1. Introduction
Synephrine is an alkaloid that was initially isolated as a synthetic organic compound, first isolated as a natural product from the leaves of various
Citrus trees, and which presence has been noted in different
Citrus juices [
1,
2]. The peel of unripe
Citrus fruits is the part of the plant that has the highest level of synephrine [
3]. The concentration of synephrine is higher in fruits of small diameter, when compared to larger ones, probably due to the higher levels found in their peels, which decrease with the maturation of the fruit [
4,
5]. Synephrine is also found in the human organism, where it is considered a trace amine, due to its low plasmatic levels [
6]. The preparations used in Traditional Chinese Medicine (TCM) are the immature and dried whole fruits from
Citrus aurantium[
7]. Extracts of
Citrus fruits or purified synephrine are also marketed in the US, sometimes in combination with caffeine, as a weight-loss-promoting dietary supplement for oral consumption. Chemically, synephrine is similar to adrenaline or epinephrine. Synephrine activates several types of adrenergic receptors [
8,
9]. Therefore, it is used pharmacologically as a sympathomimetic agent, with vasoconstrictor and bronchiectatic agent.
Eosinophils are crucial effector cells, in the inflammatory reactions associated with allergic-inflammatory diseases and parasitic infections. Their products, such as cytotoxic granule proteins, leukotrienes, and cytokines, are involved in the pathological changes seen at the sites of inflammation. Eosinophils are recruited to the sites of inflammation by locally released chemotactic agents. The CC chemokine, eotaxin-1, a potent chemoattractnat for eosinohpils, is a critical mediator for the development and perpetuation of allergen-induced eosinophilic inflammation [
10]. Eotaxin-1 is produced at high levels in patients with atopic dermatitis, and is localized to the inflammation site. This concentrated expression may preferentially target eosinophils to the epithelium, and induce degranulation, leading to the release of epithelium-damaging proteins [
11].
The JAK-STAT signaling pathway becomes activated, in response to cytokines like interleukins (IL), interferons (IFN), and certain peptide hormones [
12,
13,
14], all of which are thought to have biologically significant roles in inflammatory diseases [
15]. IL-4 is a pivotal cytokine associated with allergic disease that supports Th2 development, and that is produced by Th2 cells, basophils, and mast cells [
16,
17,
18]. The transcription factor STAT6 was shown to be responsible for the IL-4-mediated induction of eotaxin-1 [
19], and could contribute to allergen-induced airway eosinophilia, and eosinophilic inflammation [
20,
21]. Therefore, investigating the possible involvement of various JAK-STAT regulators in immune disease and the associated molecular mechanisms is an important area of research.
In the present study, we investigated the effects of synephrine on IL-4-induced eotaxin-1 production, and its mechanisms of action. In addition, we demonstrated that synephrine inhibits STAT6 phosphorylation, during IL-4-induced upregulation of eotaxin-1 in fibroblasts.
3. Experimental Section
3.1. Cell Culture and Materials
NIH/3T3 mouse fibroblast cell line and Normal human fibroblasts (NHFs) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Carlsbad, CA, USA), containing 10% Fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), at 37 °C, under 5% CO2. The EoL-1 human eosinophilic leukemia cell line was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). EoL-1 cells were cultured in RPMI1640 containing 10% FBS and 1% penicillin/streptomycin, at 37 °C, under 5% CO2.
3.3. Cytokines and Pharmacological Inhibitors
Synephrine was purchased from Sigma Aldrich (St. Louis, MO, USA). Recombinant mouse IL-4, human IL-4, human eotaxin-1, human CCR3-PE, and mouse IgG2b κ isotype PE were all purchased from eBioscience (San Diego, CA, USA). Pyridone 6 (JAK Inhibitor I) was purchased from Calbiochem (Darmstadt, Germany). Phospho-STAT6 and total-STAT6 antibody were purchased from Abcam (Cambridge, UK), and STAT6 inhibitor was purchased from Axon Medchem (Groningen, NL, USA). Phospho-JAK1 and β-actin antibody were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA).
