| 摘要: | 背景：急性呼吸窘迫症候群(ARDS)為臨床常見之呼吸道疾病，其病因為瀰漫性肺泡及微血管受損使上皮細胞通透性增加。臨床上表徵為肺內死腔及分流增加、肺順應性下降等現象，進而氧合狀況降低造成呼吸窘迫。ITIH4是一種發抗炎蛋白，具有抗細胞凋亡和基質穩定的功能。然而，ITIH4在肺部疾病中的角色及其潛在機轉仍有許多待研究之處。
目的：本研究旨在探討ITIH4透過Hippo pathway調控ARDS中第2型肺泡上皮細胞凋亡的作用。
方法：本研究利用人類的第二型肺泡上皮細胞(A549)分別暴露於濃度0、10、50、100 ng/mL之ITIH4，暴露24小時後分析其細胞毒性、細胞存活率、發炎反應。使用Western blot測定A549細胞中YAP、p-YAP、TAZ、p-TAZ、E-cadherin、β catenin、ATM、p53、SIRT1和caspase-3的蛋白表現。同時也利用LPS建立ARDS之細胞模式，研究設計利用A549於濃度10 μg/mL的LPS中暴露6小時後加入ITIH4，經24小時後收集細胞裂解液。另外將A549細胞分別暴露於ITIH4 siRNA 48小時或ITIH4 DNA 24小時，分別分析ITIH4基因缺失或過度表現對於第二型肺泡上皮細胞的影響。此外，本研究利用免疫螢光染色分析在LPS誘導ARDS的小鼠和呼吸器導致之肺損傷小鼠的肺泡區域中，第二型肺泡上皮細胞中YAP的表現。
結果：本篇研究結果指出，ITIH4在Hippo pathway參與了調控ARDS中第二型肺泡上皮細胞的凋亡。GEO數據集的分析結果顯示ARDS患者中ITIH4基因表現上升。我們發現當ITIH4的基因缺陷或過度表現時會影響Hippo pathway的下游因子YAP和TAZ的活性。而在經LPS誘導的第二型肺泡上皮細胞損傷後，暴露於ITIH4可使p-YAP/YAP的表現有下降之趨勢以及p-TAZ/TAZ的表現顯著上升（p<0.05）。此外，經LPS刺激而受損的第二型肺泡上皮細胞中，SIRT1的表現顯著增加（p<0.05），並且E-cadherin和β-catenin呈上升趨勢。同時也顯著降低了ATM和caspase-3的表現（p<0.05），並且p53呈現下降之趨勢。在動物實驗中，免疫螢光染色的結果顯示，在LPS誘導急性肺部損傷的第三天，與未受損區域相比，受損區和嚴重區的第二型肺泡上皮細胞中YAP表現呈上升趨勢。而在急性肺部損傷的第七天，隨著ITIH4的給予和濃度增加，受損區的第二型肺泡上皮細胞中YAP表現呈現下降的趨勢。最後在呼吸器導致之肺損傷小鼠的實驗模型中，免疫螢光染色的結果顯示，第二型肺泡上皮細胞上的YAP表現在接受高潮氣容積通氣的小鼠中增加，而預先?予ITIH4的小鼠在高潮氣容積通氣後YAP表現?少。
結論：我們的研究結果指出ITIH4參與了在ARDS中Hippo signaling pathway調控第二型肺泡上皮細胞的凋亡過程。
Background: Acute respiratory distress syndrome (ARDS) is a severe inflammatory lung injury, characterized by diffuse alveolar damage and increased alveolar endothelial cell permeability. Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) is an anti-inflammatory protein and functions as an antiapoptotic and matrix-stabilizing molecule. However, the role of ITIH4 in the lungs and its underlying mechanisms remains unclear.
Objective: This study aims to investigate the role of ITIH4 in regulating apoptosis of type 2 alveolar epithelial cells via the Hippo signaling pathway in ARDS.
Materials and Methods: Alveolar epithelial cell type II (AECII) A549 cells were treated with different concentration of ITIH4 (0, 10, 50, or 100 ng/mL) for 24?h. Next, cells were exposed to lipopolysaccharides (LPS) at 10 μg/mL for 6 hours, followed by adding ITIH4 (0, 10, 50, or 100 ng/mL) for 24?h. In addition, A549 cells were transfected with ITIH4 siRNA or DNA sequences. LDH and IL-6 were examined in the supernatant. Expression of E-cadherin, β-catenin, ATM (Ataxia-telangiectasia mutated kinase), p53, SIRT1 (Sirtuin 1), caspase-3, Yes-associated protein (YAP), and transcriptional co-activator with PDZ-binding motif (TAZ) was examined using Western Blot. The expression of YAP+ SPC+ cells in alveolar regions of LPS-induced ARDS mice and ventilator-induced lung injury (VILI) mice was observed by immunofluorescence staining.
Results: Our results indicated that ITIH4 was involved in Hippo signaling pathway-regulated apoptosis during the acute phase of ARDS in AECII cells. Through analysis of GEO datasets, we found that the ITIH4 gene was upregulated in ARDS patients. Furthermore, we observed that the deficiency or overexpression of the ITIH4 gene could impact the activity of downstream factors of the Hippo pathway, such as YAP and TAZ. Following LPS-induced injury in AECII cells, subsequent exposure to ITIH4 was found to modulate the downregulation of p-YAP/YAP and the significant upregulation of p-TAZ/TAZ (p<0.05). Moreover, it led to significantly increased expression of SIRT1 (p<0.05) and has an upward trend in E-cadherin and β-catenin. While significantly decreasing the expression of ATM and caspase-3 (p<0.05), and has a downtrend in p53. The immunofluorescence staining results revealed that on the third day after LPS-induced injury, AECII cells in the damaged zone and severe zone showed an upward trend in YAP expression compared to the undamaged area. On the seventh day after LPS-induced injury, the administration of ITIH4 resulted in a decreasing trend in YAP expression within the damaged zone of AECII cells. Immunofluorescence staining revealed increased YAP expression in AECII cells in mouse lung tissues exposed to high tidal volume ventilation. However, administration of ITIH4 to the mice resulted in decreased YAP expression in AECII cells following high tidal volume ventilation.
Conclusion: Our findings provide evidence that ITIH4 involves in the Hippo signaling pathway-regulated apoptosis in AECII of LPS-induced injury. ITIH4 mediates the expression of SIRT1, ATM, p53, and Caspase-3 via the Hippo signaling pathway. This study offers a unique perspective on the potential use of ITIH4 in the management of ARDS in the future. |
