Asthma is a chronic inflammatory lung disease characterized by airway hyperresponsiveness, inflammation, and respiratory remodeling, often observed in pediatric and adult patients, causing significant morbidity, mortality, and high economic burden worldwide (Gans and Gavrilova, 2020). There are several types of asthma, including allergic, seasonal, non-allergic, occupational, exercise-induced, difficult-to-control, and severe, each with its own set of triggers (Padem and Saltoun, 2019).

Allergic asthma is the most common form of asthma and is present in 50-80% of all asthma cases. Common triggers of allergic asthma are mostly attributed to interactions between environmental exposure and genetic susceptibility. These include environmental factors (dust mites, animal hair, pollen, mold, obesity, stress, and other allergic sensitizations) and genetic factors (asthma susceptibility loci) (Toskala and Kennedy, 2015). When patients with asthma are exposed to allergens, the rapid release of pro-inflammatory mediators causes contraction of airway smooth muscle, swelling of the mucosal lining, and oversecretion of mucus, thereby blocking the narrowed airway and inducing difficulty in breathing up to extreme levels (Fixman et al., 2007).

The pathophysiology of asthma involves a complex interplay between the innate and adaptive immune systems, thereby stimulating chronic airway inflammation (Kim et al., 2013). Dendritic cells present inhaled allergens to naïve T cells, thereby activating the production of type 2 helper T (Th2) cells. Th2 lymphocytes are currently recognized as the primary orchestrators of allergic airway inflammation in asthma and are known to play a triggering role in the activation and recruitment of B cells, mast cells, and eosinophils. Th2 cells secrete interleukin-4 (IL-4), IL-5, IL-10, and IL-13 which are involved in stimulation of B cells to release immunoglobulin E (IgE). IgE induces mast cell degranulation and the release of inflammatory mediators (histamine and leukotrienes), causing bronchoconstriction. IL-4, a typical kind of Th2 cytokine, induces asthma-specific IgE synthesis In addition, IL-5 recruits eosinophilic infiltration into the airway (Deo et al., 2010). The recruitment of granulocytes results in chronic airway inflammation, which subsequently leads to airway edema and mucus hypersecretion that blocks the airway. Failure to relieve inflammatory responses and apoptosis occurs in airway epithelial cells, which may result in airway remodeling and ultimately contribute to severe lung injury. Failure to relieve inflammatory responses leads to apoptosis of airway epithelial cells, which may result in airway remodeling, ultimately contributing to severe lung injury.

Neurotrophins mediate the differentiation, growth, and survival of responsive neurons by binding to two types of cell surface receptors: Trk tyrosine kinase receptors and p75 neurotrophin receptor (p75NTR) (Kaplan and Miller, 2000). Nerve growth factor (NGF) is a member of the neurotrophin family that induces bronchial hyperresponsiveness by increasing sensory innervation. These changes are expected to induce the migration and activation of inflammatory cells (Quarcoo et al., 2004). The pro-NGF prodomain masks the binding domain of NGF to the Trk A receptor, which, in the absence of Trk signaling, increases the selectivity of NGF for the p75NTR receptor, thereby preferentially inducing p75 programmed cell death in response to NGF binding (Barrett, 2000).

Asthma is a lifelong condition that can be managed with medication and avoidance of triggers that initiate asthma attacks. Glucocorticoid anti-inflammatory drugs are currently recommended in clinical practice as anti-asthma drugs for patients with mild-to-moderate asthma; however, the daily inhalation of glucocorticoids can lead to poor patient compliance and life-threatening events. Moreover, the long-term use of steroidal anti-inflammatory drugs can cause serious complications such as pneumonia, fractures, hyperglycemia, and cataracts (Li et al., 2017). These side effects limit their prolonged use in clinical asthma treatment, especially in younger patients. Therefore, new therapeutic agents with high potency against inflammation and asthma symptom relief and fewer side effects are required.

Natural products have been widely used in the treatment and prevention of human diseases for thousands of years owing to their effectiveness, wide margins of safety, and multi-targeting properties. Previously, our group investigated the composition and efficacy of a traditional herbal medicine called ‘So Cheong Ryong Tang’ which is the water extract of various herbs including Ephedra sinica, Paeonia lactiflora, Schisandra chinensis, Pinellia ternate, Asiasarum sieboldii (AS), Zingiberis officinale, Cinnamomum cassia (CC). and Glycyrrhiza uralensis. Focusing on anti-allergic and anti-inflammatory effects, the herbal composition was modified to contain three herbs: AS, CC, and Platycodon grandiflorum (PG). Among these natural products, PG has been extensively used as a medicinal herb to treat pulmonary and respiratory allergic disorders and exhibits strong antitussive, expectorant, and anti-asthmatic effects, with reported beneficial effects in relieving airway inflammation through the suppression of several pro-inflammatory cytokines (Lee et al., 2020). Another component, CC, is a traditional Oriental medicine with anti-inflammatory, immunomodulatory, and protective effects in allergic asthma disease models (Lim et al., 2022). AS is a commonly used medicinal herb in Asian countries and its beneficial effects have been demonstrated in pulmonary symptoms, such as cough, allergy, chronic bronchitis, and anti-allergic activity, owing to its ability to inhibit IgE production by B cells (Han et al., 2022). This modified herbal composition was tested in various ratios and extractions in allergic models, including asthma and atopic dermatitis, and the 70% ethanol extract of three herbal mixtures in a ratio of 2:2:1 (PG:CC:AS) was selected for its best efficacy and named BS012.

With our recent successful application of BS012 in an atopic dermatitis experimental model (Lee et al., 2023), the present study utilized BS012 in an ovalbumin (OVA)-induced allergic asthma mouse model to investigate the respiratory anti-allergic properties and underlying protective mechanisms of BS012.

Preparation of BS012 and its active component N-isobutyl-dodecatetraenamide

BS012 comprises a combination of three herbal medicines: PG, CC, and AS. The three herbal medicines were purchased from Kyungdong Market (Woori Herb), Seoul, Korea. The materials were verified and prepared as BS012 by our research collaborator Prof. Hocheol Kim at the Department of Traditional Medicine, Kyung Hee University (Seoul, Korea). The BS012 extract was prepared as described in our previous report (Lee et al., 2023). Briefly, each sample was grounded and extracted with 70% ethanol and then concentrated at 60-70°C under reduced pressure. Each lyophilized extract was weighed and mixed based on the ratio of 2:2:1 (PG, CC, and AS, respectively) and stored at –20°C until further use. Phytochemical analysis of BS012 and identification of marker compounds were performed as described in our previous reports (Jeong et al., 2018; Lee et al., 2023).

