Effects of Corni fructus on ovalbumin-induced airway inflammation and airway hyper-responsiveness in a mouse model of allergic asthma

Abstract

Background: Allergic asthma is a chronic inflammatory lung disease that is characterized by airway hyperresponsiveness (AHR) to allergens, airway oedema, increased mucus secretion, excess production of T helper-2 (Th2) cytokines, and eosinophil accumulation in the lungs. Corni fructus (CF) is a fruit of Cornus officinalis Sieb. Et. Zucc. (Cornaceae) and has been used in traditional Korean medicine as an anti-inflammatory, analgesic, and diuretic agent. To investigate the anti-asthmatic effects of CF and their underlying mechanism, we examined the influence of CF on the development of pulmonary eosinophilic inflammation and airway hyperresponsiveness in a mouse model of allergic asthma.

Methods: In this study, BALB/c mice were systemically sensitized to ovalbumin (OVA) by intraperitoneal (i.p.), intratracheal (i.t.) injections and intranasal (i.n.) inhalation of OVA. We investigated the effect of CF on airway hyperresponsiveness, pulmonary eosinophilic infiltration, various immune cell phenotypes, Th2 cytokine production, and OVA-specific immunoglobulin E (IgE) production.

Results: The CF-treated groups showed suppressed eosinophil infiltration, allergic airway inflammation, and AHR via reduced production of interleuin (IL) -5, IL-13, and OVA-specific IgE.

Conclusions: Our data suggest that the therapeutic effects of CF in asthma are mediated by reduced production of Th2 cytokines (IL-5), eotaxin, and OVA-specific IgE and reduced eosinophil infiltration.

Figure 1

Effect of CF on airway hyperresponsiveness in OVA-challenged mice. Mice were sensitized and challenged by OVA as described in the Materials and methods. The airway responsiveness to aerosolized methacholine was measured with a Buxco box, as described in the Materials and methods. The mice were placed in the main chamber and were nebulized first with PBS and then with increasing doses (3.125 to 50 mg/mL) of methacholine for 3 min for each nebulisation (A). Schematic representation showing the protocol to induce asthma model (B). The data represent the mean ± SEM from 5 independent experiments. *P < 0.05, **P < 0.01 for the control goup versus the CF-treated groups.

Figure 2

Effect of CF on airway inflammation (H&E, M-T, and PAS staining) in lung tissue and BALF of OVA-induced asthmatic mice. At the end of the experiment, the lungs were fixed, and a histologic analysis was performed. Lung sections were obtained for normal and asthmatic mice treated with vehicle (control), CF (50 mg/kg), and CF (200 mg/kg). H&E: hematoxylin-eosin stain, M-T: Masson trichrome stain, PAS: periodic acid-Schiff Stain, N: Normal BALB/c mice, CT (control): Ovalbumin inhalation + vehicle, OVA + CF (50, 200 mg/kg).

Figure 3

Effects of CF on total lung cells, total BALF cells, eosinophils in the BALF, and PBMCs. As described in the Materials and methods, the lung and BALF were harvested 24 h after the last OVA challenge. The total inflammatory cell numbers in the lung and BALF were counted, and cell classification was performed on a minimum of 200 cells to classify lymphocytes. The results are expressed as the mean ± SEM (n = 5). The statistical significance of differences between control and treatment groups was assessed by ANOVA or the nonparametric Mann-Whitney test followed by Dunnett's multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001). N: Normal BALB/c mice, CT: Ovalbumin inhalation + vehicle, OVA + CF (50, 200 mg/kg).

Figure 4

FACS analysis of various immune cell sub-types in the lung and BALF. The absolute numbers of various immune cell subtypes in the lung were counted (A, B) (described in Materials and methods). The lung and BALF cells were stained with FITC-conjugated mAb to CD3 and CD11b as well as PE-conjugated mAb to Gr-1 and CCR3. A dot-plot pattern is shown for the representative flow cytometric profile for the gated CD3-/CCR3+ and Gr-1+/CD11b + cells (C, D). The results are expressed as the mean ± SEM (n = 5). The statistical significance of differences between the control and treatment groups was assessed by ANOVA or the nonparametric Mann-Whitney test followed by Dunnett's multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001). N: Normal BALB/c mice, CT: Ovalbumin inhalation + vehicle, OVA + CF (50, 200 mg/kg).

Figure 5

Effect of CF on Th2 cytokines (IL-5, IL-13) in the BALF and OVA-specific IgE in the serum, and immunomodulatory effects of CF on OVA-specific Th1/Th2 cytokines produced by spleen cells (described in Materials and methods). The results are expressed as the mean ± SEM (n = 5). The statistical significance of differences between the control and treatment groups was assessed by ANOVA or the nonparametric Mann-Whitney test followed by Dunnett's multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001). N: Normal BALB/c mice, CT: Ovalbumin inhalation + vehicle, OVA + CF (50, 200 mg/kg).

Figure 6

HPLC chromatogram of standard mixture (A) and C. officinalis (B) at 240 nm. Morroniside (1), loganin (2). C. officinalis and 2 standards were subjected to HPLC analysis. An Optimpak C18 (4.6 × 250 mm) column was eluted with water and acetonitrile (86:14) at flow rate of 1.0 mL/min. The HPLC chromatogram is shown for the ursolic acid standard (C) and C. officinalis (D) at 200 nm. C. officinalis and ursolic acid were subjected to HPLC analysis. The Optimpak C18 (4.6 × 250 mm) column was eluted with water and acetonitrile (10:90) at flow rate of 1.0 mL/min.