The Inhibition of Mast Cell Activation of Radix Paeoniae alba Extraction Identified by TCRP Based and Conventional Cell Function Assay Systems

Chinese herbs have long been used to treat allergic disease, but recently the development was greatly impeded by the lack of good methods to explore the mechanism of action. Here, we showed the effects of Chinese herb Radix Paeoniae alba were identified and characterized by a mast cell activation assay that involves electronic impedance readouts for dynamic monitoring of cellular responses to produce time-dependent cell responding profiles (TCRPs), and the anti-allergic activities were further confirmed with various conventional molecular and cell biology tools. We found Radix P. alba can dose-dependently inhibit TCPRs, and have anti-allergic function in vitro and in vivo. Radix P. alba suppressed mast cell degranulation not only inhibiting the translocation of granules to the plasma membrane, but also blocking membrane fusion and exocytosis; and that there may be other anti-allergic components in addition to paeoniflorin. Our results suggest that Radix P. alba regulated mast cell activation with multiple targets, and this approach is also suitable for discovering other mast cell degranulation-targeting Chinese herbs and their potential multi-target mechanisms.


Introduction
Traditional Chinese Medicine (TCM) has been used as front-line pharmacotherapy for various diseases for many millennia in China. However, the development of TCM was greatly impeded owing to the lack of modern standards for the complexity of Chinese herb ingredient [1]. While no matter a single herb or herbal formula contains thousands of components that simultaneously modulate multiple pharmacological targets, which may be one of the main reasons for their observed therapeutic effects beyond the capabilities of a single compound, as well as the less adverse effects for disease prevention and chronic conditions compared to those of Western medicine [2][3][4][5]. Thus, methods for the assessment of the efficacy of TCM with multiple, interactive measures are required in order to promote its modernization.
To address this issue, several cell-based, high-throughput phenotypic approaches have been developed to determine the global responses of the targets of natural products to specific perturbations. The living cell morphological profiling method, can be used to dynamically monitor the cellular response to treatments by producing time-dependent cell response profiles (TCRPs) [6]. With this method, cells were seeded onto the surface of microelectronic cell sensor arrays integrated into the bottom of microtiter plates, and dynamically monitored cell-substrate impedance as reflected with Cell Index (CI) [7]. It is suggested the CI value basically correlates with three cellular parameters, namely cell number, morphology and attachment quality. All three parameters are intricately linked to signaling pathways regulating various facets of cellular physiology, and therefore this approach allows for expansion of the "biological space" at which a Chinese herb is screened and provides ample opportunity detect and identify biological activity associated with drugs in an unbiased manner [6,8,9].
Radix Paeoniae alba (Radix P. alba) is a Chinese herb, which has been documented to treat allergic disorders in traditional medical clinics [10,11]. The extraction of total glucosides from peony was approved as an anti-inflammatory drug by the China Food and Drug Administration. However, no data are available on the mechanism of action of the anti-allergic effect of Radix P. alba. We therefore set out to analyze the anti-allergic effect of Radix P. alba with TCRP technology and determine the potential mechanisms.

Reagents and Antibodies
All the reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless indicated otherwise. Phorbol 12-myristate 13-acetate (PMA) was obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Adenosine was obtained from the National Institutes Food and Drug Control, China (NIFDC). Gab2 short interfering RNA (siRNA) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Cyto Tox96 Non-Radioactive Cytotoxicity assay kit was obtained from Promega (Madison, WI).

Extraction and Separation
Radix P. alba (see S1 Fig) was cleaned, and crushed, and added with distilled water to ultrasonic extraction and decoction treatments for 30 min each. After centrifugation at 10000 rpm for 15 min, the supernatant was used for screening with the xCELLignece system (ACEA, CA).
To isolate the various ingredients of Radix P. alba, a dried sample (6 g) was pulverized and extracted three times with boiled water (80:1, v/w). The water solution was further extracted with petroleum ether (1:1, v/v). The petroleum ether extract was evaporated to dryness to obtain the petroleum ether component (PE). The water solution was sequentially extracted with ethyl acetate, n-butyl alcohol, and ethanol (1:1, v/v each), and the extracts were evaporated to dryness to obtain the ethyl acetate (EA), n-butyl alcohol (NBA), and ethanol (EtOH) components, respectively. The ethanol precipitate was evaporated to obtain the H 2 O components.

