FIGURE LEGENDS
Figure 1. Chemical structure of sophoricoside and HPLC
chromatograms. Chemical structure of sophoricoside and its HPLC
chromatogram (A). HPLC chromatograms of EtOH extracts of seeds (B),
leaves (C), stems (D), and roots (E) of S. japonica . * indicates
sophoricoside.
Figure 2. Sophoricoside reduces allergic airway inflammation in
OVA-induced mice. Experimental scheme for OVA-induced allergic airway
inflammation in BALB/c mice (A). Number of nasal rubbing events was
determined for 10 min on the day 26 (B). Histology of lung (upper),
liver (middle), and spleen (bottom) was assessed with H&E staining.
Scale bars: 100 or 200 µm or 1.0 mm (C). AHR was measured by Penh value
under inhalation with indicated concentrations of methacholine (D).
#p < 0.005 versus vehicle-treated group; **p< 0.005 versus OVA-induced group (n = 6). SOP,
sophoricoside.
Figure 3. Sophoricoside inhibits recruitment of immune cells
into allergic airway in OVA-induced mice. Cells prepared from BALF were
applied to a slide by cytospinning and stained with Diff-Quick or
Wright-Giemsa solution. The number of total cells, macrophages,
eosinophils, neutrophils, and lymphocytes was determined by counting
within a 1 mm2 area (A). #p < 0.005
versus vehicle-treated group; **p < 0.005 versus
OVA-induced group (n = 6). (B and C) Cells were stained with
either Diff-Quick or Wright-Giemsa solution, and total cells (B) and
eosinophils (C) were observed under light microscope. Scale bars: 50 or
20 µm. SOP, sophoricoside.
Figure 4. Sophoricoside inhibits release of pro-inflammatory
cytokines in OVA-induced mice. Amounts of pro-inflammatory cytokines
were measured by ELISA from serum of OVA-induced mice (A). #p< 0.005 versus vehicle-treated group; *p <
0.05 and **p < 0.005 versus OVA-induced group (n= 6). (B and C) Draining lymph node cells (B) and BALF cells (C) were
stained with PE-conjugated anti-CD4 antibody, APC-conjugated anti-IL-4
antibody, or FITC-conjugated anti-IFN-γ antibody, and populations of
CD4-, IL-4-, and IFN-γ-positive cells were counted by FACS analysis.
SOP, sophoricoside.
Figure 5. Sophoricoside inhibits secretion of immunoglobulins in
OVA-induced mice. (A-D) Amounts of total IgE (A), OVA-specific IgE (B),
OVA-specific IgG1 (C), and OVA-specific IgG2a (D) were measured by ELISA
from serum of OVA-induced mice. #p < 0.005 versus
vehicle-treated group; **p < 0.005 versus OVA-induced
group (n = 6). (E and F) Total cells prepared from BALF were
stained with FITC-conjugated anti-IgE or PE-conjugated anti-IgG1
antibody, and populations of IgE- and IgG1-positive cells were counted
by FACS analysis. SOP, sophoricoside.
Figure 6. Sophoricoside inhibits allergic response in PCA mice.Experimental scheme for PCA experiment in C57BL/6 mice (A). (B-D) Mice
were passively sensitized with 100 ng anti-DNP IgE. Twenty-four hours
after intradermal injection of anti-DNP IgE, sophoricoside was topically
applied to mouse ears 1 h before intravenous injection of 100 µg DNP-HSA
containing 5% Evans’ blue dye. One hour after DNP-HSA injection, images
of the ears (B), amounts of Evans blue dye (C), and ear swelling (D)
were measured. #p < 0.005 versus vehicle-treated
group; *p < 0.05 and **p < 0.005
versus IgE/antigen-induced group (n = 5). SOP, sophoricoside.
Figure 7. Sophoricoside inhibits release of histamine and
arachidonic acid metabolites. (A and B) Amounts of histamine (A) and
LTC4 (B) were measured by ELISA from BALF. #p< 0.005 versus vehicle-treated group; **p <
0.005 versus OVA-induced group (n = 6). (C-F) HMC-1 cells were
sensitized with 1 µg/mL anti-DNP-IgE overnight. Cells were washed twice
and treated with sophoricoside for 30 min, followed by activation with
DNP-HAS (10 µg/mL). Amounts of histamine (C), PGD2 (D),
LTB4 (E), and LTC4 (F) were measured by
ELISA. #p < 0.005 versus vehicle-treated group;
*p < 0.05 and **p < 0.005 versus
IgE/antigen-induced group (n = 3). (G) HMC-1 cells were incubated
with various concentrations of sophoricoside for 24 h, and their
viability was measured. SOP, sophoricoside.
Figure 8. Sophoricoside inhibits CD4+ T cell
differentiation. CD4+ T cells were incubated with
various concentrations of sophoricoside for 24-72 h, and their viability
was measured (A). (B-D) Naïve CD4+ T cells were
differentiated under appropriate differentiation conditions for 5 days
in the presence or absence of sophoricoside (30 µM), as described in
METHODS. Total RNA was isolated, and qRT-PCR was performed to analyze
levels of CD4+ T cell lineage-specific master
regulators (B) and cytokines (C). Cytokine amounts were measured by
ELISA using cultured supernatants (D). **p < 0.005
versus differentiated group (n = 3). SOP, sophoricoside.