在肺部的氧化反映被认为是一个关键组成部分，英语论文，相关于慢性阻塞性肺疾病(COPD)的发病机制[1]。动物探讨也支持一个人的能力。下面的范文进行详述。

Abstract
This study aimed to investigate bronchiolar catalase expression and its relationship with smoking and/or chronic obstructive pulmonary disease (COPD) in humans and to determine the dynamic change of bronchiolar catalase expression in response to cigarette smoke in mice.

Lung tissue was obtained from 36 subjects undergoing surgery for peripheral tumours, consisting of life-long nonsmokers and smokers with or without COPD. Male C57BL/6 mice were subjected to cigarette smoke exposure for up to 3 months followed by a 28-day cessation period. We quantified bronchiolar catalase mRNA using laser capture microdissection and quantitative reverse transcription-polymerase chain reaction. C22 club cells (Clara cells) in culture were exposed to cigarette smoke extract and monitored for viability when catalase expression was decreased by siRNA.

Catalase was decreased at mRNA and protein levels in bronchiolar epithelium in smokers with COPD. In mice, bronchiolar catalase is temporarily upregulated at 1 day after cigarette smoke exposure but is downregulated by repeated cigarette smoke exposure, and is not restored long after withdrawal once emphysema is developed. Decreasing catalase expression in C22 cells resulted in greater cigarette smoke extract-induced cell death.

Bronchiolar catalase reduction is associated with COPD. Regulation of catalase depends on the duration of cigarette smoke exposure, and plays a critical role for protection against cigarette smoke-induced cell damage.

Introduction
Oxidative stress in the lungs has been believed to be a key component of the pathogenesis of chronic obstructive pulmonary disease (COPD) [1]. Animal studies also support an individual ability to defend against cigarette smoke (CS)-induced oxidative stress by upregulation of lung antioxidant defences representing it as a critical event in development of emphysema [2].

Due to their direct contact with the environment, epithelial cells located along the respiratory tract are exposed to CS; therefore, they are likely to be involved in the pathogenesis of smoking-related diseases. In particular, terminal bronchioles are known to play a critical role in a variety of smoking-related lung diseases, and they are the major sites of airflow limitation in COPD [3]. However, there is limited information about bronchiolar epithelial antioxidant defences in smokers, and even less about those in patients with COPD. We have recently investigated the cellular and molecular changes in bronchiolar epithelium in smoking mice [4, 5], as well as in human smokers, and their relationships with the development of COPD [6–8]. In this study, we used laser capture microdissection (LCM) to isolate the terminal bronchiolar epithelium and performed cDNA array analysis, focusing on stress and toxicity pathways, for screening. These data revealed that catalase was an abundantly expressed antioxidant gene in the bronchiolar epithelium of normal (nonsmoker) adult lungs, and furthermore only catalase mRNA was remarkably decreased in the bronchiolar epithelium of patients with COPD.

Catalase, a 240-kDa tetrameric heme protein, plays a central role in the antioxidant screen of the lungs by virtue of its ability to convert hydrogen peroxide to oxygen and water [9]. Catalase is expressed during the later stages of lung development, and is constitutively expressed in airway and alveolar epithelial cells and in macrophages in adults [10]. To date, several studies have focused on catalase in pathological lung status [9–11]. However, no studies have been comprehensively conducted in association of catalase and smoking or COPD. The significance of catalase in pulmonary defence, especially at the bronchiolar level, thus has possibly been underestimated. Unlike most antioxidants, catalase is not elevated in bronchiolar epithelium in healthy human smokers [12].

Accordingly, we have examined the effects of CS exposure on the catalase expression of the cells in the mouse distal airway epithelial cells. Furthermore, we have also investigated whether catalase might play a role in protection against CS-induced damage of immortalised murine club cells (Clara cells) C22.

Collection of human tissue specimens
Lung tissue specimens were obtained from 36 patients undergoing lung resection for small peripheral tumours. COPD patients were chosen based on the guidelines of the Global Initiative for Obstructive Lung Disease [13]. Informed consent was obtained from each subject and the Ethics Committee of Hokkaido University School of Medicine, Sapporo, Japan, approved the study protocols. Some patients had been subjects in our previous study [6–8, 14].

cDNA array analysis of LCM-retrieved bronchiolar epithelial cells
Human bronchiolar epithelial cells were harvested by LCM and total RNA was extracted as described previously [6]. The non-radioactive GEArray Q series cDNA expression array filter containing 96 genes whose expression levels are indicative of stress and toxicity pathways (HS-012N; SuperArray Inc., Bethesda, MD, USA) was applied.

Immunohistochemistry for catalase and semi-quantitative scoring
Immunostaining for catalase in human lungs was performed using rabbit anti-catalase polyclonal antibody (Calbiochem-Novabiochem, San Diego, CA, USA). The catalase immuno-intensity was semi-quantified as described previously by two independent observers in a blind manner, and average scores were ed.

Mouse cigarette smoking models
Male C57BL/6J mice, 9–10 weeks of age (Charles River, Atsugi, Japan), were exposed to whole body mainstream CS for 90 min per day [7] or nose-only mainstream CS for 60 min per day [15]. 3 months of repeated CS exposure using either exposure system results in significant airspace enlargement [7, 15]. Mice were sacrificed and lung samples were collected at several time-points indicated (n=4–6 in each group), and bronchiolar epithelial cells were harvested by LCM as described previously [5].

