| 摘要: | 急性肺損傷(Acute lung injury, ALI)是一個加護病房常見的疾
病，研究急性肺損傷的致病機轉是需要的。肺泡上皮細胞(Alveolar
epithelial cells) 是第一層的抵禦細胞， 肺泡巨噬細胞(Alveolar
macrophages)也是在肺裏第一線防禦發炎細胞，當細菌侵入，肺泡
表面張力蛋白(Surfactant protein)會受到破壞而分解進一步喪失肺泡黏膜的完整性。一旦肺泡表面張力蛋白被破壞後，巨噬細胞會跟細菌產生一連串的免疫反應，第二型肺泡上皮細胞(Alveolar type II
epithelial cells)分泌的肺泡表面張力蛋白亦會繼續產生藉以補充被消耗的部分，但如果病情沒受到控制，最終病人會造成呼吸衰竭甚
至死亡。革蘭氏陰性菌(Gram negative bacteria)是造成急性肺損傷常見的原因之一，類鐸受體(Toll-like receptors, TLRs)是人類免疫系統重要的一環，他們偵測出外來侵入的致病菌，引發先天性免疫(Innateimmunity)。脂多醣（Lipopolysaccharide, LPS）是革蘭氏陰性菌細胞壁上的重要致病物質，最近的研究顯示出類鐸受體第四型（TLR4）是脂多醣的重要受體。有關急性肺損傷近年來的研究顯示出，轉錄因子（Transcription factor）Nuclear factor kappa B (NF-κB)在急性肺損傷的轉錄機轉(Transcriptional mechanisms)中公認為最重要的因子。不過當科學家拿NF-κB 的拮抗劑用於人類急性肺損傷的病人時，其結果未盡理想。所以，急性肺損傷的疾病機轉是需要被重新檢視的。
本篇論文中的第一部份就是研究脂多醣刺激人類第二型肺泡上
皮細胞株A549 後，對於肺泡表面張力蛋白的生成影響及可能的機
轉。第二個部份，用RNA 干擾技術(RNA interference, RNAi)去評估
鼠類巨噬細胞株RAW264.7 上的類鐸受體第四型在被脂多醣刺激後
造成巨噬細胞活化的影響。第三部分用生物資訊資料庫(Bioinformatics research)，發現許多細胞激素的啟動區(Promoter
region)，有很多GATA-2 轉錄因子的鍵結片段(Binding elements)，
故探討GATA-2 轉錄因子在產生細胞激素中所扮演的角色為何。最
後再使用急性肺損傷小鼠尾靜脈注射脂多醣動物模式(Animal
model of ALI)來印證上述的細胞實驗。
用脂多醣(100 ng/ml)處理人類第二型肺泡上皮細胞株A549，類
鐸受體第二型（TLR2）及肺泡表面張力蛋白A 的蛋白質及信使RNA
量都增加。分析NF-κB 質跟核的量，發現經過脂多醣處理後的人類
第二型肺泡上皮細胞株，NF-κB 轉位和DNA 鍵結的能力也都增加，
同時發現到脂多醣會刺激人類第二型肺泡上皮細胞株的ERK1/2
(Extracellular signal-regulated kinase), p38 (p38 mitogen-activatedprotein kinase, p38MAPK), 和MEK1(MAPK kinase-1) 的磷酸化作用(Phosphorylation)。用PD98059 可有效的降低脂多醣刺激人類第二型肺泡上皮細胞株的類鐸受體第二型及肺泡表面張力蛋白A 的信使RNA 產量。
加入類鐸受體第四型的微小干擾訊息核醣核酸(Small
interference RNA，siRNA）於鼠類巨噬細胞株後會有意義降低類鐸
受體第四型的蛋白質產量。同時也發現脂多醣(100 ng/ml)刺激下鼠
類巨噬細胞株產生的介白質-1β（Interleukin-1β, IL-1β）和介白質-6
（IL-6）信使RNA 產量也下降。Transwell 檢查中發現脂多醣會刺
激鼠類巨噬細胞株的趨化性，用螢光顯微鏡可觀察到脂多醣也會刺
激巨噬細胞的細胞吞噬作用，流式細胞儀（Flow cytometry）可辨別
出脂多醣可有效增加巨噬細胞的氧化能力。同樣用類鐸受體第四型
RNA 干擾技術去處理鼠類巨噬細胞，都可以有效的降低脂多醣刺激
下產生的趨化作用，細胞吞噬作用及細胞氧化能力。
同樣濃度的脂多醣去刺激鼠類巨噬細胞，用即時聚合連鎖反應
（Real-time PCR）去測量，介白質-1β 信使RNA 量會上升。脂多醣
同時也能促使GATA-2 轉錄因子從細胞質轉位(Translocate）到細胞
核。用Electrophoretic mobility shift assay（EMSA）發現轉位後的GATA-2 轉錄因子能和DNA 片段更有效的鍵結。最重要的是加入了