TCRPs Detected Using the xCELLigence System
TCRPs were recorded with the xCELLigence system as described previously [6,12,13], with 3 independent experiments run in triplicate for each test. Briefly, 50 μL of complete DMEM was added to 96-well E-Plates to obtain background readings before the addition of 100 μL of the cell suspension. After incubating at room temperature for 30 min, the E-Plates were placed onto the RTCA SP station in the incubator and the CI was recorded. The cells were allowed to grow for 18-24 h before the addition of the indicated extracts of Radix P. alba or compounds. The cells were monitored every 2 min for more than 3 h after treatment in order to capture the short-term TCRPs.
Cell Morphology Analysis Based on an Immunofluorescence Assay RBL-2H3 cells were seeded in 24-well tissue culture plates containing glass coverslips and allowed to attach and spread for 24 h. The cells were treated with various antigens, including dinitrophenyl-bovine serum albumin (DNP-BSA; Invitrogen Molecular Probes; Carlsbad, CA), adenosine, and PMA, for the indicated times, and then fixed with 4% paraformaldehyde for 15 min. The cells were permeabilized in phosphate-buffered saline (PBS) containing 0.

Degranulation Assay
RBL-2H3 cells were cultured in 24-well plates for 24 h, washed, incubated in Tyrode buffer, and then stimulated with the following antigen: 100 ng/mL DNP-BSA (after incubation with 100 ng/mL anti-DNP-IgE for 12 h), PMA, or adenosine for 30 min. Degranulation was determined based on β-hexosaminidase release as described previously [14].

Animal Model and Administration
Adult male ICR mice (SLRC Laboratory Animals, Shanghai, China) weighing 18-22 g were used in the experiments. The protocol was approved by the Committee on the Ethics of Animal Experiments of Zhejiang Chinese Medical University. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Radix P. alba at various concentrations (800 mg/kg, 400 mg/kg, 200 mg/kg) or dexamethasone (1.0mg/kg) was administered intragastrically for 6 days following sensitization with dinitrofluorobenzene (DNFB) (Shanghai Chemical Reagent Company, Shanghai, China) onto the shaved abdominal skin of the mice. The negative control mice were normally sensitized and painted with olive oil alone when challenged. The positive control mice with contact dermatitis were given normal saline. Indexes, including immunohistochemistry of the ear with hematoxylin and eosin staining (original magnification ×200), and ear weights were assayed after the second challenge on the ears with DNFB [15,16].

Calcium Measurements
Calcium levels were assayed using the Fluo4-AM Calcium Indicator (Dojindo; Kumamoto, Japan). The cells were grown in a 96-well plate for 24 h, the various extractions of Radix P. alba were added for 2 h, and then the plates were washed with Hanks' balanced salt solution (HBSS) three times. The cells were loaded with Fluo4-AM dye for 30 min at 37°C and washed with HBSS three more times, followed by the addition of stimulants. The changes in dye fluorescence were monitored on the SpectraMax M5 Multifunctional microplate reader (Molecular Devices; Sunnyvale, CA). [Ca 2+ ] i mobilization was represented as expressed as the measured of fluorescence intensity value.

Immunoblotting
IgE-sensitized RBL-2H3 cells were pretreated with Radix P. alba and its fractions for 2 h and then stimulated with 100 ng/mL DNP-BSA for 30 min. Equal amounts of cellular protein were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After blocking, the membranes were incubated with the primary antibodies, p-Erk/Erk and p-Akt/Akt (Cell Signaling Technology), and IRDye800 goat anti-Rabbit IgG secondary antibody (Li-Cor; Lincoln, NE). Immunoreactive proteins were visualized using the Odyssey Infrared Imaging System (Li-Cor).

