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

<div><p>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 <i>Paeoniae alba</i> 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 <i>P</i>. <i>alba</i> can dose-dependently inhibit TCPRs, and have anti-allergic function <i>in vitro and in vivo</i>. Radix <i>P</i>. <i>alba</i> 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 <i>P</i>. <i>alba</i> 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.</p></div

Mast cell TCRP-based identification of the bioactive ingredients of Radix <i>P</i>. <i>alba</i>.

<p>(A-B) Adenosine-mediated TCRP (A) and PMA-mediated TCRP (B) for functional screening of various Radix <i>P</i>. <i>alba</i> components. For details about the five various components, see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155930#sec002" target="_blank">Materials and Methods</a>. (C) Immunoblotting analysis of the phosphorylation of Akt and Erk1/2 in RBL-2H3 cells treated with different Radix <i>P</i>. <i>alba</i> components. (D) Effects of the five fractions on [Ca²⁺]_i mobilization. RBL-2H3 cells were treated with PE, EA, EtOH, and H₂O extractions, respectively, for 1 h and incubated with 50 μL of 5 mM Fluo-4/AM for 30 min at 37°C after washing three times with HBSS. The cells were monitored in real-time by spectra Max M5 pre- and post-stimulation with PKC. The [Ca²⁺]_i mobilization was statistically analyzed by comparing the peak fluorescence intensity value which were normalized to the control values. * P < 0.05, ** P < 0.01 vs. control.</p

Mast cell TCRPs-based screening and identification of multi-targets of Radix <i>P</i>. <i>alba</i>.

<p>(A) Adenosine-mediated TCRP-based functional screening of Radix <i>P</i>. <i>alba</i>. RBL-2H3 cells were seeded on the E-plates overnight and pretreated with various concentrations of Radix <i>P</i>. <i>alba</i> for 1 h before the addition of 10 μg/mL adenosine. (B) PMA TCRP-based functional screening of Radix <i>P</i>. <i>alba</i>. RBL-2H3 cells were seeded on E-plates overnight and pretreated with various concentrations of Radix <i>P</i>. <i>alba</i>, as indicated, for 1 h before the addition of 1 μm PMA. (C) Immunoblotting analysis of the phosphorylation of Akt and Erk1/2 in RBL-2H3 cells. IgE-sensitized RBL-2H3 cells were pre-treated with Radix <i>P</i>. <i>alba</i> for 1 h and with 100 ng/mL DNP-BSA for 30 min. The cells were then lysed and immunoblotted with anti-p-Akt, anti-Erk1/2, and anti-p-Erk1/2 antibodies. (D-E) Effects of Radix <i>P</i>. <i>alba</i> on [Ca²⁺]_i mobilization. RBL-2H3 cells were treated with Radix <i>P</i>. <i>alba</i> for 1 h and with 50 μL of 5 mM Fluo-4/AM for 30 min at 37°C. The cells were monitored in real-time by spectra Max M5 after stimulation with PKC. The [Ca²⁺]i mobilization is expressed as the relative fluorescence intensity and the values were normalized to the control values. * P < 0.05, *** P < 0.01 vs. control.</p

Construction of IgE-mediated mast cell TCRPs.

<p>(A) IgE-mediated TCRPs for functional monitoring of mast cell degranulation. RBL-2H3 mast cells were cultured on E-plates at a density of 10,000 cells/well, and incubated with 100 ng/mL anti-DNP IgE approximately 22 h later. After sensitization overnight, the cells were stimulated with 100 ng/mL DNP-BSA. The impedance expressed as the cell index (CI), was continuously monitored. (B) Pharmacological inhibitors of FcεRI receptor-activated downstream signal pathways that significantly attenuated IgE-mediated TCRPs. IgE sensitized RBL-2H3 cells were seed on the E-plates overnight at a density of 20,000 cells/well, and pre-treated with the inhibitors PP1, PD98059, bisindoylmaleimide I and <i>Gab2</i> siRNA, before stimulation with DNP-BSA. (C) Mediator release assay of RBL-2H3 mast cell activation. RBL-2H3 cells were sensitized with 100 ng/mL IgE and activated by the addition of 100 ng/mL DNP-BSA. β-hexosaminidase activities were measured at the indicated time. **, P < 0.01 vs. 0 min. Bar 10 μm. (D) RBL-2H3 mast cell activation was accompanied by morphological changes. The cells were sensitized with IgE and stimulated with DNP-BSA, and were then fixed with paraformaldehyde at the indicated time points. F-actin was stained with rhodamine-pholloidin as described in the Methods.</p

Adenosine and PMA-induced mast cell TCRPs.