3.4. Cell Viability Assay
Cell viability was measured using the MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; USB Corp., Cleveland, OH, USA) assay. Cells were plated in triplicate wells of 12-well plates, and incubated overnight. The cells were then treated with synephrine for 24 h, under a serum-free condition. Then, MTT reagent (1 mg/mL) was added to each well, and the cells were incubated for 3 h. The medium was removed, and the cells were solubilized with dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA). The absorbance was measured by spectrophotometer at a wavelength of 570 nm.
3.5. Enzyme-Linked Immunosorbent Assay (ELISA)
Eotaxin-1 concentrations were quantified in cell culture supernatants, after treatment with synephrine induced by IL-4, using a commercially available ELISA kit (eBioscience). Cell culture supernatants were collected 24 h after treatment with 200 μM synephrine, and assayed for eotaxin-1. The standard curve was linearized, and subjected to regression analysis. Eotaxin-1 concentrations were determined, using a standard curve. All samples and standards were measured in duplicate.
3.6. Transient Transfection and Luciferase Assay
NIH/3T3 cells were transfected with the eotaxin-1 luciferase reporters, using the SuperFect®Transfection Reagent (Qiagen, Hilden, Germany). After 24 h incubation, cells were incubated in the presence or absence of synephrine induced by IL-4, for 24 h. The cells were then harvested and lysed, and supernatants were assayed for luciferase activity. Luciferase activity was determined, using a Dual Luciferase Assay System (Promega, Madison, WI, USA), and an Infinite® 200 PRO luminometer (Tecan, AG, Männedorf, Switzerland). Activity was expressed as the ratio of STAT6-dependent firefly luciferase activity, to control thymidine kinase Renilla luciferase activity.
3.7. Immunohistochemistry
STAT6 translocation was determined by immunohistochemistry. An aliquot of 1 × 102 cells was seeded in 96-well plates, cultured for 24 h, and then treated with IL-4 in the presence or absence of synephrine, for 60 min. The cells were fixed in 4% paraformaldehyde, washed with PBS, permeabilized with 0.2% Triton X-100, and incubated with a rabbit polyclonal antibody to STAT6 (1:200, 5% goat serum in PBS) for 2 h, followed by fluorescein isothiocyanate (Santa Cruz Biotechnology, Santa Cruz, CA, USA) goat anti-rabbit IgG (1:200), for 1 h. After three washes with PBS, the cells were incubated with 0.5 μg/mL Hoechst 33342 (Invitrogen), for 5 min. The cells were then examined, using the IN Cell Analyzer 1000 (GE Healthcare Lifescience, Uppsala, Sweden).
3.8. Total RNA Extraction, cDNA Synthesis, and Quantitative PCR
Total RNA extraction was carried out using the RNeasy kit (Qiagen), according to the manufacturer’s instructions. The first cDNA was synthesized using a PrimeScript First Strand cDNA Synthesis kit (Takara Bio, Japan), according to the manufacturer’s instructions. Eotaxin-1 mRNA expression was measured by real-time quantitative PCR, using a specific TaqMan probe (CCL11, Applied Biosystems, Foster City, CA, USA). TaqMan gene expression assays were performed on the ABI PRISM 7300 System (Applied Biosystems), according to the manufacturer’s instructions. All samples were analyzed in triplicate, and the levels of the detected mRNAs were normalized to control mouse beta-2-microglobulin (Applied Biosystems) values. The normalized data were used to quantify the relative levels of a given mRNA, according to ΔCt analysis.
3.9. Cell Migration Assay
We used the Cytoselect Cell Migration Assay kit (Cell Biolabs, San Diego, CA, USA), according to the manufacturer’s instructions. Briefly, differentiated eosinophilic EoL-1 cells and human fibroblasts were co-cultured in two-compartment Transwell system. For eosinophilic EoL-1 cells, 2 × 106 cells in 400 μL serum free medium were added to the upper chamber of the Transwell plates, with 3-μm-pore filters. Lower chambers were first seeded with fibroblasts at 5 × 104 cells in 500 μL serum free medium, for 24 h. Synephrine and/or IL-4 were added to the lower wells, for 24 h. After 24 h incubation, migratory EoL-1 cells were subsequently lysed, and detected by fluorescence measurement.
3.10. Statistical Analysis
All data are expressed as means ± standard deviations. Differences between the control and the treatment group were evaluated by one way ANOVA. p < 0.05 was considered statistically significant.