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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Respiratory Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan (A.S., S.M., M.M.); Department of Respiratory Medicine, Ako City Hospital, Hyogo, Japan (A.S.); Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto, Japan (Y.H., H.N., J.Y.); and Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan (T.H., J.Y.)
Received November 6, 2007; accepted February 5, 2008.
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Abstract |
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was increased 15.3- and 2.4-fold, respectively. Compared with C57BL6/J mice, TRX-transgenic mice had significantly less inflammation, oxidative damage, and apoptosis, as well as decreased levels of matrix metalloprotease-12 mRNA and serum TNF-. When recombinant human TRX (40 µg/body/day, 3 days) was injected i.p. into CS-exposed C57BL6/J mice, a significant effect to offer protection against CS-induced lung injury was observed through suppression of neutrophil influx. In the chronic study, TRX-transgenic mice and C57BL6/J mice were exposed to CS for 6 months. This chronic exposure caused pulmonary emphysema in C57BL6/J mice accompanying prominent infiltration of macrophages and neutrophils to lung. These pathological changes were significantly suppressed in TRX-transgenic mice. In conclusion, TRX induction ameliorated CS-induced lung inflammation and emphysema in mice. TRX-1 may therefore play a preventive or therapeutic role in lung inflammatory disorders such as chronic obstructive pulmonary disease.
; Rabe et al., 2007). The pathology of COPD is characterized as inflammation of the airways and loss of alveolar structure. The development of COPD is associated with chronic smoking that cause chronic inflammation, oxidative stress, and proteolysis. CS induces the production of large numbers of reactive oxygen species (ROS), which disturb the balance between oxidants and antioxidants, promoting lung cell apoptosis and amplifying the inflammatory responses in the lungs (Rabe et al., 2007).
The activation of alveolar macrophage by oxidants results in releasing of several inflammatory cytokines and chemotactic factors that recruit circulating inflammatory cells such as neutrophils, which are also a secondary source of ROS (Hautamaki et al., 1997; Ofulue et al., 1998). TNF- is a well documented chemotactic factor that is activated by the macrophage metalloelastase MMP-12 in a CS-induced lung inflammation (Churg et al., 2003). The recruited neutrophils and activated alveolar macrophages produce elastolytic enzymes interacting with each other that enhance breakdown of the extracellular matrix, resulting in alveolar destruction (Shapiro et al., 2003). A promising strategy for COPD therapeutics would therefore be to enhance the antioxidative capacity of lung cells or to prevent inflammatory cell influx to lungs. Glucocorticoid is one of the most potent and widely used anti-inflammatory agents but has quite a limited effect in the treatment of COPD and cannot alter the natural course of the disease progression (Niewoehner et al., 1999), indicating that the current anti-inflammatory strategy is insufficient for this disease.
Investigations of alternative strategies, including antioxidative stress therapy, are now in progress. N-Acetylcysteine is used for antioxidative stress therapy, but a large randomized controlled trial could not find a definitive effect of N-acetylcysteine to decrease the frequency of exacerbations (Decramer et al., 2005). The more selective anti-inflammatory agent phosphodiesterase-4 inhibitor is also at an early stage of investigative development (Martina et al., 2006; Calverley et al., 2007). Thioredoxin-1 (TRX) plays a variety of redox-related roles that are conserved from Escherichia coli to humans. It is a small, ubiquitous, multifunctional protein containing a redox-active disulfide/dithiol within its active site sequence, -Cys-Gly-Pro-Cys-. It functions together with NADPH and TRX reductase as a protein disulfide-reducing system (Holmgren, 1985). Enhanced TRX expression has been observed in smokers and patients with interstitial lung disease, acute lung injury, and rheumatoid arthritis (Burke-Gaffney et al., 2005). The reason why TRX increases in such diseases is poorly understood. It might be a host defense mechanism rather than a disease-promoting factor because TRX overexpression is protective against oxidative stress, inflammation, and apoptosis, which are major key components of COPD pathogenesis (Ueda et al., 1998; Tanaka et al., 2000). We therefore hypothesized that TRX has good potential for use in COPD therapeutics. Indeed, it has already been reported that TRX suppressed emphysema in mice induced by porcine pancreatic elastase (Kinoshita et al., 2007). However, emphysema in this porcine pancreatic elastase model is known to be caused by elastin degradation products (Houghton et al., 2006) and is quite different from COPD in humans, which is primary caused by oxidative stress from CS. Therefore, in this study, we explored the protective effects of TRX against CS-induced emphysema.
