急性肺損傷及肺動脈高壓動物實驗模式在呼吸治療研究之應用

Experimental Models of Acute Lung Injury and Pulmonary Hypertension in Respiratory Research

莊逸君(I-Chun Chuang)

指導教授 ： 黃明賢

高雄醫學大學/醫學院/醫學研究所博士班/博士(2015年)

https://doi.org/10.6832/KMU.2015.00022

摘要

動物模式在科學研究與臨床應用間，扮演了一個很重要的橋樑。呼吸治療是近半世紀，因醫學進步及環境日趨污染所新興起的一門專業學科，相關的應用治療尚有許多開發空間，尤其是“治療性氣體吸入治療”及“噴霧藥物吸入治療”，皆為臨床上應用最廣也最具發展潛力的呼吸治療項目。另外，急性肺損傷(acute lung injury, ALI)及其重症表現的急性呼吸窘迫症病患(acute respiratory distress syndrome, ARDS)，常伴隨之肺動脈高壓症皆為臨床重症治療最為棘手之病症。因此，我們建立了兩個實驗動物模式，並且將實驗依主題分兩個部份設計，模擬臨床上常見的重症呼吸病灶—肺損傷及肺動脈高壓，將之應用於探討兩項極具發展潛力的呼吸治療 – 主題一、 噴霧藥物吸入治療: 利用活體肺“大鼠噴霧藥物吸入模式”及“大鼠急性肺損傷模式”探討麩醯胺酸(glutamine,GLN)吸入治療之肺臟保護效應；主題二、 治療性氣體吸入治療:利用離體肺“肺高壓模式”探討二氧化碳(carbon dioxide, CO2)吸入對肺臟血液動力學的影響。
在主題一實驗，我們建立了活體肺“大鼠噴霧藥物吸入模式”及“大鼠急性肺損傷模式”，探討熱休克蛋白72 (heat shock protein, HSP72)在麩醯胺酸吸入治療之肺臟保護效應及其調控機制。熱休克蛋白已被認為具有保護細胞的功能。其潛在的臨床疾病治療效應是不容忽視的，特別是治療上最棘手的急性肺損傷。目前已知有多種誘導因子可促使熱休克蛋白合成，包括:高溫、低溫、亞砷酸鈉(sodium arsenite)、或腺簿基因轉移載體(adenoviral gene transfer)，但這些方法目前都無法在臨床上應用。故，研發一種可誘發熱休克蛋白合成的臨床治療方法具有其特殊意義。麩醯胺酸被認定是一種安全的熱休克蛋白72誘發物質，它的細胞保護效應不管是在動物實驗或臨床人體實驗都已被證實。GLN投予方法，可考文獻只有全靜脈營養輸入(total pareteral nutrition,TPN)及靜脈單一劑量注射。GLN如能結合噴霧藥物治療(aerosol therapy)有效地被呼吸道吸收，其臨床上的應用及潛在的治療效應是不容忽視的。這部份研究主要利用內毒素 (lipopolysaccharide,LPS)吸入造成的急性肺損傷大鼠實驗模式，測試我們提出之假說:(1)GLN由噴霧方式經呼吸道吸入可誘發肺內HSP72合成，進而降低LPS吸入造成的肺損傷程度。(2)吸入型GLN誘發HSP72表現之肺臟保護效應機制為- 抑制細胞間粘附分子-1 (intercellular adhesion molecule-1, ICAM-1)及巨噬細胞炎症蛋白-2 (macrophage inflammatory protein-2, MIP-2) 肺內表現量，進而降低多核形嗜中性白血球(polymorphonuclear neutrohils, PMNs) 聚集在LPS誘發損傷的肺泡內。研究結果顯示，單一劑量GLN吸入可(1)有效地誘發HSP72表現(2)降低PMNs聚集在LPS誘發損傷的肺泡內(3)降低急性肺損傷嚴重程度。
在主題二實驗，利用離體肺建立缺氧及藥物誘發之“肺動脈高壓模式”，探討二氧化碳吸入對肺臟血液動力學的影響。在臨床上，特殊的通氣策略會影響到二氧化碳在血液中的值。但CO2在肺循環所扮演的角色，至今仍無一個很明確的定論。另外，急性肺損傷及其重症表現的急性呼吸窘迫症病患，在臨床上常有的共同症狀為高二氧化碳血症、缺氧性肺血管收縮及肺動脈高壓，CO 2在肺循環所扮演的角色，需更清楚被明白，才能擬定適合此類重症病患的通氣策略。從我們前驅之研究得知CO2的血管舒張效應是在肺動脈壓過高時才會作用，在這部份研究我們主要探討CO2在不同成因及不同程度肺高壓下之血管舒張效應及探討一氧化氮(nitric oxide,NO)在其中是否扮演著調控之角色。故，以大鼠離體肺為為實驗模式，給予內皮素 (endothelin-1, ET-1)及缺氧刺激增加肺血管阻力來提昇肺動脈血管之壓力，用以觀察5％ CO 2混合在氮氣（hypoxia）或空氣（normoxia）中，在不同之肺動脈壓之下其血管舒張效應是否有所不同。
結果顯示，吸入性二氧化碳在ET-1及缺氧所引起的高肺動脈壓，具血管舒張效應；並且，二氧化碳的血管舒張效應會隨肺動脈壓的增高而加大。另外，內源性一氧化氮會減弱肺血管對缺氧氣體及ET-1所產生的收縮，但不會增強二氧化碳所產生的血管舒張效應。

