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Allergic asthma is caused by various cells, especially mast cells, eosinophils and T lymphocytes in chronic airway inflammation in susceptible in this inflammation can cause recurrent episodes of wheezing, shortness of breath, chest tightness, and (or ) cough and other symptoms, mostly at night and (or) early morning, on a variety of stimulating factor airway hyperresponsiveness. But the symptoms can be treated themselves or remission. Mast cells in the pathophysiology of asthma attacks plays an important role. When subjected to allergen stimulation, mast cells degranulation occurs, release large amounts of hormones media, such as histamine, prostaglandin D2, leukotriene C4, etc. These media can cause bronchoconstriction, mucus secretion, mucus gland edema, eventually leading to bronchial spasm, reversible dyspnea. Study confirmed that mast cell activation and cell membrane a calcium channel - calcium release-activated calcium channels (calcium release-activated calcium channel, CRAC) closely related to the subject after allergen exposure, mast cell membrane CRAC opening extracellular calcium flow induced mast cell degranulation, cytokine release a variety of enzymes, causing a series of pathophysiological changes. Therefore, inhibition of mast cell CRAC channel opening, reducing the activation of mast cell degranulation, is the treatment of allergic asthma is an important target. Resveratrol (Resveratrol, RV) is a kind of polyphenols, widely found in grapes, peanuts, etc., have important anti-inflammatory antioxidant effect on cardiac ischemia - reperfusion injury, systemic inflammatory response has a good therapeutic effect. Recent studies have found that resveratrol also inhibit the HMC-1 cells CRAC channel opening effect, suggesting RV may inhibit degranulation of mast cells, the treatment of allergic diseases. Polydatin (polydatin, PD) is a resveratrol glucoside, structure and function is very similar with the RV. Therefore, we studied PD on mast cell degranulation and the regulation of calcium signaling mechanisms, explore the PD for the treatment of allergic asthma. The results are as follows: First, can suppress autoimmune polydatin stimulus excited degranulation of RBL-2H3 cells. Immunological stimuli stimulate mast cell degranulation test: first in RBL-2H3 mast cell culture medium was added to the anti-DNP-BSA IgE, incubated overnight sensitized RBL-2H3 cells, and then combined with DNP-BSA37 ℃ 30min induce cell de- particles. We use the β-hexokinase method to reflect the release of RBL-2H3 cells degranulation situation. 1 polydatin reduced in a dose-dependent manner to stimulate immune stimulant RBL-2H3 cells degranulation. Control group RBL-2H3 cells after stimulation by DNP-BSA, the intracellular β-hexokinase release rate of 56.2% ± 2.1% (n = 40). PD acute (30 min) or chronic (overnight incubation) effects are not due to mast cell degranulation. However, the acute or chronic polydatin pretreated, immunological stimulus excited degranulation of RBL-2H3 cells was significantly reduced, and with inhibitory effects of PD concentration increases gradually. 1μM, 10μM and 100μM polydatin acute treatment, RBL-2H3 cells β-hexokinase release rates were reduced to 28.9% ± 0.9% (n = 40, p lt; 0.01 vs control group), 21.8% ± 1.1% ( n = 40, p lt; 0.01vs control group) and 16.6% ± 1.2% (n = 40, p lt; 0.01 vs control group). And acute polydatin result of the process is similar to, respectively, 1μM, 10μM and 100μM polydatin chronic treatment, DNP-BSA-induced β-RBL-2H3 cells hexokinase release rates were reduced to 27.2% ± 1.1% (n = 40, p lt; 0.01 vs control group), 18.7% ± 1.1% (n = 40, p lt; 0.01 vs control group) and 13.9% ± 1.4% (n = 40, p lt; 0.01vs control group). 2 polydatin reduces the RBL-2H3 cells on immunological stimuli sensitivity DNP-BSA dose-dependent stimulation of IgE-sensitized RBL-2H3 cells degranulation. 0.25μg/ml, 0.5μg/ml, 1μg/ml, 2μg/ml, 4μg/ml concentration of DNP-BSA induced β-RBL-2H3 cells hexokinase release rates were 19.6% ± 2.3% (n = 16), 25.7% ± 1.8% (n = 16), 53.0% ± 2.1% (n = 16), 55.5% ± 2.4% (n = 16), and 64.0% ± 3.3% (n = 16); when treatment with 10μM polydatin RBL-2H3 cells, found 0.25μg/ml, 0.5μg/ml, 1μg/ml, 2μg/ml, 4μg/ml concentration of DNP-BSA induced RBL-2H3 cells β-hexose kinase release rates were reduced to 8.7% ± 1.0% (n = 16, p lt; 0.05 vs control group), 12.6% ± 1.0% (n = 16, p lt; 0.01 vs control group), 33.6% ± 3.0% ( n = 16, p lt; 0.01 vs control group), 28.8% ± 2.4% (n = 16, p lt; 0.01 vs control group), and 37.5% ± 2.3% (n = 16, p lt; 0.01 vs control group ). 