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. 2014 Aug 8;19(8):11883-95.
doi: 10.3390/molecules190811883.

Synephrine inhibits eotaxin-1 expression via the STAT6 signaling pathway

Kyung-Baeg Roh  1 , Il-Hyun Kim  2 , Young-Soo Kim  3 , Myungjae Lee  4 , Jung-A Lee  5 , Eunsun Jung  6 , Deokhoon Park  7
Affiliations

Synephrine inhibits eotaxin-1 expression via the STAT6 signaling pathway

Kyung-Baeg Roh et al. Molecules. .

Abstract

Citrus contain various flavonoids and alkaloids that have multiple biological activities. It is known that the immature Citrus contains larger amounts of bioactive components, than do the mature plants. Although Citrus flavonoids are well known for their biological activities, Citrus alkaloids have not previously been assessed. In this study, we identified synephrine alkaloids as an active compound from immature Citrus unshiu, and investigated the effect of synephrine on eotaxin-1 expression. Eotaxin-1 is a potent chemoattractant for eosinophils, and a critical mediator, during the development of eosinophilic inflammation. We found that synephrine significantly inhibited IL-4-induced eotaxin-1 expression. This synephrine effect was mediated through the inhibition of STAT6 phosphorylation in JAK/STAT signaling. We also found that eosinophil recruitment induced by eotaxin-1 overexpression was inhibited by synephrine. Taken together, these findings indicate that inhibiting IL-4-induced eotaxin-1 expression by synephrine occurs primarily through the suppression of eosinophil recruitment, which is mediated by inhibiting STAT6 phosphorylation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Immature Citrus unshiu fruit extracts inhibit eotaxin-1 expression. (a) The eotaxin-1 gene promoter-luciferase reporter vector was transfected into 70% confluent NIH/3T3 cells, and cultured for 24 h. The cells were pretreated with immature and mature Citrus unshiu fruit extracts at 1, 10, 100 ppm, respectively, for 1 h, and then stimulated with IL-4. Luciferase activity was calculated, against IL-4-unstimulated control; (b) Cells were pretreated with immature and mature Citrus unshiu fruit extract at 1, 10, 100 ppm, respectively, for 1 h, and then further incubated with IL-4 (50 ng/mL), for 24 h. Eotaxin-1 release was then determined, using an ELISA. Data are representative of at least three independent experiments. Results are mean ± standard deviation (SD) (n = 3). ¶ p < 0.05 vs. IL-4-untreated control. ** p < 0.05 vs. IL-4-treated control.
Figure 2
Figure 2
Phytochemical analysis between immature Citrus unshiu and mature Citrus unshiu fruit extract. (a) Contents of synephrine were analyzed by high performance liquid chromatography (HPLC). (b) Quantitative analysis of synephrine in mature Citrus unshiu fruits and immature Citrus unshiu fruits extracts.
Figure 3
Figure 3
Effect of synephrine on eotaxin-1 gene expression. (a) The eotaxin-1 gene promoter-luciferase reporter vector was transfected into 70% confluent NIH/3T3 cells, and cultured for 24 h. The cells were pretreated with synephrine for 1 h, and then stimulated with IL-4. Luciferase activity was calculated against IL-4-unstimulated control; (b) Cells were cultured in serum-free DMEM, in the presence of indicated concentrations of synephrine, for 24 h. Total RNA was isolated, and eotaxin-1 mRNA levels were assayed by quantitative real-time PCR. Data are representative of at least three independent experiments; (c) Cells were pretreated with synephrine for 1 h, and then further incubated with IL-4 (50 ng/mL), for 24 h. Eotaxin-1 release was then determined, using an ELISA. (d) Cells were pretreated with the indicated concentrations of synephrine for 1 h, and then further incubated with TNF-α (50 ng/mL), for 24 h. Eotaxin-1 release was then determined, using an ELISA. Results are mean ± standard deviation (SD) (n = 3). ¶ p < 0.05 vs. IL-4-untreated control. ** p < 0.05 vs. IL-4-treated control.
Figure 4
Figure 4
Effects of synephrine on the activation of JAK1 and STAT6. NIH/3T3 cells were starved for 24 h, and then treated with IL-4 (50 ng/mL), in the presence of synephrine. Cell lysates were prepared at 10 min, and then subjected to Western blot analysis. The bands for phospho-JAK1 were detected, and normalized to that of β-actin (a); and STAT6 bands were detected, and normalized to that of total STAT6 (b). (c, d) NIH/3T3 cells were treated with synephrine for 3 h, before stimulation with IL-4 (50 ng/mL). After 1 h stimulation, the cells were fixed, permeabilized, and then incubated with rabbit antibodies against STAT6, for 2 h. Thereafter, cells were stained with FITC-conjugated anti-rabbit IgG, and nuclei were stained with Hoechst 33342. Translocation rate was calculated, using the IN Cell Analyzer 1000 instrument. Data are representative of at least three independent experiments. ¶ p < 0.05 vs. IL-4-α-untreated control. ** p < 0.05 vs. IL-4-treated control.
Figure 5
Figure 5
Effects of signal inhibitors on eotaxin-1 expression in NIH/3T3 cells. (a) NIH/3T3 cells were pretreated with JAK1 inhibitor, pyridone 6 or STAT6 inhibitor, AS1517499 for 3 h, and then further incubated with IL-4 (50 ng/mL), for 24 h. Eotaxin-1 production was analyzed by ELISA; (b) Cell viability was measured, using the MTT assay. Data are representative of at least three independent experiments. Results are mean ± standard deviation (SD) (n = 3).¶ p < 0.05 vs. IL-4-untreated control. ** p < 0.05 vs. IL-4-treated control.
Figure 6
Figure 6
Effects of synephrine on the recruitment of eoinophils. EoL-1 cells were incubated for 7 days at 37 °C, in RPMI medium containing 500 μM n-butyrate. (a) The cell surface expression of CCR3 was determined by flowcytometry. Red histograms show CCR3, and isotype controls are represented in the black histograms; (b) Cell lysates were prepared at 7 days, and then subjected to Western blot analysis. The bands for CCR3 were detected, and normalized to that of β-actin. Migration assays were performed, using CytoSelect 24-well Cell Migration Assay kits. Human normal fibroblasts, 5 × 104 cells were seeded in lower chambers, and incubated for 24 h. Lower chambers were pretreated with synephrine, JAK1 inhibitor, pyridine 6 and STAT6 inhibitor, AS1517499 respectively, and then stimulated with IL-4. Differentiated EoL-1 cells, 2 × 106 cells were added to the upper chamber of the transwell plates, with 3 μM pore-size filters. IL-4-induced eotaxin-1 was measured using ELISA, in lower chambers (c). The number of eosinophils migrating from upper chambers to lower chambers was determined by fluorescence (d). Data are representative of at least three independent experiments. Results are mean ± standard deviation (SD) (n = 2). ¶ p < 0.05 vs. IL-4-untreated control. ** p < 0.05 vs. IL-4-treated control.
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