Research article
Supplementation with vitamins C and E improves mouse lung repair

https://doi.org/10.1016/j.jnutbio.2007.08.004Get rights and content

Abstract

Cigarette smoke (CS) induces emphysema by tissue destruction through the production of oxidants and metalloproteinases [matrix metalloproteinases (MMPs)]. The possibility of lung repair after emphysema remains unclear. Our aim was to study the effects of vitamins C and E on mouse lung repair evaluated by catalase (CAT), superoxide dismutase (SOD) and MMP-9 activities; by the amount of tumor necrosis factor (TNF)-α in lung homogenates; by cell quantification in bronchoalveolar lavage (BAL) fluid; and by lung histology. Male C57BL/6 mice (n=25) were exposed to nine cigarettes per day, 7 days/week, for 60 days in a whole-body exposure chamber. The control group was sham smoked (n=10). After 60 days of CS exposure, a group of animals was sacrificed (n=5) and the others were divided into two groups: (a) CSv (n=10) supplemented with saline and olive oil (vehicles) for 60 days and (b) CSr (n=10) supplemented with vitamins C and E (50 mg/kg/day) for 60 days. These mice were then sacrificed; BAL was performed and the lungs were removed for biochemical and histological analysis. The results demonstrated that CAT activity was decreased in the CSv and CSr groups compared to the control group. SOD activity was higher in the CSv group than in the control and CSr groups. The CSv group showed a higher neutrophil count in BAL fluid, associated with more TNF-α in lung homogenates, than the control or CSr groups. Finally, emphysema in the CSv group was associated with fewer collagen and elastic fibers than in the control and CSr groups. These results indicate a possible role of vitamins C and E in lung repair after emphysema induced by long-term CS exposure in mice.

Introduction

Chronic obstructive pulmonary disease (COPD) is a major and increasing global health problem, which is predicted to become the third commonest cause of death and the fifth commonest cause of disability in the world by 2020 [1], [2]. Cigarette smoke (CS)-induced emphysema is one of the major causes of COPD [3], [4], but there are several other risk factors, including air pollution (particularly indoor air pollution from burning fuels) [5], poor diet [6] and occupational exposure [7]. Emphysema is characterized by enlargement of alveolar spaces together with destruction of alveolar walls in the absence of obvious fibrosis [8]. CS causes an inflammatory response in the lower respiratory tract characterized by accumulation of alveolar macrophages associated with a recruitment of fewer neutrophils [9], [10]. These activated inflammatory cells release a variety of mediators, including proteases, oxidants and toxic peptides, which can damage lung structures and are believed to be a major cause of the tissue destruction found in emphysema [11].

Matrix metalloproteinases (MMPs) are a large family of zinc-dependent proteinases that regulate the destruction of extracellular matrix components in lung [12]. There is increasing evidence for a role for MMPs in COPD [4]. The concentration, expression and activity of lung MMP-9 (gelatinase B) are increased in emphysema patients [13], [14]. The interest in MMPs has also been heightened by the demonstration that emphysema induced by chronic CS exposure is prevented in MMP-12−/− mice [15].

Oxidative stress plays a key role in the pathophysiology of COPD and amplifies the inflammatory and destructive process [16], [17]. Reactive oxygen species (ROS) from CS or from inflammatory cells (particularly macrophages and neutrophils) result in several damaging effects in COPD [18], including decreased antiprotease defenses [19] and antioxidants. Many of the adverse effects of smoking may result from oxidative damage to critical biological molecules [9]. Epidemiological evidence indicates that reduced dietary intake of antioxidants may be a determinant of COPD [20]; moreover, population surveys have linked a low dietary intake of the antioxidant vitamins C and E to declining lung function [21]. Supplementary treatment with vitamin E (100 mg/kg/day) reduced bacterial colonization in the lower respiratory tract of smoke-treated rats [22]. Moreover, pretreatment (100 mg/kg/day) of either ascorbic acid or α-tocopherol acetate before smoke inhalation completely prevented single-strand DNA breaks in the lung, stomach and liver [23]. Also, α-tocopherol (50 mg/kg/day) and ascorbic acid (100 mg/kg/day) protected sperm by inhibiting the induction of ROS generation by Aroclor 1254 [24]. Bezerra et al. [9] showed that inflammatory alveolar cells and levels of tumor necrosis factor (TNF)-α, NF-κB and MMP-12 in lungs of mice exposed to CS for a short period (5 days) were reduced to control levels by ascorbic acid, α-tocopherol or, more efficiently, supplementation with both vitamins. However, it remains unclear whether vitamins C and E may play a role in mouse lung repair after emphysema induced by long-term CS exposure.

