RBL-2H3 cells are rat basophil leukemia cells, purchased from the Cell Bank of Chinese Academy of Sciences, Shanghai, China. They were cultured in DMEM with 10% FBS at 37°C and 5% CO₂. Cells in the logarithmic growth period were used for the experiments. Pre-experiments testing the effect of C48/80 as the positive control on basophil granulocytes showed that the optimal density and time period for inducing degranulation were 2×10⁵ cells/ml and 60 min.

The amount of histamine secretion is the most direct and reliable indicator of degranulation in RBL-2H3 cells (18,19). However, as histamine has a short half-life, low molecular weight and poor immunogenicity, clinically it is more common to use β-hexosaminidase measurements to evaluate pseudoallergic reactions, as in vitro, β-hexosaminidase secretion by RBL-2H3 cells is concomitantly increased by degranulation. RBL-2H3 cells were plated in 24-wells cell and incubated overnight at a density of 2×10⁵ cells/well. The following day, the cells were washed three times with incubation buffer and 100 μl of incubation buffer were added to each well followed by incubation for 10 min at 37°C. Subsequently, 100 μl of various 3-caffeoylquinic acid concentrations or an equal amount of negative control (buffer) were added and the cells were incubated at 37°C for 60 min. Following incubation, the cells were placed in an ice bath for 10 min to terminate the reaction. Subsequently, 50 μl supernatant from each well were transferred to a 96-well plate and 50 μl of substrate were added (1 mM p-nitrophenyl-N-acety-β-D-glucosaminide, citrate/sodium citrate buffer, pH 4.5). The samples were incubated at 37°C for 60 min and then 150 μl of stop solution (0.1 M Na₂CO₃/NaHCO₃, pH 10.0) were added to terminate the reaction. The light absorbance at 405 nm was measured in each well as a quantitative analysis of β-hexosaminidase secretion. In the 24-well plates, the remaining supernatant was removed and 200 μl of Triton X-100 (0.5%) were added to lyse the cells. The cell lysate was centrifuged at 3,000 rpm for 1 min and 50 μl supernatant (four times) were transferred to a 96-well plate in order to measure the intracellular β-hexosaminidase concentration in the cell lysates. The following formula was used to calculate the β-hexosaminidase secretion rate: β-hexosaminidase secretion rate (%) = [supernatant light absorbance/(supernatant light absorbance + lysate light absorbance)] ×100%.

RBL-2H3 cells (1×10⁷) from the negative control group and from the group treated with various concentrations of 3-caffeoylquinic acid 1 h were collected, washed thoroughly with pre-chilled PBS and centrifuged at 1,500 rpm for 10 min at 4°C. Pre-chilled 2.5% glutaraldehyde was added to the centrifuge tubes where a cell mass had formed after centrifugation and the mixture was incubated overnight at 4°C. Finally, the samples were fixed with 1% osmium tetroxide at 4°C, then dehydrated and permeated according to normal electron microscopy sample preparation procedures. The samples were then embedded in Epon 812, cut into ultrathin slices, stained according to normal procedures and photographed under an H-600A Transmission Electron Microscope (Hitachi High-Technologies Corp., Tokyo, Japan).

Cells were plated in a petri dish and on the following day, the cells were treated with either the drug or negative control (no drug) for 1 h in incubation buffer. Subsequently, the cells were washed twice with pre-chilled PBS and an appropriate amount of IP cell lysis buffer (containing proteinase inhibitor; Amresco, Solon, OH, USA) was added and after the cells were lysed on ice for 30 min they were centrifuged at 15,000 × g for 15 min at 4°C. An aliquot of the supernatant was used to measure the protein content using a BCA Assay (Amresco). A small amount of lysate was saved for western blot analysis and to the remaining lysate, 1 μg of 4G10 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and 10–50 μl of protein G-beads were added followed by overnight incubation at 4°C with gentle agitation. After the immunoprecipitation reaction, the magnetic beads were stabilized with a magnet while the supernatant was carefully removed. Protein G-beads were washed three to four times with 500 μl lysis buffer followed by the addition of 10 μl 5X SDS loading buffer and boiling for 5 min. The cell lysis supernatants and immunoprecipitation samples were run through SDS-PAGE for 100 min at 90 V for separation and the proteins were then transferred onto a PVDF membrane for western blot analysis. TBST with 5% non-fat milk was used for blocking at room temperature for 1 h. After removing the blocking buffer, the primary antibody, anti-Syk, was added (1:500 dilution; Santa Cruz Biotechnology, Inc.) and the membranes were incubated at room temperature for 2 h with gentle agitation. The primary antibody was then removed and the membranes were washed with TBST for 3×10 min. Finally, secondary antibody coupled with horseradish peroxidase was added (1:6,000 dilution) at room temperature for 1 h with gentle agitation. Then the secondary antibody was removed and the membrane was washed with TBST for 3×10 min. Extra liquid was removed with filter paper and ECL chemiluminescence reagent was added (ECL reagents and PMSF proteinase inhibitors were both purchased from Biyuntian Biotech, Jiangsu, China). An Alpha Innotech imaging system was used for detection (20,21). For further calculations the following formula was used:

