探討第二型血管收縮素轉換酶在早期慢性阻塞性肺炎病程中扮演的角色

Abstract

慢性阻塞性肺病 (chronic obstructive pulmonary disease, COPD) 是一種因為慢性發炎造成肺部損傷導致漸進式肺部阻塞的疾病，通常是由「慢性支氣管炎」或「肺氣腫」所引起的症狀，最後可能導致全身性病變。先前的研究指出，COPD病程中的慢性發炎反應所誘發之免疫反應，會導致肺氣腫和肺纖維化，此病程可能與腎素-血管擴張素系統（renin-angiotensin system, RAS）極其相關。已知血管收縮素轉化酶（angiotensin- converting enzyme, ACE）/血管收縮素II（angiotensin II, Ang II）途徑（ACE/Ang II axis）與許多肺部疾病有關，而在RAS系統中一個可將Ang II水解成血管收縮素1-7（angiotensin 1-7, Ang-(1-7)）的第二型血管收縮素轉化酶II （angiotensin- converting enzyme II, ACE2），其功能不僅能降低Ang II濃度，其所產生的Ang-(1-7)則被認為有拮抗Ang II的生理功能，並有減緩病理性RAS所導致的肺纖維化病程。此外，在我們過去的研究結果中顯示ACE2/Ang-(1-7) axis的異常以及基質金屬蛋白酶（matrix metalloproteinases, MMPs）與基質金屬蛋白酶組織抑制因子（tissue inhibitors of MMPs, TIMPs）的調控失衡與肺臟組織纖維化病程有關。據此，在本研究中我們的主軸為探討肺部ACE2的表現與COPD病程有關之分子機轉，以及MMPs/TIMPs平衡表現與COPD的關係，並欲證實兩個假設：(1)在COPD病程中，MMPs/TIMPs平衡表現可能與ACE2/Ang-(1-7)路徑的調控有關；(2)在COPD病程中，參與RAS的因子可能會去增強或是抑制一些分子，進而影響組織中MMPs/TIMPs的平衡，造成更嚴重的炎症反應。 本研究中我們利用野生型小鼠 (WT; C57BL/6) 與ACE2 KO小鼠 (同合子ACE2-/-與半合子ACE2-/y) 建立一個藉由香菸煙燻誘發COPD早期病症的小鼠研究模式。我們將小鼠置入一實驗裝置中並給予香菸煙燻處理，令小鼠吸入香菸燃燒產生物質，小鼠每天煙燻處理4次，每週7天，並持續3週。實驗進行後，記錄小鼠的呼吸功能，並犧牲小鼠收集其血清和肺組織用於進一步的生化與病理分析。 實驗結果顯示，小鼠經香菸煙燻的期間越長，小鼠的體重明顯下降且靜息呼吸速率(resting respiratory rate, RRR) 也會隨之增加，且ACE2 KO小鼠的RRR相較WT小鼠有顯著的上升。香菸煙燻處理顯著誘發小鼠肺臟炎症反應，此得證於小鼠肺臟組織中有高濃度的介白素-6 (interleukin-6, IL-6)、腫瘤壞死因子-α (tumor necrosis factor alpha, TNF-α)以及腫瘤生長因子-β1 (tumor growth factor beta 1, TGF-β1)，以及明顯的白血球浸潤的病理現象，在ACE2 KO小鼠中這些炎症反應更為嚴重，實驗結果亦可觀察到ACE2 KO小鼠的肺臟中肺泡空洞擴大與支氣管周圍的上皮細胞顯著增厚之現象。WT小鼠經過香菸煙燻後，肺臟的ACE以及ACE2活性皆有上升趨勢，且ACE2 KO小鼠肺臟的ACE活性更比WT小鼠來的高;香菸煙燻處理同時導致小鼠肺臟MMP-2與MMP-9活性上升，而TIMP-1和TIMP-2濃度卻下降，推測此為RAS系統被不正常激化，進而導致MMPs/TIMPs活性失衡之現象。與WT小鼠相較，ACE2 KO小鼠的MMPs/TIMPs活性失衡更為嚴重。此外，香菸煙燻亦誘使ACE2 KO小鼠肺部組織中ERK1/2以及STAT-3磷酸化的程度顯著上升，但在WT小鼠之肺臟組織中，STAT-3磷酸化的程度並無顯著性的變化。 依據上述結果我們推論香菸煙燻誘發的肺部組織發炎反應與炎症相關細胞激素和RAS訊號傳遞有關，而ACE2的缺失可能會促使肺臟組織中過量的Ang II，而Ang II 相關的訊號傳遞會刺激STAT-3的磷酸化，去引起更多的發炎反應，並使MMP-2/-9活性表現增加。而在WT小鼠中，因為ACE2的存在使得Ang II被水解成Ang-(1-7)，而Ang-(1-7)的訊號傳遞會去抑制STAT-3的磷酸化，以減緩香菸煙燻造成的發炎反應，並且抑制MMPs的活性表現。總結本研究之結果將有助於研究者與臨床醫師更了解ACE2與MMPs/TIMPs的表現調控在COPD病程演進中所扮演的角色，也提供COPD在預防或治療上開創具研發潛能之新標的。

