第二型血管收縮素轉換酶於bleomycin誘發肺部纖維化病變機轉中之角色探討

Abstract

使用博萊黴素 (Bleomycin)進行癌症治療可能使呼吸系統產生嚴重的副作用，例如間質性肺炎或肺纖維化，這些副作用與患者的生理狀況、藥物在體內的累積劑量及是否接受胸部放射治療或化學治療等密切相關。肺纖維化病變與腎素-血管收縮素系統 (Renin-angiotensin system; RAS)的調節密切相關，在RAS中，已知血管收縮素轉換酶 (Angiotensin-converting enzyme; ACE)/血管收縮素II (Angiotensin II; Ang II)途徑 (ACE/Ang II axis)與多種肺部疾病的病變有密不可分的關連性，然而在RAS中，另一個類似ACE的酵素，第二型血管收縮素轉換酶(Angiotensin-converting enzyme II; ACE2) 在肺部疾病中的生理功能目前並不清楚，但已知ACE2能將Ang II水解成血管收縮素1-7 (Angiotensin 1-7; Ang-(1-7))，Ang-(1-7)則可以拮抗Ang II所造成的不良生理影響。在以往的研究中，我們發現ACE2的失調與組織基質金屬蛋白酶 (Matrix metalloproteinases; MMPs)/基質金屬蛋白酶的組織抑制因子(Tissue inhibitors of MMPs; TIMPs)表現失衡所導致的心血管和肺臟組織纖維化病變有關。 在本研究中，我們利用實驗小鼠探討ACE2調控對肺纖維化病變的分子機制。我們主要探討ACE2在肺部的細胞和生理功能，它可以水解Ang II成Ang-(1-7)去拮抗Ang II造成的異常生理作用。我們推測ACE2可能是減緩肺纖維化病變的關鍵，因此於bleomycin誘發肺纖維化病程中，我們欲測試兩個假設：(1) 肺病變組織中MMPs/TIMPs的失衡可能與ACE2/Ang-(1-7) axis調控相關；(2) 肺部細胞的MAPK和JAK-STAT3分子訊號途徑參與RAS調控MMPs/TIMPs的平衡表現。 本研究中使用野生型 (WT; C57BL /6)和ACE2基因剔除 (ACE2 KO)小鼠 [包括 (ACE2 -/- ; 雌性)和(ACE2 -/y; 雄性)]，建立一個bleomycin誘發肺纖維化的實驗模型。其處理為將小鼠麻醉後，由氣管直接給予單一劑量bleomycin溶液 (4mg/kg)，此後每週進行小鼠體重和呼吸功能量測，並分別在第3週和第6週犧牲小鼠，收集肺組織和血液用於進一步的生化、病理及分子分析。 實驗結果顯示經bleomycin處理後，小鼠的體重明顯下降，但是靜息呼吸速率 (Resting respiratory rate; RRR)逐漸增加，ACE2 KO小鼠與WT小鼠相比，其RRR增加更為顯著。肺組織病理檢查也顯示在ACE2 KO小鼠的肺部損傷和白血球細胞浸潤的炎症反應現象也較WT小鼠者顯著，再則tumor growth factor beta 1 (TGF-β1) 和interleukin-6 (IL-6)等炎症因子在ACE2 KO小鼠的肺臟中也明顯增加。小鼠的肺組織中ACE活性在bleomycin的處理後顯著增加，同時MMP-2和MMP-9活性也顯著上升，但是TIMP-1和TIMP-2的濃度卻顯著降低，這些變化在ACE2 KO小鼠處理組中更為明顯。以上結果顯示，異常RAS激活現象會造成MMPs/TIMPs的表現失衡。此外我們測定肺臟組織中結締組織生長因子(CTGF)、TGF-β1及elastin的表現，我們觀察到在bleomycin處理後第三週時，CTGF和TGF-β1的大量表現，而在第六週時elastin明顯提升。在分子檢測方面，肺臟中ERK1 / 2磷酸化的現象在bleomycin處理的WT和ACE2 KO小鼠皆顯著提升；而STAT-3的磷酸化現象在ACE2 KO小鼠第六週時顯著提升，但在WT小鼠則沒有明顯差異，根據結果我們推測ACE2能抑制IL-6進而減緩STAT-3的磷酸化現象。 我們透過實驗動物，以及病理和分子機轉的研究結果顯示，RAS失調在bleomycin誘發肺纖維化病程中有著關鍵的作用，也證明ACE2的缺乏會加速這種肺纖維化病程發展，此乃由於ACE2缺乏就無法將不正常累積於組織的Ang II降解為Ang-(1-7)，因此加速bleomycin誘發的肺臟組織病變。雖然ACE2/ Ang-(1-7) axis在肺纖維化病程機制中的重要性仍待更多實驗探討，然而我們實驗結果證明了藉由RAS的調節是可能減緩bleomycin處理可能誘發肺部炎症反應、纖維化及其他等副作用的發生。