<p>(A-B) IgE-mediated TCRP comparison with cell-electrode impedance dynamics induced by adenosine or PMA. RBL-2H3 cells were seeded on the E-plates overnight at a density of 20,000 cells/well, and were then treated with DNP-BSA (IgE-sensitized) and adenosine or PMA (no sensitization). (C) Representative distribution of CD63 stimulated by different antigens in RBL-2H3. RBL-2H3 cells with or without IgE sensitization were stimulated with DNP-BSA, adenosine, and PMA for 30 min. Bar, 5 μm. (D) Adenosine induced microtubule formation whereas PMA induced F-actin disassembly. Treated cells were fixed and processed for staining with phalloidin-rhodamine (red) and antibody to β-tubulin (green). Bar, 5 μm. (E) Adenosine together with PMA induced β-hexosaminidase release. The cells were seeded in 24-well plates overnight, pretreated with PMA or adenosine and subsequently activated with different concentrations of adenosine or PMA. *** P < 0.001 vs. with PMA or adenosine only.</p

Summary of the effects of five fractions.

<p>Summary of the effects of five fractions.</p

Validation of the anti-allergic effect of Radix <i>P</i>. <i>alba in vitro</i> and <i>in vivo</i>.

<p>(A) Typical dynamic cell response curves are indicative and predictive for inhibition of mast cell activation. IgE-sensitized RBL-2H3 cells were incubated with different concentrations of Radix <i>P</i>. <i>alba</i> 1 h prior to the addition of 100 ng/mL DNP-BSA. The cell index (CI) values were continuously assayed for over 3 h. (B) Effect of Radix <i>P</i>. <i>alba</i> on mast cell degranulation. The cells sensitized with 100 ng/mL IgE were incubated with the different concentrations of Radix <i>P</i>. <i>alba</i> and subsequently actived by 100 ng/mL DNP-BSA. β-hexosaminidase activity was measured as described in Methods. (C) TEM images of the vehicle fusion in mast cells treated with or without Radix <i>P</i>. <i>alba</i>. The cells were sensitized with 100 ng/mL IgE overnight and pre-treated with 500 μg/mL Radix <i>P</i>. <i>alba</i> before stimulation with DNP-BSA. After stimulation for 30 min, the cells were fixed with 2.5% glutaraldehyde, and then stained, dehydrated, and embedded. The ultrathin sections were observed using a Tecnai 10 transmission electron microscope. (D) Hematoxylin and eosin-stained sections of the ears prepared 24 h after the challenge. (E) Effect of Radix <i>P</i>. <i>alba</i> on the DNFB-induced ear swelling response in mice. It was determined by ear punch weight determinations. Values are means ± SEM of 12 mice; * P < 0.05, ** P < 0.01 vs. positive control, △△ P < 0.01 vs. negative control.</p

Image_1_The alteration of left ventricular strain in later-onset spinal muscular atrophy children.tif

BackgroundPatients with spinal muscular atrophy (SMA) may suffer from multisystem injury, including an impaired cardiovascular system. However, M-mode echocardiography, the current dominant echocardiographic modality, is limited in the detection of myocardial injury. We considered the use of left ventricular strain imaging in detecting myocardial injury and explored the serum lipid profile related to cardiovascular disease in later-onset SMA children.MethodsA case-control study involving 80 patients with later-onset SMA and 80 age-, gender-, and body surface area-matched control children was conducted in a single tertiary pediatric hospital in China. Data on the left ventricular strain measured using two-dimensional speckle tracking echocardiography, left ventricular function parameters assessed by M-mode echocardiography, and serum lipid profile of these two groups were retrospectively collected for differential analysis.ResultsThe mean age of the 80 SMA patients were (6.87 ± 2.87) years, of which 46 were type 2 and 34 were type 3 patients. The global longitudinal strain (GLS) of the SMA group (−18.7 ± 2.9%, p ConclusionCompared with healthy controls, later-onset SMA children presented with reduced GLS and prolonged TTPLS while the LVEF and LVFS values were within normal range. In particular, whether a reduced GLS or prolonged TTPLS in later-onset SMA compared to the control group can predict the risk of future cardiomyopathy remains to be investigated.</p