4. DISCUSSION
Asthma causes serious health and socioeconomic problems worldwide because it affects a large portion of the population, from children to adults. The prevalence, hospitalization rate and mortality of asthma have markedly increased over the past half century (Dharmage, Perret, & Custovic, 2019). Over the past few decades, asthma treatment has focused on relieving inflammation and bronchoconstriction in the respiratory system with corticosteroids, leukotriene modifiers, and anti-cholinergic agents. Recently, novel targeted therapies have been developed based on improved pathophysiologic and biologic understanding. The targets mainly include IgE, Th2 cell subset-derived cytokines, C-X-C motif chemokine receptor 2 (CXCR2), C-C motif chemokine receptor 3 (CCR3), and tyrosine kinases (Wechsler, 2018). In this study, we demonstrated the anti-allergic and anti-asthmatic effects of sophoricoside that suppress mast cell activation and CD4+ T cell differentiation.
Sophoricoside is an isoflavone substance containing genistein glycoside with a glucose moiety at position 4. Isoflavones are widely contained in soybeans and soy protein products. They play an important role in maintaining human health via prevention of various chronic human diseases, including inflammatory disorders. Sophoricoside has been isolated from S. japonica , a plant of the Leguminosae family. Sophoricoside was present in the highest amount in seeds (3.302 mg/g DW), followed by leaves (0.048 mg/g DW), and it was present in trace amounts in the stems and roots of S. japonica . Since initial isolation and hormonal effects of sophoricoside were reported (Gabor, 1961), there have been few reports of its biological activities. We were interested in the inhibitory effects of sophoricoside on IL-3, IL-5, and IL-6 bioactivities (Min et al., 1999; Yun et al., 2000) and its anti-inflammatory and immunosuppressive effects (Kim et al., 2003; Kim et al., 2013; Lee et al., 2013). Specifically, IL-3, IL-5, and IL-6 are important pro-inflammatory cytokines produced from Th2 cells that play an essential role in the pathogenesis of inflammatory, allergic, and asthmatic diseases (Zhu, Yamane, & Paul, 2010). Allergen-induced allergic and asthmatic diseases are mainly associated with type 2 inflammation, which is induced by pro-inflammatory cytokines produced from Th2 cells (Gauvreau, El-Gammal, & O’Byrne, 2015). Results suggested that sophoricoside may affect Th2 cell-mediated allergic and asthmatic diseases.
Allergic and asthmatic diseases are commonly induced by the sensitization to and challenge with foreign antigens. The patterns of airway inflammation are caused by immune cells such as eosinophils, neutrophils, basophils, and lymphocytes controlled by CD4+ T cell-subsets, including Th1, Th2, and Th17 (Lambrecht & Hammad, 2105). Since asthmatic inflammation is considered a hallmark of Th2 cells, we generated acute allergic and asthmatic airway inflammation by intraperitoneal sensitization and intratracheal challenge with OVA in BALB/c mice. OVA is widely used to induce experimental asthma in animals. OVA induces asthmatic airway inflammation features through T cell antigen receptor (TCR)-mediated Th2 type immune responses (Durrant & Metzger, 2010). The main symptoms are characterized by influx of immune cells into the lungs, excessive production of mucus and AHR in the airway, and elevated levels of serum IgE (Holgate et al., 2015; Casaro et al., 2019). The mouse model generated by OVA showed elevated allergic and asthmatic symptoms, indicating well-induced airway inflammation in the mice. Sophoricoside treatment exhibited effective anti-allergic and anti-asthmatic activities through reduced nasal rubbing, recruitment of inflammatory cells into the lungs, AHR, and incresed levels of IgE, pro-inflammatory cytokines, histamine, and arachidonic acid metabolites. Sophoricoside also effectively decreased allergic responses such as ear swelling and mast cell-mediated leakage of blood vessels in IgE-antigen reaction-induced PCA mice. The results obtained from in vivo models indicate that sophoricoside is a promising reagent for preventing allergy and asthma.
Mast cells and eosinophils are the main effector cells of allergic inflammation. Activated mast cells contribute to secretion of histamine and newly-synthesized lipid metabolites that are responsible for production of various cytokines, chemokines, and growth factors, including IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-11, IL-13, GM-CSF, TNF-α, transforming growth factor-beta (TGF-β), basic fibroblast growth factor (b-FGF), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF) (Minai-Fleminger & Levi-Schaffer, 2009; Stone, Prussin, & Metcalfe, 2010). IgE and Th2 subset signature cytokines IL-4 and IL-13 play an important role in maintaining mast cell homeostasis, and released mast cell metabolites recruit Th2 cells and eosinophils to inflammatory sites to promote allergic process (Leung, Beukema, & Shen, 2015; McLeod, Baker, & Ryan, 2015; Simon et al., 2020). To evaluate whether sophoricoside influences mast cell activation, we determined the amounts of released histamine and arachidonic acid metabolites in OVA-induced mice and human mast cells stimulated with IgE plus antigen. Sophoricoside decreased the levels of released histamine and arachidonic acid metabolites in OVA-induced mice and mast cells, indicating an inhibitory effect of sophoricoside on mast cell activation and reduced allergic responses. Histamine and arachidonic acid metabolites released from IgE-primed mast cells recruit immune cells, such as eosinophils, neutrophils, basophils, and Th2 cells (Metcalfe et al., 2016; Schauberger et al., 2016). Based on these signaling networks, the correlation between mast cell activation, immune cell recruitment, and CD4+ T cell differentiation is tightly correlated with exacerbation of allergic inflammation. In this pathogenic circumstance, sophoricoside reduced allergic airway inflammation by inhibition of signaling loops.
Naïve CD4+ T cell-derived effector subsets mainly include Th1, Th2, Th17, and Treg, which play important roles in direct immune responses. These cells aggravate allergic airway inflammation by producing lineage-specific signature cytokines and recruiting immune cells to inflammatory sites (Zhu, Yamane, & Paul, 2010; Akdis & Akdis, 2014; Lambrecht &  Hammad, 2015). Interestingly, sophoricoside more strongly inhibited the release of Th2 and Th17 specific cytokines, such as IL-4, IL-5, IL-13, and IL-17, compared to Th1 specific cytokines, such as IFN-γ and TNF-α. Consistent results were also observed within vitro differentiation experiments of naïve CD4+ T cells into Th1, Th2, and Th17 cell subsets. Sophoricoside also inhibited the mRNA levels of master regulators, T-bet, GATA-3, and RORγt, for Th1, Th2, and Th17 cell subsets, respectively, as well as the production and mRNA levels of Th cell-specific cytokines. The inhibitory effects of sophoricoside on Th2 and Th17 cell subsets were slightly stronger than those on the Th1 cell subset.
In summary, we isolated sophoricoside from mature seeds of S. japonica to investigate its anti-allergic and anti-asthmatic activities in animal models. There was higher sophoricoside content in mature seeds than in leaves, stems, and roots. Sophoricoside reduced allergic and asthmatic responses in OVA-induced allergic airway inflammation and PCA models by inhibiting mast cell activation and CD4+ T cell differentiation. Our results imply that sophoricoside may be a useful therapeutic candidate for treating allergic diseases, including allergic asthma.