Anticancer treatment with bleomycin possibly cause side effects in respiratory system, such as interstitial pneumonitis and pulmonary fibrosis. The side effects are related with the physiological condition of patient, accepting cumulative dose of bleomycin, whether received chest radiation or chemo therapy. This pathology is closely relative with the regulation of renin-angiotensin system (RAS) in the patient. In RAS, angiotensin converting enzyme (ACE)/angiotensin II (Ang II) axis is associated with the development of several pulmonary diseases. However, the role of angiotensin conversion enzyme II (ACE2) is not well known, an ACE homologue that hydrolyses Ang II to angiotensin 1-7 (Ang-(1-7)), a peptide that exerts the actions opposite to those of Ang II. In our previous studies, we showed that ACE2 dysregulation and imbalanced matrix metalloproteinases (MMPs)/tissue inhibitors of MMPs (TIMPs) are highly associated with the fibrotic damage in cardiovascular and pulmonary diseases. In this project, we aimed to study the molecular mechanism of ACE2 regulation on pulmonary fibrosis by experimental mouse models. We focus on exploring the cellular and physiological roles of ACE2 in pulmonary system, it can hydrolyze angiotensin II to Ang-(1-7). We hypothesis whether part of the anti-fibrotic effects of ACE2/Ang-(1-7) axis is via Mas receptor to anti-fibrotic in the bleomycin induced pulmonary injury. In this study, we proposed that ACE2 may play a key in the pathogenesis of pulmonary, therefore we want to test two hypothesis: (1) the imbalance regulation of MMPs/TIMPs may be associated with ACE2/Ang-(1-7) axis in pulmonary fibrosis; (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 pulmonary fibrosis. Wild-type (WT; C57BL/6) and ACE2 KO mice, including homozygotes (ACE 2 -/-; female) and homozygotes (ACE2-/y; male), were applied in the disease model of bleomycin induced pulmonary fibrosis. The mice were anesthetized and given a single dose of bleomycin solution (4 mg/kg) directly into respiratory tract through trachea. After bleomycin treatment, body weight and resting respiratory rates (RRR) of the mice were detected every week. The mice were sacrificed, and then lung tissue and plasma were collected for biomedical assays and pathological analysis at the 3rd and 6th week after bleomycin challenge. The establishment of pulmonary fibrosis mouse model in this study is successful that each mouse was intratracheal administration bleomycin to induce pulmonary fibrosis. After bleomycin challenge, body weight and RRR of the mice were decreased and increased respectively. Increasing RRR was more severe in ACE2 KO mice compared with that in WT mice. Infiltration of white blood cells, alveolar damage in the lungs of ACE2 KO mice was significantly increased compared with those in WT mice. The markedly increases of pulmonary TGF-β1 and IL-6 in ACE2 KO mice were also measured. After bleomycin challenge, ACE activity in the lungs of WT mice significantly increased and the level of ACE activity in ACE2 KO mice was 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 we examined the connective tissue growth factor (CTGF), TGF-β1 and elastin expression in lung. We observed that CTGF and TGF-β1 were highly expressed at the 3rd week and elastin was highly expressed at the 6th week after bleomycin challenge. In molecular mechanism 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 at 6th week, but not in WT mice. According to our results, we presumed ACE2 would inhibit inflammation mediator IL-6 level and down regulate STAT-3 phosphorylation. Our results imply that RAS dysregulation plays a pivotal role in the development of pulmonary fibrosis. However, the role of ACE2/Ang-(1-7) axis in the pathogenesis remains to be determined in the future. Therefore, ACE2 deficiency may enhance such injury via abnormal accumulation of Ang II. Herein, we provided experimental results that regulating RAS can possibly reduce bleomycin caused pulmonary inflammation and other side effects.