A total of 1×10⁵ cells/ml were plated in a 35-mm glass-bottom petri dish the previous day and grown overnight. The following day, the extra DMEM medium was removed, the cells were washed twice with D-Hank’s medium and 500 μl D-Hank’s medium were added. After 10 min of incubation at 37°C in a CO₂ incubator, 500 μl of 10 μM Fluo-3 AM working solution were added (final concentration 5 μM) and the samples were incubated for 30 min in an incubator. Following incubation, D-Hank’s medium was used to wash away extra probes. Fluo-3 AM is a fluorescent dye that can penetrate the cell membrane and its fluorescence is very weak without intensifying higher calcium ion concentrations. After Fluo-3 AM enters the cell and is cleaved by esterases inside the cell, it becomes Fluo-3, which is trapped inside the cell and binds to calcium ions. Fluo-3 is not fluorescent when not bound to calcium ions, but when bound to calcium ions its fluorescence (526 nm) increases by a factor of at least 40. In the actual experiment, the recommended excitation wavelength was approximately 488 nm and emission wavelength was 525–530 nm. Samples were photographed under a confocal microscope to use as a background and the changes in intracellular calcium ion concentration were observed as the appropriate drug was slowly added.

Cells were treated with the appropriate drug or an equal amount of negative control (incubation buffer) for 1 h, after which the drug or buffer was removed and the cells were washed three times with PBS. Cells were fixed with 4% paraformaldehyde at room temperature for 15 min, washed with PBS for 30 sec, permeabilized with 0.5% Triton X-100 for 10 min and washed again with PBS for 30 sec. Subsequently, 200 μl of 100 nM rhodamine-phalloidin (purchased from Cytoskeleton, Inc., Denver, CO, USA) were added, the samples were incubated in the dark at room temperature for 30 min, washed three times with PBS and photographed under a confocal laser microscope. Alternatively, 300 μl of methanol were added and the samples were incubated in the dark for 2 h. The supernatant was transferred to a black plate with a transparent bottom and fluorescence was detected at 535/590 nm.

Cells were plated the previous day. To each well 2 μM NBD-C6-HPC dye (NBD-C6-HPC fluorescent dye was purchased from Invitrogen, Carlsbad, CA, USA) was added and the samples were incubated at room temperature in the dark for 30 min, washed three times with PBS, treated with the appropriate drug or an equal amount of negative control (incubation buffer) for 1 h and washed again three times with PBS. Under a confocal laser, a circular area on the cell membrane of a fixed size was selected and changes in membrane fluidity were observed using photo bleaching. The formula used for calculating the fluorescence recovery was as follows:

where F_R is the maximum fluorescence intensity after recovery, F₀ is the fluorescence intensity after photo bleaching and F₁ is the fluorescence intensity before photo bleaching (22).

Each of the in vitro experiments presented in this study were repeated multiple (≥3) times. A one-way analysis of variance (ANOVA) was used for the comparison of multiple experimental groups. When ANOVA yielded significance, pairwise comparisons using a t-test with the Bonferroni adjustment were conducted between two groups. The results were considered statistically significant if a two-tailed P-value was <0.05.

