| 摘要: | 背景：慢性阻塞性肺部疾病（Chronic Obstructive Pulmonary Disease, COPD）為慢性發炎反應所導致的呼吸道氣流阻塞疾病，病理分型主要分為支氣管炎（Bronchitis）和肺氣腫（Emphysema）。COPD的風險因子除了常見吸菸、空污，反覆的肺部感染也有可能導致COPD發生。COPD常見症狀為喘、痰液變多、胸悶，進而使日常生活品質下降，治療此疾病的醫療花費十分可觀，也對病人的身心靈有極大影響。COPD不單單是肺部疾病，體內的發炎介質和急性蛋白釋放會影響其他器官功能，導致更複雜的疾病發生。血紅素結合蛋白（Haptoglobin, Hp）為急性蛋白的一種，受IL-6的正向調控。Hp由肝臟分泌，其他像是單核球（Monocyte）、巨噬細胞（Mcrophage）、第二型肺泡上皮細胞（Alveolar epithelial cell type 2, AEC II）亦會分泌Hp。在脾臟和肝臟中，Hp主要透過與衰老或過多的血紅蛋白（Hemoglobin, Hb）結合並經由巨噬細胞結合來代謝血紅素（Heme）和再利用鐵（Iron），從而參與紅血球（Red blood cells）的代謝。近年來有研究指出COPD病人血清中Hp濃度比正常人高，且Hp分型也影響著COPD的嚴重度。然而，Hp 在調節 COPD 中的免疫致病作用尚不清楚。因此在本研究中，我們旨在探究Hp在COPD中的角色。
研究材料與方法：本研究收案於台灣北部某家準醫學中心，收案對象為須經手術治療的肺癌患者。從中隨機挑出非合併COPD病史及合併COPD各七位。採集受試者手術切除之肺部組織，並且避開腫瘤的部分，將組織進行免疫組織化學染色法分析Hp的分布表現。另外，抽取COPD病人周邊血液，分離出人體周邊血單核細胞（Peripheral blood mononuclear cell, PBMC）以Reducing SDS-PAGE膠體電泳分析Hp的表現。肺氣腫動物模式部分，以豬胰蛋白酶（Porcine pancreatic elastase, PPE）多次由氣管給予七週大C57BL/6公鼠，於數周後使用蘇木精-伊紅染色（Hematoxylin and eosin, H&E）分析小鼠肺部病理組織染色，以及使用免疫組織染色（Immunohistochemistry, IHC）分析促發炎之誘導型一氧化氮合酶（Inducible nitric oxide synthase, iNOS）和Hp。使用西方墨點法（Western blot）分析小鼠肺部裂解液和支氣管肺泡灌洗液（Bronchoalveolar lavage fluid, BALF）中Hp及iNOS的表現。細胞實驗則是使用THP-1細胞，使用Hp-Hb complex刺激後分析iNOS、精氨酸酶（Arginase-1, Arg-1）、細胞路徑。圖表使用GraphPad Prism version. 8.0製作，數據資料使用IBM SPSS version. 19.0分析。病人資料描述性統計使用student’s t test檢定，以百分比、平均值與標準差（Mean ± SD）呈現，P < 0.05代表有統計上差異，LDH則採用one-way ANOVA分析。
研究結果：COPD病人肺部切片進行免疫組織染色顯示，Hp在肺泡壁（Alveolar wall）以及支氣管上皮細胞（Bronchiolar epithelial cell）呈現高表現。由COPD病人的PBMC中顯示，Hp在26 Kd至43 Kd有些微增加的變化。此外，透過H&E分析顯示，經由PPE刺激的小鼠出現肺氣腫伴隨肺泡擴大和肺部結構破壞的現象。使用 IHC 和西方墨點法檢測也發現 PPE小鼠的肺組織中的Hp和iNOS表現增加。由細胞實驗發現，外加純化的Hp-Hb complex會誘導THP-1細胞中iNOS和AKT活化的表現。
結論：在本研究中，我們證明了肺部Hp潛在調控COPD和 PPE 小鼠肺氣腫的疾病發展，並可能與 AKT 激活有關。迄今為止，Hp 被證明是 COPD 發病機制中潛在的正調節劑，然而Hp 參與疾病進展的機制仍需要進一步研究。
Background: Chronic Obstructive Pulmonary Disease (COPD) is the airway obstruction caused by chronic inflammation. The pathological types are mainly divided into bronchitis and emphysema. In addition to common smoking, air pollution, repeated lung infections may also cause COPD occurred. The common symptoms of COPD are wheezing, productive sputum, and chest tightness, which in turn degrade the quality of daily life. The medical cost of treating this disease is very considerable, and it also has a great impact on the patient's body and mind. COPD is not just a lung disease, inflammatory mediators and acute phase protein release can affect the functions of other organs, leading to more complex comorbidities and complications. Haptoglobin (Hp) is a kind of acute phase protein, which is positively regulated by IL-6. Hp is secreted by liver, monocytes/macrophages and alveolar epithelial cell type 2 (AEC II). In spleen and liver, Hp mainly participates in the metabolism of red blood cells through combining with aging or excessive hemoglobin (Hb) and macrophages to