GATA-2 微小干擾訊息核醣核酸於鼠類巨噬細胞株後，會有意義的
下降酯多醣刺激後增加的介白質-1β 信使RNA 的量。分別加入類鐸
受體第四型微小干擾訊息核醣核酸及銜接蛋白（Adaptor protein）骨髓分化因子八八蛋白(Myeloid differentiation factor 88, MyD88）微小干擾訊息核醣核酸於鼠類巨噬細胞株後，也觀察到GATA-2 轉錄因子從細胞質轉位到細胞核的能力都下降。進一步用有絲分裂原活化蛋白激酶（Mitogen-activated protein kinase, MAPK）的抑制劑去處理鼠類巨噬細胞株後，發現PD98059（ERK inhibitor）最為有效的抑制GATA-2 轉錄因子轉位至細胞核。從急性肺損傷小鼠尾靜脈注射脂多醣(30 mg/kg)的動物模式中，肺部切片看到肺泡充血(Alveolarcongestion)、氣道分泌物阻塞(Airway mucus plugs)、單核球增生(Monocyte prolifeation)等典型病理變化。從經過脂多醣處理過後的研磨肺組織中，GATA-2 轉錄因子會比控制組來的高，共軛交顯微鏡(Confocal microscopy)可觀察到肺部組織細胞中，GATA-2 轉錄因子的表現量也增加。
在本篇論文中可證實“脂多醣在人類第二型肺泡上皮細胞株會
刺激MEK1 磷酸化，進一步再去磷酸化ERK1/2 和p38，然後活化
NF-κB，啟動類鐸受體第二型及肺泡表面張力蛋白A 的生成” ；“類鐸受體第四型會活化巨噬細胞的功能” 和“GATA-2轉錄因子會參與脂多醣刺激介白質-1β 信使RNA 的生成”。從小鼠動物模式中，經過脂多醣處理過後，也證實GATA-2 轉錄因子在肺部組織中會有升高的現象。以上的資料，從臨床醫師的角度看，除了先前用NF-κB的拮抗劑來治療病人外，用微小干擾訊息核醣核酸去降低類鐸受體第四型及GATA-2 轉錄因子的生成，對以後革蘭氏陰性菌所引發的
急性肺損傷的病人，可提供一個新的治療方向
Acute lung injury (ALI) is a common disease in intensive care unit. Alveolar macrophages and epithelial cells are the first line immune cells and barriers in the lungs responsive to infectious agents. Sequentially, macrophages can be activated and productions of surfactant protein (SP) yielded by type II epithelial cells are also influenced. These phenomenon will lead to respiratory failure and even death. Toll-like receptors (TLRs) are essential in the innate immunity. They will trigger the immune responses when pathogens are detected. Gram negative bacterial pneumonia is a common cause of ALI. Lipopolysaacharide (LPS) is the major component on outer membrane of gram negative bacteria. Recent studies identified toll-like receptor 4 (TLR4) as a receptor of LPS. Nuclear factor-kappa B (NF-κB) is the most important transcription factor in the transcriptional mechanisms of ALI. Scientists used the NF-κB antagonist to treat the patient with ALI but the preliminary results were frustrating. To survey the pathophysiological mechanisms of ALI is indicated.
