虎杖苷抗过敏性哮喘的作用及机制
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摘要
过敏性哮喘是由多种细胞特别是肥大细胞、嗜酸性粒细胞和T淋巴细胞参与的慢性气道炎症,在易感者中此种炎症可引起反复发作的喘息、气促、胸闷和(或)咳嗽等症状,多在夜间和(或)凌晨发生,气道对多种刺激因子反应性增高。但症状可自行或经治疗缓解。
肥大细胞在哮喘发作的病理生理学机制上起着重要的作用。当受到变应原刺激后,肥大细胞会发生脱颗粒,释放大量的内分泌素介质,如组胺,前列腺素D2,白三烯C4等,这些介质会引起支气管收缩,粘液分泌,黏液腺水肿,最终导致支气管发生痉挛,出现可逆性的呼吸困难。研究证实,肥大细胞激活与细胞膜上的一种钙通道——钙释放激活钙通道(calcium release-activated calcium channel, CRAC)密切相关,在受到过敏原刺激后,肥大细胞膜上的CRAC开放,外钙内流引起肥大细胞脱颗粒,多种酶及细胞因子释放,从而引起一系列的病理生理改变。因此,抑制肥大细胞CRAC通道开放,减少活化肥大细胞脱颗粒,是过敏性哮喘的治疗的一个重要靶标。
白藜芦醇(Resveratrol, RV)是一种多酚类物质,广泛存在于葡萄、花生等等,具有重要的抗炎抗氧化的作用,对心脏缺血-再灌注损伤、全身性炎症反应等具有良好的治疗作用。最近研究发现,白藜芦醇还具有抑制HMC-1细胞CRAC通道开放的效应,提示RV可能具有抑制肥大细胞脱颗粒,治疗过敏性疾病的作用。虎杖苷(polydatin, PD)是一种白藜芦醇糖苷,结构与功能上与RV非常相似。因此我们研究PD对肥大细胞脱颗粒的调节作用及其钙信号机制,探讨PD对过敏性哮喘的治疗作用。结果如下：
一、虎杖苷能够抑制免疫性刺激物激发的RBL-2H3细胞脱颗粒。
免疫性刺激物激发的肥大细胞脱颗粒检测：首先在RBL-2H3肥大细胞培养液中加入抗DNP-BSA的IgE,过夜孵育致敏RBL-2H3细胞,然后再加DNP-BSA37℃30min诱导细胞脱颗粒。我们用β-己糖激酶释放法来反映RBL-2H3细胞的脱颗粒情况。
1.虎杖苷以剂量依赖的方式减少免疫性刺激物激发RBL-2H3细胞的脱颗粒。
对照组的RBL-2H3细胞受到DNP-BSA刺激后,其细胞内的β-己糖激酶释放率达到56.2%±2.1%(n=40)。PD急性(30 min)或慢性(过夜孵育)作用均不引起肥大细胞的脱颗粒。但是,经急性或慢性虎杖苷预处理后,免疫性刺激物激发的RBL-2H3细胞脱颗粒显著降低,而且随着PD浓度的升高抑制效应逐渐增强。1μM,10μM和100μM虎杖苷急性处理后,RBL-2H3细胞β-己糖激酶释放率依次减少到28.9%±0.9%(n=40,p<0.01 vs对照组),21.8%±1.1%(n=40,p<0.01vs对照组)和16.6%±1.2%(n=40,p<0.01 vs对照组)。与急性虎杖苷处理的结果相类似,,分别以1μM,10μM和100μM虎杖苷慢性处理后,DNP-BSA诱导的RBL-2H3细胞β-己糖激酶释放率依次减少到27.2%±1.1%(n=40,p<0.01 vs对照组),18.7%±1.1%(n=40,p<0.01 vs对照组)和13.9%±1.4%(n=40,p<0.01vs对照组)。
2虎杖苷降低了RBL-2H3细胞对免疫性刺激物的敏感性
DNP-BSA呈剂量依赖性刺激IgE致敏RBL-2H3细胞脱颗粒。0.25μg/ml, 0.5μg/ml, 1μg/ml,2μg/ml,4μg/ml浓度下的DNP-BSA所引起的RBL-2H3细胞β-己糖激酶释放率依次为19.6%±2.3%(n=16),25.7%±1.8%(n=16),53.0%±2.1%(n=16),55.5%±2.4%(n=16),和64.0%±3.3%(n=16)；当用10μM虎杖苷处理RBL-2H3细胞后,发现0.25μg/ml,0.5μg/ml, 1μg/ml,2μg/ml,4μg/ml浓度下的DNP-BSA所引起的RBL-2H3细胞β-己糖激酶释放率依次减少到8.7%±1.0%(n=16,p<0.05 vs对照组),12.6%±1.0%(n=16,p<0.01 vs对照组),33.6%±3.0%(n=16,p<0.01 vs对照组),28.8%±2.4%(n=16,p<0.01 vs对照组),和37.5%±2.3%(n=16,p<0.01 vs对照组)。
3.虎杖苷抑制免疫性刺激物引起的钙离子通过CRAC通道内流。
我们通过Fluo-4(5μM)的荧光强度来检测胞内的Ca2+浓度,DNP-BSA引起IgE致敏肥大细胞产生Ca2+瞬变,幅度(ΔF／F0)为1.58±0.06(n=22)。虎杖苷处理后,Ca2+瞬变幅度明显降低,ΔF／F0为0.61±0.05(n=51,p<0.01 vs对照组).对Ca2+瞬变动力学分析发现,PD延长Ca2+瞬变达到峰值的时间(TTP),缩短Ca2+瞬变50%衰减的时间(T0.5)。对照组TTP和T0.5分别为128.11±6.05(n=22)和616.61±13.15(n=22).经虎杖苷处理后,RBL-2H3细胞胞内Ca2+浓度到达峰值的时间(TTP)延长至212.24±5.07(n=51,p<0.01 vs对照组),RBL-2H3细胞胞内Ca2+浓度降低50%所需的时间(T0.5)缩短至372.61±6.45(n=51,p<0.01 vs对照组)。当移除细胞外液中的2mM Ca2+时,胞内Ca2+浓度的升高完全依赖于胞内的释放,虎杖苷对胞内Ca2+浓度升高无明显抑制,说明虎杖苷主要通过减少胞外Ca2+通过CRAC通道内流来减少免疫性刺激物引起的Ca2+瞬变。
