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.