白芍总苷调控肥大细胞Gab2信号抗过敏哮喘机制及其配伍研究

The prevalence of allergic asthma continues to rise, but current drugs provide symptomatic relief to some, but not all, patients. Recent studies have demonstrated Gab2 molecule as a scaffold protein played a very important role in allergic inflammatory signal transduction. Our previous study found that Radix Paeoniae Alba and total glucosides of paeony (TGP) can not only inhibit the degranulation of mast cells and inflammatory cytokine release, but also reduce the alveolar inflammation in mice with allergic asthma. Our preliminary study found that the mechanism of Radix Paeoniae Alba was that inhibiting mast cell activation downstream of P-Gab2, P-Akt and P-Erk protein levels and Gab2 / Shp2 complex formation. We speculate that the effect of TGP inhibit degranulation so as to achieve the role of anti-allergic asthma through the regulation of Gab2 signaling in mast cells. This project aims to use Gab2 transgenic mice to clear the pharmacological activity of TGP anti-allergic asthma through regulation of Gab2 signal anti-asthma model; using siRNA, iTRAQ quantitative proteomics and transcriptome expression profile microarray technology, to reveal the regulation of TGP on multiple targets with Gab2 as the center of asthma by spectral efficiency and network; and cooperative study and compatibility of the material basis of TGP. The expected results of this project not only can understand the anti-allergic asthma mechanism of TGP, but also for the development of new anti-allergic asthma medication to provide ideas and drug targets.

过敏性哮喘发病率逐年上升，但缺乏有效防治药物。最新研究证明Gab2作为细胞磷酸化信号重要支架蛋白在过敏炎症信号转导中发挥了极为重要作用。我们前期发现白芍及TGP不仅能体外抑制肥大细胞脱颗粒及炎症因子释放，还能抑制体内小鼠过敏性哮喘肺泡炎症。究其机理我们初步研究发现白芍能抑制肥大细胞活化下游P-Gab2，P-Akt和P-Erk蛋白水平及Gab2/Shp2复合物形成。据此我们推测TGP通过调控Gab2信号抑制肥大细胞脱颗粒从而达到抗过敏性哮喘作用。本项目拟采用Gab转基因小鼠构建哮喘模型来明确TGP通过调控Gab2信号抗过敏哮喘药理活性；采用siRNA、iTRAQ定量蛋白质组和全转录组表达谱芯片等技术，揭示TGP对哮喘病理网络以Gab2为中心的多靶点调控；并通过谱效和协同研究探讨TGP物质基础及最佳配伍。本项目不仅能揭示TGP抗过敏性哮喘作用机制，同时为开发新抗过敏哮喘药物提供思路和药物靶点。

1.白芍呈剂量依赖性抑制肥大细胞脱颗粒标志物β-hexosaminidase水平；白芍能显著抑制迟发型过敏反应的小鼠耳廓肿胀程度和病理改变；可显著降低肥大细胞多种炎症因子（TNFα、IL6、IL13等）mRNA水平表达；可抑制肥大细胞P-Gab2、P-Akt和P-Erk蛋白水平和降低Gab2/Shp2复合物形成。.2.模型组哮喘小鼠吸入ACh后，气道阻力明显升高；肺泡灌洗液中白细胞、嗜酸性粒细胞和中性粒细胞数量均明显升高，肺部病理表现为炎症细胞浸润、粘膜充血、杯状细胞增生、胶原沉积和支气管收缩。而哮喘小鼠灌胃给予不同剂量的白芍总苷治疗后，可以明显降低哮喘小鼠白细胞，尤其是嗜酸性粒细胞的数量，减轻肺部炎症，减少胶原沉积，逆转支气管狭窄，降低气道高压；白芍总苷可降低Eotaxin和MIP1α水平；白芍总苷能显著降低哮喘小鼠血清和肺组织中肿瘤坏死因子-α和白细胞介素-4的水平，对血清中的白介素-13水平有轻微的降低作用。提示TGP可能通过减少细胞因子和趋化因子的释放来抑制肥大细胞脱颗粒。以β-氨基己糖苷酶释放作为肥大细胞激活的标志物，我们在体内观察到白芍总苷能减少变应原诱导的肥大细胞脱颗粒。此外，31~1000μg/mL白芍总苷在体外以剂量依赖方式抑制β-己糖苷酶的释放，且呈剂量依赖性地抑制细胞内钙的释放。同时我们也发现1000μg/mL白芍总苷可明显抑制巨噬细胞分泌IL-6、IL-10、MCP-1、IFNγ和TNF水平；可明显趋化因子CCL2、CCL3、CCL4、CCL5、CCL7 mRNA表达，尤其对CCL2和CCL7抑制作用较强，可明显抑制NF-kB p65入核。.3.在芍药苷可明显治疗小鼠过敏性哮喘的作用，且通过整合转录组和代谢组学发现，芍药苷的潜在靶蛋白在脂肪酸代谢、炎症反应、氧化应激和局部粘连中发挥重要作用。