大量文献报道,内质网Ca2+-ATP酶的激动剂毒胡萝卜素(Thapsigargin,TG)通过排空胞内钙库诱导钙释放激活的钙通道(CRAC通道)开放引起胞外Ca2+内流。在胞外液内含有2mM Ca2+时,1μM TG诱导产生较大Ca2+瞬变,虎杖苷明显降低Ca2+瞬变幅度,ΔF／FO从对照组的1.44±0.08(n=36)减少到虎杖苷治疗组的0.67±0.03(n=65,p<0.01 vs对照组)。为了区分胞外Ca2+通过CRAC通道内流在虎杖苷介导的Ca2+瞬变中所起的作用,我们首先在无细胞外Ca2+时用TG刺激,此时产生的Ca2+瞬变完全由细胞内钙释放引起。然后细胞外液换成2mMCa2+的液体,诱导一个完全由胞外Ca2+通过CRAC通道的内流的Ca2+瞬变。虎杖苷能够明显降低第二个Ca2+瞬变的幅度,ΔF／FO从对照组的1.58±0.14(n=127)减少到虎杖苷处理组的0.49±0.07(n=51,p<0.01 vs对照组)。这些数据进一步证实,虎杖苷通过抑制细胞外钙通过CRAC通道内流而减少免疫性刺激物诱导的肥大细胞产生的Ca2+瞬变。
4.虎杖苷减少免疫性刺激物活化的RBL-2H3细胞胞内氧自由基(Reactive oxygen species, ROS)的产生。
已有文献报道,免疫性刺激物诱导的肥大细胞脱颗粒与ROS生成的增加有关,ROS刺激肥大细胞的CRAC通道开放,产生Ca2+瞬变,从而引起肥大细胞脱颗粒。我们采用DCFH-DA标记细胞内ROS,发现DNP-BSA增加胞内ROS的生成,而虎杖苷能够明显减少ROS的生成。对照组DNP-BSA引起ROS生成增加76%±3.0%(n=127),而虎杖苷组DNP-BSA引起ROS生成增加仅为50%±4.1%(n=137,p<0.01 vs阳性对照组)。这些数据表明：虎杖苷可能通过降低活化肥大细胞的氧化应激来抑制CRAC通道的活性。
二.虎杖苷抑制OVA诱导的Balb/c小鼠过敏性哮喘
4-6周龄的Balb/c mice小鼠,随即分成4组,即对照组,单独虎杖苷组,过敏性哮喘模型组,虎杖苷过敏性哮喘治疗组。过敏性哮喘通过OVA诱发。
1.虎杖苷降低过敏性哮喘Balb/c小鼠气道对乙酰甲胆碱的反应性
采用测量Balb/c小鼠气道高反应性的装置,给Balb／c小鼠雾化吸入乙酰甲胆碱,浓度依次为1mg/mL、10mg/mL、30mg/mL、50mg/mL、100mg/mL),观察Balb/c小鼠对不同浓度乙酰甲胆碱的反应性,在过敏性哮喘组,不同浓度乙酰甲胆碱相对应的基线增加百分比值依次为11.4%(n=6),58.3%(n=6),246.9%(n=6),492.0%(n=6),832.5%(n=6),而经虎杖苷治疗后,基线增加百分比值依次降低到9.2%(n=6,p>0.05 vs过敏性哮喘对照组),24.1%(n=6,p<0.05 vs过敏性哮喘对照组),70.1%(n=6,p<0.01 vs过敏性哮喘对照组),211.4%(n=6,p p<0.01vs过敏性哮喘对照组),和351.8%(n=6,p p<0.01 vs过敏性哮喘对照组)。这些结果提示：虎杖苷可能通过降低气道对乙酰甲胆碱的反应性来改善Balb／c小鼠过敏性哮喘症状。
2.虎杖苷明显降低支气管肺泡灌洗液(BALF)中的嗜酸性粒细胞数目
用冰冷磷酸盐缓冲液(PBS)500μL进行支气管肺泡灌洗(BALF),反复抽吸3次,然后回收支气管肺泡灌洗液(BALF),并记录回吸收量；BALF经500 g,37℃,离心5 min后,取离心后的沉淀细胞,用1mL PBS重悬细胞,进行计数；另取细胞悬液于洁净光滑载玻片上涂片,用刘氏染色液染色,计数200个细胞,进行细胞分类计数。发现过敏性哮喘小鼠支气管肺泡灌洗液内的嗜酸性粒细胞明显增加,过敏性哮喘组嗜酸性粒细胞为69.2±3.1(n=6),虎杖苷治疗组嗜酸性粒细胞的数目显著减少至11.0±0.7(n=6,p p<0.01 vs过敏性哮喘对照组),结果表明：虎杖苷具有降低免疫性刺激物引起肺组织中的嗜酸性粒细胞浸润的作用。
3.虎杖苷减轻过敏性哮喘小鼠肺组织的炎症反应,改善支气管结构的完整性
先进行支气管肺泡灌洗,接着取肺组织做石蜡组织切片,结果发现过敏性哮喘组小鼠肺间质内有大量炎性细胞浸润,肺细支气管上皮细胞不同程度增生及肺细支气管一定程度的破坏；而虎杖苷组的肺间质炎性细胞明显减少,肺细支气管结构的完整性也有明显的改善,并且上皮细胞增生明显降低。提示虎杖苷可能通过改善肺功能来治疗过敏性哮喘。
4.虎杖苷抑制过敏性哮喘小鼠肥大细胞CRAC通道的开放
腹腔分离的肥大细胞用5μM Fluo-4染料染色30min,然后加1μM毒胡萝卜素(Thapsigargin),通过Fluo-4的荧光来反映肥大细胞胞内的荧光强度变化。TG在无Ca2+情况诱导胞内较少的Ca2瞬变,然后当细胞外液变为2mM Ca2+液体时,胞内的Ca2+瞬变明显增加。后者的Ca2+瞬变增加反映的是胞外Ca2+通过CRAC通道的内流,与过敏性哮喘小鼠相比,经过虎杖苷治疗后的过敏性哮喘小鼠,肥大细胞胞内出现的第二次Ca2+瞬变明显降低,F／F0从4.3±0.1(n=106)降低到3.2±0.2(n=52,p p<0.01 vs过敏性哮喘对照组),这些数据从在体水平进一步证实：虎杖苷具有抑制胞外Ca2+经过活化肥大细胞CRAC通道内流的作用,从而减轻过敏性哮喘的发生。
