3.1. Identification and sophoricoside content of S.
japonica
The structure of sophoricoside from S. japonica was assigned by
nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses
(data not shown). Structural elucidation and identification were carried
out with MS using a JEOL JMS-600W mass spectrometer (Tokyo, Japan), and1H- and 13C-NMR spectra were created
using a Bruker AVANCE 500 NMR spectrometer (Rheinstetten, Germany). Fast
atom bombardment (FAB)-MS and NMR data were compared with values in the
literature (Wu et al., 2012). The content of sophoricoside in seeds,
leaves, stems, and roots of S. japonica by HPLC analysis are
shown in Figure 1. Sophoricoside was present in the highest amount in
seeds (3.302 mg/g DW), followed by leaves (0.048 mg/g DW). Sophoricoside
was present in trace amounts in stems and roots of S. japonica(Table 1).
3.2. Sophoricoside reduces
allergic and asthmatic features in OVA-induced mice
IL-3 and IL-5 are Th2 cytokines associated with allergic airway
inflammation
(Asquith
et al, 2008;
Dougan, Dranoff,
&
Dougan,
2019) and sophoricoside inhibited their bioactivities (Yun et al.,
2000), suggesting that allergic airway inflammation can be controlled by
sophoricoside. To demonstrate this possibility, an allergic airway
inflammation model was established in mice by sensitization and
challenge with OVA (Figure 2A). The model was used to evaluate whether
sophoricoside suppresses allergic and asthmatic symptoms. Nasal rubbing
was considered a visible allergic symptom on day 26 before sacrificing
mice. Induction of allergic asthma increased nasal rubbing compared with
the normal group, and the behavior was decreased with administration of
sophoricoside (Figure 2B). Histologic analyses showed increased
infiltrating inflammatory cells in the airways and thickened airway
walls in OVA-induced mice compared with normal mice. The administration
of sophoricoside reduced this infiltration and thickening without
cytotoxic effects in livers and spleens (Figure 2C). These results
indicate that sophoricoside has anti-allergic and anti-asthmatic
activities achieved through suppression of airway inflammation.
3.3. Sophoricoside
decreases AHR in OVA-induced mice
AHR is a cardinal feature in the pathogenesis of bronchial asthma
because allergic airway inflammation increases AHR and excessive
bronchoconstriction in asthmatic patients (Busse, 2010). Therefore, we
evaluated whether sophoricoside could improve shortness of breath and
inappropriate airflow in the airways of allergic inflammation-induced
asthmatic mice. Airway function was determined by calculating the Penh
value using a whole-body plethysmograph. Upon inhalation of
methacholine, the Penh value increased in a concentration-dependent
manner in OVA-induced mice compared with normal mice. Conversely, the
administration of sophoricoside decreased values from 7.19 ± 0.62 to
6.28 ± 0.56 and 3.93 ± 0.40 at 3 and 30 mg/kg compared with OVA-induced
asthmatic mice at 80 mg/mL inhalation of methacholine (Figure 2D),
indicating the anti-asthmatic effect of sophoricoside.
3.4. Sophoricoside
decreases recruitment of immune cells into the allergic airway
Airway inflammation, a predominant features of allergic asthma, recruits
immune cells such as eosinophils, macrophages, neutrophils, and
lymphocytes (Saglani &
Lloyd,
2015). The number of various immune cells in BALF was counted to
evaluate the anti-inflammatory effect of sophoricoside. Upon
OVA-challenge, the number of total cells and immune cells, including
macrophages, eosinophils, neutrophils, and lymphocytes, was dramatically
increased compared with normal control mice. The number of recruited
immune cells was effectively decreased by the administration of
sophoricoside in OVA-challenged mice (Figure 3). The results indicate
that sophoricoside decreases recruitment of immune cells into regions of
allergic airway inflammation.
3.5. Sophoricoside
decreases release of pro-inflammatory cytokines
Immune cells produce many inflammatory mediators that exacerbate
allergic airway inflammation by inducing histamine release through mast
cell activation (Pawankar et al., 2015;
Hirose
et al., 2017). Analysis of the released pro-inflammatory cytokines
showed consistent results regarding recruitment of total cells and
immune cells. Sophoricoside effectively decreased the amount of released
pro-inflammatory cytokines, including IFN-γ, TNF-α, IL-4, IL-5, IL-13,
and IL-17, compared with the serum of OVA-challenged mice (Figure 4A).
Interestingly, the inhibitory effect of sophoricoside on Th2 and Th17
cell-specific cytokines (IL-4, IL-5, IL-13, and IL-17) was stronger than
that of Th1 cell-specific cytokines (IFN-γ and TNF-α). Consistently, the
population of both IFN-γ- and IL-4-positive cells decreased in both
draining lymph nodes and BALF cells, and the population of IL-4-positive
cells was more effectively decreased than that of IFN-γ-positive cells
(Figure 4B and C). These results indicate that sophoricoside has
anti-inflammatory activity and may have a therapeutic effect on Th cell
subsets, in particular Th2 and Th17 cells.
3.6. Sophoricoside
decreases secretion of immunoglobulins in OVA-induced mice
Elevated levels of immunoglobulins, specifically IgE, are key upstream
players in allergic asthma. Therefore, anti-IgE therapy is a recommended
therapeutic strategy for improving severe allergic asthma
(Platts-Mills,
2001;
Perez
et al., 2017). The levels of total IgE and OVA-specific IgE, IgG1, and
IgG2a in mice serum were measured. Higher levels of all immunoglobulins
were observed in OVA-challenged mice than normal control mice.