Animal experimental design

In general, female mice have more pronounced B cell-mediated immunity than age-matched males, and therefore have higher Ig levels and stronger antibody responses to various foreign antigens. Various studies have suggested that female mice are more susceptible to allergic airway inflammation than male mice (Melgert et al., 2005). Female mice challenged with OVA showed significantly increased indicators of allergic airway inflammation and airway remodeling as well as higher levels of OVA-specific IgE than male mice, which is consistent with the epidemiology of human asthma (Takeda et al., 2013; Mostafa et al., 2022). Therefore, female mice were selected to establish the asthma model in this study. Seven-week-old female BALB/c mice were purchased from Orient Bio (Seongnam, Korea). The mice were placed under a regulated temperature (24 ± 2°C), humidity (50 ± 5%) and 12-h day/night cycle conditions with ad libitum access to normal food pellets and water. All procedures involving animals were approved by the Korea Institute of Science and Technology Animal Care Committee and performed in accordance with the ethical guidelines (KIST Approval No. KIST-2019-108).

After one week of acclimatization, the mice were randomly divided into five experimental groups (n=8 per group) as follows: control, OVA, OVA+BS012 (100 and 200 mg/kg), and OVA+dexamethasone (1.5 mg/kg). Except for the control group, all other mice were sensitized with an intraperitoneal injection of OVA (1 mg/kg) along with aluminum hydroxide (200 mg in 1 mL of saline) on days 1 and 8. Dexamethasone (1.5 mg/kg body weight) was intraperitoneally administered as a positive control from day 15, once daily, for 4 days. BS012 was orally administered at doses of 100 and 200 mg/kg body weight from day 1, once daily for 19 days. All animals, except for the control group, were challenged with 5% (w/v) OVA solution aerosolized using an ultrasonic nebulizer from day 15 onwards for 30 min per day for 4 days. On day 19, all animals were anesthetized and whole blood was collected from the abdominal aorta to separate the plasma for biochemical analysis. The lungs were lavaged three times with cold saline through a tracheal cannula to collect bronchoalveolar lavage fluid (BALF). BALF was further centrifuged to collect the supernatant, which was stored at –80°C until further analysis. Part of the lung tissue was stored in 10% formalin at room temperature for histopathological analysis and the rest was snap frozen and kept at –80°C until protein extraction. The experimental schedule is shown in Fig. 1A.

Figure 1. Scheme of the experimental procedure (A) and effect of BS012 on the levels of ovalbumin-specific IgE and Th2-specific cytokines production in ovalbumin-induced asthma mice. The level of (B) OVA-specific IgE in plasma, (C) BALF IL-4, and (D) BALF IL-5 was measured. All the data are expressed as the mean ± SEM (n=5 per group). *** Significantly different (p<0.001) from Control group. ^##, ^### Significantly different (p<0.01, 0.001) from OVA group.

Measurements of inflammatory cytokines and immunoglobulin production

Levels of IL-4 (R&D Systems, MN, USA, Cat. M4000B) and IL-5 (cat. M5000) in BALF, and OVA-specific IgE (BioLegend, San Diego, CA, USA, Cat. 439807) in the plasma were measured using an enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s instructions. Specific monoclonal antibodies were pre-coated onto the microplates. Standards and samples were pipetted into the wells and any specific antigen present was bound by the immobilized antibody. After washing away unbound substances, a specific enzyme-linked monoclonal antibody was added to the wells. Following washing to remove any unbound antibody-enzyme reagent, a colorimetric substrate was added to the wells to form a colored solution when catalyzed by the enzyme. The absorbance was measured at 450 nm using a microplate reader.

Histological analysis of lung tissue

Lung samples fixed in 10% formalin were embedded in paraffin and sliced into 4 μm thick sections. Lung histology was further confirmed using hematoxylin and eosin (H&E) staining and the thickness of the bronchial epithelium was measured using ImageJ software. Additionally, to identify the degree of goblet cell hyperplasia and mucus production in the airway, Periodic Acid Schiff (PAS) staining was performed, and the number of PAS-positive cells was counted using ImageJ software (Figi, Paris, France).

Western blot analysis of lung tissue

Total protein was extracted from lung tissue using PRO-PREP™ protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) by centrifuging the lysate at 13,000 rpm for 15 min at 4°C to collect the protein-containing supernatant in a new tube. The protein concentration was quantified using a bovine serum albumin (BSA) kit (Cat. #5000207) following the manufacturer’s instructions. Protein samples were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and then transferred to 0.2 μM poly-vinylidene difluoride membrane. After blocking with Every Blot Blocking Buffer (Bio-Rad, CA, USA, Cat. #12010020), the membranes were incubated with primary antibodies overnight at 4°C and incubated with appropriate secondary antibodies for 1 h at room temperature. The signal intensity was imaged and quantified using a LAS-4000 system (Fuji Film, Tokyo, Japan). The primary antibodies (NGF (#52918, Abcam), p75NTR (#4201), P-JNK (#9251), JNK (#9252), P-c-jun (#3270), c-jun (#9165), Bcl-xl (#2764), P-Bad (#5284), Bad (#9292), Bax (#2772), Cytochrome c (#11940), cleaved Caspase-9 (#9509), Caspase-9 (#9504), cleaved Caspase-3 (#9664), Caspase-3 (#9662), and GAPDH (#2118)) and secondary antibodies (rabbit IgG (#7074)) were purchased from Cell Signaling Technology (MA, USA).