5.虎杖苷降低过敏性哮喘小鼠血清中的IgE水平,升高血清中的IgG2a水平
OVA致敏Balb/c小鼠。过敏性哮喘对照组小鼠血清IgE水平为0.54±0.14(n=18),当接受虎杖苷治疗后,血清中的IgE水平明显减少至0.32±0.08(n=18,p<0.01 vs过敏性哮喘对照组)；另外,过敏性哮喘对照组小鼠血清IgG2a水平为0.79±0.17(n=18),在接受虎杖苷治疗后,血清中的IgG2a水平明显增加至1.15±0.36(n=18,p<0.01 vs过敏性哮喘对照组)。IgE抗体为致敏性抗体已得到公认,而IgG抗体被大多文献证实是一种保护性抗体,它可以竞争性抑制IgE与其受体结合,从而减少肥大细胞脱颗粒,进而减轻过敏性哮喘症状。这些数据表明：虎杖苷可能通过减少血清中的IgE水平,增加血清中的IgG2a水平来减轻过敏性哮喘症状。
结论：
1虎杖苷剂量依赖性抑制免疫性刺激物引起的RBL-2H3肥大细胞脱颗粒。
2虎杖苷通过抑制胞外Ca2+经过CRAC通道内流减少免疫性刺激物诱导的Ca2+瞬变。
3虎杖苷抑制活化RBL-2H3细胞胞内ROS的产生。
4虎杖苷对OVA诱导的过敏性哮喘具有治疗作用,表现为气道反应性降低、肺泡组织炎症细胞的浸润减少、血清中IgE水平降低,而IgG2a水平升高。
5虎杖苷对过敏性哮喘治疗的一个重要机制：减少免疫性刺激物引起的ROS产生,从而抑制CRAC通道激活造成的细胞外Ca2+内流,进而抑制肥大细胞脱颗粒。
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 manner
In 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 stimulation
DNP-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 cells
It 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 mice
Balb/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 methacholine
With 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 bronchial
Firstly,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 cells
Isolated 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 mice
Balb/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.
肥大细胞在哮喘发作的病理生理学机制上起着重要的作用。当受到变应原刺激后,肥大细胞会发生脱颗粒,释放大量的内分泌素介质,如组胺,前列腺素D2,白三烯C4等,这些介质会引起支气管收缩,粘液分泌,黏液腺水肿,最终导致支气管发生痉挛,出现可逆性的呼吸困难。研究证实,肥大细胞激活与细胞膜上的一种钙通道——钙释放激活钙通道(calcium release-activated calcium channel, CRAC)密切相关,在受到过敏原刺激后,肥大细胞膜上的CRAC开放,外钙内流引起肥大细胞脱颗粒,多种酶及细胞因子释放,从而引起一系列的病理生理改变。因此,抑制肥大细胞CRAC通道开放,减少活化肥大细胞脱颗粒,是过敏性哮喘的治疗的一个重要靶标。
白藜芦醇(Resveratrol, RV)是一种多酚类物质,广泛存在于葡萄、花生等等,具有重要的抗炎抗氧化的作用,对心脏缺血-再灌注损伤、全身性炎症反应等具有良好的治疗作用。最近研究发现,白藜芦醇还具有抑制HMC-1细胞CRAC通道开放的效应,提示RV可能具有抑制肥大细胞脱颗粒,治疗过敏性疾病的作用。虎杖苷(polydatin, PD)是一种白藜芦醇糖苷,结构与功能上与RV非常相似。因此我们研究PD对肥大细胞脱颗粒的调节作用及其钙信号机制,探讨PD对过敏性哮喘的治疗作用。结果如下：
一、虎杖苷能够抑制免疫性刺激物激发的RBL-2H3细胞脱颗粒。
免疫性刺激物激发的肥大细胞脱颗粒检测：首先在RBL-2H3肥大细胞培养液中加入抗DNP-BSA的IgE,过夜孵育致敏RBL-2H3细胞,然后再加DNP-BSA37℃30min诱导细胞脱颗粒。我们用β-己糖激酶释放法来反映RBL-2H3细胞的脱颗粒情况。
1.虎杖苷以剂量依赖的方式减少免疫性刺激物激发RBL-2H3细胞的脱颗粒。
对照组的RBL-2H3细胞受到DNP-BSA刺激后,其细胞内的β-己糖激酶释放率达到56.2%±2.1%(n=40)。PD急性(30 min)或慢性(过夜孵育)作用均不引起肥大细胞的脱颗粒。但是,经急性或慢性虎杖苷预处理后,免疫性刺激物激发的RBL-2H3细胞脱颗粒显著降低,而且随着PD浓度的升高抑制效应逐渐增强。1μM,10μM和100μM虎杖苷急性处理后,RBL-2H3细胞β-己糖激酶释放率依次减少到28.9%±0.9%(n=40,p<0.01 vs对照组),21.8%±1.1%(n=40,p<0.01vs对照组)和16.6%±1.2%(n=40,p<0.01 vs对照组)。与急性虎杖苷处理的结果相类似,,分别以1μM,10μM和100μM虎杖苷慢性处理后,DNP-BSA诱导的RBL-2H3细胞β-己糖激酶释放率依次减少到27.2%±1.1%(n=40,p<0.01 vs对照组),18.7%±1.1%(n=40,p<0.01 vs对照组)和13.9%±1.4%(n=40,p<0.01vs对照组)。