In the present study, mice overexpressing human TRX were used to examine whether high level of inducible TRX could ameliorate CS-induced inflammation, apoptosis, and oxidative stress. In addition, recombinant human TRX was used to test its extracellular effect against CS-induced inflammation. Finally, we evaluated whether TRX ameliorates CS-induced emphysema in mice.
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Materials and Methods |
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). Mice were housed in a temperature-controlled room and supplied with laboratory chow and water ad libitum. The animal research committee of Kyoto University approved this animal experiment for this article.
Cigarette Smoke Exposure
In the acute study, 8-week-old male WT and TRX-Tg mice were used. Mice were divided into three groups, each of two sets. The first group consisted of TRX-Tg and WT mice, and the second group contained mice receiving recombinant human (rh)-TRX i.p. injections and mice receiving control saline injections.
Mice were exposed to mainstream CS (Kentucky 2R4F reference cigarette, Cigarette Laboratory at the Tobacco and Health Research Institute, University of Kentucky, Lexington, KY) in a Plexiglas box for 1 h daily for 3 days (40 cigarettes daily). CS was generated by burning filter-cut standard cigarettes using a smoke generator (SG-200; Shibata Scientific Technology Ltd., Tokyo, Japan). CS was diluted to 3% with air to reduce toxicity. In the chronic study, 12-week-old mice (n = 5 per group) were exposed to CS of 10 cigarettes daily, 5 days a week for 24 weeks. Experiments were performed safely, and no mice were killed by smoke exposure. Blood COHb levels were approximately at 30% in the acute study and approximately at 10% in the chronic study immediately after the exposure. It was reduced to 0 to 1% after 24 h of exposure, and there was no daily accumulation by repeated CS exposure both in acute and chronic exposure. Besides COHb, concentration of total particle matter in the mainstream CS was monitored. The levels of total particle matter were 395.8 mg/m3 in the acute study and 445.3 mg/m3 in the chronic study. These values were the same as the previous report (Gebel et al., 2006).
Acute Study
Bronchoalveolar Lavage and Cell Differential. After 3 h from first exposure, mice were anesthetized with 20 mg/kg pentobarbital by i.p. injection. Lungs were lavaged through an intratracheal cannula one time with 1 ml of cold saline. Twenty-four hours after the last CS exposure, lungs were lavaged five times with 1 ml of cold saline. The lavage fluid was collected and centrifuged for determination of the inflammatory cell differential (Shandon Scientific Ltd., Cheshire, UK). Supernatants were stored at –80°C until required. At least 600 cells were counted on each cytospin slide stained with Diff-Quik (Dade Behring, Inc., Deerfield, IL) under a light microscope.
Lung Pathology. The left lungs were then fixed with 10% formalin at a constant pressure of 25 cm of H2O, cut sagittally in 4-µm sections, and stained with hematoxylin and eosin for histological analysis. At least three sections were used for analysis in each mouse. Findings were quantified using a four-point scoring system (0, normal; 3, severe finding) by two analysts blinded to the groups according to the previous method (Matthew et al., 2001).
RNA Isolation and Real-Time PCR. Total RNA was extracted from right lung tissue using TRIzol (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. Single-stranded cDNA was synthesized from 1 µg of total RNA using the SuperScript III Reverse Transcription Kit (Invitrogen). cDNA was amplified and quantified using the ABI PRISM 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) with oligonucleotide PCR primer pairs and fluorogenic probes (TaqMan Gene Expression Assay; Applied Biosystems) for TNF-, MIP-2/CXCL2, and MMP-12 (catalog nos. Mm00443258_m1, Mm00436450_m1, and Mm00500554_m1, respectively). 18S rRNA (catalog no. 4310893E) was used as an endogenous control (Applied Biosystems). Primer sequences for real-time PCR were not disclosed by the company.