Conversely, the administration of sophoricoside effectively decreased
levels of all immunoglobulins (Figure 5A-D). Consistent results were
also observed by FACS analysis in BALF cells showing that elevated
populations of IgE- and IgG1-positive cells with OVA-induction were
significantly decreased by sophoricoside treatment (Figure 5E and F).
These results indicate that sophoricoside can successfully reduce
secretion of immunoglobulins, including IgE, and may improve the
symptoms of allergic asthma.
3.7. Sophoricoside reduces
allergic response in PCA mice
To further demonstrate the anti-allergic effect of sophoricoside by
reducing IgE release and mast cell activation, acute allergic skin
response was induced in a PCA mouse model by passive sensitization with
intradermal injection of DNP-specific IgE into mice ears and intravenous
challenge with soluble DNP-HSA antigen containing 5% Evans blue dye
(Figure 6A). Topical application of sophoricoside decreased mast
cell-mediated leakage of blood vessels, resulting in decreased amount of
diffused dye (Figure 6B). Dye intensity was decreased by about 24.6 ±
2.4% and 90.9 ± 1.0% with 3 and 30 mg/kg sophoricoside treatment,
respectively, compared with DNP-specific IgE-induced PCA mice (Figure
6C). The increased ear swelling also reduced from 16.5 ± 1.9 µm to 14.2
± 1.2 and 9.4 ± 0.8 µm with 3 and 30 mg/kg sophoricoside, respectively,
compared with DNP-specific IgE-induced PCA mice (Figure 6D). These
results indicate that sophoricoside enacts an anti-allergic effect
through suppression of IgE-mediated antibody-antigen reactions.
3.8. Sophoricoside
decreases release of histamine and lipid metabolites
Mast cells are responsible for immediate allergic reactions initiated by
cross-linking of allergens to high-affinity receptor-bound surface IgE.
Released histamine and lipid metabolites, such as PGD2and LTs, from IgE-primed mast cells induce allergic reactions and
inflammation by recruiting immune cells such as neutrophils,
eosinophils, basophils, and Th2 cells
(Metcalfe
et al., 2016;
Schauberger
et al., 2016). Therefore, the amounts of released histamine was
measured to determine mast cell activation and degranulation in
OVA-induced mice. OVA-induction leads to dramatically elevated histamine
release in BALF compared with normal mice, and the amounts were markedly
decreased by sophoricoside treatment (Figure 7A). Sophoricoside also
decreased the amount of LTC4, an arachidonic acid
metabolite associated with allergy and asthma, in BALF of OVA-induced
mice (Figure 7B). The release of histamine and arachidonic acid
metabolites, including PGD2, LTB4, and
LTC4, in HMC-1 cells was further observed after
activation with anti-DNP-IgE plus DNP-HSA. Consistent results were seen
with OVA-induced mice, and treatment with sophoricoside effectively
decreased histamine release in activated HMC-1 cells (Figure 7C). In
addition, sophoricoside treatment decreased the amounts of
PGD2, LTB4, and LTC4compared to untreated HMC-1 cells (Figure 7D-F) without affecting cell
viability (Figure 7G). These results indicate that sophoricoside
inhibits allergic reactions by preventing release of histamine and
arachidonic acid metabolites via inhibition of mast cell activation.
3.9. Sophoricoside
suppresses CD4+ T cell differentiation
Differentiated CD4+ T cell driven airway inflammatory
responses also play an important role in allergen-mediated allergic
asthma. In particular, Th2 cells promote IgE-mediated sensitization,
airway hyperreactivity, and eosinophilia by producing Th2 cell-specific
cytokines
(Ling
&
Luster,
2016;
, , &
2017). Figure 4 shows results indicating that sophoricoside inhibited
production of Th cell-specific cytokines in serum of OVA-challenged
mice, suggesting that sophoricoside inhibits CD4+ T
cell differentiation. The cytotoxic activity of sophoricoside was
evaluated before determining the effect of sophoricoside on
CD4+ T cell differentiation. Sophoricoside exhibited
weak to no cytotoxic activity in cultured mouse CD4+ T
cells in vitro at concentrations of up to 300 µM for 24 to 72 h
(Figure 8A). To confirm whether sophoricoside affects
CD4+ T cell differentiation, qRT-PCR analysis was
performed to determine expression of Th cell lineage-specific master
regulators in cultured naïve CD4+ T cells under the
appropriate differentiation conditions. Sophoricoside markedly
suppressed mRNA levels of T-bet, GATA-3 and RORγt, which are the master
regulators for Th1, Th2, and Th17 cell subsets (Figure 8B). In addition,
the mRNA levels of Th1 lineage-specific cytokine IFN-γ, Th2
lineage-specific cytokine IL-4, and Th17 lineage-specific cytokine
IL-17A were effectively decreased by sophoricoside treatment (Figure
8C). Consistent with the mRNA levels of master regulators and cytokines,
sophoricoside significantly decreased the amounts of released IFN-γ,
TNF-α, and IL-17A (Figure 8D). These results indicate that sophoricoside
might improve Th cell-mediated airway inflammatory responses, including
allergic asthma, by inhibiting CD4+ T cell
differentiation.