2虎杖苷降低了RBL-2H3细胞对免疫性刺激物的敏感性
DNP-BSA呈剂量依赖性刺激IgE致敏RBL-2H3细胞脱颗粒。0.25μg/ml, 0.5μg/ml, 1μg/ml,2μg/ml,4μg/ml浓度下的DNP-BSA所引起的RBL-2H3细胞β-己糖激酶释放率依次为19.6%±2.3%(n=16),25.7%±1.8%(n=16),53.0%±2.1%(n=16),55.5%±2.4%(n=16),和64.0%±3.3%(n=16)；当用10μM虎杖苷处理RBL-2H3细胞后,发现0.25μg/ml,0.5μg/ml, 1μg/ml,2μg/ml,4μg/ml浓度下的DNP-BSA所引起的RBL-2H3细胞β-己糖激酶释放率依次减少到8.7%±1.0%(n=16,p<0.05 vs对照组),12.6%±1.0%(n=16,p<0.01 vs对照组),33.6%±3.0%(n=16,p<0.01 vs对照组),28.8%±2.4%(n=16,p<0.01 vs对照组),和37.5%±2.3%(n=16,p<0.01 vs对照组)。
3.虎杖苷抑制免疫性刺激物引起的钙离子通过CRAC通道内流。
我们通过Fluo-4(5μM)的荧光强度来检测胞内的Ca2+浓度,DNP-BSA引起IgE致敏肥大细胞产生Ca2+瞬变,幅度(ΔF／F0)为1.58±0.06(n=22)。虎杖苷处理后,Ca2+瞬变幅度明显降低,ΔF／F0为0.61±0.05(n=51,p<0.01 vs对照组).对Ca2+瞬变动力学分析发现,PD延长Ca2+瞬变达到峰值的时间(TTP),缩短Ca2+瞬变50%衰减的时间(T0.5)。对照组TTP和T0.5分别为128.11±6.05(n=22)和616.61±13.15(n=22).经虎杖苷处理后,RBL-2H3细胞胞内Ca2+浓度到达峰值的时间(TTP)延长至212.24±5.07(n=51,p<0.01 vs对照组),RBL-2H3细胞胞内Ca2+浓度降低50%所需的时间(T0.5)缩短至372.61±6.45(n=51,p<0.01 vs对照组)。当移除细胞外液中的2mM Ca2+时,胞内Ca2+浓度的升高完全依赖于胞内的释放,虎杖苷对胞内Ca2+浓度升高无明显抑制,说明虎杖苷主要通过减少胞外Ca2+通过CRAC通道内流来减少免疫性刺激物引起的Ca2+瞬变。
大量文献报道,内质网Ca2+-ATP酶的激动剂毒胡萝卜素(Thapsigargin,TG)通过排空胞内钙库诱导钙释放激活的钙通道(CRAC通道)开放引起胞外Ca2+内流。在胞外液内含有2mM Ca2+时,1μM TG诱导产生较大Ca2+瞬变,虎杖苷明显降低Ca2+瞬变幅度,ΔF／FO从对照组的1.44±0.08(n=36)减少到虎杖苷治疗组的0.67±0.03(n=65,p<0.01 vs对照组)。为了区分胞外Ca2+通过CRAC通道内流在虎杖苷介导的Ca2+瞬变中所起的作用,我们首先在无细胞外Ca2+时用TG刺激,此时产生的Ca2+瞬变完全由细胞内钙释放引起。然后细胞外液换成2mMCa2+的液体,诱导一个完全由胞外Ca2+通过CRAC通道的内流的Ca2+瞬变。虎杖苷能够明显降低第二个Ca2+瞬变的幅度,ΔF／FO从对照组的1.58±0.14(n=127)减少到虎杖苷处理组的0.49±0.07(n=51,p<0.01 vs对照组)。这些数据进一步证实,虎杖苷通过抑制细胞外钙通过CRAC通道内流而减少免疫性刺激物诱导的肥大细胞产生的Ca2+瞬变。
4.虎杖苷减少免疫性刺激物活化的RBL-2H3细胞胞内氧自由基(Reactive oxygen species, ROS)的产生。
已有文献报道,免疫性刺激物诱导的肥大细胞脱颗粒与ROS生成的增加有关,ROS刺激肥大细胞的CRAC通道开放,产生Ca2+瞬变,从而引起肥大细胞脱颗粒。我们采用DCFH-DA标记细胞内ROS,发现DNP-BSA增加胞内ROS的生成,而虎杖苷能够明显减少ROS的生成。对照组DNP-BSA引起ROS生成增加76%±3.0%(n=127),而虎杖苷组DNP-BSA引起ROS生成增加仅为50%±4.1%(n=137,p<0.01 vs阳性对照组)。这些数据表明：虎杖苷可能通过降低活化肥大细胞的氧化应激来抑制CRAC通道的活性。
二.虎杖苷抑制OVA诱导的Balb/c小鼠过敏性哮喘
4-6周龄的Balb/c mice小鼠,随即分成4组,即对照组,单独虎杖苷组,过敏性哮喘模型组,虎杖苷过敏性哮喘治疗组。过敏性哮喘通过OVA诱发。
1.虎杖苷降低过敏性哮喘Balb/c小鼠气道对乙酰甲胆碱的反应性
采用测量Balb/c小鼠气道高反应性的装置,给Balb／c小鼠雾化吸入乙酰甲胆碱,浓度依次为1mg/mL、10mg/mL、30mg/mL、50mg/mL、100mg/mL),观察Balb/c小鼠对不同浓度乙酰甲胆碱的反应性,在过敏性哮喘组,不同浓度乙酰甲胆碱相对应的基线增加百分比值依次为11.4%(n=6),58.3%(n=6),246.9%(n=6),492.0%(n=6),832.5%(n=6),而经虎杖苷治疗后,基线增加百分比值依次降低到9.2%(n=6,p>0.05 vs过敏性哮喘对照组),24.1%(n=6,p<0.05 vs过敏性哮喘对照组),70.1%(n=6,p<0.01 vs过敏性哮喘对照组),211.4%(n=6,p p<0.01vs过敏性哮喘对照组),和351.8%(n=6,p p<0.01 vs过敏性哮喘对照组)。这些结果提示：虎杖苷可能通过降低气道对乙酰甲胆碱的反应性来改善Balb／c小鼠过敏性哮喘症状。