TNF- Enzyme-Linked Immunosorbent Assay. To evaluate early elevation of TNF- in serum and BALF, samples were collected at 3 h after first exposure on day 1. TNF- levels in serum and BALF were determined using TNF- Quantikine ELISA System (R&D Systems, Minneapolis, MN) following the manufacturer's instruction.
Total Antioxidant Capacity. Total antioxidant capacity was evaluated in lung homogenates using the Quantitative Assay for Total Antioxidant Potential kit (OXIS Research, Inc., Portland, OR) according to the manufacturer's instructions. In brief, after tissue homogenate and Cu2+-containing buffer were incubated, reduced form of copper complexed with chromogenic reagent, which has an absorption maximum at 490 nm, was measured. A known concentration of uric acid was used as reference. Data were expressed in micromolar copper-reducing equivalents per microgram.
Reduced and Oxidized Glutathione Content. The ratio of reduced to oxidized glutathione (GSH) was measured in lung tissue using GSH/GSSG-412 (OXIS Research, Inc.) according to manufacturer's instructions.
Immunohistochemistry. In the acute study, apoptosis and oxidative stress was assessed by immunohistochemistry. In brief, formalin-fixed lung sections were incubated with a rabbit polyclonal anti-single-stranded DNA (ssDNA) primary antibody (1:100 dilution; Dako North America, Inc., Carpinteria, CA) and a rabbit polyclonal anti-cleaved caspase-3 primary antibody (1:400 dilution; Cell Signaling, Danvers, MA) (Kasagi et al., 2005) or with a monoclonal anti-8-hydroxy-2'-deoxyguanosine (8-OHdG) antibody (1:100 dilution; Japan Institute for the Control of Aging, Shizuoka, Japan). Staining was performed using the Dako EnVision+ system (peroxidase/DAB; Dako, Kyoto, Japan) and counterstained with 1% methylgreen or Gill's hematoxylin. Immunoreactive cells were counted in at least eight fields and expressed as the positive cell ratio to the length of alveolar septa.
Endogenous murine-TRX expression was evaluated by immunohistochemistry with anti-mouse TRX antisera (1:500 dilution; Redox Bioscience, Kyoto, Japan) with a detection system of Vectastatin ABC-AP reagent (Vector Laboratories, Burlingame, CA).
rhTRX Challenge in the Acute Study. Because both intracellular and extracellular levels of TRX are high in TRX-Tg mouse, we studied the independent effect of extracellular TRX in vivo. Mice received i.p. injections with 20 µg of rhTRX (a gift from Ajinomoto Co., Tokyo, Japan) or saline just before and 1.5 h after CS exposure on each day of the experiment. The severity of CS-induced inflammation was evaluated by neutrophil counts in BALF as before.
Chronic Study
Lung Morphometry. After 6 months of CS exposure, mice were killed by exsanguinations. The left lung was inflated with 50% optimal cutting temperature fluid through tracheal cannulation at a constant pressure of 25 cm of H2O. Frozen lung sections were prepared and stained with hematoxylin. Airspace enlargement and alveolar destruction were evaluated by mean linear intercepts (Lm) and destructive index (DI) according to our previously published methods, respectively (Sato et al., 2007).
Immunohistochemistry. In the chronic study, neutrophil and macrophage infiltration was quantified by counting cells identified by using anti-murine neutrophil antibody and anti-Mac-3 antibody (1:50 dilution; Serotec, Oxford, UK) as primary antibodies. Staining was performed with Vectastatin ABC-AP reagent (Vector Laboratories) or DAB Reagent (Dako) according to the manufacturer's instructions. The positive-stained cell ratio was counted as same as above.
Statistics. Results are expressed as means ± S.D. Statistical analysis was performed using StatView software version 5 (SAS Institute, Cary, NC). Groups were compared by two-way analysis of variance followed by Bonferroni's post-hoc test. Nonparametric data were compared with the use of the Mann-Whitney test. p Values of less than 0.05 were considered to indicate statistical significance.
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Anti-Inflammatory Effects of Overexpressed TRX on CS-Exposed Mice. Inflammation was evaluated at two time points. First, after 24 h from last CS exposure, BALF total cell, macrophage, and neutrophil counts were significantly increased by CS exposure in WT and TRX-Tg mice (p < 0.001; Fig. 2, A and B). BALF neutrophil counts were increased to a significantly lower level in TRX-Tg mice compared with WT mice (p < 0.001; Fig. 2B).