Isolation of bone marrow dendritic cells and co-culture assays

Bone marrow cells were obtained from the femurs and tibias of naïve mice and cultured in complete RPMI medium supplemented with 2.5% HEPES and 20 ng/mL GM-CSF to induce differentiation. Flow cytometry confirmed that more than 90% of the cells were CD11c⁺ dendritic cells (DC). Dendritic cells (DC) were prepared for co-culture by CD11c MACS enrichment of bone marrow-derived dendritic cells. CD4⁺ T cells were enriched by collecting and pooling spleens and peripheral lymph nodes from 6-week male C57BL/6N mice, followed by CD4 MACS enrichment (Miltenyi Biotec, Bergisch, Germany). The enriched samples were identified and flow-sorted for naïve CD4⁺-gated CD4⁺CD44^lowCD25-CD62L^hi cells using a FACSAria III cell sorter. For DC - CD4⁺ T cells co-culture assays, cells were co-cultured in complete media with soluble anti-CD3ε Ab (clone 145-2C11), anti-IFNγ (clone XMG1.2), IL-2, IL-4 in the presence of N-isobutyl-dodecatetraenamide for 3 days. For intracellular cytokine analysis, the cell suspension was incubated with PMA (Cat# P1585, Sigma-Aldrich, St. Louis, MO, USA), ionomycin (Cat# I9657, Sigma-Aldrich), GolgiStop (Cat#00450551, Thermo Fisher, MA, USA), and GolgiPlug (Cat# 00450651, Thermo Fisher) for 3 h. Fixation and permeabilization were performed using an Intracellular Fixation & Permeabilization Buffer set (Cat# 88-8824-00, Thermo Fisher) following the manufacturer’s protocol. The samples were analyzed using a FACSLyric (BD Biosciences, CA, USA). The antibodies used for flow cytometry analysis are listed in Supplementary Table 1.

Statistical analysis

The data were presented as the mean ± standard error of the mean (SEM). Statistical analyses were performed using the Prism 10 software (GraphPad Software, Inc., San Diego, CA, USA). After the normal distribution test, differences among the groups were analyzed using one-way analysis of variance, followed by Tukey’s multiple comparison post-hoc test. Statistical significance was set at 0.05.

UPLC-Orbitrap-MS based metabolomic profiling

The plasma metabolome was extracted according to the protocol outlined by Lee (Lee et al., 2021). Briefly, 40 µL of plasma was mixed with 120 µL of methanol and centrifuged at 14,000 rpm and 4°C for 10 minutes. Subsequently, 100 µL of the supernatant was diluted with 50 µL of distilled water containing 4 µg/mL reserpine as an internal standard (IS). Lung tissue (150 mg) was homogenized with 450 μL of 0.9% sodium chloride solution, followed by the transfer of 250 µL of homogenate to a new tube. Acetonitrile (1 mL) was added for extraction. After vortexing for 1 min, the mixture was centrifuged for 10 min. Following this, 800 μL of the supernatant was transferred into a new tube, evaporated, and reconstituted with 80 μL of acetonitrile for concentration.

Prepared samples were analyzed using Orbitrap Exploris™ 120 Mass spectrometer coupled with Vanquish Flex UHPLC system (Thermo Fisher Scientific, San Jose, CA, USA) at the Biopolymer Research Center for Advanced Materials (BRCAM). Samples were injected into a randomized analytical batch along with quality control (QC) samples. The QC samples consisted of equal volumes of samples for conditioning and were included in every 10 samples throughout the batch. The instrumental parameters of UPLC-Orbitrap-MS have been described previously (Lee et al., 2021). Raw MS data were preprocessed using Xcalibur version 2.2 and Compound Discoverer 3.3 (Thermo Fisher Scientific). Compound intensities were normalized using internal standards (ISs) and multivariate analysis was performed using SIMCA 17 software (Umetrics, Inc., Ume, Sweden). The Wilcoxon rank-sum test was used to evaluate the statistical significance between the control and OVA-induced asthma model groups, as well as between the asthma model and groups treated with the positive control, dexamethasone, or BS012. Statistical significance was set at p<0.05. Compound identification was conducted by matching the m/z, retention time (RT), and MS/MS fragmentation patterns of compounds with databases or by confirming identity using standard substances.

Effect of BS012 on IgE Production and Th2-specific Cytokines Secretion

To investigate the effects of BS012 on OVA-mediated allergic responses in mice, plasma OVA-specific IgE levels were measured. As Fig. 1B shows, secretion of OVA-specific IgE in the plasma was significantly increased in the OVA-induced group (21.8 ± 1.6 ng/mL) compared with the control group (0.1 ± 0.0 ng/mL). The mice treated with BS012 100 and 200 mg/kg showed significantly down-regulated levels of OVA-specific IgE (14.6 ± 0.9 ng/mL and 13.8 ± 1.0 ng/mL, respectively) in the plasma compared with the OVA group mice.

Among the various Th2-specific cytokines secreted, the levels of IL-4 (Fig. 1C) and IL-5 (Fig. 1D) were measured in BALF samples. The level of IL-4 was significantly increased in the OVA-induced mice (188.2 ± 31.9 ng/mL) compared with the control group (4.4 ± 0.4 ng/mL). The mice treated with BS012 100 (123.5 ± 21.0 ng/mL) and 200 (88.0 ± 14.1 ng/mL) mg/kg showed a significant reduction in IL-4 level compared with the OVA group mice in a dose-dependent manner. Similar to the IL-4 results, IL-5 level in BALF was also increased significantly in the OVA group (313.1 ± 31.3 ng/mL) compared with the control group (10.2 ± 1.2 ng/mL). IL-5 level was markedly decreased in the mice treated with BS012 at doses of 100 (86.4 ± 14.5 ng/mL) and 200 (45.7 ± 10.9 ng/mL) mg/kg compared to the control group. As expected, dexamethasone injection, which was used as a positive control, significantly suppressed the levels of OVA-specific IgE, IL-4, and IL-5 by 65.6%, 81.0%, and 84.0%, respectively, compared to the OVA group.

Effect of BS012 on lung histology in OVA-induced asthma mice

Structural changes were observed by the histological evaluation of H&E-stained lung tissue sections (Fig. 2A). The mice in the control group had smooth and intact bronchial structures. The thickness of the bronchus was normal (11.24 ± 1.5 μm) without inflammatory infiltration around the bronchioles. Compared with the control group, there was a significant increase in the infiltration of inflammatory cells around the bronchioles in the OVA-induced group. Such change was accompanied by prominent thickening (three times) of the bronchiole walls in the OVA-induced group (35.9 ± 5.3 μm) compared to the control group (Fig. 2B). BS012 treatment significantly reduced these histological changes in a dose-dependent manner. Reduced inflammatory cell infiltration was visible and reduced thickening of the bronchial epithelium (16.0 ± 2.6 μm) was statistically significant in the BS012 200 mg/kg treatment group. Such a reduction in the epithelium was also observed in the dexamethasone-treated group.

