DMEM/F12 was provided by the Amicogen Inc. (Jinju, Republic of Korea). All the other reagents were purchased unless otherwise specified. Fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA were obtained from Lonza (Walkersville, MD, USA). Inhibitor of nuclear factor kappa-B-α (IκB-α) and β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), while phosphorylated NF-κB p65 and NF-κB antibodies were obtained from Cell Signaling (Beverly, MA, USA). Essential fatty acid-free bovine serum albumin and skim milk were sourced from Becton Dickinson Company (Sparks, MD, USA). The low-molecular-weight COS used in the experiments were produced directly by Amicogen Inc. (Jinju, Republic of Korea).

Human bronchial epithelial cells (BEAS-2B) were obtained from the American Type Culture Collection (CRL-3588, ATCC) and maintained in DMEM/F12 medium containing 10% FBS and 1% penicillin-streptomycin in a CO₂ incubator. The MTT assay was performed to assess cytotoxic effects. The cells were seeded at a density of 1 × 10⁴ cells/well in a 96-well plate and treated with HDM or COS. After 24 h of incubation, 5 mg/ml of MTT reagent was added, and the cells were incubated for an additional 2 h. Subsequently, the culture was centrifuged at 2,000 rpm for 10 min at 4°C, and the supernatant was discarded. DMSO was then added to each well, and the absorbance was measured at 540 nm using a microplate reader (Thermo Scientific, Waltham, MA, USA).

This study was approved by the Institutional Animal Care and Use Committee of Jeonbuk National University Hospital (JBUH-IACUC-2022-28) and was conducted in compliance with the University's guidelines for the care and use of experimental animals. The following conditions were maintained in the experimental environment: temperature of 23°C ± 3°C and relative humidity of 55% ± 15%.The lighting cycle lasted 12 h, at an intensity between 150 and 300 Lux. These environmental parameters were regularly monitored throughout the breeding period. The animals were provided with food obtained from the Central Experimental Animal Center (Seoul, Republic of Korea) and had free access to it. Filtered water was supplied in polycarbonate bottles, and was also freely available. Bedding materials were sourced from Coretech Co., Ltd. (Pyeongtaek, Republic of Korea). During the adaptation, treatment, and blood collection phases, the rodents were housed in polycarbonate cages (235 mm wide × 380 mm long × 175 mm high), with a maximum of five animals per cage. Cages, bedding, and water bottles were changed at least once a week. Prior to infection, the animals were weighed, ranked, and randomly assigned to groups to ensure uniform average body weight within each group.

To establish an HDM-induced airway allergic inflammation model, freeze-dried HDM extract (14-4107-01, Thermo fisher) was reconstituted in saline at a concentration of 2 μg/μl. Following acclimation, the animals were anesthetized, and the extract was administered four times at 1-week intervals at a dose of 100 μg/50 μl per mouse using a small animal ventilator (SAR-1000, CWE, Inc.) maintaining a respiratory rate of 80 bpm and a tidal volume of 0.18 ml. The experiment was conducted for five groups, each consisting of eight mice and defined as follows: control, HDM, HDM + 10 mg/kg/body weight (BW), HDM + 20 mg/kg/BW, and HDM + 100 mg/kg/BW. For the control group, which did not receive any disease-inducing treatment, an equivalent volume of saline solution (50 μl) was administered using the same equipment.

After administration, the trachea of each mouse was intubated, and the lungs and airways were rinsed with 1 ml of PBS, before collecting a sample of bronchoalveolar lavage fluid (BALF). The number of inflammatory cells was determined using a Hemavet HV950 multispecies hematologic analyzer (Drew Scientific, Oxford, CT, USA). The right lung was collected, frozen in liquid nitrogen, and stored at −80℃ until use for real time (RT)-PCR and Western blotting. The left lung was fixed in 4% paraformaldehyde and used for immunohistochemical analysis.