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and MIP-2 were significantly up-regulated by CS exposure (2.4- and 15.3-fold, respectively) in WT mice (Fig. 3, A and B). There was no significant difference between expression levels in WT or TRX-Tg mice. The mRNA expression level of MMP-12 was also up-regulated by CS exposure (7.7-fold) but to a significantly lesser extent in TRX-Tg mice (3.8-fold; Fig. 3C).
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was not detectable in serum or BALF. On day 1, at 3 h after the first exposure, serum TNF- was significantly lower in TRX-Tg than in WT mice (Fig. 3D; WT, TRX-Tg = 0.0 ± 0.0: 20.0 ± 12.4, mean ± S.D.) The elevation of TNF- in BALF was observed in two of four WT mice (8.7 ± 11.1, mean ± S.D.) and none of four in TRX-Tg mice. BALF TNF- tended to be higher in WT than in TRX-Tg (p = 0.17), but no significant difference was found between them (Fig. 3D). TRX Ameliorated Lung Cell Death Induced by CS Exposure. Lung histological feature of WT mice exposed to CS revealed severe lung injury in the form of cytoplasmic vacuolization and cytoplasmic blebbing of bronchial epithelium. CS-induced lung injury was less prominent in TRX-Tg mice (Fig. 4). Cytoplasmic vacuolization and blebbing were significantly ameliorated in TRX-Tg mice (Table 1).
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ssDNA-positive or cleaved caspase-3-positive apoptotic cells increased after CS exposure and were localized to the alveolar septa (Table 2). TRX-Tg mice had significantly fewer ssDNA-positive and cleaved caspase-3-positive cells compared with WT mice (64.0 and 67.8% in the alveolar septa, respectively). Representative images of apoptotic cells are shown in an on-line supplement (Supplemental Fig. 1).
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Lung Oxidative Stress Was Suppressed in TRX-Tg Mice. CS exposure caused a marked increase in oxidative stress levels of WT mouse lungs as represented by 8-OHdG immunoreactivity in the alveolar cells (Fig. 5A). Almost no positive cell was observed in nonsmoked strains. These findings were less prominent in TRX-Tg mice than WT mice, with fewer 8-OHdG-positive cells in TRX-Tg mice (p < 0.01). Representative damaged cells by oxidative stress were shown (Fig. 5A). Total antioxidant capacity was significantly increased in TRX-Tg mice, but the reduced/oxidized GSH ratio was not different between WT and TRX-Tg mice (Fig. 5, B and C).
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Effect of rhTRX against CS-Induced Lung Injury. Administration of exogenous rhTRX significantly suppressed the increase total cell counts and neutrophil counts in BALF (Fig. 6A). The numbers of macrophages were not different between the two smoked strains. Lung injury by CS exposure was also ameliorated by injected exogenous rhTRX in histology when evaluated by lung injury score (data not shown).
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Chronic Study
Overexpression of TRX Ameliorated CS-Induced Emphysema. Figure 7A shows lung histology of mice exposed to CS for 6 months in binary images. In WT mice, the chronic CS exposure caused pulmonary emphysema as demonstrated by airspace dilatation and alveolar destruction in WT mice. In nonsmoked mice, there was no difference in Lm (Fig. 7B) and DI (Fig. 7C) between TRX-Tg and WT mice (Lm, WT, TRX-Tg = 70.9 ± 2.8: 68.4 ± 1.0; DI, WT, TRX-Tg = 3.7 ± 2.0: 3.3 ± 1.0). Both Lm and DI were significantly increased in CS-exposed WT mice than in TRX-Tg mice (Lm, WT smoke, TRX-Tg smoke = 97.3 ± 3.8: 70.1 ± 2.0, p < 0.001; DI, WT, TRX-Tg = 34.4 ± 5.5: 4.7 ± 1.17). Lm and DI in TRX-Tg mice were not different from nonsmoked mice.