3 polydatin suppress autoimmune irritants calcium influx through the CRAC channel. We Fluo-4 (5μM) to detect the fluorescence intensity of the concentration of intracellular Ca2, DNP-BSA IgE-sensitized mast cells induced Ca2 transient amplitude (ΔF/F0) was 1.58 ± 0.06 (n = 22). Polydatin treatment, Ca2 transient amplitude decreased, ΔF/F0 was 0.61 ± 0.05 (n = 51, p lt; 0.01 vs control group). On Ca2 transient dynamics analysis, PD prolonged time to reach peak transient Ca2 (TTP), transient Ca2 50% shorter decay time (T0.5). TTP and T0.5 control group were 128.11 ± 6.05 (n = 22) and 616.61 ± 13.15 (n = 22). Treated by polydatin, RBL-2H3 cells intracellular Ca2 concentration time to peak (TTP) was extended to 212.24 ± 5.07 (n = 51, p lt; 0.01 vs control group), RBL-2H3 cells in the concentration of intracellular Ca2 reduced by 50% of the time (T0.5) reduced to 372.61 ± 6.45 (n = 51, p lt; 0.01 vs control group). When you remove the extracellular fluid 2mM Ca2 when the concentration increased intracellular Ca2 totally dependent on the intracellular release of Polydatin on intracellular Ca2 concentration were not suppressed, indicating polydatin primarily by reducing extracellular Ca2 through CRAC channel flow to reduce irritation caused by autoimmune Ca2 transients. A lot of literature, the endoplasmic reticulum Ca2-ATP enzyme agonist thapsigargin (Thapsigargin, TG) through the emptying of intracellular calcium stores induced calcium release-activated calcium channels (CRAC channels) open causing extracellular Ca2 influx. In the extracellular fluid containing 2mM Ca2 when, 1μM TG induced larger Ca2 transients polydatin significantly reduced Ca2 transient amplitude, ΔF / FO control group from 1.44 ± 0.08 (n = 36) treatment group reduced to polydatin of 0.67 ± 0.03 (n = 65, p lt; 0.01 vs control group). To distinguish extracellular Ca2 influx through the CRAC channel mediated Ca2 polydatin role transients, we first time in the absence of extracellular Ca2 stimulated by TG, when Ca2 transients generated entirely by intracellular calcium release cause. Then replaced 2mMCa2 extracellular fluid, induces an entirely extracellular Ca2 influx through the CRAC channel of Ca2 transients. Polydatin can significantly reduce the amplitude of the second Ca2 transients, ΔF / FO control group from 1.58 ± 0.14 (n = 127) was reduced to polydatin treated group 0.49 ± 0.07 (n = 51, p lt; 0.01 vs control group). These data further confirmed polydatin by inhibiting extracellular calcium influx through the CRAC channel decreased immunological stimuli induced Ca2 transients produced by mast cells. 4 polydatin reduce immune stimulant RBL-2H3 cells activated intracellular oxygen free radicals (Reactive oxygen species, ROS) generation. It has been reported immunological stimuli-induced mast cell degranulation and increased ROS generation, ROS stimulate mast cells CRAC channel opening, Ca2 transients generated, causing mast cell degranulation. We use DCFH-DA labeled intracellular ROS, found DNP-BSA increased intracellular ROS production, while polydatin can significantly reduce the generation of ROS. Control group DNP-BSA induced ROS generation increased 76% ± 3.0% (n = 127), while polydatin group DNP-BSA induced ROS production increase of only 50% ± 4.1% (n = 137, p lt; 0.01 vs positive control group). These data show that: polydatin mast cells may be activated by reducing oxidative stress to inhibit the activity of CRAC channel. Two. Polydatin OVA-induced inhibition of Balb / c mice allergic asthma 4-6 weeks old Balb / c mice mice were then divided into four groups, namely control group, separate polydatin group, allergic asthma model group, Polygonum glycosides allergic asthma treatment groups. By OVA-induced allergic asthma. 1 polydatin reduce allergic asthma Balb / c mice airway responsiveness to methacholine by measuring the Balb / c mice airway hyperresponsiveness device to Balb / c mice inhaled methacholine alkali concentration were 1mg/mL, 10mg/mL, 30mg/mL, 50mg/mL, 100mg/mL), observe Balb / c mice with different concentrations of methacholine reactivity in allergic asthma, different methacholine concentration corresponding to the percentage increase from baseline values ??were 11.4% (n = 6), 58.3% (n = 6), 246.9% (n = 6), 492.0% (n = 6), 832.5% (n = 6), as amended by polydatin treatment, the percentage increase from baseline values ??were reduced to 9.2% (n = 6, p gt; 0.05 vs control group allergic asthma), 24.1% (n = 6, p lt; 0.05 vs allergy Asthma control group), 70.1% (n = 6, p lt; 0.01 vs control group allergic asthma), 211.4% (n = 6, pp lt; 0.01vs allergic asthma control group), and 351.8% (n = 6, pp lt; 0.01 vs control group allergic asthma). These results suggest that: polydatin possible by reducing airway responsiveness to methacholine to improve the Balb / c mice allergic asthma symptoms. 