尘螨、豚草、曲霉粗提物诱导的慢性气道炎症及以Syk为靶点化合物Vam3抗炎机制研究

发布时间：2018-05-14 10:42

本文选题：哮喘 + 尘螨　；参考：《北京协和医学院》2013年博士论文

【摘要】：目前全球大约有3亿人患哮喘,其中多数患者通过吸入免疫抑制剂(如糖皮质激素)和p-肾上腺素受体激动剂治疗急性哮喘,获得良好的效果。然而大约5%～10%的慢性哮喘患者却对此类治疗反应效果较差。基于类固醇拮抗型气道炎症严重影响人类的健康,且发病机制尚未明确,因此在此类疾病的预防、诊断、治疗上还存在很大的缺口,现有的部分临床治疗药物也存在长期的副作用或难吸收等各种各样的问题,研究类固醇拮抗型气道炎症机制和开发针对此类疾病有效的药物成为当前急需解决的问题。本研究的目的是探索慢性气道炎症机制,并评价以Km(米氏常数)原理高通量筛选的Syk激酶特异性抑制剂Vam3的抗炎机制,以期开发出具有治疗慢性气道炎症的一类新药。大多数哮喘患者的致敏原为环境中的多种抗原,但在多数实验动物模型中只使用一种过敏原,不能反映患者发病机制的本质。本研究通过采用患者常见的尘螨、豚草和曲霉菌属组成的3类过敏原复合物(DRA)免疫动物,建立了慢性哮喘模型,并发现在慢性气道炎症中通过NF-κB影响炎症因子IL-8表达与活性的作用机制。首先,将雌性BALB/c小鼠分为3组,模型组和药物组中尘螨、豚草和曲霉菌属(DRA)作为抗原,生理盐水作为对照,重复免疫动物10周。其次,药物组中地塞米松作为治疗药物,对照组和模型组中生理盐水作为安慰剂,口服灌胃两周,每天1次。结果观察到DRA增加了小鼠支气管肺泡灌流(BAL)液中的IL-10、TGF-β水平和中性粒细胞在肺组织中募集。地塞米松降低了炎症相关细胞因子的水平,抑制了肺组织中肥大细胞的浸润和粘液高分泌。但是,地塞米松并没有降低反而增加了肺组织中性粒细胞浸润和与人类功能同源的IL-8的水平。体外研究发现,尘螨主要抗原组分Der p1可刺激人肺泡上皮A549细胞,增强了IL-8的基因表达和NF-κB的活性。然而,未见地塞米松抑制A549细胞中升高的IL-8水平,但NF-κB抑制剂BAY11-7082却降低了IL-8的高表达。实验结果表明IL-8水平的增加可能参与类固醇拮抗型中性粒细胞型气道炎症,而这种机制可能与通过激活NF-κB途径有关。脾酪氨酸激酶(Syk)可表达于T细胞、B细胞、肥大细胞等多种免疫细胞内,在急性和慢性炎症中,通过细胞外抗原诱导的IgE受体FcεRI介导的免疫细胞活化中发挥重要作用。本研究的目的是观察二苯乙烯类化合物Vam3对免疫复合物诱导炎症的抑制及其机制。首先,建立了以Km原理高通量化合物筛选模型,观察Vam3对Syk激酶的抑制作用。其次,通过活化IgE受体FcεRI介导的免疫细胞活化,评价Vamm3在细胞水平上的抗炎作用。最后,应用蛋白免疫印迹技术评价Vam3对Syk激酶下游信号通路的影响。结果观察到二苯乙烯类化合物Vam3竞争性抑制了Syk激酶的催化活性,其Ki和IC50值分别为61.09nM和62.95nM。 Vam3在细胞水平上以剂量依赖的方式降低了大鼠腹腔肥大细胞和RBL-2H3细胞内Ca2+的水平。同时,Vam3抑制了多种炎症介质的产生,如肿瘤坏死因子TNF-α, IL-6, IL-8等,其IC50值分别为3.2μM、2.43μM、1.3μM。此外,通过蛋白免疫印迹技术发现Vam3能够抑制Syk蛋白激酶的Y525/526位点、下游分子LAT的Y191位点、cPLA2的S505位点、PLCγ-1的Y783位点和S1248位点的磷酸化。实验结果表明了Vam3是通过抑制Syk的催化活性,导致下游分子LAT、cPLA2、PLCγ-1活化的抑制,进而抑制炎症细胞的活化和下游炎症因子的释放。本研究所建立的高通量筛选Syk激酶特异性抑制方法为以Syk为靶点的抗哮喘药物筛选及其抗哮喘慢性气道炎症机制研究提供了实验依据。
[Abstract]:At present, about 300 million people worldwide suffer from asthma, most of them have achieved good results in the treatment of acute asthma by inhaling immunosuppressive agents (such as glucocorticoid) and p- adrenergic receptor agonists. However, about 5% to 10% of chronic asthma patients have poor response to this kind of treatment. There is still a great gap in the prevention, diagnosis and treatment of this kind of disease. There are also various problems, such as long-term side effects or difficult absorption, and the study of the mechanism of steroid antagonistic airway inflammation and the development of effective drugs for such diseases. The aim of this study is to explore the mechanism of chronic airway inflammation and to evaluate the anti-inflammatory mechanism of the Syk kinase specific inhibitor, Vam3, which is a high throughput screening of Km (Michaelis constant) principle, in order to develop a new class of drugs for the treatment of chronic airway inflammation.
The sensitization of most asthmatic patients is a variety of antigen in the environment, but only one kind of allergen is used in most experimental animal models. It does not reflect the nature of the patient's pathogenesis. This study established a chronic asthma model by using 3 types of allergen complex (DRA) immune animals, which were composed of common dust mites, ragweed and Aspergillus. And we found that NF- kappa B affects the expression and activity of inflammatory factor IL-8 in chronic airway inflammation. First, female BALB/c mice are divided into 3 groups: dust mites in model group and