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In the present study, we examined the protective effect of TRX at two different time points of CS exposure, 3 days and 6 months. As observed in the previous studies, 6 months of CS exposure was required to develop emphysema in C57BL6/J mice, and chronic impairment of alveolar repair mechanism exceeded pulmonary emphysema (Guerassimov et al., 2004; Rennard et al., 2006). It was hard to evaluate initial and dynamic inflammatory mechanism in our chronic model because the degree of inflammation was weak, and the phenotypes depend on damage summation. In the acute study, strong neutrophil inflammation was observed, but we regarded this setting as one-sidedly toxic for long-term use and as different from the chronic model revealing pulmonary emphysema. Upon discussing the protective effect of TRX, we assume that events leading to pulmonary emphysema in this mouse model are as follows; the initial step is oxidative stress to the alveolar cells, such as alveolar macrophages. This oxidative stress activates alveolar cells to produce inflammatory mediators, leading to infiltration of inflammatory cells, one of which is MMP-12/TNF- cascade. The recruited inflammatory cells generate secondary oxidative stress and promote lung cell death. The lung cell death as well as matrix degradation promoted by excessive proteases such as MMPs and norepinephrine enhance loss of alveolar structure and eventually lead to emphysema development. We then discuss the protective effects of TRX in each component leading to emphysema, oxidative stress, inflammation, and apoptosis.
Oxidative stress after lung inflammation is a major pathogenesis of emphysema that causes cell death and enhances the production of inflammatory mediators (Rabe et al., 2007). In the acute study, overexpression of TRX ameliorated oxidative stress, which was observed as a decreased number of 8-OHdG-positive cells. TRX-Tg mice also demonstrated reduced CS-induced oxidative stress in alveolar septa in lung. The oxidative stress in this model is probably to be caused directly from CS and indirectly from accumulated inflammatory cells. In transgenic mice, additional intracellular human TRX could scavenge oxidative stimuli (Nakamura et al., 1997), whereas additional extracellular human TRX could suppress neutrophil chemotaxis and reduce secondary oxidative stimuli. We confirmed basal level of total antioxidant capacity and reduced/oxidized GSH ratio in lung tissues. Greater antioxidant capacity, which is independent of GSH, was shown in TRX-Tg mice than WT mice.
In the acute study, as demonstrated by ssDNA and cleaved caspase-3 immunostaining, apoptosis of alveolar septal cells caused by CS exposure was also attenuated by TRX overexpression. In general, CS leads to apoptosis by means of oxidative stress, mitochondrial and nuclear damage, and TNF receptor signaling (Tsuda et al., 2000; Carnevali et al., 2003; Tuder et al., 2003). CS-induced single strand DNA break by acute CS exposure is caused by stimulation of free radicals (Tsuda et al., 2000). In this in vivo smoke model, TNF- and ROS from inflamed cells could activate the apoptosis signal-regulating kinase 1 (ASK1). Alternatively, TRX has been shown to induce ASK1 ubiquitination and degradation, resulting in the inhibition of ASK1-induced apoptosis (Liu and Min, 2002; Hsieh and Papaconstantinou, 2006). TRX also plays a critical role in redox regulation of intracellular caspase-3-like activity, which is associated with mitochondrial apoptosis (Ueda et al., 1998). Further investigation will be required to clarify the precise mechanism of apoptosis suppression in our murine model.
Moreover, in the present study, TRX overexpression suppressed lung MMP-12 transcripts that were enhanced by CS exposure. MMP-12 enhances elastin breakdown and promotes neutrophil chemotaxis by cleaving membrane-bound TNF-, acting as a "TNF- converting enzyme" (Black et al., 1997; Moss et al., 1997). MMP-12 is predominantly produced by alveolar macrophages in lung tissue but also by bronchial epithelial cells and dendritic cells (Bracke et al., 2005; Lavigne and Eppihimer, 2005). As shown in the result of immunohistochemistry of TRX, alveolar macrophages expressed TRX strongly. Considering most of MMP-12 is derived from alveolar macrophage in lung, TRX might affect mRNA expression of MMP-12 in alveolar macrophage, finally its activation. In this study, after 3 h from first exposure, serum TNF- was elevated in WT mice and was suppressed in TRX-Tg mice. At the same time, TNF- in 1 ml of BALF was elevated in two of four samples in WT mice but zero of four in TRX-Tg mice. In our preliminary study, TNF- levels in BALF and serum elevated simultaneously (data not shown); therefore, serum TNF- might reflect lung inflammation. Although the results were statistically negative, it remains possible that overexpressing TRX ameliorates activation of TNF- in lung. In the present study, overexpression of TRX could not regulate TNF- mRNA expression. We speculated that not all lung cells could respond to TRX. Nevertheless, membrane-bound TNF- should be activated primarily by MMP-12; thus, mRNA expression of TNF- might not reflect inflammation.
