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研究生:陳煒
研究生(外文):Wei Chen
論文名稱:抗血小板功能的阿斯匹靈對於急性呼吸窘迫症候群的預防功效評估
論文名稱(外文):Evaluation of prevention effects on aspirin-based antiplatelet therapy for the acute respiratory distress syndrome
指導教授:陳全木陳全木引用關係
指導教授(外文):Chuan-Mu Chen
口試委員:吳誠中陳春榮徐武輝葛其梅
口試委員(外文):Cheng-Chung WuChun-Jung ChenWu-Huei HsuChi-Mei Hsueh
口試日期:2016-07-27
學位類別:博士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:104
中文關鍵詞:阿斯匹靈急性呼吸窘迫症候群高濃度氧抗發炎
外文關鍵詞:AspirinAcute respiratory distress syndromeHyperoxiaAnti-inflammation
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急性呼吸窘迫症候群(ARDS)是一種常見的重症疾病常導致病人的死亡。目前無特殊藥物可以治療這疾病。唯一有效的治療是使用呼吸器低潮氣量的設定來保護肺部。血小板扮演急性呼吸窘迫症候群的致病機轉中關鍵的角色。因此抗血小板治療可能是有機會對於改善此疾病的藥物。在第一章中,我們介紹了急性呼吸窘迫症候群的致病幾轉,然後再更深討論血小板對於相關急性呼吸窘迫症候群機制的影響,其中主要機制包括血小板黏附到受損血管壁,血小板和白血球細胞併內皮細胞的相互作用,血小板相關脂類介質和中性粒細胞胞外陷阱(neutrophil extracellular traps)。我們還總結了抗血小板治療,包括阿斯匹靈,糖蛋白IIb / IIIa受體受體拮抗劑,和P2Y12抑製劑,應用在實驗和臨床的急性呼吸窘迫症候群。在第二章,由於急性呼吸窘迫症候群的流行病學研究大多數是在西方國家進行的,而在亞洲的研究是有限的。因此我們使用台灣全民健康保險研究數據庫統計,發現於1997年和2011年之間,台灣的急性呼吸窘迫症候群的總發生率為每10萬人有15.74人/年,和整體院內死亡率為57.8%,顯示急性呼吸窘迫症候群在台灣亦為不可忽視的重要疾病。在第三章中,研究是否到院前服用阿斯匹靈的患者,比較不會得到急性呼吸窘迫症候群。我們使用美國范德堡大學附設醫院重症病人的前瞻性收集資料庫,發現到院前服用阿斯匹靈使急性呼吸窘迫症候群的風險降低。在第四章中,進一步使用NF-κB/螢光素酶轉基因小鼠合併活體影像系統(IVIS),研究在高濃度氧引起急性肺損傷中,阿斯匹靈的抗炎和抗氧化作用的機制。研究結果顯示不論是前處理(預防性)或後處理(治療性)阿斯匹靈,對於ARDS病症的小鼠,均可透過抑制NF-κB的抗發炎途徑而減少嗜中性白血球的浸潤與肺水腫的症狀。因此,阿斯匹靈可用於保護或治療ARDS肺損傷之病症上。

Acute respiratory distress syndrome (ARDS) is a common and devastating syndrome contributes to serious morbidities and mortality in critically ill patients. No pharmacologic therapy is beneficial to ARDS and the only effective management is protective lung strategy. Platelet plays a crucial role the pathogenesis of ARDS and antiplatelet therapy may be a potential medication for ARDS. In the Chapter 1, we introduce the overall pathogenesis of ARDS first and then focus the platelet related mechanisms for development of ARDS, including platelet adhesion to the injured vessel wall, platelet-leukocyte-endothelium interactions, platelet related lipid mediators and neutrophil extracellular traps. We further summarize the antiplatelet therapy, including aspirin, Glycoprotein IIb/IIIa receptor antagonists, and P2Y12 Inhibitors for ARDS in experimental and clinical studies. In the Chapter 2, because of most epidemiological studies of ARDS have been conducted in western countries, and studies in Asia are limited. We evaluated the incidence, in-hospital mortality and one-year mortality of ARDS in Taiwan by using a nationwide inpatient cohort study based on the Taiwan National Health Insurance Research Database between 1997 and 2011. It showed that overall incidence of ARDS was 15.74 per 100,000 person-years, and overall in-hospital mortality was 57.8%. Thus, ARDS in Taiwan also cannot be ignored important diseases. In the Chapter 3, we aimed to determine whether prehospital aspirin use is associated with decreased ARDS in patients at high risk for ARDS after adjusting for the propensity to receive aspirin by using secondary analysis of patients enrolled prospectively in the Validating Acute Lung Injury Markers for Diagnosis (VALID) study. It showed that prehospital aspirin use was independently associated with a decreased risk of ARDS even after adjusting for the propensity of pre-hospital aspirin use. In the Chapter 4, we investigated the mechanisms of the anti-inflammatory and anti-oxidant effect of aspirin in NF-κB/luciferase transgenic mice on hyperoxia-induced acute lung injury using in vivo imaging system (IVIS). These results showed that either pretreatment with aspirin (protective effect) or post treatment with aspirin (therapeutic efficacy) obviously reduced neutrophil infiltration and lung edema compared to treatment with hyperoxia treated alone. Thus, aspirin has a protective effect or therapeutic efficacy on hyperoxia-induced lung injury.

