Reduction of Asthmatic Parameters by Sea Hare Hydrolysates in a Mouse Model of Allergic Asthma

Abstract

Sea hare has a variety of biological activities. However, little is known regarding the anti-asthmatic effects of sea hare. This study was performed to identify the effect of sea hare hydrolysates (SHH) on an ovalbumin (OVA)-induced allergic asthma model. The experimental asthma model was sensitized and challenged with OVA. We found that a high-dose of SHH (HSHH) significantly inhibited OVA-induced airway inflammation and mucus production around the airway in lung sections, while low- and medium-dose SHH showed an insignificant effect. In addition, HSHH highly reduced OVA-induced production of interleukin-4, -5, -13, leukotriene D4, E4, and histamine in bronchoalveolar lavage fluid. HSHH decreased the histamine-induced increase in the intracellular Ca²⁺ level and contractions in asthmatic smooth muscle cells. Furthermore, HSHH did not affect the weights of the spleen nor thymus, whereas dexamethasone (DEX), a steroidal anti-inflammatory drug, reduced them. Taken together, these results showed that HSHH reduced asthmatic parameters in a mouse model of allergic asthma, and suggest that SHH could be used as a potential therapeutic agent for asthma.

Keywords: airway smooth muscle cell; asthma; cytokines; sea hare hydrolysates.

Figure 1

Modeling allergic asthma in mice. Mice were sensitized on days 0 and 7 with an intraperitoneal (IP) injection of 75 μg of ovalbumin (OVA) emulsified with 2 mg of aluminum hydroxide (alum) in 100 μL of phosphate-buffered saline (PBS). After the second sensitization, mice were challenged intranasally (IN) with 50 μg of OVA on days 14, 15, 21, and 22. Control mice were sensitized and challenged with PBS at the time of OVA injection. Control, OVA, and dexamethasone (DEX) groups received distilled water at the time of sea hare hydrolysates (SHH) administration. DEX and SHH were received orally 1 h before each OVA challenge (10:00–11:00 a.m.). The low-, medium-, and high-dose (LSHH, MSHH, and HSHH) groups orally received 0.1 g/kg, 0.5 g/kg, and 1.0 g/kg of SHH daily from day 14 to day 22. Animal experiments were performed in triplicate.

Figure 2

HSHH-induced reduction of inflammation in OVA-challenged asthmatic mice. (A) Total cell counts in bronchoalveolar lavage fluid (BALF); (B) Differential cell counts in BALF. Total cell counts were determined with 1 mL of BALF, and differential cell counts were assessed by Wright-Giemsa staining; (C) Representative images of Hematoxylin and Eosin (H&E) staining of lung tissue. The remarkable increase in cellular infiltration and airway thickness in the OVA group was not observed in the OVA + DEX nor OVA + HSHH groups. The scale bar represents 200 μm. The bar graphs show the inflammation score in each experimental group (see Materials and Methods 2.7); (D) Reduction of OVA-challenged goblet cell hyperplasia by HSHH. Representative images of periodic acid–Schiff (PAS) staining of goblet cells in the experimental groups. The scale bar represents 200 μm. The bar graphs show the number of PAS-positive mucus-producing cells (see Materials and Methods 2.7). In the bar graphs, data are shown as the means ± SE. * p < 0.05, compared to control. ^† p < 0.05, compared to OVA. ^# p < 0.05, compared to DEX.

Figure 3

Inhibitory effect of HSHH on total and OVA-specific Immunoglobulin E (IgE) levels in serum. Levels of (A) total; and (B) OVA-specific IgE. Data are shown as means ± SE. * p < 0.05, compared to control. ^† p < 0.05, compared to OVA. ^# p < 0.05, compared to DEX.

Figure 4

Inhibitory effect of HSHH on T helper cell type 2 (Th2) cytokines, leukotrienes, and histamine levels in BALF obtained from asthmatic mice. (A) Levels of IL-4, IL-5, and IL-13; (B) Levels of LTD4 and LTE4; (C) Levels of histamine. Data are shown as means ± SE. * p < 0.05, compared to control. ^† p < 0.05, compared to OVA. ^# p < 0.05, compared to DEX.

Figure 5

Reduction in histamine-induced contraction of ABSMCs by SHH. (A) Histamine-induced Ca²⁺ increase and reduction of the Ca²⁺ response by SHH in MABSMCs. Arrow indicates the addition of 100 μM histamine. Bar graph shows the effect of SHH on basal Ca²⁺ level in cells, which is the [Ca²⁺]_i before treatment with chemicals. FI represents the fluorescence intensity; (B) Representative images of collagen matrices in a collagen gel contraction assay. Cells were exposed to medium with or without chemicals and SHH for 24 h. Acetylcholine (ACh) and 2,3-butanedione monoxime (BDM) were used as a positive control and a negative control, respectively. Bar graphs show the contractility of ABSMCs induced by treatment with chemicals and SHH. Data are shown as means ± SE. * p < 0.05, compared to control. ^† p < 0.05, compared to histamine.

Figure 6

No changes in the spleen nor thymus weights by HSHH treatment. (A) Changes in the size of spleens and thymi in asthmatic mice; (B) Spleen and thymus weights in asthmatic mice. Bar graphs show the spleen and thymus weights isolated from the control, OVA, OVA + DEX, and OVA + HSHH groups. The ratios of organ weight to body weight were calculated. Data are shown as means ± SE. * p < 0.05, compared to control. ^† p < 0.05, compared to OVA. ^# p < 0.05, compared to OVA + DEX.