關鍵字

急性肺損傷 ； 肺動脈高壓 ； 呼吸治療

並列摘要

Experimental animals play a very important bridge between science and clinical application. In nearly half of century, the profession of respiratory therapy has grown so quckly with great potential in developing treatment-related application. Especially, gas inhalation therapy and aerosolized medication therapy are the most widely used in clinical respiratory therapy.
In part I“aerosolized medication therapy”study, we established the experimental model of aerosolized medication and model of acute lung injury to examine the effect of L-alany-L glutamine (GLN) inhalation in enhacing pulmonary heat shock protein 72 (HSP 72) expression and attenuating lung damage in acute lung inury induced by LPS inhalation. Studies have demonstrated that HSP72 plays an important role in the protection of stressed organisms. The development of strategies for enhancing HSPs expression may provide novel means of minimizing inflammatory lung conditions, such as acute lung injury. GLN, a nonessential amino acid, is the most abundant in the healthy human being and has also been considered a “conditionally essential” amino acid in states of serious illness or injury. GLN appears to be a possible candidate for novel cytoprotective drugs by enhancement of HSP72 expression. To date, reported routes of administration for exogenous glutamine to prevent glutamine-deficiency in critically ill patients are mainly oral, parenteral, enteral and intravenous. Therefore, we designed a practical method to administer GLN via airway inhalation. Utilizing the experimental model of aerosolized medication and model of acute lung injury, the study aimed to examine the following hypothesis that: (1) GLN inhalation would enhance pulmonary HSP72 expression and attenuate lung damage in a model of acute lung inflammation induced by Lipopolysaccharide (LPS) inhalation, (2) Anti-inflammatory effect of GLN inhalation induced HSP72 is related to prevention of PMNs (polymorphonuclear neutruphils) accumulation through inhibition of ICAM-1(intercellular adhesion molecule-1) and MIP-2 (macrophage inflammatory protein-2). The results imply that prophylactic inhalation of nebulized glutamine (1) enhanced lung tissue HSP72 synthesis; (2) attenuated severity of lung injury; (3) reduced PMNS accumulation in the alveoli. The study indicates that prophylactic glutamine inhalation associated with the enhancement of HSP72 synthesis attenuates tissue damage in experimental lung injury.
In the part II “therapeutic gas inhalation”stydy, we established an ex vivo lung pulmonary hypertension model to investigate effect of carbon dioxide on pulmonary vascular tone at various pulmonary arterial pressure levels induced by enothelin-1(ET-1) and hypoxia. There have been contradictory reports suggesting that carbon dioxide (CO2) may constrict, dilate or have no action on pulmonary vessels. Permissive hypercapnia has become a widely adopted ventilatory technique to avoid ventilator-induced lung injury, particularly in patients with acute respiratory distress syndrome (ARDS). On the other hand, respiratory alkalosis produced by mechanically induced hyperventilation, is the mainstay of treatment for newborn infants with persistent pulmonary hypertension. Also, in clinical observations, hypoxia and hypercapnia often coexist with ARDS and other forms of acute or chronic lung disease. It is important to clarify the vasomotor effect of CO2 on pulmonary circulation in order to better evaluate the strategies of mechanical ventilation in intensive care. In the present study, pulmonary vascular responses to CO2 were observed in isolated rat lungs under different levels of pulmonary arterial pressure (PAP) induced by various doses of ET-1. The purposes of this study were to investigate: (1) the vasodilatory effect of 5% CO2 in either N2 (hypoxic-hypercapnia) or air (normoxic-hypercapnia) at different PAP levels induced by various doses of endothelin-1; and (2) the role of nitric oxide in mediating the pulmonary vascular response to hypercapnia and hypoxia. The result provide evidence that (1) CO2 produces pulmonary vasodilatation at high PAP under ET-1 and hypoxic vasoconstriction; (2) the vasodilatory effects of CO2 at different pressure levels vary in accordance with the levels of PAP - the dilatory effect tends to be more evident at higher PAP; and (3) endogenous NO attenuates ET-1 and hypoxic pulmonary vasoconstriction but does not augment the CO2-induced vasodilatation.

並列關鍵字

acute lung injury ； pulmonary hypertension ； respiratory therapy

參考文獻

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