Effects Of Polydatin On Allergic Asthma And The Underlying Mechanism

Allergic asthma is a chronic airway inflammation involving in many cells, especially mast cells, eosinophils, T cells, Such inflammation can be induced in susceptible patients with recurrent episodes of wheezing, shortness of breath, chest tightness, and (or) cough and other symptoms, and more at night and (or) in the early morning, airway responsiveness to many stimulating factor increased. But the symptoms can be treated on their own or mitigation.Mast cells play an important role in the pathogenesis of asthma. When stimulated by the allergen, mast cells degranulate and secrete the autacoid mediators, such as histamine, prostaglandin (PG) D2, and leukotriene (LT) C4, which are capable of inducing bronchoconstriction, mucus secretion, and mucosal edema, all features of asthma. This is particularly evident during experimental allergen challenge, in which blockade of these mediators attenuates the early fall in lung function. However, mast cells also synthesize and secrete a large number of proinflammatory cytokines (including IL-4, IL-5, and IL-13), which regulate both IgE synthesis and the development of eosinophilic inflammation, in addition to these, the proteases tryptase and chymase are being demonstrated to have a range of actions consistent with key roles in inflammation, tissue remodeling, and bronchial hyperresponsiveness. It has been widely recognized that when the mast cells are stimulated by the allergen, a calcium channel called calcium release-activated calcium channels (CRAC) opens and calcium ion entries, causing mast cell degranulation, and the simultaneous release of a variety of enzymes and cytokines, which leads to a series of chain reactions. When using this channel-specific blockers (such as:La3+,2-APB, etc.), the influx of extracellular calcium significantly reduced and the degranulation of mast cells was also significantly reduced. Therefore, the development of potent and specific inhibitors of CRAC channel in mast cells should prevent the cell degrannulation and offer a novel approach to the treatment of asthma.A large number of papers had demonstrated that Resveratrol has anti-inflammatory and anti-allergic effects. Recently, a paper reported that Resveratrol suppresses the activity of CRAC in HMC-1 cells, highlighting the important role of Resveratrol in anti-allergy. Polydatin (PD) is a derivative of resveratrol and has similar structure as that of resveratrol. Furthermore, Polydatin can be metabolized to resveratrol in vivo. suggesting that polydatin also have anti-inflammatory and anti-allergic effects.Based on the above considerations, we explored the effect of PD on mast cell degrannulation and Ca2+ signaling involving in mast cell degrannulation in RBL-2H3 cell line and found that PD diminished the degranulation of mast cells through suppressing the Ca2+ influx through CRAC. Based on the in vitro findings, we investigated the role of PD in the pathogenesis allergic asthma in vivo and demonstrated that PD had dramatic effect in treatment of allergic asthma. The results are as follows:一,Polydatin can inhibit degranulation of the sensitized RBL-2H3 cells.IgE-dependent mast cell degranulation was produced in RBL-2H3 cell lines, in which the cells were sensitized overnight with anti-DNP IgE and stimulated for 30min at 37℃with DNP-BSA. Degranulation of RBL-2H3 cells was indexed byβ-hexokinase release.1 PD decreased the IgE-dependent mast cell degranulation in a dose-dependent mannerIn control cells, stimulation with DNP-BSA resulted in the release of 56.2% (n=40) of the cell content ofβ-hexosaminidase. PD itself had no effect on the cell degranulation. However, pretreatment with PD acutely (for 30 min) and chronically (overnight) both decreased the IgE-dependent mast cell degranulation in a dose-dependent manner. Acute application of PD at the concentration of 1μM,10μM, 100μM reduced DNP-BSA inducedβ-hexokinase release to 28.9%(n=40, p<0.01 vs control),21.8%(n=40, p<0.01 vs control) and 16.6%(n=40, p<0.01 vs control), respectively. Similarly to the acute effect of PD, chronic application of PD at the concentration of 1μM, 10μM, 1000μM reduced DNP-BSA inducedβ-hexokinase release to 27.2%(n=40, p<0.01 vs control),18.7%(n=40, p<0.01 vs control) and 13. 