Chronic obstructive pulmonary disease (COPD) is a long term disease in which the lungs were damaged and airways were obstructed, leading to impaired breathing. Unfortunately, the mechanisms leading to COPD and its sequelae are poorly understood at the molecular level, and there is still lacking in effective treatment. Previous studies demonstrated that the chronic inflammation in COPD derived from abnormal immune mechanism, and the inflammatory response could cause emphysema and pulmonary fibrosis; these mechanisms may be associated with renin- angiotensin system (RAS). Angiotensin converting enzyme (ACE) / angiotensin II (Ang II) axis in RAS has been recognized associated with the development of several pulmonary diseases; however, the role of angiotensin converting enzyme II (ACE2) is still not well known. Some previous studies have shown that imbalanced matrix metalloproteinases and their tissue inhibitors (MMPs/TIMPs) are highly associated with the lung damage in COPD. In addition, it is well known that cigarette smoke could induce a lot of inflammatory responses induced by many cytokines and chemokine, such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and tumor growth factor beta (TGF-β), and by extracellular-signal-regulated kinase (ERK1/2) and activator of transcription-3 (STAT-3) signaling pathways. The purpose of this study is to investigate the molecular mechanism of ACE2 in regulating MMPs/TIMPs balance in early-stage COPD by cigarette smoke exposure. In this study, we proposed that ACE2 may play a key role in the pathogenesis of COPD, therefore we want to test two hypothesis：(1) the imbalance regulation of MMPs/TIMPs may be associated with ACE2/Ang-(1-7) axis in COPD; (2) MAPK and JAK-STAT3 pathways, may be enhanced or inhibited by ACE/Ang II axis to influence the balance of MMPs/TIMPs in the pathogenesis of COPD. Wild-type (WT; C57BL/6) and ACE2 KO mice, including homozygotes (ACE2-/-；female) and hemizygotes (ACE2-/y；male), were applied in the disease model of cigarette smoke-induced COPD. The mice were exposed to cigarette smoke four times per day and 7 days per week for 1 to 3 weeks by using a smoke-exposure system. After smoke exposure, resting respiratory rates (RRR) of the mice were detected and the mice were sacrificed to collect the lung tissue for biochemical and pathological assays by ELISA, zymography assay and western blot. The establishment of COPD mouse model in this study is successful that each mouse was intensively exposed cigarette smoke to induce COPD. Body weight and RRR of the mice were decreased and increased respectively which were correlated with the exposure time of cigarette smoke, respectively. Increasing RRR was more severe in ACE2-/- and ACE2-/y mice compared with that in WT mice. Infiltration of white blood cells, alveolar damage and airway epithelial thickening in the lungs of ACE2 KO mice was significantly increased compared with those in WT mice. The markedly increases of pulmonary TGF-1, TNF-α and IL-6 in ACE2 KO mice were also measured. After cigarette smoke exposure, ACE activity in the lungs of WT mice significantly increased and the level of ACE activity in ACE2 KO mice was more higher than that in WT mice. The markedly increases of pulmonary MMP-2 and MMP-9 activity in ACE2 KO mice were also observed simultaneously, and the pulmonary TIMP-1 and TIMP-2 levels in WT and ACE2 KO mice were markedly decreased. The results indicates that an imbalance of MMPs/TIMPs induced by abnormal RAS. In addition, ERK1/2 phosphorylation of lung tissue was increased in both of WT and ACE2 KO mice. In addition, STAT-3 phosphorylation was increased obviously in ACE2 KO mice, but not in WT mice. Based on our results, we presumed ACE2 would play a protective role in the pathogenesis of early-stage COPD. To supress the inflammation, ACE2 in RAS could degrade Ang II into Ang-(1-7) to inhibit STAT-3 phosphorylation to downregulate the gelatinase activity after smoke exposure in WT mice in the pathogenesis of early-stage COPD. But in ACE2 KO mice, ACE2 deficiency might lead to elevated Ang II, and excessive Ang II could consistently enhance STAT-3 phosphorylation and increase more gelatinase. Therefore, it is indicated that more severe inflammatory responses were observed in ACE2 KO mice than in WT mice when exposed to cigarette smoke. It implies that RAS dysregulation plays a pivotal role in the development of COPD. However, the role of ACE2/Ang-(1-7) axis in the COPD pathogenesis remains to be determined in the future.