Because of the enormous burden of COPD and escalating health care costs, there is now renewed interest in the underlying cellular and molecular mechanisms and a search for new treatment strategies. Vitamins C and E are antioxidants and can therefore be expected to change the oxidant–antioxidant imbalance induced in lungs by CS. Our aim was to study the effects of vitamins C and E on mouse lung repair as evaluated by catalase (CAT) and superoxide dismutase (SOD) activities, by quantification of TNF-α and MMP-9 zymography in lung homogenates, by cell quantification in bronchoalveolar lavage (BAL) fluid and by morphometric and stereological parameters.

Section snippets

Reagents and animals

Thiobarbituric acid, adrenaline, acrylamide, gelatin, sodium dodecyl sulfate (SDS), Triton X-100, Tris–HCl, CaCl2, ZnCl2, Coomassie Blue, hematoxylin–eosin, orcein and Sirius red were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Specific enzyme-linked immunosorbent assay (ELISA) for TNF-α was purchased from R&D Systems (Minneapolis, MN, USA). Diff-Quik was purchased from Baxter Dade AG (Dudingen, Switzerland). Bradford reagent was purchased from Bio-Rad (Hercules, CA, USA). Formalin,

Vitamins C and E improved lung repair after emphysema

Histological changes are illustrated in Fig. 1. The control group lungs were histologically normal, with parenchyma consisting of alveoli connected to alveolar ducts, separated from each other only by thin alveolar septa (Fig. 1A). Collagen and elastic fibers in the alveolar septa were characterized by delicate branching fibers (Fig. 1B and C).

The lungs of all CSv mice showed emphysematous lesions, areas of alveolar septa disruption and enlarged air spaces (Fig. 1D). Collagen and elastic fibers

Discussion

The present study reports lung repair after emphysema induced by long-term CS exposure in mice supplemented with vitamins C and E. A marked neutrophilia associated with high TNF-α content and emphysema was observed in mice previously exposed to smoke for 60 days (CSv group), but the inflammatory status was reduced by vitamins C and E with improved lung histology.

In COPD, MMPs play an important role in the destruction of lung tissue [3], [11]. MMPs attack and degrade extracellular matrix

Acknowledgments

Funding for this study was provided by FAPERJ, CNPq and UERJ.

References (42)

  • S. Hurd

    Global impact of COPD

    Exp Lung Res

    (2005)
  • J.R. Spurzem et al.

    Pathogenesis of COPD

    Semin Respir Crit Care Med

    (2005)
  • M.G. Belvisi et al.

    The role of matrix metalloproteinases (MMPs) in the pathophysiology of chronic obstructive pulmonary disease (COPD): a therapeutic role for inhibitors of MMPs?

    Inflamm Res

    (2003)
  • P. Martin et al.

    Chronic obstructive pulmonary disease (COPD): smoking remains the most important cause

    N Z Med J

    (2005)
  • P. Boschetto et al.

    Chronic obstructive pulmonary disease (COPD) and occupational exposures

    J Occup Med Toxicol

    (2006)
  • R.A. Pauwels et al.

    Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary

    Am J Respir Crit Care Med

    (2001)
  • F.S. Bezerra et al.

    Alpha-tocopherol and ascorbic acid supplementation reduced acute lung inflammatory response by cigarette smoke in mouse

    Nutrition

    (2006)
  • A.I. D'Hulst et al.

    Time course of cigarette smoke-induced pulmonary inflammation in mice

    Eur Respir J

    (2005)
  • R.E. Russell et al.

    Release and activity of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by alveolar macrophages from patients with chronic obstructive pulmonary disease

    Am J Respir Cell Mol Biol

    (2002)
  • K. Ohnishi et al.

    Matrix metalloproteinase-mediated extracellular matrix protein degradation in human pulmonary emphysema

    Lab Invest

    (1998)
  • R.D. Hautamaki et al.

    Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice

    Science

    (1997)
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