drug group, ragweed and Aspergillus (DRA) as antigen, saline as control, repeated immune animals for 10 weeks. Secondly, dexamethasone in the drug group is treated as a treatment. The treatment, the control group and the model group were treated with saline as a placebo, two weeks after oral administration of the stomach, 1 times a day. The results showed that DRA increased the IL-10, TGF- beta level and neutrophils in the lung tissue of the bronchoalveolar perfusion (BAL) fluid in mice. The infiltration of large cells and hypersecretion of mucus. However, dexamethasone did not decrease and increased the level of neutrophils infiltration and human function homologous IL-8. In vitro studies have found that Der P1 of the main antigen of dust mite can stimulate human alveolar epithelial A549 cells and enhance the gene expression of IL-8 and the activity of NF- kappa B. Dexamethasone inhibited the elevated IL-8 level in A549 cells, but NF- kappa B inhibitor BAY11-7082 reduced the high expression of IL-8. The experimental results showed that the increase of IL-8 level may be involved in steroid antagonistic neutrophil type airway inflammation, which may be related to the activation of NF- kappa B pathway.
Splenic tyrosine kinase (Syk) can be expressed in a variety of immune cells, such as T cells, B cells, mast cells, and in acute and chronic inflammation, and plays an important role in the activation of immune cells mediated by the IgE receptor Fc e RI induced by extracellular antigen. The purpose of this study was to observe the inflammation induced by the two phenyl ethylene compound Vam3 on immune complexes. Inhibition and its mechanism. First, a high throughput screening model based on Km principle was established to observe the inhibitory effect of Vam3 on Syk kinase. Secondly, the anti-inflammatory effect of Vamm3 on cell level was evaluated by activating IgE receptor Fc e RI mediated immune cell activation. Finally, protein immunoblotting technique was used to evaluate the downstream signal pathway of Vam3 against Syk kinase. The results showed that the two styrene compound Vam3 competitively inhibited the catalytic activity of Syk kinase, and its Ki and IC50 values were 61.09nM and 62.95nM. Vam3, respectively, at a dose dependent manner at the cell level, reducing the level of Ca2+ in rat peritoneal mast cells and RBL-2H3 cells. Meanwhile, Vam3 inhibited the production of a variety of inflammatory mediators. Such as tumor necrosis factor TNF- alpha, IL-6, IL-8 and so on, their IC50 values are 3.2 mu M, 2.43 mu M, 1.3 mu M., and Vam3 can inhibit the Y525/526 loci of Syk protein kinase by protein immunoblotting. Inhibition of the activation of the downstream molecules, LAT, cPLA2, and PLC gamma -1 by inhibiting the catalytic activity of Syk, thus inhibits the activation of inflammatory cells and the release of downstream inflammatory factors. The high throughput screening of Syk kinase specific inhibition methods established by this study is the screening of anti asthma drugs with Syk as the target and the mechanism of chronic airway inflammation in asthma. The experimental basis is provided.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R562.25

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