Statistical Analysis
All data are reported as the means ± S.E.M. Data were analyzed using standard statistical methods of one-way ANOVA followed by Student's t-tests using SPSS 15.0 software. P < 0.05 was considered as statistically significant.

Recording Mast Cell Degranulation with TCRPs
In mast cells, crosslinking high-affinity IgE receptor (FcεRI) stimulation with an antigen results in the degranulation of secretory vesicles accompanied by dramatic remodeling of the cellular cytoskeleton [17][18][19], and this process can be recorded and characterized using TCRPs. Representative dose-dependent TCRPs produced by IgE-mediated RBL-2H3 cell activation were shown in Fig 1A, which displayed the impedance between the cell memberane and the sensor surface correlating with cell morphology and adhesion. Activation of the FcεRI receptor results in subsequent activation of downstream signaling pathways involving phosphorylation and dephosphorylation of a number of signal proteins that are indispensable for mast cell degranulation. Therefore, we selected some reported pharmacological inhibitors to pretreat IgE-sensitized RBL-2H3 cells, which were then stimulated with DNP-BSA. As shown in Fig 1B, the TCRPs revealed that certain protein kinase inhibitors impaired IgE-mediated mast cell degranulation, which is in agreement with previous reports [20][21][22]. The Gab2 mRNA expression level and mast cell degranulation was decreased by Gab2 siRNA treatment (see S2 Fig). To determine whether the IgE-mediated TCRP was associated with RBL-2H3 mast cell activation, IgE-mediated morphological changes and mediators release were measured. As seen in Fig 1C  and 1D, aggregation of the IgE-bound FcεRI receptor triggered the release of β-hexosaminidase, a marker for mast cell degranulation, as well as time-dependent dynamic reorganization of the actin cytoskeleton. Taken together, these results show that impedance values are in line with the change of morphological dynamics and mediator release, and the IgE-mediated impedance-based mast cell TCRP could fully reflect the mast cell degranulation process. Therefore, this approach is suitable for high-throughput screening of compounds or Chinese herbs that target mast cell degranulation. Mast Cell TCRP-Directed Screening of Radix P. alba Then we use this mast cell TCPR to identify the effects of Radix P. alba. As shown in Fig 2A, Radix P. alba dose-dependently attenuated the impedance response of the IgE-mediated TCRP under the non-cytotoxic dose (S3 Fig). To verify the hypothesis that a decrease of the CI in TCPR is predictive of inhibiting mast cell activation. The activity of β-hexosaminidase released by activated mast cells was further determined. The results showed Radix P. alba significantly reduced the release of β-hexosaminidase (Fig 2B), and inhibited vesicle fusion in activated mast cells (Fig 2C).
Furthermore, we evaluated the anti-allergic activity of Radix P. alba in vivo using a DNFB induced ear swelling mouse model. Ear swelling was induced by placing DNFB into the ear and was assessed by measuring ear weight 24 h later. The inflamed auricles of the mice were examined histologically following hematoxylin and eosin staining. Severe inflammation such as edema, epidermal hyperplasia, and infiltration of inflammatory cells was observed in DNFBtreated mouse ears ( Fig 2D). As expected, Radix P. alba treatment ameliorated inflammation of the epidermis of mice just as well as treatment with the positive control drug dexamethasone ( Fig 2D). Application of DNFB resulted in a significant increase in ear weight, which was greatly reduced by Radix P. alba treatment (Fig 2E).

