Basic Nutritional Investigationα-Tocopherol and ascorbic acid supplementation reduced acute lung inflammatory response by cigarette smoke in mouse
Introduction
Chronic obstructive pulmonary disease (COPD) represents a worldwide leading cause of morbidity and mortality and it is predicted to rank as the third most common cause of death by 2020 [1], [2]. Cigarette smoke (CS) is the major etiologic factor in the pathogenesis of COPD [3], [4], and thus the majority of these cases can be prevented by quitting smoking.
Cigarette smoke contains a large number of oxidants, and it has been hypothesized that many of the adverse effects of smoking may appear as a result of oxidative damage to critical biological substances [5], [6], [7]. CS exposure increases the amount of alveolar oxidants, not only because CS itself contains an expressive number of free radicals but also because it increases the number of inflammatory cells in alveoli, which spontaneously release oxidants [8]. These oxidants inactivate α1-antitrypsin and other protease inhibitors such as secretory leukoprotease inhibitor [5], [9]. Further, the recruitment of inflammatory cells increases the protease burden, thus tipping the protease-antiprotease balance further toward the protease side [10], [11], [12]. In addition, evidence suggests that oxidative stress enhancement is associated with inflammatory cell influxes to the lung followed by lipid peroxidation and by an increase in the amount of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) [13], [14]. Matrix metalloproteinases (MMPs) consist of a number of structurally related enzymes capable of digesting extracellular matrix and basement membrane components [15]. Oxidants induced by CS can directly damage components of the lung extracellular matrix such as elastin and collagen or even modify the matrix to make it more susceptible to protease attack [16]. Specific proteases derived from alveolar macrophages (AMs) and polymorphonuclear cells (PMNs) are responsible for lung injury [10], [11], [12], [17]. Extracellular matrix degradation is a complex multistep process that involves a family of zinc-dependent endopeptidases known as matrix metalloproteinases. MMPs consist of a number of structurally related enzymes capable of digesting extracellular matrix and basement membrane components [15]. MMP activity is regulated at different levels, including transcriptional control, extracellular activation of proenzymes, and active enzyme inhibition [10]. The tissue inhibitors of metalloproteinases are a family with four members identified; they form complexes with active enzymes and inhibit MMP activity [18].
Vitamins are important for lung development, being involved in several reactions regarding alveoli growth and regeneration [19] and lung protection [20]. Antioxidant vitamins such as ascorbic acid (vitamin C) and α-tocopherol (vitamin E) have been described as potent substances due to their interaction with oxidizing radicals [21]. Vitamin C is known to scavenge aqueous reactive oxygen species by rapid electron transfer and thus inhibits lipid peroxidation and reduces the level of oxidized vitamin E [22], [23] and vitamin E terminates the chain reaction of lipid peroxidation in membranes and lipoproteins [23], [24]. Daily oral administration of 10 mg/kg of all-trans retinoic acid in feed induced an inhibition in the growth of squamous cell carcinoma [25]. Moreover, 5 consecutive days of vitamin C or E (100 mg/kg daily) pretreatment completely prevented the formation of DNA single-strand breaks induced by CS in the lung, stomach, and liver of mice [26]. These vitamins also have beneficial effects over other mechanisms such as infectious diseases [27], and doses of 20, 30, and 50 mg/kg of food of tocopherol were equally effective in preventing aspirin-induced gastric lesions in rats [28], although it is not clear if vitamin C or E supplementations decrease lung inflammation induced by CS. After setting up an acute model of CS-induced inflammation in mice [29], our aim in this study was to analyze the effects of vitamin C and/or E supplementations on AM and PMN influxes. The following procedures were performed: Western blot to determine levels of nuclear factor-KB (NFKB), immunohistochemistry to quantify MMP-12 and TNF-α and biochemical analysis for lipid peroxidation, with the purpose of understanding the mechanisms of lung inflammation induced in this model.
Section snippets
CS exposure
A three-step experiment was undertaken to analyze the effect of antioxidant vitamins E and C over the inflammatory cells influx into pulmonary parenchyma. All procedures were carried out in accordance with conventional guidelines for experimentation with animals and the local committee approved the experimental protocols.
Results
Figure 1 shows the effects of cigarette doses in BAL 24 h after the last smoke exposure (n = 5 each for group). The number of AMs increased significantly according to cigarette doses of 6cig, 9cig, and 15cig (P < 0.05) and was 3.4 times more significant with 12cig (P < 0.001) when compared with the control group. The number of PMNs increased only in mice exposed to 6cig (P < 0.01) compared with the control group.
Figure 2 shows the effects of 6cig exposure from 1 to 5 d (n = 5 each for group).
Discussion
Cigarette smoke has been implicated as the major risk factor for the development of COPD [7], [12]. The adverse action of the CS is due to the presence of a large variety of compounds such as nicotine, benzo(a)pyrene, oxidants, and free radicals that could initiate, promote, and/or amplify oxidative damage [34], [35]. CS is the major determinant of diseases related to oxidative stress and the individual variations in the incidence and extension of COPD are numerous [36]. Specific reasons such
Conclusion
Alveolar macrophages, NFκB, MMP-12, and TNF-α significantly increase in response to CS exposure. The antioxidant nutrients vitamins C and E prevent this response to some degree. The inflammatory process after cigarette exposures was reduced by ascorbic acid, α-tocopherol, or more efficiently by both vitamin supplementations.
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This study was supported with a grant from the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro. Manuella Lanzetti and Wagner Alves Pimenta received academic bursaries from the Conselho Nacional de Desenvolvimento Científico e Tecnológico and UERJ.