Figure 2. Assessment of effect of BS012 on histological examination of lung tissues by H&E staining. (A) H&E-stained sections of lung tissues (original magnification ×200). (B) Measured the thickness of bronchial epithelium. Data are presented as mean ± SEM (n=5 per group). *** Significantly different (p<0.001) from Control group. ^#, ^## Significantly different (p<0.05, 0.01) from OVA group. Assessment of effect of BS012 on goblet cell hyperplasia in lung tissue by PAS staining. (C) PAS staining results of the lung tissues (original magnification ×200), mucus is stained as a purple color. (D) Quantitative analyses of PAS positive cells in bronchial. Data are presented as mean ± SEM (n=5 per group). *** Significantly different (p<0.001) from Control group. ^### Significantly different (p<0.001) from OVA group.

OVA-induced goblet cell hyperplasia and mucus production in lung tissue were examined by PAS staining. As shown in Fig. 2C, the control group exhibited a smooth mucosal layer at the bronchioles with no signs of mucus formation. However, with the OVA challenge, mucus hypersecretion was observed, with a marked increase in goblet cell hyperplasia in the bronchioles. In addition, as shown in Fig. 2D, significantly increased PAS-positive cells were counted in the OVA-induced group, and approximately 45 PAS-positive cells/mm² were detected compared to the control group (3 PAS-positive cells/mm²). Dexamethasone and BS012 200 mg/kg treatments both showed a substantial reduction in PAS-positive cell counts (5 and 4 PAS-positive cells/mm², respectively) to the normal level along with decreased mucus secretion and goblet cell hyperplasia in the bronchioles.

Effect of BS012 on NGF-mediated JNK signaling pathway in OVA-induced asthma mice

To determine the effect of BS012 on the apoptotic pathway in OVA-induced asthmatic mice, proteins related to the NGF-mediated JNK signaling pathway were measured in the lung tissue using western blotting (Fig. 3). Challenge with OVA increased the protein level of NGF up to 184.8 ± 5.8% in the OVA group compared with the control group (Fig. 3A). BS012 treatment at 200 mg/kg significantly decreased the NGF protein expression level to 134.4 ± 4.7% when compared with the control group. Dexamethasone injection showed a similar effect as the BS012 200 mg/kg treatment reaching 132.0 ± 6.6% compared to the control group. However, a lower decrease was observed in the BS012 100 mg/kg treated group, at 161.1 ± 18.7% of the control group. Meanwhile, the protein expression level of p75NTR was also significantly upregulated with OVA challenge when compared to the control group exhibiting 392.2 ± 27.4% of the control group (Fig. 3B). Dexamethasone and BS012 200 mg/kg treatment significantly reduced this expression level down to 224.0 ± 15.6% and 230.0 ± 16.1% of the control group, respectively. The p75NTR protein expression level also showed a reducing trend in the BS012 100 mg/kg treatment group (324.0 ± 12.0% of the control group); however, this was not statistically significant. Under the challenge of OVA, the protein expression level of JNK was also significantly enhanced, up to 305.4 ± 32.8% of the control group (Fig. 3C). JNK protein expression level was significantly suppressed down with dexamethasone and BS012 200 mg/kg treatment, down to 133.3 ± 8.1% and 169.3 ± 21.1% of the control group, respectively. Administration of 100 mg/kg BS012 resulted in a slight decrease in JNK levels, with no statistical significance, compared to the OVA group. For the c-Jun protein expression as shown in Fig. 3D, exposure to OVA up-regulated its expression level to 169.6 ± 7.0% of the control group. BS012 treatment diminished the c-Jun protein expression in a dose-dependent manner, 100 and 200 mg/kg BS012 both significantly reduced its expression level down to 103.8 ± 5.6% and 55.3 ± 3.5% of the control group, respectively. Injection with dexamethasone also significantly suppressed c-Jun expression down to 85.8 ± 13.6% that of the control group.

Figure 3. Effect of BS012 on OVA-induced activation of NGF-mediated JNK signaling pathway in the lungs. The protein expression levels of (A) NGF, (B) p75NTR, (C) P-JNK/JNK, and (D) P-c-Jun/c-Jun activation in the lung tissues were determined via western blot analysis. Quantitative analysis was performed by densitometric analysis. All the data are expressed as the mean ± SEM (n=5 per group). *** Significantly different (p<0.001) from Control group. ^#, ^##, ^### Significantly different (p<0.05, 0.01, 0.001) from OVA group.

Effect of BS012 on the activation of Bcl-2 family proteins in OVA-induced asthma mice

We measured the expression of several proteins belonging to the Bcl-2 family (Fig. 4A-4C). We found that protein expression of P-Bad was highly upregulated by the OVA challenge, up to 307.0 ± 6.3% compared with the control group (Fig. 4A). Dexamethasone injection significantly reduced its expression level down to 158.5 ± 18.1% compared to the control group. Administration with BS012 100 and 200 mg/kg suppressed the Bad protein expression in a dose-dependent manner, but only the BS012 200 mg/kg treated group showed statistical significance against the OVA-induced group, at 259.8 ± 9.4% of the control group. Exposure to OVA also highly increased the expression level of Bax protein to 166.5 ± 7.4% of the control group (Fig. 4B). This up-regulation was significantly suppressed in the group injected with dexamethasone, at 95.2 ± 9.1% of the control group. BS012 100 and 200 mg/kg treatment suppressed the expression of Bax down to 130.8 ± 5.5% and 123.9 ± 5.5% of the control group, respectively, statistical significance against the OVA-induced group was found both in these two groups. As Fig. 4C shows, anti-apoptotic protein Bcl-xl was decreased by the OVA challenged, down to 52.8 ± 2.4% of the control group, while dexamethasone injection significantly enhanced its expression level up to 135.0 ± 12.9% of the control group. No apparent difference was observed in the BS012 100 mg/kg treated group when compared to the OVA group. In contrast, administration with BS012 200 mg/kg showed statistical significance when compared with the OVA group, at 97.5 ± 7.2% of the control group.

Figure 4. Effect of BS012 on regulating (A-C) Bcl-2 family related apoptosis proteins and (D-F) cytochrome c-initiated caspase activation pathway in ovalbumin-induced asthma mice lung tissues. Expression levels of (A) P-Bad/Bad, (B) Bax, and (C) Bcl-xl, (D) cytochrome C, (E) cleaved-caspase 9/caspase-9, and (F) cleaved-caspase 3/caspase-3 proteins in mice lung tissues were detected by western blot analysis. Quantified protein expression levels were performed by densitometric analysis. Data are presented as mean ± SEM (n=5 per group). **, *** Significantly different (p<0.01, 0.001) from Control group. ^#, ^##, ^### Significantly different (p<0.05, 0.01, 0.001) from ovalbumin group.

