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.