Effects of vitamin E on mitochondrial dysfunction and asthma features in an experimental allergic murine model

Abstract

We showed recently that IL-4 causes mitochondrial dysfunction in allergic asthma. IL-4 is also known to induce 12/15-lipoxygenase (12/15-LOX), a potent candidate molecule in asthma. Because vitamin E (Vit-E) reduces IL-4 and inhibits 12/15-LOX in vitro, here we tested the hypothesis that Vit-E may be effective in restoring key mitochondrial dysfunctions, thus alleviating asthma features in an experimental allergic murine model. Ovalbumin (OVA)-sensitized and challenged male BALB/c mice showed the characteristic features of asthma such as airway hyperresponsiveness (AHR), airway inflammation, and airway remodeling. In addition, these mice showed increase in the expression and metabolites of 12/15-LOX, reduction in the activity and expression of the third subunit of mitochondrial cytochrome-c oxidase, and increased cytochrome c in lung cytosol, which indicate that OVA sensitization and challenge causes mitochondrial dysfunction. Vit-E was administered orally to these mice, and 12/15-LOX expression, key mitochondrial functions, ultrastructural changes of mitochondria in bronchial epithelia, and asthmatic parameters were determined. Vit-E treatment reduced AHR, Th2 response including IL-4, IL-5, IL-13, and OVA-specific IgE, eotaxin, transforming growth factor-beta1, airway inflammation, expression and metabolites of 12/15-LOX in lung cytosol, lipid peroxidation, and nitric oxide metabolites in the lung, restored the activity and expression of the third subunit of cytochrome-c oxidase in lung mitochondria and bronchial epithelia, respectively, reduced the appearance of cytochrome c in lung cytosol, and also restored mitochondrial ultrastructural changes of bronchial epithelia. In summary, these findings show that Vit-E reduces key mitochondrial dysfunctions and alleviates asthmatic features.

Fig. 1.

Vitamin E (Vit-E) treatment reduced airway hyperresponsiveness in asthmatic mice. A: in dose-titration experiments, partial concentration of methacholine required to increase baseline enhanced pause (Penh) to 200% (MCh PC₂₀₀ Penh) was determined in 6 groups of mice: Sham/PBS/Veh [PBS sensitized, PBS challenged, and orally administered 50% ethanol (vehicle, Veh)], OVA/OVA/Veh [ovalbumin (OVA) sensitized, OVA challenged, and orally administered vehicle], and OVA/OVA/Vit-E 5 IU, 10 IU, 15 IU, and 20 IU (OVA sensitized, OVA challenged, and orally administered 5, 10, 15 and 20 IU/kg of Vit-E). In verification experiments, % baseline Penh (B), specific airway resistance (sRaw; C), and specific airway conductance (sGaw; D), indicators of bronchoconstriction, were determined in 3 groups of mice: Sham/PBS/Veh, OVA/OVA/Veh, and OVA/OVA/Vit-E (15 IU/kg Vit-E). Each mouse was challenged with increasing concentrations of MCh (0–16 mg/ml), and final results for single-chamber plethysmography are expressed either in MCh PC₂₀₀ Penh or in % baseline Penh; double-chamber plethysmography results are expressed in % baseline sGaw and sRaw, respectively, while the PBS aerosol values are considered as baseline. Values are means ± SE of 3 independent experiments. *P < 0.05 vs. Sham/PBS/Veh; †P < 0.05 vs. OVA/OVA/Veh.

Fig. 2.

Vit-E treatment reduced airway inflammation and expression of 12/15 lipoxygenase (12/15-LOX). A: to determine the effect of Vit-E on airway inflammation, hematoxylin and eosin staining was performed in lung tissue sections. Representative photographs are shown; all photographs are at ×10 magnification. Br, bronchus. B: Western blot for 12/15-LOX was performed in lung cytosols and compared with α-tubulin. C: signals were estimated with spot densitometry. Results shown are representative of at least 3 independent experiments. *P < 0.05 vs. Sham/PBS/Veh; †P < 0.05 vs. OVA/OVA/Veh.

Fig. 3.

Vit-E treatment reduced IL-4 and metabolites of 12/15-LOX. IL- 4 (in lung homogenates; A), 13-(S)-hydroxyoctadecaenoic acid (HODE) (B), and 12-(S)-hydroxyeicosatetraenoic acid (HETE) (C) (lung cytosols) were estimated. *P < 0.05 vs. Sham/PBS/Veh; †P < 0.05 vs. OVA/OVA/Veh.

Fig. 4.

Vit-E treatment reduced nitrooxidative stress in lung. After euthanasia of mice, 8-isoprostane (A), nitrates/nitrites (B), and nitrotyrosine (C) levels were measured in lung homogenates. Data are means ± SE of 3 independent experiments. *P < 0.05 vs. Sham/PBS/Veh; †P < 0.05 vs. OVA/OVA/Veh.

Fig. 5.

Vit-E treatment restored activity of cytochrome-c oxidase of electron transport chain (COX_ETC) and expression of subunit III of COX_ETC. A: after euthanasia, mitochondria were isolated from fresh lung tissue; COX_ETC activity was estimated and normalized by respective citrate synthase activity. Data are means ± SE of 3 independent experiments. *P < 0.05 vs. Sham/PBS/Veh; †P < 0.05 vs. OVA/OVA/Veh. B: immunohistochemistry (IHC) was performed for subunit III of COX_ETC in lung tissue sections. Brown color indicates positive expression of respective subunit. Br, bronchus. Primary antibody against subunit III of COX_ETC was omitted in IHC negative control. Representative photomicrographs from 3 independent experiments are shown at ×40 magnification.

Fig. 6.

Vit-E treatment reduced levels of cytochrome c in lung cytosol and restored mitochondrial ultrastructural changes in bronchial epithelia. A: cytochrome c levels were measured in cytosolic fractions by ELISA. Data are means ± SE of 3 independent experiments. *P < 0.05 vs. Sham/PBS/Veh; †P < 0.05 vs. OVA/OVA/Veh. B: transmission electron microscopy was performed with first-generation bronchi to determine the ultrastructural changes of mitochondria with Vit-E treatment. Representative images are shown from each group.

Fig. 7.

Vit-E treatment reduced subepithelial fibrosis and goblet cell metaplasia. To determine the effect of Vit-E on subepithelial fibrosis and goblet cell metaplasia, Masson trichrome (A) and periodic acid-Schiff (B) staining were performed in lung tissue sections. All photographs are at ×10 magnification. Br, bronchus.