Cell and lung tissue samples were processed to synthesize cDNA from 5 μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The reaction mixture for RT-PCR contained 1 μl of the synthesized cDNA, 1 μl of TaqMan probes, 10 μl of TaqMan Universal Master Mix II (Thermo Fisher Scientific), and 8 μl of nuclease-free water. The TaqMan genes used for quantitative PCR (qPCR) were obtained from Applied Biosystems. The RT-PCR conditions included an initial step at 50°C for 2 min, followed by one cycle at 95°C for 10 min, and then 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 15 s. PCR primers were used based on the information provided in Table 1.

Cell and lung tissue samples were washed twice with PBS, and proteins were extracted using NE-PER nuclear and cytoplasmic extraction reagents (Cat#: 78835, Thermo Fisher). The extracted proteins were quantified using Bradford reagent (Bio-Rad, Hercules, CA, USA). Then, they were mixed with sample buffer and heated at 95°C for 5 min, followed by separation using 10%–12% SDS-PAGE. After separation, the proteins were transferred to a PVDF membrane using a semi-dry transfer system at 15 V for 60 min. The membrane was then incubated with blocking buffer (5% skim milk in 1 × TBST) for over 1 h, followed by incubation with the primary antibody at 4°C overnight. The membrane was washed five times with 1 × TBST at 7-min intervals. Subsequently, it was incubated with the secondary antibody at room temperature for over 1 h, followed by another five washes with 1 × TBST at 7-min intervals. Finally, the membrane was developed using an enhanced chemiluminescent (ECL) reagent and exposed to an x-ray film.

Mice that had fasted for more than 14 h, all the mice, were anesthetized with ketamine, and 1 ml of blood was collected via cardiac puncture. The collected blood was centrifuged at 3,000 rpm for 10 min to obtain serum, which was transferred to EP tubes and stored at −80°C. Biochemical analyses for triglycerides, total cholesterol, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were performed. The reagents for these analyses, i.e., triglyceride measurement reagent (AM-157S-K), cholesterol measurement reagent (AM 202-K), GOT reagent (AM-103 K), and GPT reagent (AM-102 K), were purchased from Asan Pharmaceutical (Seoul, Republic of Korea). The levels of IgE in the collected plasma were measured using the mouse IgE ELISA kit (ab157718, Abcam).

Mice were anesthetized with a mix of ketamine (125 mg/kg) and medetomidine 0.4 mg/kg) and ventilated with O₂/air (1:2) at a rate of 150 beats/min (TV = 0.3 ml). Pressure was measured by placing a small catheter in the trachea of each mouse. Methacholine (acetyl-β-methyl-choline chloride; Sigma) was administered in increasing doses (0–50 mg/ml, delivered as 10% puffs over 10 s) via an aerosol generated by a nebulizer (EMKA Technologies, Paris, France). The nebulizer was connected to the animal the FlexiVent device (Flexivent FV-FX2, SCIREQ). After the initial dose of methacholine, lung resistance was measured for 3 min, and this procedure was repeated for each subsequent dose (22).

Small lung tissue samples were collected at the end of the experiments and fixed in 10% formalin for histological analyses. Once embedded in paraffin, these samples were cut into sections with a thickness of 5 μm and deparaffinized. Depending on the specific staining required, the sections were subjected to one of the following staining procedures: (1)

Harris hematoxylin and eosin Y (H&E) staining: The sections were first stained with Harris hematoxylin for 2 min and then with eosin Y.

In the statistical analyses, the interquartile range method was employed to exclude outliers. The interquartile range is defined as the difference between the first quartile (Q1) and the third quartile (Q3). Values that exceeded the established criteria for outliers were excluded from the analysis. Results are presented as the mean ± SE for each treatment group across all experiments. Statistical analyses were performed in SPSS v. 18.0 (IBM Corp., Armonk, NY, USA). All data measurements were tested for normality using the Kolmogorov-Smirnov and Shapiro-Wilk methods, followed by comparisons between groups. Based on the normality results, significance was assessed using the ANOVA method, with post hoc testing performed via the t-test. In cases where normality was not met, significance was evaluated using the Kruskal-Wallis test, with post hoc testing conducted using the Mann-Whitney U-test. Significance levels for the t-test were defined as ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001, while significance for the U test was defined as *p < 0.05, **p < 0.01, and ***p < 0.001.