Effect of BS012 on cytochrome-c-initiated caspase pathway activation in OVA-induced asthma mice

Next, we analyzed the protein expression levels of cytochrome c, caspase-9, and caspase-3 to further confirm the protective effect of BS012 on the cytochrome c-initiated caspase activation pathway following OVA (Fig. 4D-4F). Significant upregulation of cytochrome-c protein expression was observed in OVA-induced group (491.5 ± 13.8%) compared to the control group (Fig. 4D). High expression level of cytochrome-c was prevented by dexamethasone treatment with statistical significance compared to the OVA group, exhibited 148.3 ± 12.4% of the control group. Even though the cytochrome-c level was not much altered by BS012 100 mg/kg treatment (441.4 ± 17.0%) when compared to the OVA group, BS012 200 mg/kg treatment significantly decreased its level down to 341.4 ± 29.5% of the control group, which was significantly different to the OVA group. For the caspase-9 protein expression as shown in Fig. 4E, exposure to the OVA increased its expression level up to 199.6 ± 10.3% of the control group. Treatment of dexamethasone and BS012 100 mg/kg slightly decreased the caspase-9 level without statistical significance when compared to the OVA group (162.1 ± 5.6% and 185.8 ± 14.4% compared to the control group, respectively). However, a significant decrease in caspase-9 level was found in the BS012 200 mg/kg treated group, down to 130.1 ± 11.8% of the control group. The expression level of caspase-3 protein (Fig. 4F) was significantly increased in the OVA group, up to 1953.0 ± 104.3% of the control group. BS012 200 mg/kg significantly reduced the caspase-3 protein expression down to 975.8 ± 158.7% compared to the control group and it showed statistical significance against the OVA-induced group. Dexamethasone and BS012 100 mg/kg treated groups showed less reduction level of caspase-3 protein expression, at 1188.0 ± 290.8% and 1238.3 ± 138.6% of the control group, respectively.

Metabolic effects of BS012 on plasma metabolome in OVA-induced asthma mice

The plasma metabolite profiles of the OVA-induced asthmatic mice were characterized using UPLC-Orbitrap-MS in both positive and negative ionization scan modes. Multivariate statistical analysis was used to visualize the cross-group comparison of the metabolic patterns of OVA-treated mice following BS012 administration (Fig. 5A, 5B). Clustering of metabolic patterns was not only visible between the control group and OVA-induced asthmatic mice but also among groups treated with dexamethasone and BS012 at different doses, suggesting that they have distinct metabolic profiles. Univariate analysis was used to identify metabolites that showed significant differences between the OVA-induced and BS012-treated groups. As a result, 20 metabolites were identified and most of them belonged to lipid and amino acid metabolism (Fig. 5C). After BS012 administration, the metabolic changes between the OVA-induced and control groups showed an inverse relationship (Supplementary Table 2). Graphs of each metabolite change in plasma are presented in Supplementary Fig. 1.

Figure 5. Changes in plasma metabolome in OVA-induced asthma mice administered with BS012 at 100, 200 mg/kg, and dexamethasone 1.5 mg/kg. The partial least squares discriminant analysis (PLS-DA) score plot derived from (A) positive and (B) negative ionization mode. (C) Heatmap summarizing the relative levels of metabolites significantly changed in the OVA-induced group compared to the control group or BS012 treated group compared to the OVA-induced group. Relative levels represent the standardized intensity values by auto scaling (the value divided by the mean center and the standard deviation of each variable).

Metabolic effects of BS012 on lung tissue metabolome in OVA-induced asthma mice

The lung tissue metabolite profiles of the OVA-induced asthmatic mice were analyzed using the same method (Fig. 6A, 6B). In the lung tissue, the metabolic profile of the OVA-induced asthma group showed significant changes compared with that of the control group. The groups administered dexamethasone and 100 mg/kg BS012 showed similar metabolic profiles, whereas the group administered 200 mg/kg BS012 was clustered. Upon identifying the metabolites contributing to these changes, a total of 26 metabolites were found to be significantly altered in the OVA-induced group compared to the control group and BS012 treated group compared to the OVA-induced asthma group (Supplementary Table 3). The identified metabolites were classified into four classes based on their related metabolism, with the majority categorized as lipid metabolites (Fig. 6C). Most metabolites showed an increasing trend after asthma induction, followed by a decrease after BS012 or Dexamethasone administration, whereas TG levels showed the opposite trend. Graphs of changes in each metabolite in the lung tissue are shown in Supplementary Fig. 2. The overall pathways of metabolic changes induced by OVA and BS012 in the plasma and lung tissues are summarized in Fig. 7.

Figure 6. Changes in lung tissue metabolome in OVA-induced asthma mice administered with BS012 at 100, 200 mg/kg, and dexamethasone 1.5 mg/kg. The partial least squares discriminant analysis (PLS-DA) score plot derived from (A) positive and (B) negative ionization mode. (C) Heatmap summarizing the relative levels of metabolites significantly changed in the OVA-induced group compared to the control group or BS012 treated group compared to the OVA-induced group. Relative levels represent the standardized intensity values by auto scaling (the value divided by the mean center and the standard deviation of each variable).

Figure 7. Overall metabolic pathways altered in systemic (plasma) and lung tissue of OVA-induced asthma mice after BS012 administration. Red arrows indicate significant changes between the OVA-induced group and the control group, while blue arrows denote significant changes between the BS012 treated group and the OVA-induced group.

Effect of N-isobutyl-dodecatetraenamide, an active compound of BS012, on isobutylTh2-specific cytokines secretion in bone marrow dendritic cell culture

Among the main components of BS012, N-isobutyl-dodecatetraenamide had an inhibitory effect on the Th2 immune response. The active compound, N-isobutyl-dodecatetraenamide, was identified and isolated through successive column chromatography over silica gel, RP-C18 silica gel, and Sephadex LH-20, with preparative HPLC (Jeong et al., 2018). N-isobutyl-dodecatetraenamide was identified based on a comparison of its NMR spectroscopic data with previously reported values, as well as the results of the MS analysis (Lopes-Lutz et al., 2011; Jeong et al., 2018). While N-isobutyl-dodecatetraenamide did not alter IL-4 levels, it significantly decreased the production of IL-13 in a concentration-dependent manner under Th2-skewing conditions (Fig. 8A, 8B). Additionally, in the DC-T cell co-culture assay, N-isobutyl-dodecatetraenamide potently inhibited IL-13 expression compared to the vehicle-treated group (Fig. 8C). After BS012 administration, the identification of N-isobutyl-dodecatetraenamide was confirmed by LC-MS analysis in the positive ionization MRM mode, which demonstrated the absorption of the active compound into the bloodstream via blood plasma (Supplementary Fig. 3).

