Allergic Airway-Induced Hypersensitivity Is Attenuated by Bergapten in Murine Models of Inflammation

Abstract

Bergapten (5-methoxypsoralen, 5-MOP) is a plant-derived furocoumarin with demonstrated anti-inflammatory action. The present study investigated its effects on allergic inflammation in two related pathways of mast cell degranulation. Compound 48/80 and lipopolysaccharide (LPS) were used to activate the IgE-independent pathway while bovine serum albumin (BSA) was used as allergen for the IgE-dependent pathway. The modulatory effect of bergapten on mast cell degranulation, neutrophil extravasation, protein concentration, lung histopathology, and oxidative stress was assessed. Bergapten at 10, 30, and 100 μg/ml for 15 min stabilized mast cells in rat mesenteric tissue from disruption in vitro and when administered in vivo at 3, 10, and 30 mg kg^-1 for 1 h protected mice from fatal anaphylaxis induced by compound 48/80. Similarly, treatment of LPS-challenged mice with bergapten (3, 10, and 30 mg kg^-1) for 24 h significantly decreased neutrophil infiltration into bronchoalveolar lavage fluid, mean protein concentration, and inflammatory cell infiltration of pulmonary tissues when compared to the saline-treated LPS-challenged control. In addition, lung histology of the bergapten-treated LPS-challenged mice showed significantly less oedema, congestion, and alveolar septa thickening when compared to the saline-treated LPS-challenged disease control. LPS-induced oxidative stress was significantly reduced through increased tissue activities of catalase and superoxide dismutase and reduced malondialdehyde levels on treatment with bergapten. In the triple antigen-induced active anaphylaxis, daily administration of bergapten at 3, 10, and 30 mg kg^-1 for 10 days, respectively, protected previously sensitized and challenged mice against anaphylactic shock. Overall, our study demonstrates the ability of bergapten to attenuate allergic airway-induced hypersensitivity in murine models of inflammation, suggesting its possible therapeutic benefit in this condition.

Figure 1

Effect of bergapten on C48/80-induced rat mesenteric mast cell degranulation. A Sprague Dawley rat was sacrificed; pieces of its mesentery excised and treated with either Ringer–Locke (a), normal saline 10 ml kg⁻¹ (b), disodium cromoglycate, DSC 10 μg ml⁻¹ (c), or bergapten 10, 30 and 100 μg ml⁻¹, respectively (d–f). Tissues except the naïve control were challenged with C48/80 (1 μg) for 15 min and stained with Toluidine blue (0.1%, pH 2.5) for 30 min. Representative micrographs of stained mast cells are shown (a–f) and degranulation quantified (g). Data are expressed as mean ± SEM (n = 5). ^∗∗∗∗p < 0.0001 compared to the saline-treated C48/80-challenged group. ^####p < 0.0001 compared to naive control group (One-way ANOVA followed by Dunnett's post hoc test). Micron bar represents 100 μm.

Figure 2

Effect of bergapten on C48/80-induced systemic anaphylaxis. C57BL/6 mice received either saline 10 ml kg⁻¹, disodium cromoglycate 50 mg kg⁻¹, or bergapten 3–30 mg kg⁻¹, p.o., and challenged with C48/80 (8 mg kg⁻¹, i.p.) 1 h later. Mortality was monitored for 1 h. Data were analyzed using the log-rank (Mantel–Cox) test. Survival curves were significant p < 0.0001.

Figure 3

Effect of bergapten on neutrophil extravasation and total protein in BALF in LPS-induced lung inflammation. C57BL/6 mice were treated with either saline 10 ml kg⁻¹, dexamethasone 10 mg kg⁻¹, or bergapten 3–30 mg kg⁻¹ for 1 h. Test mice were challenged with LPS and sacrificed 24 h later. BAL fluid was collected for neutrophil cell count (a) or centrifuged and supernatant used for total protein determination (b). Data are presented as cell mean count (10³/µl) ± S.E.M (n = 5). ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 compared to the saline-treated LPS-challenged disease control. ^####p < 0.0001 compared to the naive control (one-way ANOVA followed by Dunnett's post hoc test).

Figure 4

Effect of bergapten on lung damage in LPS-induced pulmonary inflammation. C57BL/6 mice were grouped and treated as naïve control, saline 10 ml kg⁻¹ (a), disease control, saline 10 ml kg⁻¹ (b), dexamethasone 10 mg kg⁻¹ (c), or bergapten 3, 10, and 30 mg kg⁻¹ (d–f) for 1 h. Test mice were challenged with LPS while naïve control received PBS only. Mice were sacrificed 24 h later. Lungs were fixed in 10% formalin and embedded in paraffin. 3 μm sections were stained with H&E for histopathological examination. Degree of cell infiltration was quantified using an infiltration score described by Zare et al., (2008) with slight modifications. (g). Data are expressed as mean cell infiltration score ± SEM (n = 5). ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 compared to saline-treated LPS-challenged disease control. ^####p < 0.0001 compared to naïve control (one-way ANOVA followed by Dunnett's post hoc test). Micron bar represents 500 μm.

Figure 5

Effect of bergapten on oxidative stress markers in LPS-induced lung inflammation. C57BL/6 mice were treated with either saline 10 ml kg⁻¹, dexamethasone 10 mg kg⁻¹, or bergapten 3–30 mg kg⁻¹ for 1 h. Test mice were challenged with LPS and sacrificed 24 h later. Lungs were harvested, processed, and supernatant analyzed quantitatively for catalase (CAT) (a), superoxide dismutase (SOD) (b), and malondialdehyde (MDA) (c). Data are expressed as mean ± SEM (n = 5). ^∗p < 0.01, ^∗∗p < 0.01, and ^∗∗∗∗p < 0.0001 compared to saline-treated LPS-challenged disease control. ^####p < 0.0001 compared to naïve control (one-way ANOVA followed by Dunnett's post hoc test). ns is not significant.