Contents
Chapter 1. Literature Review: Antiplatelet therapy for acute respiratory distress syndrome
1-1 Abstract..………………………………………………………………………… .1
1-2 Introduction……………………………………………………………………......1
1-2-1 Definition and epidemiology…………………………………………………1
1-2-2 Pathogenesis of ARDS………………………………………………………..2
1-3 The role of platelet in ARDS…………………………………………………...…3
1-3-1 Platelet adhesion to the injured vessel wall…………………………………..4
1-3-2 Platelet-leukocyte-endothelium interactions…………………………………4
1-3-3 Lipid mediators and platelet aggregation…………………………………….5
1-3-4 Neutrophil extracellular traps (NETs)………………………………………..6
1-4 Antiplatelet agents in experimental studies…………………………………...…...7
1-4-1 Aspirin………………………………………………………………………..7
1-4-2 Glycoprotein IIb/IIIa receptor (GP IIb/IIIa) antagonists……………………..9
1-4-3 P2Y12 Inhibitors……………………………………………………………...9
1-5 Antiplatelet agents in clinical studies……………………………………...……..10
1-6 Conclusions……………………………………………………………….……...11
Chapter 2. Incidence and outcomes of acute respiratory distress syndrome – a nationwide study in Taiwan, 1997–2011
2-1 Abstract…………………………………………………………………………..14
2-2 Introduction………………………………………………………………………15
2-3 Materials and Methods…………………………………………………………...16
2-3-1 Study subjects and definition...……………………………………………...16
2-3-2 Statistical analysis....................……………………………………….……..16
2-4 Results……………………………………………………………………………17
2-4-1 The trend of incidence of ARDS...………..………………………………...17
2-4-2 In-hospital mortality...............……………………………………….……...18
2-4-3 One-year mortality...............…………...…………………………….……...18
2-5 Discussion………………………………………………………………………..18
Chapter 3. Prehospital aspirin use is associated with reduced risk of acute respiratory distress syndrome in critically Ill patients: a propensity-adjusted analysis
3-1 Abstract………………………………………………….……………………….26
3-2 Introduction ……………………………………………………………………..26
3-3 Materials and Methods…………………………………………………………..28
3-3-1 Description of the VALID cohort…………………...……………………...28
3-3-2 Study population.....................……………………………………………...29
3-3-3 Statistical analysis....................……………………………………………..29
3-4 Results……………………………….…………………………………………...30
3-5 Discussion…….………………………………………………………………….32
Chapter 4. Protective effect of aspirin against hyperoxia-induced acute lung injury using NF-κB/luciferase inducible transgenic mice
4-1 Abstract………………………………………………………….……………….44
4-2 Introduction………………………………………………………………………45
4-3 Materials and Methods…………………………………………………………..46
4-3-1 Animals........................................…………………...……………………....47
4-3-2 Murine model of hyperoxia-induced acute lung injury...…………………...47
4-3-3 Western blot analysis………………………………………………………..48
4-3-4The imaging of luciferase activity...…………………....................................48
4-3-5 Pathological histology......…………………………………………………..48
4-3-6 Measurement of reactive oxygen species (ROS) generation..………….…..49
4-3-7 Analysis of airway inflammation in bronchoalveolar lavage fluid (BALF)...........…………………………………………………..…………..49
4-3-8 Immunohistochemistry staining…………………..………………… ……..49
4-3-9 Statistical analysis…………………………………………………………..49
4-4 Results…………………………………………………………….……..….…. 50
4-4-1 Part I: The effect of time course in mouse lungs induced by hyperoxia..….50
4-4-2 Part II: Protective effect of aspirin against hyperoxia-induced acute lung injury using NF-κB/luciferase inducible transgenic mice....……………...51
4-4-3 Part III: Therapeutic efficacy of aspirin against hyperoxia-induced acute
lung injury using NF-κB/luciferase inducible transgenic mice.............…..52
4-5 Discussion…………………………………………………………….………….56
Chapter 5. General conclusion……………………………………………………..83
References……………………………………………………………………..……..85
Appendix……………………………………………………………………………100
Figure List
Figure 1. The role of platelets in acute respiratory distress syndrome………….....…12
Figure 2. Protective effects of aspirin against lung inflammation………………..…..13
Figure 3. Incidence of ARDS in Taiwan……………………………….……….…….23
Figure 4. In-hospital mortality rates of ARDS in Taiwan………………………...…..24
Figure 5. One-year mortality rates of ARDS between 1997 and 2011 in Taiwan…....25
Figure 6. Study flow diagram……….………………………………………………..41
Figure 7. Timing of onset of ARDS during the first four days of ICU admission among the 368 patients in the cohort who developed ARDS……..……….42