Figure 8. Effect of N-isobutyl-dodecatetraenamide on cytokine production in CD4⁺ T cells under Th2-skewing condition. Bone marrow cells were obtained from the femur and tibia of naïve mice. Dendritic cells (DC) were prepared for co-culture by performing CD11c MACS enrichment in bone marrow derived dendritic cells. CD4⁺ T cells were enriched by collecting and pooling spleens and peripheral lymph nodes from 6-week male C57BL/6N mice, followed by CD4 MACS enrichment. The enriched samples were identified and flow-sorted for naïve CD4⁺ gated as CD4⁺CD44^lowCD25-CD62L^hi using FACSAria III cell sorter. For DC - CD4⁺ T cells co-culture assays, cells were co-cultured in complete media with soluble anti-CD3ε Ab (clone 145-2C11), anti-IFNγ (clone XMG1.2), IL-2, IL-4 in the presence of N-isobutyl-dodecatetraenamide for 3 days. Intracellular cytokines were analyzed in CD4⁺ T cells under Th2-skewing condition targeting (A) IL-4 and (B) IL-13. From the CD4⁺ T cells co-cultured with dendritic cells, (C) IL-13 production was analyzed. All the data are expressed as the mean ± SEM (n=5 per group). *, **, *** Significantly different (p<0.05, 0.01, 0.001) from Control group.

BS012 improves respiratory allergic immune responses in OVA-induced asthma mice

As a complex and chronic inflammatory disorder, several key cellular and molecular mediators are involved in the airway inflammatory response during the progression of allergic asthma. Th2 cells contribute to the immunopathology of allergic asthma by producing various inflammatory mediators, including IL-4 and IL-5, which further contribute to the hallmark features of asthma, such as IgE production, airway inflammation, and remodeling (Lambrecht et al., 2019). Upon initial exposure to the allergen, antigen-presenting dendritic cells sensitize naïve T cells and direct their development of Th2 cells. Activated Th2 cells induce the production of inflammatory cytokines, such as IL-4 and IL-13, which trigger the production of allergen-specific IgE by B cells, stimulate eosinophil production, and promote mast cell growth. Repeated exposure to allergens results in the cross-linking of membrane-bound IgE in mast cells and basophils, which induces cellular degranulation and the release of pro-inflammatory mediators. These cytokines influence early allergic reactions that manifest as edema, vasodilation, and bronchoconstriction. The events induced in the early response lead to the production and release of cytokines and chemokines, such as IL-3, IL-4, IL-5, and IL-13, and inflammatory cells, such as neutrophils, eosinophils, and T cells, which are recruited to the site of inflammation. This process is known as late-stage hypersensitivity and is characterized by mucus hypersecretion, airway inflammation, hyperresponsiveness, and airway remodeling (Humbert et al., 2019). IL-4 promotes co-stimulatory interactions between T and B cells, which are involved in the class switching of B cells to IgE synthesis (Steinke and Borish, 2001), IL-5 plays an important role in the growth, differentiation, and activation of eosinophils (Murdoch and Lloyd, 2010). Therefore, regulating Th2 cytokine secretion and IgE production is an important therapeutic approach for the treatment of allergic asthma. In this study, we demonstrated that BS012 effectively suppressed the expression levels of Th2-related cytokines (IL-4 and IL-5) and OVA-specific IgE, which were highly increased in OVA-treated mice. Taken together, these results indicate that BS012 exerts anti-asthmatic effects by downregulating Th2 cytokines and OVA-specific IgE in a dose-dependent manner.

Airway remodeling is a pathological feature of chronic asthma that contributes to the clinical manifestations of the disease, such as persistent airflow obstruction, which can be attributed to goblet cell hyperplasia, airway smooth muscle cell proliferation, and fibroblast activation with increased bronchial epithelium thickness (Hough et al., 2020). Under normal conditions, the mucous lining of the airway protects the airway epithelium from inhaled irritants (Rogers, 2004). However, increased goblet cell numbers lead to mucus hypersecretion, mucus plugging, and airflow obstruction, which contribute to the development of the pathophysiology (Rogers, 2002). Therefore, goblet cell hyperplasia is thought to have a pathophysiological significance in airway mucus hypersecretion. This study demonstrated that BS012 treatment significantly attenuated the histological changes caused by the OVA attack in a dose-dependent manner, with evidence of reduced inflammatory cell infiltration and thickness of the bronchial epithelium, decreased PAS-positive cells, and mucus secretion along with the BS012 treatment. These findings are in mechanistic correlation with those of previous studies involving the effect of AS essential oil in asthmatic and allergic rhinitis animal models, in which the inhibition of cytokines and reduction of Th2-mediated allergic responses have been reported (Zhang and Kang, 2020; Han et al., 2022). CC and PD have also been reported to have anti-allergic effects against atopic dermatitis in NC/Nga mice, where Th2-specific cellular responses were attenuated and hyperimmune responses were downregulated, as observed in the current study (Kim et al., 2011; Sung et al., 2011).

BS012 improves NGF-mediated apoptotic pathway in lungs of OVA-induced asthma mice