9%(n=40, p<0.01 vs control), respectively.2 PD decreased the sensitivity of mast cell to degranulation stimulationDNP-BSA stimulate the degranulation of IgE sensitized RBL-2H3 cells in a dose-dependent manner, in which 0.25μg/ml,0.5μg/ml, 1μg/ml,2μg/ml,4μg/ml DNP-BSA led to 19.6%(n=16),25.7%(n=16),53.0%(n=16),55.5%(n=16), and 64.0%(n=16),β-hexokinase release, respectively. In the presence of 10μM PD,β-hexokinase release stimulated by 0.25μg/ml,0.5μg/ml,1μg/ml,2μg/ml,4μg/ml DNP-BSA was decreased to 8.7%(n=16, p<0.05 vs control),12.6%(n=16, p<0.01 vs control),33.6%(n=16, p<0.01 vs control),28.8%(n=16, p<0.01 vs control), and 37.5%(n=16, p<0.01 vs control), respectively.3 Polydatin inhibited extracellular calcium influx in activated RBL-2H3 cells The intracellular Ca2+ was visualized by Fluo-4 fluorescence. Consistent with previous report, DNP-BSA caused a Ca2+ transient in IgE-sensitized cells. The amplitude of Ca2+ transient indexed by theΔF/FO was 1.58±0.06 (n=22) in control group. With the treatment of PD, the amplitude of Ca2+ transient was significantly reduced to 0.61±0.05 (n=51, p<0.01 vs control). Furhtermore, the kinetics of Ca2+ transient indexed by time to peak (TTP) was 128.11±6.05 (n=22).and half-time of decay (T0.5) was 616.61±13.15 (n=22) in control group, With the treatment of PD, the kinetics of Ca2+ transient indexed by time to peak (TTP) was significantly increased to 212.24±5.07 (n=51).and half-time of decay (TO.5) was significantly reduced to 372.61±6.45 (n=51).When the 2mM extracellular Ca2+ was removed from extracellular solution, the Ca2+ transient attributed completed by intracellular Ca2+ release was dramatically decreased. In contrast to the effect of PD at 2 mM extracellular Ca2+, PD had no effect on Ca2+ transient at 0 mM extracellular Ca2+. The data suggested that PD reduced IgE-stimulated Ca2+ transient through reducing extracellular Ca2+ influx.It is well established that thapsigargin (TG), the agonist of Ca2+- ATP enzyme in endoplasmic reticulum (ER) induced CRAC channel opening and consequent Ca2+ influx by empting intracellular Ca2+ store. Under 2 mM extracellular Ca2+, 1μM TG induced a big Ca2+ transient which cannot distinguish the intracellular Ca2+ release and extracellular Ca2+ influx. PD significantly reduced the TG-induced Ca2+ transient. The amplitude was decreased from 1.44±0.08 (n=36) in control cells to 0.67±0.03 in PD treated cells (n=65, p<0.01 vs control). To distinguish the role of extracellular Ca2 + influx through CRAC channel in PD-mediated reduction of Ca2+ transient, we applied TG under extracellular Ca2+-free circumstances, followed by restoring the extracellular Ca2+ to 2 mM. A small Ca2+ transient was induced with the application of TG under 0 extracellular Ca2+, while a big Ca2+ transient appeared when the 0 extracellular Ca2+ was replaced to 2 mM extracellular Ca2+. The later Ca2+ transient reflect the extracellular Ca2+ influx through CRAC channel. PD dramatically decreased the amplitude of the second Ca2+ transient, from 1.58±0.14 (n=127) in control cells to 0.49±0.07 in PD treated cells (n=51, p<0.01 vs control). The data further confirmed that PD has a suppression effect on Ca2+ influx through CRAC channel in stimulated mast cells.4 Polydatin reduce intracellular ROS (Reactive oxygen species) generation in stimulated RBL-2H3 cellsIt has been suggested that IgE-dependent mast cell degranulation is related to increased ROS production, which is linked to activity of CRAC channel in stimulated mast cells. Consistent with previous report, DNP-BSA increased intracellular ROS level, as indexed by DCFH-DA fluorescent intensity. PD significantly decreased ROS increment. With PD treatment, DNP-BSA decreased ROS production by 49%(n=137 p<0.01 vs control), which was much lower than 76%(n=127) without PD treatment. The data suggest that PD might suppress the activity of CRAC channel by decreasing the oxidative stress in stimulated mast cells.二,The therapeutic effect of Polydatin in the OVA-induced allergic asthma in miceBalb/c mice were divided into control, PD control, allergic asthma control and allergic asthma with PD treatment groups. Allergic asthma was produced by OVA induced method.1 Polydatin decreased airway hyperresponsiveness of allergic asthma in Balb/c mice on methacholineWith airway hyperresponsiveness instrument,by inhalation of methacholine to Balb/c mice, the concentration followed by Omg/mL, 1mg/mL, 10mg/mL,30mg/mL, 50mg/mL, 100mg/mL, And Percent Above Baseline(%) was 11.4%(n=6), 58.3%(n=6),246.9%(n=6),492.0%(n=6),832.5%(n=6) in allergic asthma group, respectively., With the treatment of PD, Percent Above Baseline(%) was decreased to 9.2%(n=6, p>0.05 vs allergic asthma control),24.1%(n=6, p<0.05 vs allergic asthma control),70.1%(n=6, p<0.01 vs allergic asthma