metabolize heme and reuse iron. Recently, Hp is reported that the concentration of serum Hp in patients with COPD is higher than healthy donor, where different Hp phenotypes correlates the severity of COPD as well. However, the
immunopathogenic role of Hp in regulating COPD remains unclear. In this study, we thus aim to investigate the role of Hp in COPD.
Materials and Methods: This study was admitted to a would-be medical center in northern Taiwan, and the subjects of enrollment were lung cancer patients who required surgical treatment. Seven people with a history of COPD and other seven people without COPD were randomly selected. Collections of lung tissues of the subjects were avoided the parts of tumor for applying immunohistochemistry (IHC) of Hp. In addition, the peripheral blood mononuclear cells (PBMC) from COPD patients were collected for Hp analysis by the reducing SDS-PAGE. For the animal model of emphysema, porcine pancreatic elastase (PPE) was administered to seven-week-old C57BL/6 male mice through the trachea for several times. A few weeks later, histopathologic examination of lung, immunohistochemical staining of iNOS and Hp were analysed. Western blotting was used to explore the expression of iNOS and Hp in mice lung lysate and in bronchoalveolar lavage fluid (BALF). Human THP-1 cells were stimulated by Hp-Hb complex followed by the analysis of iNOS, arginase-1 (Arg-1), AKT, and MAPK pathways. GraphPad Prism version 8.0 was used to make graphs, and statistical analysis was examined by IBM SPSS version 19.0. The descriptive statistics of the patient data were determined by student's t test, presented in percentage, mean and standard deviation (Mean ± SD), P <0.05 means there is a statistical difference, LDH was analyzed by one-way ANOVA.
Results: In lung samples obtained from COPD patients, higher expression of Hp were detected in alveolar wall and bronchial epithelial cells. In PBMC of patients with COPD, the similar upregulation of Hp could be measured where Hp expression in molecular weight of 26 Kd to 43 Kd was shown a slight increase. Moreover, PPE mice developed lung emphysema with the enlarged and destructive structure of air sac by H&E analysis. The increase expression of Hp and iNOS also could be detected in the lung tissues of PPE mice using IHC and western blot analysis. Extracelluar stimulations of purified human Hp-Hb complex showed that Hp-Hb complex partly induced iNOS expression and AKT activation in human THP-1 cells.
Conclusion: In the present study, we demonstrated that pulmonary Hp expression is potentially upregulated in patients with COPD and PPE mice with emphysema development, in which may be possibly correlated with AKT activation. To date, Hp is shown as a potential positive modulator in the pathogenesis of COPD, however, the mechanisms of Hp involved in disease progression still need further invesegations. |