2.虎杖苷明显降低支气管肺泡灌洗液(BALF)中的嗜酸性粒细胞数目
用冰冷磷酸盐缓冲液(PBS)500μL进行支气管肺泡灌洗(BALF),反复抽吸3次,然后回收支气管肺泡灌洗液(BALF),并记录回吸收量；BALF经500 g,37℃,离心5 min后,取离心后的沉淀细胞,用1mL PBS重悬细胞,进行计数；另取细胞悬液于洁净光滑载玻片上涂片,用刘氏染色液染色,计数200个细胞,进行细胞分类计数。发现过敏性哮喘小鼠支气管肺泡灌洗液内的嗜酸性粒细胞明显增加,过敏性哮喘组嗜酸性粒细胞为69.2±3.1(n=6),虎杖苷治疗组嗜酸性粒细胞的数目显著减少至11.0±0.7(n=6,p p<0.01 vs过敏性哮喘对照组),结果表明：虎杖苷具有降低免疫性刺激物引起肺组织中的嗜酸性粒细胞浸润的作用。
3.虎杖苷减轻过敏性哮喘小鼠肺组织的炎症反应,改善支气管结构的完整性
先进行支气管肺泡灌洗,接着取肺组织做石蜡组织切片,结果发现过敏性哮喘组小鼠肺间质内有大量炎性细胞浸润,肺细支气管上皮细胞不同程度增生及肺细支气管一定程度的破坏；而虎杖苷组的肺间质炎性细胞明显减少,肺细支气管结构的完整性也有明显的改善,并且上皮细胞增生明显降低。提示虎杖苷可能通过改善肺功能来治疗过敏性哮喘。
4.虎杖苷抑制过敏性哮喘小鼠肥大细胞CRAC通道的开放
腹腔分离的肥大细胞用5μM Fluo-4染料染色30min,然后加1μM毒胡萝卜素(Thapsigargin),通过Fluo-4的荧光来反映肥大细胞胞内的荧光强度变化。TG在无Ca2+情况诱导胞内较少的Ca2瞬变,然后当细胞外液变为2mM Ca2+液体时,胞内的Ca2+瞬变明显增加。后者的Ca2+瞬变增加反映的是胞外Ca2+通过CRAC通道的内流,与过敏性哮喘小鼠相比,经过虎杖苷治疗后的过敏性哮喘小鼠,肥大细胞胞内出现的第二次Ca2+瞬变明显降低,F／F0从4.3±0.1(n=106)降低到3.2±0.2(n=52,p p<0.01 vs过敏性哮喘对照组),这些数据从在体水平进一步证实：虎杖苷具有抑制胞外Ca2+经过活化肥大细胞CRAC通道内流的作用,从而减轻过敏性哮喘的发生。
5.虎杖苷降低过敏性哮喘小鼠血清中的IgE水平,升高血清中的IgG2a水平
OVA致敏Balb/c小鼠。过敏性哮喘对照组小鼠血清IgE水平为0.54±0.14(n=18),当接受虎杖苷治疗后,血清中的IgE水平明显减少至0.32±0.08(n=18,p<0.01 vs过敏性哮喘对照组)；另外,过敏性哮喘对照组小鼠血清IgG2a水平为0.79±0.17(n=18),在接受虎杖苷治疗后,血清中的IgG2a水平明显增加至1.15±0.36(n=18,p<0.01 vs过敏性哮喘对照组)。IgE抗体为致敏性抗体已得到公认,而IgG抗体被大多文献证实是一种保护性抗体,它可以竞争性抑制IgE与其受体结合,从而减少肥大细胞脱颗粒,进而减轻过敏性哮喘症状。这些数据表明：虎杖苷可能通过减少血清中的IgE水平,增加血清中的IgG2a水平来减轻过敏性哮喘症状。
结论：
1虎杖苷剂量依赖性抑制免疫性刺激物引起的RBL-2H3肥大细胞脱颗粒。
2虎杖苷通过抑制胞外Ca2+经过CRAC通道内流减少免疫性刺激物诱导的Ca2+瞬变。
3虎杖苷抑制活化RBL-2H3细胞胞内ROS的产生。
4虎杖苷对OVA诱导的过敏性哮喘具有治疗作用,表现为气道反应性降低、肺泡组织炎症细胞的浸润减少、血清中IgE水平降低,而IgG2a水平升高。
5虎杖苷对过敏性哮喘治疗的一个重要机制：减少免疫性刺激物引起的ROS产生,从而抑制CRAC通道激活造成的细胞外Ca2+内流,进而抑制肥大细胞脱颗粒。
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 manner
In 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 stimulation
DNP-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 cells
It 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 mice
Balb/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 methacholine
With 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 bronchial
Firstly,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 cells
Isolated 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 mice
Balb/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.
