探討第二型血管收縮素轉換酶於PM2.5誘導肺臟急性受損病程中扮演的角色

Abstract

隨著科技與產業的進步，環境汙染的程度也隨之上升，在這之中又以空氣汙染的影響最廣闊。細懸浮微粒 (Particulate matter, PM2.5) 為近年來新興的空氣汙染指標之一，能夠停滯於肺臟組織中，且可以穿透血管壁進入循環系統的特性，PM2.5的重要性節節攀升。在過去的研究中，我們了解到PM2.5與肺臟和心血管系統疾病有莫大的關聯。同樣地，腎素-血管收縮素系統 (Renin-angiotensin system, RAS) 也與肺臟和心血管系統疾病有極大關係。然而PM2.5與RAS之間的關係性卻鮮少被人探討。因此本研究欲瞭解受到PM2.5的影響下，肺部RAS因子的變化與肺臟病程的關係，並探討缺少第二型血管收縮素轉換酶 (angiotensin converting enzyme II, ACE2) 下病程發展的情形。 我們利用C57BL/6野生型 (Wild-type, WT) 小鼠與ACE2基因剔除 (ACE2 KO) 小鼠建立一個PM2.5誘發小鼠肺臟急性受損的動物研究模式。實驗小鼠經過氣管給予之方式，每天給予PM2.5 (6.25 mg/kg/day) 1次，並連續給予3天。小鼠在結束給予PM2.5後的第2天與第5天犧牲，並採集肺臟組織樣本進行後續生化、組織病理及分子檢測。實驗期間內，每日偵測小鼠呼吸速率與體重變化。 實驗結果顯示，WT小鼠在PM2.5刺激的期間體重明顯下降，而呼吸速率隨著給予時間上升。在停止給予PM2.5後，WT小鼠體重緩慢增加，且呼吸速率也維持在一定的區間內不再增高。WT小鼠在給予PM2.5後產生嚴重的炎症反應，體內的炎症因子如：TNF-α、TGF-β1及IL-6表現量提升，但在給予PM2.5的5日後有下降至正常水平。肺臟組織中的血管收縮素轉換酶 (angiotensin conversing enzyme, ACE) 與ACE2的濃度在給予PM2.5後顯著提升，但ACE濃度卻在處理的5天後顯著下降，而ACE2濃度則維持提升的趨勢。在PM2.5給予的2日後，MMP-2與MMP-9表現量皆增加，而在PM2.5給予後的5日，MMP-9雖然是有恢復正常表現量的趨勢，但MMP-2依然保持著高表現的情形。而ACE2 KO小鼠在經過PM2.5誘導肺部受損後產生的情形與WT小鼠類似，但顯得更為嚴重，且在PM2.5給予後的5日恢復的程度也較差。 我們的結果顯示，在缺乏ACE2的情況下PM2.5誘發的組織炎症反應與組織重塑變化較為嚴重，且在停止PM2.5暴露後之肺臟修復的程度也較差。因此我們認為ACE2在肺臟扮演重要的角色，其除了能夠減緩PM2.5對肺臟產生的傷害外，也對肺臟修復具有一定程度的影響。本研究結果為將來在防治環境中高濃度PM2.5所引起的肺臟疾病提供一個新的預防與治療標的藥物。

The level of environmental pollution is increased with the improvement of technology and industry, and air pollution is one of the pollutions, which influences widely. Particulate matter (PM2.5) is one of the emerging air pollution indicators in recent years. PM2.5 becomes more and more important with the particulate size that can stagnate in the lungs and penetrate the vascular wall into the circulatory system. In the past researches, we know that PM2.5 has a great association with lung and cardiovascular diseases. Renin-angiotensin system (RAS) is also associated with lung and cardiovascular diseases. However, there are few studies about the relationships between PM2.5 and RAS. Therefore, we want to understand the role and changes of RAS factors after PM2.5 challenge, and to explore the disease progression in lacking of angiotensin converting enzyme II (ACE2). Animal model of acute lung injury induced by PM2.5 was established by using wild type (C57BL/6, WT) and ACE2 gene knockout (ACE2 KO) mice. The mice were treated with PM2.5 via intratracheal instillation once a day for a total of three days and sacrificed at the 2nd and the 5th day after the PM2.5 treatment. We detected the changes in respiratory rates and body weight, and collected the lung tissues for further biochemical, pathologic and molecular assays after the animal sacrificed. The results show that body weight of WT mice was significantly decreased and respiration rate was increased during the period of PM2.5 challenge. The body weight was slowly increased and the respiratory rate was maintained within a stable range in WT mice after stopping the PM2.5 treatment. Serious inflammation was induced by PM2.5 administration in WT mice, the expression of inflammatory TNF-α, TGF-β1 and IL-6, were significantly increased, but obviously recovered to almost normal level at the 5th day after PM2.5 treatments. Angiotensin conversing enzyme (ACE) and ACE2 were significantly increased after the administration of PM2.5. At the 5th day after PM2.5 treatment, ACE was rapidly declined but ACE2 still maintained the increasing level. Both of matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9) were increased at the 2nd day after PM2.5 treatment, and MMP-9 was recovered but MMP-2 was still keeping in a high expression at the 5th day. In ACE2 KO mice, there were more serious damages on biochemical, molecular and pathologic changes compared with those in WT mice after PM2.5 treatment. Additionally, ACE2 KO mice presented less recovery of damages at the 5th day after PM2.5 challenge. Our results indicate that the effects of injury causing by PM2.5 are more serious, and the level of pulmonary repair is also poor after stopping PM2.5 exposure when the absence of ACE2. Therefore, we suggest that ACE2 plays a critical role in the lungs, besides to inhibit the injury induced by PM2.5 and also impact on the process of lung repair. Our results hint that the patients with discomfort by PM2.5 might be treated with ACE2 regulation, and ACE2 is a potential candidate of new targets for pulmonary diseases.