Inhalations with Brine Solution from the 'Wieliczka' Salt Mine Diminish Airway Hyperreactivity and Inflammation in a Murine Model of Non-Atopic Asthma

Abstract

Inhalations with brine solutions are old but underestimated add-ons to pharmacological treatments of inflammatory lung diseases. Although widely used, not all features underlying their action on the respiratory system have been explored. The aim of the present study was to elucidate the mechanism of the beneficial action of inhalations of brine solution from the 'Wieliczka' Salt Mine, a Polish health resort, in a murine model of non-atopic asthma. Asthma was induced in BALB/c mice by skin sensitization with dinitrofluorobenzene followed by an intratracheal challenge of cognate hapten. All animals underwent 12 inhalation sessions with brine solution, pure water or physiological saline. Control mice were not inhaled. We found that brine inhalations reduced, as compared to non-inhaled mice, the typical asthma-related symptoms, like airway hyperreactivity (AHR), the infiltration of pro-inflammatory cells into the bronchial tree, and the inflammation of the airways at the level of pro-inflammatory cytokines IL-1α, IL-1β and IL-6. The level of the anti-inflammatory IL-10 was elevated in brine-inhaled mice. Inhalations with pure water increased AHR, whereas saline had no influence, either on AHR or cytokine concentrations. These observations indicate that inhalations with a brine solution from the 'Wieliczka' Salt Mine diminish the asthma-related symptoms, mostly by reducing the inflammatory status and by decreasing AHR.

Keywords: add-on therapy; airway hyperreactivity; airway inflammation; brine inhalations; murine model; non-atopic asthma.

Figure 1

Upper panel: Airway hyperreactivity to methacholine. # p < 0.05 vs. VEH+S (Mann–Whitney U test); * p < 0.05 vs. DNFB (Mann–Whitney U test); means ± SE, n = 5–10. Lower panel: Airway hyperreactivity for the concentration of methacholine 20 and 40 mg/mL before and after inhalations. & p < 0.05 before vs. after inhalation (Wilcoxon test); means ± SE, n = 5–10. VEH+S, sham-sensitized saline-inhaled; VEH+B, sham-sensitized brine-inhaled; DNFB+W, sensitized pure water inhaled; DNFB+S, sensitized saline inhaled; DNFB+B, sensitized brine inhaled; DNFB, sensitized non-inhaled.

Figure 2

Total number of inflammatory cells per lung. # p < 0.05 vs. VEH+S (Mann–Whitney U test); * p < 0.05 vs. DNFB (Mann–Whitney U test); means ± SE, n = 6–10; VEH+S, sham-sensitized saline-inhaled; VEH+B, sham-sensitized brine-inhaled; DNFB+W, sensitized pure water inhaled; DNFB+S, sensitized saline inhaled; DNFB+B, sensitized brine inhaled; DNFB, sensitized non-inhaled.

Figure 3

Total number of neutrophils per lung. # p < 0.05 vs. VEH+S (Mann–Whitney U test); * p < 0.05 vs. DNFB (Mann–Whitney U test); means ± SE, n = 6–10 VEH+S, sham-sensitized saline-inhaled; VEH+B, sham-sensitized brine-inhaled; DNFB+W, sensitized pure water inhaled; DNFB+S, sensitized saline inhaled; DNFB+B, sensitized brine inhaled; DNFB, sensitized non-inhaled.

Figure 4

IL-1α (A), IL-6 (B), IL-10 (C) and total glutathione (D) levels in lung tissue. # p < 0.05 vs. VEH+S (Mann–Whitney U test); * p < 0.05 vs. DNFB (Mann–Whitney U test); means ± SE, n = 5–7 VEH+S, sham-sensitized saline-inhaled; VEH+B, sham-sensitized brine-inhaled; DNFB+W, sensitized pure water inhaled; DNFB+S, sensitized saline inhaled; DNFB+B, sensitized brine inhaled; DNFB, sensitized non-inhaled.

Figure 5

IL-1α (A), IL-1β (B) and IL-6 (C) levels in BALF. # p < 0.05 vs. VEH+S (Mann–Whitney U test); * p < 0.05 vs. DNFB (Mann–Whitney U test); means ± SE, n = 5–8; VEH+S, sham-sensitized saline-inhaled; VEH+B, sham-sensitized brine-inhaled; DNFB+W, sensitized pure water inhaled; DNFB+S, sensitized saline inhaled; DNFB+B, sensitized brine inhaled; DNFB, sensitized non-inhaled.

Figure 6

The outline of the experiment. DNFB, skin sensitization with 1-fluoro-2, 4-dinitrobenzene; VEH, skin sensitization with vehicle (acetone: olive oil 4:1 v/v); DNS, intratracheal challenge with dinitrobenzene sulfonic acid; MCh, test with increasing concentrations of metacholine.