In situ hybridisation for catalase
Mouse lungs were inflation-fixed with 10% neutral-buffered formalin, paraffin-embedded, and cut into 5-μm sections. Deparaffinised sections were hybridised with a digoxigenin-labelled RNA probe corresponding to nucleotides 1215–1533 of the mouse catalase gene. After hybridisation, digoxigenin detection was performed using the alkaline phosphate-conjugated anti-digoxigenin antibody (Roche, Basel, Switzerland).

Real-time RT-PCR
RNA purification, reverse transcription and quantitative PCR were carried out as described previously [6]. A Taqman Gene Expression Assays probe Hs00156308_m1 was used for human catalase and levels were normalised against glyceraldehyde-3-phosphatase dehydrogenase mRNA, while Mm00437992_m1 was used for mouse catalase and levels were normalised against beta2-microglobulin mRNA (Applied Biosystems, Foster City, CA, USA).

Exposure of CS extract to C22 cells and inhibition of catalase by siRNA
C22 cells were transfected with 20 nM catalase siRNA duplex (Sigma-Aldrich, St. Louis, MO, USA) using INTERFERin siRNA transfection reagent (Polyplus-Transfection Inc., San Marcos, CA, USA) and exposed to diluted CS extract in serum-free media as described previously [4]. In order to assess the cell viability, the cell-free media was assayed for lactate dehydrogenase (LDH) activity as previously described [4].

Data presentation and statistical analysis
All data are expressed as the mean±SEM or the median, as appropriate. In humans, differences were analysed using single factor analysis of variance followed by Fisher's protected least significant difference test as a post hoc test or the Kruskall–Wallis test and Mann–Whitney test. In mice, statistical significance was determined by Dunnet multiple comparative analyses.

Characteristics of human subjects
We collected the subjects of three groups: 13 life-long nonsmokers, 13 smokers without COPD and 10 smokers with COPD. Clinical characteristics of the subjects are summarised in table 1. None of the subjects had a history of asthma and none had suffered from acute respiratory infections in the preceding month. Both groups of smokers were of similar pack-years of smoking with various durations of smoking and cessation (table 2). All of the COPD patients exhibited forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) lower than the lower limit of normal [16].

Discussion
This study has important findings about catalase in bronchiolar epithelium in humans and mice. In humans, catalase was an antioxidant gene most abundantly expressed in bronchiolar epithelium of adult nonsmokers, whereas bronchiolar epithelial catalase was markedly decreased in the lungs of patients with COPD. The experiments in mice demonstrated that the effects of smoking on bronchiolar catalase expression are time-dependent, increasing early after initial smoke exposure but falling with chronic exposure and remaining low even long after smoke exposure has terminated. We also found that the depletion of catalase in C22 cells increased the susceptibility to CS-induced cell death, implying a critical role of catalase in bronchiolar epithelial cells against CS-induced cell damage.

In the human study, the period of smoking cessation was variable among the smokers. On the one hand, it is possible that the changes in gene expression would have been more substantial if measurements were performed while the subjects were still smoking [18]. On the other hand, this observation particularly acknowledges the fact that not all gene expressions are restored to normal in airway epithelium as a result of giving up smoking. Some nonreversible changes in gene expression, e.g. catalase, might be linked to the progression of the disease even after smoking cessation and/or high risk of development of lung cancer in COPD patients [19]. Having COPD and smoking has a bigger effect than smoking without COPD on the suppression of bronchiolar catalase at the protein level (fig. 2), whereas the statistical difference was not significant between smokers with and without COPD at the mRNA level (fig. 1). This discrepancy suggests that COPD, for unknown reasons, is also associated with a faster turnover or impaired synthesis of catalase protein.

Our time course study in mice indicates that the acute effects of CS exposure cannot be extrapolated confidently to the chronic effects of smoking. The effects of CS exposure on bronchiolar epithelial cells over time may result from several processes having different time frames. Interestingly, the downregulation of bronchiolar catalase persists in mice after the withdrawal of CS exposure once airspace enlargement has developed. These features may mimic the status in former smokers with mild COPD in humans. At transcriptional levels, catalase is directly regulated by FoxO3a and co-activator, peroxisome proliferator-activated receptor γ co-activator 1-α [20]. HWANG et al. [21] recently ed that FoxO3 was predominantly localised in airways/alveolar epithelium in nonsmokers, which was decreased both in lungs of smokers and patients with COPD, and also was decreased in lungs of mice exposed to CS. In that study, the catalase upregulation in mice lungs, in response to CS exposure for 3 days was significantly impaired in FoxO3-deficient mice, although there is no difference in the level of catalase expression in the lungs at steady state between wild-type and Foxo3-deficient mice, suggesting the pivotal role of FoxO3 in transcriptional regulation of catalase. Catalase can also be affected by nuclear factor (NF)-E2-related factor 2 (Nrf2) in responses of the lung to CS [4]. Although the levels of Nrf2 expression were not decreased in bronchiolar epithelial cells [8], genetic or epigenetic inactivation of those transcriptional factors might be involved in the mechanism by which catalase downregulation persists in bronchiolar epithelial cells. These studies emphasise the importance of antioxidant-mediated cell response for protection against CS-induced lung epithelial cell damage. After chronic CS exposure, the oxidative stress becomes greater than the antioxidant potential, along with a downregulation of catalase, and CS-induced apoptosis occurs in bronchiolar epithelial cells.

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