To explore the specific protective mechanism of BS012 in allergic asthma, we investigated its role BS012 in OVA-induced lung cell apoptosis. Previous studies have shown that NGF tends to increase at the site of inflammation in inflammatory and autoimmune diseases, especially those characterized by the abnormal activation of immune cells and increased cytokine production (Minnone et al., 2017). NGF, a member of the neurotrophic family, can induce the migration and activation of inflammatory cells, thereby infiltrating the bronchial mucosa, which helps mediate inflammatory responses in the airways and promotes the progression of allergic asthma progression (Frossard et al., 2004). A study using OVA-sensitized rats found that NGF can regulate inflammatory responses by inducing the migration and activation of inflammatory cells in the bronchial mucosa, thereby exacerbating allergic inflammation in asthma. Moreover, NGF affects the contraction, migration, differentiation, and proliferation of airway structural cells, thereby linking the NGF airway to the airway remodeling mechanism (Yang et al., 2013). Furthermore, NGF regulates neuronal survival and cell death by activating two types of receptors in the death receptor family, TrkA and p75NTR. TrkA mediates survival and differentiation, and p75NTR signals to initiate apoptosis in the absence of Trk receptors. NGF is initially synthesized as a precursor of pro-NGF. Under normal conditions, pro-NGF is neurotrophic; however, in response to injury, TrkA loss in the presence of p75NTR transfers pro-NGF, but not NGF, signaling a shift from cell survival to cell death. The pro-NGF prodomain masks the binding domain of NGF to the TrkA receptor, thus increasing its binding affinity for the p75NTR receptor and inducing apoptosis (Ioannou and Fahnestock, 2017). In addition to promoting the survival effects in conjunction with TrkA in response to NGF, p75NTR can also signal to mediate apoptosis, since NGF also shows the ability to promote cell death through a ligand-dependent signaling mechanism involving the p75 neurotrophin receptor (Barrett, 2000). The binding of NGF to the p75 neurotrophin receptor leads to a sustained increase in JNK activity and enhancement of c-Jun phosphorylation, which is thought to be involved in the signal transduction pathway leading to apoptosis (Kenchappa et al., 2010). In our study, NGF, P75NTR, JNK, and c-Jun protein expression were downregulated by BS012 treatment, especially at 200 mg/kg. Bcl-2 family proteins play a critical role in the apoptotic response under various pathophysiological conditions (Czabotar et al., 2014). Bax and Bad are members of the pro-apoptotic group of Bcl-2-related proteins and play a positive role in the process of cell apoptosis, which is essential for the (c-J)un N-terminal kinase JNK-stimulated release of cytochrome c. Bax activation is thought to be mediated in part by the isolation of multidomain anti-apoptotic Bcl2 proteins (such as Bcl-2 and Bcl-xl) by BH3-only members of the Bcl2 family (Lei and Davis, 2003). We found that BS012 significantly inhibited OVA-induced Bax and Bad protein activation. BS012 administration up-regulated the protein expression of Bcl-2. Previous studies have shown that the activation of JNK is necessary for the release of mitochondrial pro-apoptotic molecules (including cytochrome c) and cell apoptosis (Lei et al., 2002). Under homeostatic conditions, cytochrome c is located in the mitochondria and acts as an electron carrier in the mitochondrial respiratory chain. Exposure to proapoptotic stimuli causes permeabilization of the outer membrane, which allows for the release of cytochrome c. After being released from the mitochondria, cytochrome c mediates the allosteric activation of apoptosis-protease activating factor 1 in the cytosol, which is required for the activation of caspase-9 and caspase-3, the activated caspase cascade eventually leading to cell death (Elena-Real et al., 2018). Our results showed that the expression levels of cytochrome c, caspase-9, and caspase-3 were downregulated by BS012 treatment and exhibited a greater effect at a dose of 200 mg/kg. These results confirmed the protective role of BS012 against OVA-induced asthma by suppressing the apoptotic pathway.

BS012 improves systemic inflammation and energy-related metabolic abnormalities in OVA-induced asthma mice

In plasma metabolomics, which reflects systemic changes in the whole body, amino acid metabolism related to inflammation and energy metabolism was mainly altered after BS012 administration. Phenylalanine metabolism increased after OVA induction and decreased after BS012 administration. This disruption in phenylalanine metabolism is noted not only as a hallmark of atopic asthma exacerbations in children but is also significantly correlated with lung damage in patients with acute respiratory distress syndrome (Cottrill et al., 2023). Phenylalanine promotes inflammation by enhancing the innate immune response and increasing the release of inflammatory cytokines (Tang et al., 2023). The reduction in phenylalanine levels by BS012 indicates a potential relationship with the mitigation of pulmonary inflammation and shows a clear reduction pattern, distinct from that of dexamethasone, the positive control. Homoarginine is a less efficient substrate for nitric oxide synthase (NOS) than arginine and plays a regulatory role in the pathways leading to NO production and inflammation by inhibiting arginase, thereby improving the availability of arginine to NOS (Servillo et al., 2013). Therefore, decreased levels of homoarginine after dexamethasone and BS012 administration may be associated with a reduction in responses related to NO production due to inflammation. Another amino acid, L-tryptophan, plays an important precursor role in energy metabolism and has been reported to be negatively correlated with the severity of allergic asthma in patients (van der Sluijs et al., 2013). Therefore, elevated tryptophan levels after high-dose BS012 administration are associated with the restoration of systemic energy metabolism and amelioration of asthma severity. In addition, changes in plasma lactate levels related to energy and inflammatory metabolism were observed. In asthma, increased lactate levels are associated with enhanced aerobic glycolysis, which promotes T-cell activation and IL-17 secretion (Shime et al., 2008). Thus, the decrease in plasma lactate levels after BS012 administration indicated the restoration of energy metabolism and inhibition of inflammation-related metabolism.

Another significant metabolic change observed in plasma is related to lipid metabolism. Decreased sphingolipid metabolism is associated with an increased risk of developing asthma in children and lower concentrations of key phosphosphingolipids, such as sphingosine-1-phosphate, are associated with increased airway resistance (Rago et al., 2021). In our study, the decrease in sphingomyelin observed after OVA induction, along with the upregulation of sphingosine-1-phosphate and several species of sphingolipids after BS012 administration, was linked to a potential improvement in asthma-related airway symptoms, as shown in Fig. 2. Disturbances in glycerophospholipid metabolism can lead to changes in energy metabolism, endothelial dysfunction, and inflammation (Shevchenko and Simons, 2010). Specifically, polyunsaturated acyl-lysophosphatidylcholines (LPCs), which are upregulated by BS012 administration, act as lipid mediators that combat inflammation by inhibiting the effects of saturated LPCs. These compounds are crucial for triggering anti-inflammatory responses, as they suppress various inflammatory mediators, including the activation of inflammatory cells, IL-6, and NO (Liu et al., 2020). In the present study, LPC 20:4 and 18:3 showed notable increases when high doses of BS012 were administered. Improvements in glycerophospholipid metabolism were observed in both the LPC and phosphatidylcholine (PC) species. A decrease in PC levels in bronchoalveolar lavage fluid (BALF) has been associated with reduced pulmonary surfactant levels and lung function (Wright et al., 2000). Similarly, modulations in PC levels have been observed in the serum profiles of patients with asthma, correlating with asthma susceptibility genes (Ried et al., 2013). Our results indicated that the anti-inflammatory effect of BS012 contributed to the restoration of PC metabolic imbalance observed in OVA-induced asthma, resulting in a significant increase in PC levels. BS012 has been shown to regulate amino acid, glycerophospholipid, and lipid metabolism in the plasma, inducing a systemic anti-asthmatic effect by restoring the balance between inflammation and energy metabolism disrupted by asthma.