引文
[1]CHEN PENG, YANG L ICHUAN, LEIWEIYA, et al.Effects of polydatin on platelet aggregation and platelet cytosolic calcium [J]. Natural Product R&D, 2005,17(1):21-25
[2]Rennard, S. I. Repair mechanisms in asthma. J. Allergy Clin. Immunol. 98:S278-286; 1996.
[3]Alma J. Nauta, Ferdi Engels, Leon M. Knippels, et al. Mechanisms of allergy and asthma. European Journal of Pharmacology 585 (2008) 354-360
[4]Yukiko I. Nigo, Masakatsu Yamashita, Kiyoshi Hirahara, et al. Regulation of allergic airway inflammation through Toll-like receptor 4-mediated modification of mast cell function. Proc Natl Abad Sci USA.2006 Feb 14;103(7):2286-91.
[5]Kampe M,Janson C, Stalenheim G, et al. Experimental and seasonal exposure to birch pollen in allergic rhinitis and allergic asthma with regard to the inflammatory response. Clin Respir J.2010 Jan;4(1):37-44.
[6]Hawrylowicz C,Ryanna K. Asthma and allergy:the early beginnings. Nat Med. 2010 Mar; 16(3):274-5.
[7]Togias A, Fenton MJ, Gergen PJ, et al. Asthma in the inner city:the perspective of the National Institute of Allergy and Infectious Diseases. J Allergy Clin Immunol.2010 Mar;125(3):540-4.
[8]Ozol D, Mete E. Asthma and food allergy. Curr Opin Pulm Med.2008 Jan;14(1):9-12.
[9]Meeyoung Lee, Soyoung Kim, Ok-Kyoung Kwon, et al. International Immunopharmacology, April 2009,9(4):418-424
[10]Hofmann AM, Abraham SN. New roles for mast cells in pathogen defense and allergic disease. Discov Med 2010 Feb;9(45):79-83
[11]Hofmann AM, Abraham SN. New roles for mast cells in modulating allergic reactions and immunity against pathogens. Curr Opin Immunol,2009, Dec; 21(6):679-86
[12]Palker TJ, Dong G, Leitner WW, et al. Mast cells in innate and adaptive immunity to infection. Eur J Immunol.2010 Jan;40(1):13-8.
[13]Kondo N, Matsui E, Nishimura A, et al. Pharmacogenetics of asthma in children. Allergy Asthma Immunol Res.2010 Jan;2(1):14-9.
[14]Halken S. Prevention of allergic disease in childhood:clinical and epidemiological aspects of primary and secondary allergy prevention. Pediatr Allergy Immunol.2004 Jun;15 Suppl 16:4-5,9-32.
[15]Bierbaum S, Heinzmann A.The genetics of bronchial asthma in children. Respir Med.2007 Jul;101(7):1369-75.
[16]Barthwal MS, Katoch CD, Marwah V. Impact of optimal asthma education programme on asthma morbidity, inhalation technique and asthma knowledge. J Assoc Physicians India.2009 Aug;57:574-6,579.
[17]Warman KL, Silver EJ, Stein RE. Asthma symptoms, morbidity, and antiinflammatory use in inner-city children. Pediatrics.2001 Aug; 108(2): 277-82.
[18]Stelekati E, Orinska Z, Bulfone-Paus S. Mast cells in allergy:innate instructors of adaptive responses. Immunobiology.2007;212(6):505-19.
[19]Metz M, Maurer M. Mast cells--key effector cells in immune responses. Trends Immunol.2007 May;28(5):234-41.
[20]Rao KN, Brown MA. Mast cells:multifaceted immune cells with diverse roles in health and disease. Ann N Y Acad Sci.2008 Nov;1143:83-104.
[21]Heib V, Becker M, Taube C, et al. Advances in the understanding of mast cell function. Br J Haematol.2008 Sep;142(5):683-94.
[22]Taube C, Stassen M. Mast cells and mast cell-derived factors in the regulation of allergic sensitization. Chem Immunol Allergy.2008;94:58-66.
[23]Stassen M, Hultner L, Miiller C, et al. Mast cells and inflammation. Arch Immunol Ther Exp (Warsz).2002;50(3):179-85.
[24]Vliagoftis H, Befus AD. Rapidly changing perspectives about mast cells at mucosal surfaces. Immunol Rev.2005 Aug;206:190-203.
[25]Frossi B, De Carli M, Pucillo C. The mast cell:an antenna of the microenvironment that directs the immune response. J Leukoc Biol.2004 Apr;75(4):579-85.
[26]Ryan JJ, Fernando JF. Mast cell modulation of the immune response. Curr Allergy Asthma Rep.2009 Sep;9(5):353-9.
[27]MacGlashan D Jr. IgE receptor and signal transduction in mast cells and basophils. Curr Opin Immunol.2008 Dec;20(6):717-23.