We reported that TRX overexpression ameliorated systemic inflammation in smoking model and in inflammatory bowel disease model by down-regulating TNF- and macrophage migration inhibitory factor, which counteracts to TRX (Sato et al., 2006; Tamaki et al., 2006). It is thus speculated that migration inhibitory factor inhibition might also account for an anti-inflammatory effect of TRX in our present study.
The protective effects observed in TRX-Tg mice were partially confirmed by i.p. injection of rhTRX, which attenuated BALF neutrophilia and lung cell injury. However, the mechanism of this efficacy is probably different from that of TRX-Tg. It is assumed that "extracellular TRX," especially in the blood vessels, acts directly on neutrophils or endothelial cells to suppress neutrophil adhesion or extravasation. We previously reported that the protease activity for L-selectin on the neutrophil cell surface is regulated by TRX (Nakamura et al., 2001). It has recently been shown that exogenous TRX is barely incorporated into cells and that injected TRX is quickly removed from blood vessels by renal excretion (Ueda et al., 2006). This extracellular mechanism might also account for the effects of overexpressed human TRX in TRX-Tg mice, where serum TRX levels are approximately 10 times higher than basal mouse TRX levels (Takagi et al., 1999).
The protective effect of TRX against CS-induced emphysema was confirmed pathologically in the chronic study. Less neutrophil invasion to lung parenchyma was observed in TRX-Tg mice. At the same time, less invasion of macrophage was observed. In a previous report suggesting interaction between proteases, macrophage elastase contributes to activating neutrophil elastase and is important for developing emphysema (Shapiro et al., 2003), and they also reported that macrophage accumulation and emphysema were significantly suppressed, but neutrophil counts in BALF were not decreased in CS-exposed MMP-12–/– mice. In the present study, suppressing invasion both of macrophage and neutrophil by TRX might ameliorate protease interaction. However, in the acute study, the number of macrophages in BALF was increased, and there was no difference between smoked strains. TRX suppresses macrophage activation but might not suppress accumulation. Further investigation is needed.
The beneficial aspects described suggest that TRX might be a good candidate for COPD therapeutics. However, COPD is a chronic lung disease, so it would not be practical to administer such an expensive protein compound for a prolonged period. An alternative solution would be to identify low-molecular weight TRX inducers; indeed, we previously reported that geranylgeranylacetone, temocapril, and sulforaphane have the potential to induce TRX (Hirota et al., 2000; Yuan et al., 2002; Tanito et al., 2005). Otherwise, recombinant human TRX could be applied as short-term therapy for acute COPD exacerbation, where TNF- or neutrophil-derived inflammation is dysregulated. Clinical trials of i.v. TRX administration against acute respiratory distress syndrome/acute lung injury are now underway in our institution. This method could be applied for COPD exacerbation once drug safety and kinetics have been established.
In conclusion, TRX provides a protective effect against lung injury caused by CS exposure by suppressing excessive oxidative stress, apoptosis, and neutrophil influx. The exact protective mechanism depends on whether the molecule is applied internally or externally. These results suggest a potential use of TRX in COPD therapeutics.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CS, cigarette smoke; COPD, chronic obstructive pulmonary disease; ROS, reactive oxygen species; TNF, tumor factor; MMP, matrix metalloprotease; TRX, thioredoxin-1; WT, wild-type; Tg, transgenic; rh, recombinant human; PCR, polymerase chain GSH, glutathione; ssDNA, single-stranded DNA; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; Lm, mean linear intercept(s); DI, destructive ASK1, apoptosis signal-regulating kinase 1: MIP, macrophage inflammatory protein.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Yuma Hoshino, Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 6068507 Japan. E-mail: yuma{at}kuhp.kyoto-u.ac.jp
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