BS012 regulated inflammation-related metabolism in lung tissues of OVA-induced asthma mice

We investigated the effect of BS012 on lung tissue metabolism, which is directly influenced by asthma symptoms, and observed clear changes in inflammation-related lipid metabolism. Notably, the upregulation of glycerophospholipids was observed after OVA induction. Several studies have reported the involvement of LPCs in acute lung injury and chronic inflammatory lung diseases such as asthma. Elevation of LPC in the lungs of asthmatic subjects and the promotion of inflammatory responses by exogenous LPC administration in the lungs of animal models have shown a correlation between LPC and inflammatory damage in the lungs (Nishiyama et al., 2004; Yoder et al., 2014). Interestingly, alterations in glycerophospholipids within the lung tissue exhibited an opposite trend to those observed in plasma. This observation suggests that the increase in LPC is noticeable in the local metabolic environment of the lung tissue, where symptoms and inflammation occur directly but do not have a significant effect on the systemic environment. A significant negative correlation between glycerophospholipid levels in the extracellular (BALF) and intracellular (lung tissue) compartments was previously identified in a house dust mite-induced asthma model, suggesting that these differences may be related to choline levels, which are linked to its protective anti-inflammatory effects in airway inflammation (Mehta et al., 2010; Ho et al., 2014). These changes indicate a crucial role for glycerophospholipid metabolism in both the plasma and lung tissue, which was effectively recovered by BS012 administration. However, follow-up studies with larger numbers of tissue samples are required to further understand and validate these dynamics. Our results suggest that BS012-induced glycerophospholipid changes in plasma and lung tissues represent a potential key target for a better understanding of the local environment of asthma.

l-Carnitine plays a crucial role in enabling the transport of fatty acyl-CoA into the mitochondria for fatty acid oxidation. Inhibition of this process results in the accumulation of acylcarnitine derivatives (Al-Biltagi et al., 2012). Previous studies have shown that increased levels of acetylcarnitine in the lung tissues of asthma models indicate a systemic shift in energy metabolism owing to decreased fatty acid oxidation (Yu et al., 2017). The observed significant reduction in acetylcarnitine levels following BS012 administration could be related to the potential of BS012 to restore energy metabolism in the body.

In addition to changes in lipid metabolism, alterations in purine metabolism have also been observed in the lung tissue. The observed increase in inosine levels in bronchial asthma models can occur when adenosine is metabolized to inosine, potentially because of the breakdown of ATP induced during inflammation in allergic asthma (Moon et al., 2010). Furthermore, in bronchial asthma models, an increase in adenosine deaminase, an enzyme that catabolizes purines and protects against the inflammatory response, is correlated with the inflammatory response (Barankiewicz and Cohen, 1985). Therefore, the significant reduction observed following the administration of 200 mg/kg BS012 suggests that it may modulate the inflammatory response through mechanisms involving both anti-allergic and tissue-protective effects. These findings revealed that BS012 could effectively modulate inflammation-related metabolism in the lung tissues of OVA-induced asthmatic mice, significantly impacting lipid and purine metabolism.

N-isobutyl-dodecatetraenamide, a major component of BS012, suppresses Th2 immune response

We performed additional experiments to determine which of the major components of BS012, identified in our previous study, influenced this mechanism of action (Lee et al., 2023). We considered compounds that inhibit the Th2 immune response, which is a major factor in allergic diseases, to be active compounds. Th2-specific cytokines such as IL-4 and IL-13 play crucial roles in the pathophysiology of allergic asthma (Leon and Ballesteros-Tato, 2021). Therefore, we assessed the inhibitory effects of IL-4 and IL-13 on Th-2 cells and DC-T cells. We identified N-isobutyl-dodecatetraenamide, one of the major components of BS012, which suppresses the expression of IL-13 at the cellular level. This result demonstrates that N-isobutyl-dodecatetraenamide plays a role in the anti-allergic effect of BS012 and further studies are needed to confirm its efficacy and the underlying mechanism.

In conclusion, this study demonstrated that BS012, an herbal remedy comprising Asarum sieboldii, Platycodon grandiflorum, and Cinnamomum Cassia, exhibited anti-allergic and protective effects against OVA-induced allergic asthma in mice. Oral administration of BS012 suppressed lung inflammation by reducing the release of Th2-specific cytokines, such as IL-4, IL-5, and IgE, as well as by inhibiting inflammatory cell recruitment. Additionally, BS012 alleviated the thickening of the bronchial epithelium and goblet cell hyperplasia and regulated the levels of mucus production, thereby alleviating airway obstruction and relieving the structural changes found in allergic asthma. BS012 treatment led to a dose-dependent decrease in the expression of apoptotic proteins in lung tissue. We also investigated the effects of BS012 on systemic inflammation and metabolic dysregulation in OVA-induced asthmatic mice. BS012 administration restored systemic imbalances in inflammation, energy metabolism-related amino acids, and glycerophospholipid metabolism in the plasma, while reversing sphingolipid metabolism disturbances, indicating the potential recovery of markers associated with asthma-related airway symptoms. The metabolic effect of BS012 in lung tissues suggests the regulation of lipid and purine metabolism, hinting at the alleviation of inflammatory responses. We identified N-isobutyl-dodecatetraenamide, a major component of BS012, which exhibited remarkable inhibition of Th2-specific cytokine IL-13 expression. This requires further in-depth investigation. In summary, we have demonstrated that BS012 is a promising anti-inflammatory and anti-apoptotic therapeutic agent for improving the pathophysiology of allergic asthma. This is performed not only through molecular markers and mechanisms but also through systemic and tissue-specific metabolic pathways. Furthermore, by identifying the key components responsible for such effects, we aimed to facilitate the development of BS012 as a clinical treatment for asthma-related conditions through subsequent research efforts.

This research was funded by the Bio-Synergy Research Project [NRF2013M3A9C4078145] [NRF 2012M3A9C4048794] of the Ministry of Science, ICT, and Future Planning through the National Research Foundation of the Korea Institute of Science and Technology Institutional Program [2E31623].

The authors declare that they have no competing interests.