin-1 (rhSTC1) on LPS-induced lung injury. C57BL/6 mice were challenged with LPS (2 mg/kg intratracheally) with or without concurrent rhSTC1 (250 ng intratracheally, 1 h after LPS administration). (A) The lung weight to body weight ratio (LW/BW) was recorded and (B) total protein concentration, lactate dehydrogenase (LDH) activity, (C) total cell count (TCC), and enzyme-linked immunosorbent assay analysis of CXCL2 were measured in BALF 24 h after LPS. The data are expressed as the mean 7SD (n ¼6 per group). nSignificantly different from the LPS group (p o 0.05).

rhSTC1 was administered 4 h after LPS (data not shown). These results are not surprising because at this time point, both inflammatory cytokines and endogenous STC1 have increased to high levels, suggesting the occurrence of the inflammatory stress response. In summary, we have demonstrated that STC1 is a stress response protein with cytoprotective functions in response to LPSinduced oxidative and inflammatory stress. Intrapulmonary pretreatment with rhSTC1 significantly suppressed the LPS-induced inflammatory response and apoptotic injury in mice. This protective effect was associated with upregulation of UCP2 expression and reduction in ROS generation (Supplementary Fig. S4). Compared with rhSTC1 pretreatment, early treatment after LPS produced similar protective effects. Our results suggest that exogenous rhSTC1 has potential as a novel therapeutic agent in the treatment

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