Treating RBL-2H3 basophil granulocytes with C48/80 as the positive control showed that the RBL-2H3 cells secreted β-hexosaminidase in a dose- and time-dependent manner (Table I). After 60 min of C48/80 incubation, the relative β-hexosaminidase secretion rate by RBL-2H3 cells reached a maximum, which dropped to lower levels after an additional hour in all groups. Treating RBL-2H3 cells with C48/80 can be used as a positive control for pseudoallergic reactions. We used chlorogenic acid and its analogues (3-caffeoylquinic acid, 4-caffeoylquinic acid, 3,4-dicaffeoylquinic acid and 1,5-dicaffeoylquinic acid) to test whether a similar reaction occurs with caffeoylquinic acids. Both mono-caffeoylquinic acids and dicaffeoylquinic acids induced RBL-2H3 cells to secrete β-hexosaminidase in a concentration-dependent manner after 60 min of incubation. The pseudoallergic reactions induced by dicaffeoylquinic acids were relatively weaker compared with those induced by mono-caffeoylquinic acids (Table II). These results revealed that 3-caffeoylquinic acid induces pseudoallergic reactions in RBL-2H3 cells.

Since RBL-2H3 is a rat basophilic leukemia cell line, the cells have high affinity IgE receptors on their surface and they can undergo degranulation following activation. Thus, in this study, we performed a NBD-C6-HPC assay experiment to monitor the cell membrane fluidity of the RBL-2H3 cells. 3-Caffeoylquinic acid (1.0 mmol/l) was added to the RBL-2H3 cells for 60 min and by comparing the fluorescence intensity before photo bleaching, after photo bleaching and after recovery, we found that cells in the 3-caffeoylquinic acid-treated group showed significantly lower fluorescence recovery compared with the cells in the negative control group (Fig. 1). This indicates a change in the fluidity of the cell membrane molecules. We also used electron microscopy to observe the ultrastructures of the cell membranes (Fig. 2). The results revealed that in the negative control group, the RBL-2H3 cells had a clear nucleolus, the cell membranes had more microvilli and in the cytoplasm there were grain-like structures of varying sizes, while the cells contained dense granules. By contrast, in the cells treated with 3-caffeoylquinic acid, there was a marked increase in the number of empty vesicles in the cytoplasm, the chromatin in the nucleus was more agglomerated and most significantly, the number of granules in the cells were reduced, the cytoplasm was thinner, and the number of vesicles had increased. In addition, a large number of empty bubble-like structures appeared and the granule-containing vesicles migrated towards the cell membrane, fused with it for exocytosis; these features are typical of degranulation (Fig. 2).

Since a change in the fluidity of the cell membrane can affect the concentrations of calcium ions inside and outside the membrane, we measured the concentration of calcium ions inside the RBL-2H3 cells treated with 3-caffeoylquinic acid. We found that after treatment with 5.0 mmol/l 3-caffeoylquinic acid, the calcium ion level in the RBL-2H3 cells increased and binding with Fluo-3 caused a 300% higher fluorescence signal, indicating that a high 3-caffeoylquinic acid concentration can induce changes in the calcium ion concentration; furthermore, the rate of calcium ion influx increased in a basically linear fashion along with the drug treatment time (Fig. 3).

Syk is an important kinase for signal transduction pathways of B cell activation and an important signaling molecule for FcγR and FcɛR signal transduction. RBL-2H3 is a rat basophilic leukemia cell line with high affinity IgE receptors on the surface of the cells and the FcR of IgE can be recognized by FcγR and FcɛR. Thus, when a multivalent antigen (such as 3-caffeoylquinic acid) binds with IgE and causes two or more FcɛRI to couple, it can activate mast cells, leading to Syk phosphorylation, which activates PKC. We measured phospho-Syk and total Syk protein 60 min after the addition of 1 mmol/l of 3-caffeoylquinic acid to the cells and found that 3-caffeoylquinic acid induced Syk phosphorylation in the RBL-2H3 cells (Fig. 4).

The polymers of the cytoskeleton control the morphology and kinetics of eukaryotic cells and consist of three major forms: actin filaments (AFs), MTs and intermediate filaments (IFs). These three components are organized into a network to resist changes in cell shape, but in response to an external force, they can reorganize themselves, thus playing an important role in maintaining cell integrity. Our quantitative F-actin measurements in the RBL-2H3 cells treated with 3-caffeoylquinic acid revealed that in the 3-caffeoylquinic acid-treated group, compared with the negative control group, significantly more actin depolymerization occurred (Fig. 5), indicating that 3-caffeoylquinic acid induces the reorganization of the cytoskeleton.