The toxicity assessment was conducted on airway epithelial cells treated with HDM and COS. Specifically, BEAS-2B cells were treated with HDM at concentrations ranging from 12.5 to 1,000 μg/ml for 24 h, and cell viability was then evaluated using the MTT assay. As shown in Figure 1A, cell viability was 77% after treatment with 100 μg/ml HDM. Since HDM are known to simultaneously induce cell death and inflammation, their concentration was set at 100 μg/ml in subsequent experiments (23, 24). No toxicity was observed at COS concentrations below 1,000 μg/ml (Figure 1B). To evaluate the safety of orally administering COS to animals, body weight was measured at the end of the 4-week experiment. No significant differences in body weight were observed between groups, and while overall lung weight increased in the HDM-exposed group, no significant changes were noted after COS ingestion. Among the measured hematological indexes, which included aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TG), and total cholesterol (TC), AST decreased in animals treated with high doses of COS, and the same tendency was observed for TG and TC in this group. These findings confirmed that COS have a slight impact on hematological indexes but remain safe (Table 2).

The IgE levels in mouse plasma samples obtained via blood centrifugation were analyzed using the mouse IgE ELISA kit. The HDM-treated mice exhibited significantly higher IgE levels than the control group. In contrast, a significant decrease in this parameter was observed in the group administered with COS at 20 mg/kg/BW compared with the HDM-treated group (Figure 2A). Elevated IgE levels are known to cause mucus hypersecretion and increased airway resistance (25). The latter reaction was specifically assessed by administering methacholine, which induces airway constriction. The results showed that airway resistance increased in the HDM-treated mice, while it significantly decreased in the group treated with COS concentrations > 20 mg/kg/BW (Figure 2B).

The effect of COS in terms of suppressing the excessive secretion of airway mucus was evaluated through PAS staining of lung tissue sections. Mucus secretion increased with HDM exposure; however, microscopic examination showed that mucus accumulation decreased in the groups that received COS at doses > 20 mg/kg/BW. Airway sclerosis is characterized by the thickening of airway tissues due to inflammation and fibrosis, with collagen fiber accumulation playing a crucial role. Sirius Red or Masson's trichrome staining can be employed to observe increases in collagen fibers or their abnormal arrangement, which are associated with the thickening of the airway wall. The infiltration of inflammatory cells and the increase in collagen fibers occur simultaneously, reflecting the pathological features of airway sclerosis (Figure 3A) (26, 27). In the HDM-treated group, fibrosis was noted around the smooth muscle of the airway, while in the group administered with COS, airway sclerosis was suppressed (Figure 3B,C).

H&E staining revealed the differences in the infiltration of inflammatory cells in lung tissues between the control and HDM-treated mice. Significant pathological features of inflammatory cell infiltration were observed around the small airways in the mice exposed to HDM. The evaluation was conducted by three experts, who scored the level of inflammation on a 0–3 scale. Better scores were assigned to the group treated with COS at > 20 mg/kg/BW (Figure 4A). The number of inflammatory cells measured in the BALF, including eosinophils, neutrophils, lymphocytes, and macrophages, decreased under COS administration at doses greater than 10 mg/kg/BW (Figures 4B–F). This suggested that the reduction in the number of inflammatory cells was associated with decreased inflammatory cell infiltration.

In the airway epithelial cells treated with HDM, the RNA expressions of TNF-α, IL-1β, and IL-6 increased, while they began to decrease at COS concentrations > 50 µg/ml (Figures 5A–C). The mRNA expression of inflammatory cytokines induced by HDM was significantly suppressed in the lung tissues of experimental mice treated with COS (Figures 5D–F). It was also found that HDM promoted the phosphorylation of IκB, leading to increased nuclear translocation of NF-κB, which regulates a key mechanism related to inflammation. The COS treatment inhibited the phosphorylation of IκB and NF-κB, thereby counteracting the inflammatory signaling pathway of NF-κB and ultimately reducing inflammation in the airways (Figures 5G,H).