Microtubule-Dependent Granule Translocation and Calcium-Dependent Membrane Fusion and Exocytosis Based on TCRP
Recent studies have demonstrated that FcεRI-dependent degranulation involves at least two processes: (1) the calcium-independent, microtubule-dependent translocation of granules to the plasma membrane and (2) calcium-dependent membrane fusion and exocytosis [18,23]. The two main events of mast cell degranulation involve different signaling cascades. The microtubule-dependent translocation of granules to the plasma membrane depends on the phosphoinositide 3 kinase (PI3K)/AKT signaling pathway, whereas calcium-dependent membrane fusion and exocytosis require protein kinase C (PKC) signaling [18]. We examined TCRPs treated with adenosine (Binding to A3R GPCRs and leading to the activation of PI3Kγ) [24] and PMA (a PKC agonist). As shown in Fig 3A and 3B, both adenosine and PMA caused an immediate and transient increase with a subsequent decrease in the CI, but there were differences in the duration time to complete the response. Therefore, each treatment showed its own characteristic 'signature' pattern. To determine whether adenosine and PMA stimulated mast cell degranulation, we examined the staining distribution pattern of CD63, a broadly expressed transmembrane protein [25], and determined the level of β-hexosaminidase release. After stimulation with DNP-BSA or adenosine, the granules labeled with CD63 were translocated to the plasma membrane in RBL-2H3 cells, but this did not occur in cells treated with PMA ( Fig 3C). Furthermore, neither adenosine nor PMA induced β-hexosaminidase release, which agrees with previous reports by Yanase et al. [19] and Nunomura et al. [26] (S4A Fig). In addition, we observed enhancement of the formation of microtubule in cells treated with adenosine, and membrane ruffling of the cortical F-actin in cells treated with PMA, as shown in Fig  3D. Moreover, adenosine amplified the effects of DNP-BSA-stimulated mast cell degranulation [26]; however, this result was not observed in PMA-treated cells (S4B Fig). Because vesicle translocation and PKC-dependent Ca 2+ activation are two of the most important processes for mast cell degranulation, we examined whether RBL-2H3 cells stimulated with both adenosine and PMA showed induced degranulation. Indeed, regardless of the order of pre-treatment or concentration of adenosine and PMA, the combined treatment induced mast cell degranulation in all cells (Fig 3E). These results indicated that the adenosine and PMA TCRPs could respectively record mast cell-activated processes based on the translocation of vesicles and exocytosis-mediated cytoskeleton rearrangement.

Characterization of Multi-Targets in the Chinese Herb Radix P. alba
The effectiveness of TCM for the treatment and prevention of various diseases has been well documented, and tends to show a multi-target pattern in its mechanism. Indeed, the underlying theory of TCM is rooted in the fundamental principle that Chinese herbs regulate and restore the balance of a disorder of the human body through multiple targets and components [3,27]. We next investigated whether Radix P. alba could suppress mast cell degranulation in a multi-targeted manner using various mast cell activation TCRPs. First, we tested the effect of Radix P. alba on the adenosine and PMA TCRPs. As shown in Fig 4A and 4B, Radix P. alba inhibited the mast cell activation that was stimulated by either adenosine or PMA in a dosedependent manner. To determine the specific activity of Radix P. alba, we further detected the changes of phosphorylated Akt and Erk (p-Akt and p-Erk, respectively) levels in mast cell degranulation. The results showed a dose-dependent decrease of p-Akt and p-Erk levels in Radix P. alba-treated cells (Fig 4C). Measurement of Ca 2+ influx also showed that stimulation of IgE-sensitized mast cells with DNP-BSA induced a rapid Ca 2+ flux, which was dose-dependently inhibited by treatment of Radix P. alba (Fig 4D and 4E). These results demonstrated that Radix P. alba inhibits mast cell degranulation with a multi-target pattern, and that the application of TCRP technology represents a rapid analytical tool for elucidating the potential cellular mechanisms of Chinese herbs.