control),211.4%(n=6, p<0.01 vs allergic asthma control),351.8%(n=6, p<0.01 vs allergic asthma control) respectively.The date suggest that Polydatin might improve allergic asthma in Balb/c mice by decreasing airway hyperresponsiveness on methacholine.2 Polydatin decreased greatly the number of eosinophils of bronchoalveolar lavage fluid (BALF)With ice-cold phosphate buffered saline (PBS) 500μL for bronchoalveolar lavage (BALF), repeatedly for three times, and then recovered in bronchoalveolar lavage fluid (BALF), and record the remained fluid; BALF was centrifugated for 5 min after 500 g,37℃, the precipitate obtained after centrifugation, cells were re-suspended with 1mL PBS,\cell suspension were put on clean and smooth glass slide smears, stained with Liu Staining, counted 200 cells and counted every species cells. Eosinophils in bronchoalveolar lavage fluid of allergic asthma in mice were found within the marked increase, the number of eosinophils was 69.2±3.1 (n=6) in control group. With the treatment of PD,, the number of eosinophils was significantly reduced to 11.0±0.7 (n=6, p<0.01 vs allergic asthma control). These findings suggest that Polydatin might improve the allergic asthma by reducing the number of eosinophils of lung tissue.3 In lung tissue slices, Polydatin reduced allergic inflammation in lung tissue of asthmatic mice and improve the structural integrity of bronchialFirstly,we conducted a bronchoalveolar lavage, followed done the paraffin lung tissue biopsy. and found that allergic asthma groups of mice lung slices:the pulmonary interstitial infiltrated a large number of inflammatory cells, lung epithelial cells in bronchioles occurred a certain degree of hyperplasia and damage; while pulmonary interstitial inflammatory cells with treatment of Polydatin significantly reduced,the structural integrity of lung bronchioles had marked improvement, and epithelial cell proliferation decreased significantly. These results indicated that Polydatin may have some therapeutic effect in improving lung function.4 Polydatin inhibited extracellular calcium influx in isolated peritoneal mast cellsIsolated peritoneal mast cells were stained with fluo-4 dye for 30min, the intracellular Ca2+ was visualized by Fluo-4 fluorescence. It is well established that thapsigargin (TG), the agonist of Ca2+- ATP enzyme in endoplasmic reticulum (ER) induced CRAC channel opening and consequent Ca2+ influx by empting intracellular Ca+ store.. A small Ca2+ transient was induced with the application of TG under 0 extracellular Ca2+, while a big Ca2+ transient appeared when the 0 extracellular Ca2+ was replaced to 2 mM extracellular Ca2+. The later Ca2+ transient reflect the extracellular Ca2+ influx through CRAC channel. allergic asthma with PD treatment groups dramatically decreased the amplitude of the second Ca2+ transient, from 4.3±0.1 (n=106) in control cells to 3.2±0.2 in PD treated cells (n=52, p<0.01 vs allergic asthma control). The data further confirmed that PD has a suppression effect on Ca2+ influx through CRAC in stimulated Isolated peritoneal mast cells.5 Polydatin reduced the serum IgE levels, but elevated levels of serum IgG2a of allergic asthma in miceBalb/c mice were sensitized by OVA. The serum IgE levels was 0.54±0.14 (n=18) in allergic asthma control group; With the treatment of PD, The serum IgE levels was significantly reduced to 0.32±0.08 (n=18, p<0.01 vs allergic asthma control). Furhtermore, The serum IgG2a levels was 0.79±0.17 (n=18) in allergic asthma control group; With the treatment of PD, The serum IgG2a levels was significantly increased to 1.15±0.36 (n=18, p<0.01 vs allergic asthma control). The data suggested that Polydatin alleviated the symptoms of allergic asthma by decreasing the serum IgE levels, but elevating levels of serum IgG2a of allergic asthma in mice. Conclusion:1. PD inhibited FcεR-mediated degranulation of mast cells, in a dose-dependent manner.2. PD suppressed FcεR-mediated Ca2+ transient by decreasing Ca2+ influx through CRAC channels.3. PD inhibited FcεR-mediated ROS production in mast cells.4. PD has therapeutic effect on OVA-induced allergic asthma, manifested by decrease of airway hyperactivity, reduction of infiltration of inflammatory cells in bronchia and alveolae, decrease of serum IgE and increase of serum IgG2a, etc.5. An important pathway underlying PD mediated therapeutic effect on allergic asthma:PD reduces FcεR-mediated ROS production, resulting in decreased Ca2+ influx through CRAC and subsequently diminished degranulation in mast cells.