氣喘為一種呼吸道慢性發炎，而造成氣喘的過敏原最主要為塵蟎Dermatophoides pteronyssinus (Der p)。這些環境中的過敏原含有許多的蛋白分解酵素，其可造成氣道上皮障璧受到破壞，並使得在氣道黏膜中的肥大細胞釋放引起發炎的媒介物如：組織胺和胰蛋白酶 ( tryptase )，使得氣道產生發炎反應。肥大細胞所釋放的胰蛋白酶具有很強的生物活性，可能是進一步造成氣喘中的發炎反應和氣道過度反應。而廣泛存在於肺部內皮或上皮細胞上屬於G protein- coupled receptor的protease activated receptor 2 (PAR-2)可被胰蛋白酶活化，會去影響過敏原誘發的呼吸道發炎反應，在過敏性疾病和呼吸道發炎反應中扮演某種重要的角色，所以猜測一些蛋白酶抑制劑，特別是胰蛋白酶抑制劑 ( tryptase inhibitor )，可能減緩過敏性發炎反應。所以我們利用3種不同的蛋白酶抑制劑，觀察在氣喘老鼠模式上面是否具有抑制發炎的現象。在實驗設計上主要去探討這些抑制劑在預防、治療與過敏原的致過敏性 (allergenicity)的作用，在預防的實驗設計1中，分別看到了給予FUT (nafamostat mesilate)和FOY (gabexate mesilate)後，肺泡沖洗液中的總白血球數、嗜中性白血球和嗜酸性白血球有明顯的降低，而且也抑制了免疫球蛋白IgE的產生，同時也看到發炎性細胞激素和Th2細胞激素下降的情形，也在給予FUT的組別中看到減弱呼吸道過度反應(AHR)，也抑制了肥大細胞的活化作用，在肺組織染色下(Hematoxylin and Eosin stain, H-E stain )，也看到嗜伊紅性白血球的浸潤也有明顯的減少，而在給予FUT和FOY的組別中，肺泡沖洗液中細胞PAR-2的表現有明顯的下降，最後發現塵蟎刺激巨噬細胞後抑制輔助刺激因子B7.2 (CD86)和iNOS的表現，而UTI (Ulinastatin, urinary trypsin inhibitor)的組別中並無看到任何顯著的差異，而在第2個治療的實驗設計中，也看到和實驗設計1相似的情形，給予FUT和FOY的組別中，減少了肺泡沖洗液中發炎細胞的產生，也降低血清中Der p 特異性抗體IgG1的產生，同時抑制了發炎性細胞激素TNF-α的產生，並增加IL-12的產生，FUT和FOY都可抑制肥大細胞的活化，也降低了呼吸道的過度敏感性反應，而給予FUT使得細胞表現PAR-2有明顯的下降，而UTI並無看到任何的差異，在H-E stain也看到FUT和FOY減少肺部嗜伊紅性性白血球的浸潤。而在第3個實驗設計，探討這些蛋白酶抑制劑是否會去干擾這些過敏原本身的酵素活性，進而影響過敏原的過敏原性 (allergenicity)，結果發現給予FUT、FOY降低了肺泡沖洗液中的嗜伊紅性白血球、嗜中性白血球，也降低Der p特異性抗體IgG1的產生，FUT明顯的抑制細胞激素IL-4及IL-6，而UTI反而使得TNF-α大量的產生， FUT和FOY同時也降低了呼吸道敏感性的反應，FUT和FOY同時也抑制肺部沖洗液中細胞PAR-2的表現。
　　論文第二部份則以MH-S(小鼠肺泡巨噬細胞)和P815(小鼠肥大細胞)這兩種細胞株來觀察蛋白酶抑制劑在Der p、LPS、AP (PAR-2 agonist peptide)、RP (antagonist peptide)、trypsin (APR-2 activator)刺激後的PAR-2的影響，給予DP、LPS、AP、RP刺激12和24小時之後對這2種細胞表現PAR-2並無明顯差異。而利用10μM、100μM之AP和RP分別刺激1、6、24之後，發現在AP 10μM刺激MH-S細胞6小時後，PAR-2的表現最高，在100μM刺激後則看到在24小時表現最高。但在P815細胞株並未觀察到這些差異。總而上述實驗，我們認為這些蛋白酶抑制劑FUT和FOY在氣喘老鼠的模式下，所造成的抑制發炎的效果，可能是對於老鼠在致敏的階段中去影響發炎細胞的活化，而使得發炎反應受到影響而減弱，也使得肥大細胞的活化受到抑制，影響了過敏原與肥大細胞活化之間的反應，或是去干擾了過敏原的本身的酵素活性，進而影響過敏原的過敏原性 (allergenicity)，我們也發現這些抑制劑會使得PAR-2的活化受到抑制，細胞發炎的訊息傳遞受到影響，最後減弱氣喘老鼠產生發炎物質與細胞激素的產生。但是這些蛋白酶抑制劑的詳細作用機制目前還不是很清楚，所以必須更進一步的去探討，未來相信這些結果將可提供在過敏性氣喘疾病上一個新的治療方式。

　Asthma is a chronic inflammatory disease of the bronchial airways orchestrated by Th2 T cells and their secreted cytokines. In previous studies, major allergens of Dermatophoides pteronyssinus (Der p) have been identified as a group of proteolytic enzymes, which may cause airway epithelial barrier disruption to contact with airway mucosa mast cell, and releasing preformed or newly synthesis, mediators, such as histamine, tryptase, and evoke the allergen-induced airway inflammation. Mast cell tryptase has potent biologic activities that may contribute to the inflammatory response and airway hyper-reactivity seen in asthma. Moreover, tryptase can activate protease-activated receptor-2 (PAR2), a G protein-coupled receptor that expressed on lung endothelial or epithelial cells, which has important role in the control of airway inflammation and smooth muscle cells proliferation. It is reasonable to suggest that protease inhibitors, particularly for tryptase inhibitor, may attenuate allergenic inflammatory response. In this study, we investigated the effects of protease inhibitors: Nafamostat Mesilate (FUT) and Gabexate Mesilate (FOY), both are serine protease inhibitors and Ulinastatin (UTI), a human urinary trypsin inhibitor, on airway inflammation in a mouse model of allergic asthma. We