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.