TCRP-Directed Isolation of Radix P. alba
To investigate the active ingredients inhibiting the mast cell activation in Radix P. alba, we introduced mast cell TCRPs for identifying active components in combination with chemical separation techniques. The Radix P. alba crude exact was serially extracted with organic solvents and separated into five fractions. The fractions extracted with PE and EtOH dramatically decreased the peak values of adenosine-mediated TCRP (Fig 5A), which correspondingly down-regulated the p-Akt and p-Erk levels (Fig 5C). The EA, NBA, and EtOH fractions significantly reduced the peak values of the PMA-mediated TCRP (Fig 5B). To further verify these effects, we analyzed the Ca 2+ influx of RBL-2H3 cells triggered by DNP-BSA in the presence or absence of the different fractions. The EA, NBA, and EtOH fractions significantly decreased the Ca 2+ influx (Fig 5D), which was consistent with the TCRPs. We identified the components of the five fractions with HPLC analysis, and found that the PE fraction did not contain paeoniflorin, a known anti-allergic ingredient of Radix P. alba [28][29][30][31] (S5A Fig). This suggested there may be other active components present. We further examined the effects of the different fractions on β-hexosaminidase release. The PE, EA, and NBA fractions all significantly decreased the β-hexosaminidase release levels, while the EtOH fraction only slightly inhibited the degranulation level ( S5B Fig). This may due to the relative differences in the inhibiting power of the different signaling pathways involved. Together, these results demonstrate the multi-targeted function of Radix P. alba produced by its various components (Table 1).

Discussion and Conclusion
In this work, we used mast cell TCRPs to screen anti-allergic action of Chinese herb Radix P. alba. There are several important features that are critical for their application to natural product screening. First, the TCRP technology has the capacity to quantitatively measure minute changes in cell morphology with a very specific manner depending on the Rho family GTPase being activated. As we have known, IgE-mediated mast cell degranulation usually accompanies  the actin cytoskeleton and cell morphology changes. While the actin cytoskeleton is that of a dynamic and plastic system that controls the intracellular functional signaling and not simply a static framework assigned to maintain cellular architecture. Therefore, the profiles generated with IgE-mediated mast cell degranulation are amenable to modulation by biologically active natural products in a manner that is dependent on mechanism and concentration of the sample being tested. Radix P. alba initially screened by TCRP was suggested that it inhibited mast cell activation. We confirmed this hypothesis by in vitro and in vivo experiments, and the results showed Radix P. alba decreased allergic response by inhibiting mast cell degranulation. The mast cell degranulation process can be dissected into two main events: the microtubule-dependent translocation of granules to the plasma membrane, and actin-dependent granule-plasma membrane fusion and exocytosis; and these two processes respectively depend on Gab2/PI3K/AKT signaling and calcium signaling, they allow for a second feature, which is that IgE-mediated TCRP can also be decomposed into two components: (1) granule-translocation TCRP stimulated by adenosine and (2) granule-plasma membrane fusion and exocytosis TCRP induced by PMA. Our results showed individually activating any of them couldn't induce mast cell degranulation, but simultaneously could, which suggested these two pathways are essential for mast cell activation. We also found Radix P. alba not only inhibited adenosine TCRPs but also attenuated PMA TCRPs, which indicated Radix P. alba regulated mast cell degranulation by inhibiting the translocation of granules to the plasma membrane as well as membrane fusion and exocytosis, as further was confirmed with well-orchestrated traditional tools.
Radix P. alba as traditional medicine has the role of anti-pyretic and anti-inflammatory activity. Recent years, studies showed it and its formulas have anti-allergic effects, inhibiting ovalbumin-induced allergic rhinitis [10], IgE-mediated type I hypersensitivity response [32], and decreasing histamine release from rat mast cells. Bomi et al. found the main constituents paeonol and paeoniflorin both can inhibit the mast cell degranulation, while paeonol was more powerful one [32]. Myungsuk et al. detected the anti-allergic effects of seventeen isolated compounds from Paeonia anomala used in Mongolian traditional medicine, and found 7 compounds can inhibit mast cell degranulation [33]. These studies demonstrated there are multicomponent involved in inhibiting mast cell activation, as is also consistent with our results.
Together, Radix P. alba inhibited mast cell activation with multi-target, and this approach is also suitable for discovering other mast cell degranulation-targeting Chinese herbs and their potential multi-target mechanisms.
Supporting Information S1 Data. Raw data.