hypothesized that serine protease inhibitors might inhibit Der p-elicited airway inflammation in a mouse model of asthma. The purpose of this study was to determine the prevention and therapeutic potential or influence of allergenicity of Der p of protease inhibitors on allergy airway inflammation in a mouse model of asthma. The preventive protocol 1 results showed that both FUT and FOY could inhibit total cells counts、eosinophils and neutrophils infiltration and inhibition PAR-2 expression in the BALF, and attenuated Der p-induced Th2 cytokines production and reduced serum IgE levels. In addition, FUT inhibited the iNOS and CD86 expression of alveolar macrophage stimulated with Der p as shown by fluorescence microscope. But, only FUT-treated mice showed a significant change of mast cell activation and airway hyperresponsiveness (AHR). However, UTI-treated mice did not have significantly change in a mouse model of asthma. In the therapeutic experiment of protocol 2, the results showed similar pattern as protocol 1 in decreasing total cell counts, neutrophils, and eosinophils in the BALF of FUT- and FOY-treated mice. The concentrations of Der p-specific IgG1 also showed markedly decreased pro-inflammatory cytokines TNF-α, and IL-12 increased in the BALF on FUT- and FOY-treated mice. Both FUT- and FOY-treated mice showed significantly decrease of mast cell activation of mMCP-1 release and airway hyperresponsiveness (AHR). Only FUT-treated mice showed a significant decrease of PAR-2 expression in the BALF. However UTI-treated mice did not have significantly change in a mouse model of asthma. The protocol 3 results showed a similar pattern as protocol 2 in decreasing total cell counts, neutrophils, and eosinophils in the BALF of FUT- and FOY-treated mice. Only FUT-treated mice showed attenuated IL-4 and IL-6 production, while UTI-treated mice showed increase TNF-α production. Both FUT- and FOY-treated mice showed significantly decreased AHR and inhibited PAR-2 expression in the BALF.
　The second part of our studies was focus on MH-S (mouse alveolar macrophage) and P815 (mouse mast cell) cell lines that stimulated with Der p、LPS、AP (PAR-2 agonist)、RP (PAR-2 antagonist)、trypsin (PAR-2 activator) to observe the effects of protease inhibitors on PAR-2 expression. Both of cell lines stimulated with Der p、LPS、AP、RP in 12 or 24 hours did not have significantly difference in PAR-2 expression. When stimulated with AP 10μM 6 hours and 100μM 24 hours, MH-S cells showed PAR-2 highest expression. Stimulation on P815 cell with above reagents did not have significantly difference in PAR-2 expression. Therefore, we suspect that the anti-inflammatory effect in a asthma model of FUT and FOY may via the influence of allergenicity of Der p、mast cell activation in the airways to reduce allergic airway inflammation and cytokine production. We also found FUT and FOY inhibited PAR-2 activation in BALF from the animal model of asthma. The mechanism of this suppression effect of serine protease inhibitors on allergic airway inflammation is still unclear. Such properties of protease inhibitors might be useful in combination, or as an alternative treatment on allergic disease.

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