The first part of these studies was designed to examine the effects of LPS on the regulation of SP in human alveolar epithelial cell line.
The second part of these studies used the RNA interference technique to evaluate the roles of TLR4 in LPS-activated macrophages. A search using bioinformatics approach found that there are several GATA-2-binding elements in the promoter region of the cytokines. The third part of these studies specifically investigated the role and molecular mechanisms of GATA-2 transcription factor on cytokine production. Finally, we wanted to confirm the experimental results using ALI model through LPS intravenous administration.
Exposure of A549 cells to LPS (100 ng/ml) increased the
expressions of TLR2 and SP-A mRNA and protein. Immunoblotting analyses revealed that exposure to LPS enhanced the translocation of NF-κB from the cytoplasm to nuclei. EMSA further showed that LPS augmented the transactivation activity of NF-κB in A549 cells.
Sequentially, treatment of A549 cells with LPS increased
phosphorylation of ERK1/2 (Extracellular signal-regulated kinase), pP38MAPK (p38-mitogen-activated protein kinase), and MEK1 (mitogen-activated protein kinase, MAPK kinase-1). Pretreatment with PD98059, an inhibitor of ERK1/2, significantly decreased LPS-induced TLR2 and SP-A mRNA expressions.
Application of TLR4 small interference (si)RNA into macrophages decreased the levels of this receptor, and simultaneously ameliorated LPS-induced IL1-β and IL-6 mRNA production (100 ng/ml). Transwell analysis showed that LPS increased chemotactic activity of macrophages. Phagocytic activities of macrophages were significantly augmented following LPS treatment. Flow cytometry revealed that LPS increased oxidative ability of macrophages. The addition of TLR4 siRNA to macrophage lowered the LPS-enhanced chemotacic activity, phagocytic activity, and oxidative ability significantly.
Administration of LPS (100 ng/ml) to Raw 264.7, IL-1β mRNA
level was apparently elevated analyzed by real-time polymerase chain reaction. LPS also significantly enhanced GATA-2 protein translocation.
LPS-activated GATA-2 translocation was accompanied with significant increasing the DNA-binding activity of GATA-2 using electrophoretic mobility shift assay (EMSA). Strikingly, GATA-2 siRNA markedly downregulated the levels of LPS-stimulated IL-1β mRNA.
LPS-activated GATA-2 translocation were lowered by respective addition of TLR4 siRNA and myeloid differentiation factor 88 (MyD88) siRNA. MAPK (Mitogen-activated protein kinase）Inhibitors, especially PD98059, definitely alleviated the increase of LPS-activated GATA-2 translocation. Mouse ALI model was performed using intravenous adminisation of LPS (30 mg/kg) through tail vein. Typical pathological findings were seen in lung tissue, including alveolar congestion, airway mucus plugs, and monocyte proliferation. More importantly, immunoblotting analyses disclosed GATA-2 elevation in homoginized lung tissue and confocal microscopy also showed increased GATA-2 expressions in the lungs of LPS-treated mice.
We concluded that LPS induces tlr2 and SP-A gene expression in A549 through upregulating phosphorylation of MEK1-ERK1/2 and consequent activation of NF-κB; TLR4 modulated the functions of LPS-activated macrophages and GATA-2 is involved in the process of LPS-stimulated IL-1β production. In animal model, GATA-2 elevation was documented in homogenized lung tissue from LPS-treated mice. To the clinician’s concern, besides NF-κB antagonist, downregulating TLR4 and GATA-2’s translation as therapeutic strategy may be applied to the patients with ALI caused by Gram-negative bacteria. |