Figure 8. Incidence of ARDS in patients with and without prehospital aspirin,
statin or combined aspirin and statin use……………………...…………...43
Figure 9. Study flow chart of part II………………………………………….………59
Figure 10. Study flow chart of part III……………………………….….…..……….60
Figure 11. Effect of lung damage in mouse lungs after induction of hyperoxia
for 0, 24, 48 and 72 h……………………………………………………...61
Figure 12. Effect of alveolar growth in mouse lungs after induction of hyperoxia
for 0, 24, 48 and 72 h……………………………...……………………....62
Figure 13. Effect of apoptosis in mouse lungs after induction of hyperoxia
for 0, 24, 48 and 72 h…………………………………….……………..…63
Figure 14. Effect of inflammation in mouse lungs after induction of hyperoxia
for 0, 24, 48 and 72 h…………..……………….……………………...….64
Figure 15. Bioluminescence imaging of lung tissue from NF-κB/luciferase
transgenic mice after induction of hyperoxia for 72 h in the N.C.,
Mock, Aspirin-L and Aspirin-H groups…………….…………….………65
Figure 16. (A) Gross appearance of lung and (B) Lung wet-to-dry weight ratio analysis in the N.C., Mock, Aspirin-L and Aspirin-H groups…..….……..66
Figure 17. Histopathological analyses of lung tissue in the N.C., Mock,
Aspirin-L and Aspirin-H groups…………………………………….…….67
Figure 18. The number of macrophages in bronchoalveolar lavage fluid (BALF)
in the N.C., Mock, Aspirin-L and Aspirin-H groups…….………..………68
Figure 19. Bioluminescence imaging of lung tissue in the N.C., Mock, A25
and A50 groups………………………………………………….….……..69
Figure 20. (A) Gross appearance of lung, (B) Histopathological analyses of lung tissue and (C) Lung wet-to-dry weight ratio analysis in the N.C., Mock, A25 and A50 groups…………………………………….…….……..……70
Figure 21. The number of (A) total cell, (B) macrophage and (C) lymphocyte of bronchoalveolar lavage fluid (BALF) in the N.C., Mock, A25 and A50 groups…………………………………………………………………...…71
Figure 22. Quantification of ROS productions in the (A) extracellular fluid
and (B) intracellular fluid of the BALF in the N.C., Mock, A25 and
A50 groups.…………………………………………...……………….…..72
Figure 23. The CXCL4 protein of the immunohistochemical (IHC) staining in
the lung tissues in the N.C., Mock, A25 and A50 groups…………….…..73
Figure 24. The CC10 protein of the immunohistochemical (IHC) staining in the
lung tissues in the N.C., Mock, A25 and A50 groups………………...…..74
Figure 25. Effect of alveolar growth by hyperoxia in the N.C., Mock, A25 and
A50 groups…………………………………………………………...……75
Figure 26. Effect of alveolar growth by hyperoxia in mouse lungs………………….76
Figure 27. Changes in protein expression levels of p-AKT and p-MAPK in the
N.C., Mock, A25 and A50 groups………………..……………………….77
Figure 28. Changes in protein expression level of NF-kappa B in the N.C.,
Mock, A25 and A50 groups…………………………………………..…..78
Figure 29. Changes in protein expression level of Bax and Bcl-2 in the N.C.,
Mock, A25 and A50 groups…………...………………………..………..79
Figure 30. Changes in protein expression level of Nrp-1 in the N.C., Mock,
A25 and A50 groups……………………………………..………….……80
Figure 31. Changes in protein expression level of TNF-α, IL-8, IL-1β and
IL-6 in the N.C., Mock, A25 and A50 groups…………………….……...81
Figure 32. Scheme of aspirin regulatory pathway………………………………..…..82
Table List
Table 1. Demographic characteristics, clinical features, and resource utilizations
of patients with adult respiratory distress syndrome………………………..22
Table 2. Comparison of demographic data and outcomes between patients
with and without ARDS………………………………………………...…..36
Table 3. Comparison of demographic data and outcomes between patients
with prehospital aspirin use and without aspirin use…………………..……37
Table 4. Logistic regression analysis of prehospital aspirin use and development
of ARDS in all enrolled patients (n=1149)……………………………….....38
Table 5. Logistic regression analysis of prehospital aspirin use and development
of ARDS in sepsis patients (n=725)………………………………………...39
Table 6. Multivariate logistic regression model for mortality (aspirin adjusted
by propensity) in 1149 patients……………………………………….....….40
Table 7. Lung damage frequency in the N.C., Mock, A25 and A50 group of
the mouse lung tissues (n=6) using histopathological image analysis...……58


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