[28]Metcalfe DD, Peavy RD, Gilfillan AM. Mechanisms of mast cell signaling in anaphylaxis. J Allergy Clin Immunol.2009 Oct;124(4):639-46;
[29]Felaco P,Toniato E,Castellani ML, et al. Infections and mast cells. J Biol Regul Homeost Agents.2009 Oct-Dec;23(4):231-8.
[30]Putney JW Jr. A model for receptor-regulated calcium entry. Cell Calcium.1986 Feb;7(1):1-12.
[31]Hoth M,Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells[J].Nature,1992,355(6358):353-356
[32]Putney JW Jr,Bird GS. The inositol phosphate-calcium signaling system in nonexcitable cells[J].Endocr Rev,1993,14(5):610-631
[33]Parekh AB,Putney JW Jr. Store-operated calcium channels[J].Physiol Rev,2005,85(2):757-810
[34]Di Capite J, Parekh AB. CRAC channels and Ca2+ signaling in mast cells. Immunol Rev.2009 Sep;231(1):45-58.
[35]Inoue T, Suzuki Y, Yoshimaru T, et al. Nitric Oxide positively regulates Ag (I)-induced Ca2+ influx and mast cell activation:role of a Nitric Oxide Synthase-independent pathway. J Leukocyte Biol.2009 Dec;86(6):1365-75.
[36]Ma HT, Beaven MA. Regulation of Ca2+ signaling with particular focus on mast cells. Crit Rev Immunol.2009;29(2):155-86.
[37]Ng SW, di Capite J, Singaravelu K, et al Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels. J Biol Chem.2008 Nov 14;283(46):31348-55.
[38]Chang WC, Di Capite J, Nelson C, et al. All-or-none activation of CRAC channels by agonist elicits graded responses in populations of mast cells. J Immunol.2007 Oct 15;179(8):5255-63.
[39]Parekh AB. Local Ca2+ influx through CRAC channels activates temporally and spatially distinct cellular responses. Acta Physiol (Oxf).2009 Jan;195(1):29-35.
[40]Wenting ZHANG, Qing LI, Ming ZHU, et al. Direct Determination of Polydatin and Its Metabolite in Rat Excrement Samples by High-Performance Liquid Chromatography. Chem. Pharm. Bull.56(11) 1592-1595 (2008)
[41]SIYUAN ZHOU, RUNTAO YANG, ZENGHUI TENG, et al. Dose-Dependent Absorption and Metabolism of trans-Polydatin in rats. J. Agric. Food Chem. 2009,57,4572-4579
[42]Chibuike C Udenigwe, Vanu R Ramprasath, Rotimi E Aluko, et al.Potential of resveratrol in anticancer and anti-inflammatory therapy. Nutrition Reviews Vol. 66(8):445-454
[43]Catalina Alarc_n de la Lastra and Isabel Villegas. Resveratrol as an anti-inflammatory and anti-aging agent:Mechanisms and clinical implications. Mol. Nutr. Food Res.2005,49,405-430
[44]Stephanie C. Eisenbarth, Damani A.et al. Lipopolysaccharide-enhanced, Toll-like Receptor 4-dependent T Helper Cell Type 2 Responses to Inhaled Antigen. Brief Definitive Report,2002 December
[45]D. Murakami, H. Yamada, T. Yajima,et al. Lipopolysaccharide inhalation exacerbates allergic airway inflammation by activating mast cells and promoting Th2 responses. Clinical and Experimental Allergy,2006 37,339-347
[2]Rennard, S. I. Repair mechanisms in asthma. J. Allergy Clin. Immunol. 98:S278-286; 1996.
[3]Alma J. Nauta, Ferdi Engels, Leon M. Knippels, et al. Mechanisms of allergy and asthma. European Journal of Pharmacology 585 (2008) 354-360
[4]Yukiko I. Nigo, Masakatsu Yamashita, Kiyoshi Hirahara, et al. Regulation of allergic airway inflammation through Toll-like receptor 4-mediated modification of mast cell function. Proc Natl Abad Sci USA.2006 Feb 14;103(7):2286-91.
[5]Kampe M,Janson C, Stalenheim G, et al. Experimental and seasonal exposure to birch pollen in allergic rhinitis and allergic asthma with regard to the inflammatory response. Clin Respir J.2010 Jan;4(1):37-44.
[6]Hawrylowicz C,Ryanna K. Asthma and allergy:the early beginnings. Nat Med. 2010 Mar; 16(3):274-5.
[7]Togias A, Fenton MJ, Gergen PJ, et al. Asthma in the inner city:the perspective of the National Institute of Allergy and Infectious Diseases. J Allergy Clin Immunol.2010 Mar;125(3):540-4.
[8]Ozol D, Mete E. Asthma and food allergy. Curr Opin Pulm Med.2008 Jan;14(1):9-12.
[9]Meeyoung Lee, Soyoung Kim, Ok-Kyoung Kwon, et al. International Immunopharmacology, April 2009,9(4):418-424
[10]Hofmann AM, Abraham SN. New roles for mast cells in pathogen defense and allergic disease. Discov Med 2010 Feb;9(45):79-83
[11]Hofmann AM, Abraham SN. New roles for mast cells in modulating allergic reactions and immunity against pathogens. Curr Opin Immunol,2009, Dec; 21(6):679-86
[12]Palker TJ, Dong G, Leitner WW, et al. Mast cells in innate and adaptive immunity to infection. Eur J Immunol.2010 Jan;40(1):13-8.
[13]Kondo N, Matsui E, Nishimura A, et al. Pharmacogenetics of asthma in children. Allergy Asthma Immunol Res.2010 Jan;2(1):14-9.
[14]Halken S. Prevention of allergic disease in childhood:clinical and epidemiological aspects of primary and secondary allergy prevention. Pediatr Allergy Immunol.2004 Jun;15 Suppl 16:4-5,9-32.