2 polydatin significantly reduced bronchoalveolar lavage fluid (BALF) in the number of eosinophils with ice-cold phosphate-buffered saline (PBS) 500μL bronchoalveolar lavage (BALF), repeated aspiration three times, and then recovered in bronchoalveolar lavage lavage fluid (BALF), and record back to the absorption capacity; BALF after 500 g, 37 ℃, centrifuged for 5 min, take precipitate after centrifugation cells were resuspended with 1mL PBS cells were counted; Another cell suspension in clean smooth slide smears stained with Liu stain, counting 200 cells for cell count. Allergic asthma in mice found in bronchoalveolar lavage fluid within the marked increase in eosinophils, allergic eosinophilic asthma group was 69.2 ± 3.1 (n = 6), polydatin eosinophils treatment group significantly reduced the number of to 11.0 ± 0.7 (n = 6, pp lt; 0.01 vs control group allergic asthma), the results showed that: polydatin with reduced immune stimuli cause lung tissue eosinophil infiltration role. 3 polydatin alleviate allergic asthma mouse lung tissue inflammation, improve the structural integrity of the bronchial lavage performed first, followed by the lung tissues were paraffin tissue sections and found that allergic asthma, interstitial lung of mice There are a large number of inflammatory cell infiltration, pulmonary bronchiolar epithelial cell proliferation and lung bronchioles varying degrees of damage to a certain degree; while polydatin group of interstitial lung inflammatory cells was significantly reduced, the structural integrity of the lung bronchioles there was a marked improvement, and epithelial cell proliferation was significantly reduced. Tip polydatin possible through improvements in lung function to treat allergic asthma. 4 polydatin inhibition of allergic asthma mouse mast cell CRAC channel opening peritoneal mast cells isolated with 5μM Fluo-4 dye 30min, then add 1μM thapsigargin (Thapsigargin), through Fluo-4 fluorescence to reflect the mast cells intracellular fluorescence intensity. In the case of TG-induced intracellular free Ca2 fewer Ca2 transient, and when the extracellular fluid into liquid 2mM Ca2, intracellular Ca2 transient increased significantly. The latter reflects the Ca2 transient increase of extracellular Ca2 influx through the CRAC channel, compared to mice with allergic asthma, after polydatin after treatment of allergic asthma in mice, mast cells appear in the second intracellular significantly reduced Ca2 transients, F/F0 from 4.3 ± 0.1 (n = 106) is reduced to 3.2 ± 0.2 (n = 52, pp lt; 0.01 vs control group allergic asthma), these data further confirmed from the in vivo level: Polygonum glycosides inhibit extracellular Ca2 mast cells through activation of the role of CRAC channel flow, thereby reducing the incidence of allergic asthma. 5 polydatin reduce allergic asthma in mice serum IgE levels, elevated serum levels of IgG2a OVA sensitized Balb / c mice. Allergic serum IgE levels in control mice was 0.54 ± 0.14 (n = 18), while receiving polydatin treatment, serum IgE levels were significantly reduced to 0.32 ± 0.08 (n = 18, p lt; 0.01 vs allergic asthmatic control group); addition, allergic asthma control level of serum IgG2a 0.79 ± 0.17 (n = 18), receiving polydatin treatment, serum IgG2a levels were significantly increased to 1.15 ± 0.36 (n = 18, p lt; 0.01 vs control group allergic asthma). IgE antibody sensitization antibodies have been recognized, and IgG antibodies are proven to be a most literature protective antibodies, which can be competitive inhibition of IgE binding to its receptor, thereby reducing the mast cell degranulation, and thus reduce the symptoms of allergic asthma. These data show that: polydatin possible by reducing serum IgE levels, increased serum IgG2a levels to alleviate the symptoms of allergic asthma. Conclusion: a dose-dependent inhibition of autoimmune polydatin irritants RBL-2H3 mast cell degranulation. 2 polydatin by inhibiting the extracellular Ca2 influx through the CRAC channel decreased immune stimulant-induced Ca2 transients. 3 polydatin RBL-2H3 cells inhibited the activation of intracellular ROS generation. 4 polydatin on OVA-induced allergic asthma has a therapeutic effect, manifested as decreased airway hyperresponsiveness, inflammatory cell infiltration of alveolar tissue decreased serum IgE levels decreased, while IgG2a levels. 5 polydatin treatment of allergic asthma is an important mechanism: reducing autoimmune irritants ROS production, thereby inhibiting the activation of CRAC channel caused by extracellular Ca2 influx, thereby inhibiting mast cell degranulation.
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