[15]Bierbaum S, Heinzmann A.The genetics of bronchial asthma in children. Respir Med.2007 Jul;101(7):1369-75.
[16]Barthwal MS, Katoch CD, Marwah V. Impact of optimal asthma education programme on asthma morbidity, inhalation technique and asthma knowledge. J Assoc Physicians India.2009 Aug;57:574-6,579.
[17]Warman KL, Silver EJ, Stein RE. Asthma symptoms, morbidity, and antiinflammatory use in inner-city children. Pediatrics.2001 Aug; 108(2): 277-82.
[18]Stelekati E, Orinska Z, Bulfone-Paus S. Mast cells in allergy:innate instructors of adaptive responses. Immunobiology.2007;212(6):505-19.
[19]Metz M, Maurer M. Mast cells--key effector cells in immune responses. Trends Immunol.2007 May;28(5):234-41.
[20]Rao KN, Brown MA. Mast cells:multifaceted immune cells with diverse roles in health and disease. Ann N Y Acad Sci.2008 Nov;1143:83-104.
[21]Heib V, Becker M, Taube C, et al. Advances in the understanding of mast cell function. Br J Haematol.2008 Sep;142(5):683-94.
[22]Taube C, Stassen M. Mast cells and mast cell-derived factors in the regulation of allergic sensitization. Chem Immunol Allergy.2008;94:58-66.
[23]Stassen M, Hultner L, Miiller C, et al. Mast cells and inflammation. Arch Immunol Ther Exp (Warsz).2002;50(3):179-85.
[24]Vliagoftis H, Befus AD. Rapidly changing perspectives about mast cells at mucosal surfaces. Immunol Rev.2005 Aug;206:190-203.
[25]Frossi B, De Carli M, Pucillo C. The mast cell:an antenna of the microenvironment that directs the immune response. J Leukoc Biol.2004 Apr;75(4):579-85.
[26]Ryan JJ, Fernando JF. Mast cell modulation of the immune response. Curr Allergy Asthma Rep.2009 Sep;9(5):353-9.
[27]MacGlashan D Jr. IgE receptor and signal transduction in mast cells and basophils. Curr Opin Immunol.2008 Dec;20(6):717-23.
[28]Metcalfe DD, Peavy RD, Gilfillan AM. Mechanisms of mast cell signaling in anaphylaxis. J Allergy Clin Immunol.2009 Oct;124(4):639-46;
[29]Felaco P,Toniato E,Castellani ML, et al. Infections and mast cells. J Biol Regul Homeost Agents.2009 Oct-Dec;23(4):231-8.
[30]Putney JW Jr. A model for receptor-regulated calcium entry. Cell Calcium.1986 Feb;7(1):1-12.
[31]Hoth M,Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells[J].Nature,1992,355(6358):353-356
[32]Putney JW Jr,Bird GS. The inositol phosphate-calcium signaling system in nonexcitable cells[J].Endocr Rev,1993,14(5):610-631
[33]Parekh AB,Putney JW Jr. Store-operated calcium channels[J].Physiol Rev,2005,85(2):757-810
[34]Di Capite J, Parekh AB. CRAC channels and Ca2+ signaling in mast cells. Immunol Rev.2009 Sep;231(1):45-58.
[35]Inoue T, Suzuki Y, Yoshimaru T, et al. Nitric Oxide positively regulates Ag (I)-induced Ca2+ influx and mast cell activation:role of a Nitric Oxide Synthase-independent pathway. J Leukocyte Biol.2009 Dec;86(6):1365-75.
[36]Ma HT, Beaven MA. Regulation of Ca2+ signaling with particular focus on mast cells. Crit Rev Immunol.2009;29(2):155-86.
[37]Ng SW, di Capite J, Singaravelu K, et al Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels. J Biol Chem.2008 Nov 14;283(46):31348-55.
[38]Chang WC, Di Capite J, Nelson C, et al. All-or-none activation of CRAC channels by agonist elicits graded responses in populations of mast cells. J Immunol.2007 Oct 15;179(8):5255-63.
[39]Parekh AB. Local Ca2+ influx through CRAC channels activates temporally and spatially distinct cellular responses. Acta Physiol (Oxf).2009 Jan;195(1):29-35.
[40]Wenting ZHANG, Qing LI, Ming ZHU, et al. Direct Determination of Polydatin and Its Metabolite in Rat Excrement Samples by High-Performance Liquid Chromatography. Chem. Pharm. Bull.56(11) 1592-1595 (2008)
[41]SIYUAN ZHOU, RUNTAO YANG, ZENGHUI TENG, et al. Dose-Dependent Absorption and Metabolism of trans-Polydatin in rats. J. Agric. Food Chem. 2009,57,4572-4579
[42]Chibuike C Udenigwe, Vanu R Ramprasath, Rotimi E Aluko, et al.Potential of resveratrol in anticancer and anti-inflammatory therapy. Nutrition Reviews Vol. 66(8):445-454
[43]Catalina Alarc_n de la Lastra and Isabel Villegas. Resveratrol as an anti-inflammatory and anti-aging agent:Mechanisms and clinical implications. Mol. Nutr. Food Res.2005,49,405-430
[44]Stephanie C. Eisenbarth, Damani A.et al. Lipopolysaccharide-enhanced, Toll-like Receptor 4-dependent T Helper Cell Type 2 Responses to Inhaled Antigen. Brief Definitive Report,2002 December
[45]D. Murakami, H. Yamada, T. Yajima,et al. Lipopolysaccharide inhalation exacerbates allergic airway inflammation by activating mast cells and promoting Th2 responses. Clinical and Experimental Allergy,2006 37,339-347