研究發炎刺激物(TNF-α和空氣懸浮微粒)對肺上皮細胞的影響及相關機制

To study the effects of inflammatory stimuli (TNF-α and particulate matters) on lung epithelial cells and the related mechanisms

陳又溱(Yu-Chen Chen)

指導教授 ： 陳玉怜

國立臺灣大學/醫學院/解剖學暨細胞生物學研究所/博士(2021年)

https://doi.org/10.6342/NTU202101727

摘要

肺臟組織受外在刺激所產生的發炎反應是導致呼吸系統衰竭的主要因素。因此，關於肺臟組織損傷的研究及其相關治療方案是迫切需要的。現今已有越來越多的證據顯示，維生素D3的缺乏與發炎相關疾病具有密切相關性。然而，維生素D3的抗發炎作用及其機轉仍有待釐清。在第一部分研究中，我們利用細胞及動物實驗模型來探討維生素D3 (Vit. D)對於腫瘤壞死因子α (TNF-α)刺激肺泡上皮細胞後，影響細胞間黏附因子1 (ICAM-1)的表現及其相關機轉。經由TNF-α刺激後，第二型肺泡上皮細胞(A549細胞)中ICAM-1的表現、粒線體活性氧化物(mtROS)、粒線體分裂和粒線體自噬皆顯著性地增加。在此研究中，我們發現維生素D3透過調節粒線體功能進而預防TNF-α所誘導之肺臟發炎反應。在A549細胞中，維生素D3經由減少TNF-α所誘導之DRP1、Mff和 Bnip3的表現以抑制粒線體分裂和粒線體自噬發生。此外，維生素D3透過抑制AKT/NF-κB的磷酸化進而減少粒線體分裂、粒縣體自噬及發炎反應的產生。根據以上的實驗結果，維生素D3提供治療肺臟發炎相關疾病的一種治療方式。此外，我們利用空氣懸浮微粒(PMs)作為發炎刺激劑進行肺臟上皮細胞的損傷和機制之研究。PMs很容易經由呼吸道進入人體循環系統，並引起全身發炎和免疫反應。然而，PMs 在上皮間質轉化(EMT)和肺纖維化中的作用仍需要進一步的釐清。在第二部分研究中，我們利用細胞及動物實驗模型來探討PMs對EMT的形成、肺纖維化發展的影響及其相關機轉。在本研究中，我們發現經由 PMs處理A549細胞後，EMT相關蛋白、纖連蛋白、細胞移行和EMT相關轉錄因子(NF-κB p65和ETS-1)的表現量增加。此外，實驗小鼠經由氣管內注射PMs後 第7天和第14天，我們發現小鼠肺臟組織中EMT相關蛋白、纖連蛋白和ETS-1的表現量皆增加。經由人類肺纖維化組織微陣列的免疫組織染色結果顯示，ETS-1與纖連蛋白的表現具有密切相關性。由此可知，PMs所誘導之EMT的形成和肺纖維化的產生是經過ETS-1的活化。根據以上的實驗結果，我們提供經由發炎刺激劑(TNF-α及PMs)所引起肺損傷之呼吸系統疾病的相關機制。此外，我們也為此種傷害提供了一種新穎且實用的治療方法。

關鍵字

發炎 ； 纖維化 ； 腫瘤壞死因子α ； 空氣懸浮微粒 ； 粒線體 ； 上皮間質轉化

並列摘要

Inflammation is considered to be a key factor in lung injury leading to respiratory system failure. Therefore, there is an urgent need for new research related to lung injury to generate new insights into the potential treatment of lung injury. A growing evidence showed that vitamin D3 deficiency is closely related to inflammatory diseases. However, the anti-inflammatory effects of vitamin D3 and their underlying mechanisms remained to be determined. In the part I study, we aimed to examine the effects and mechanisms of vitamin D3 (Vit. D) on the expression of intercellular adhesion molecule-1 (ICAM-1) in vitro and in vivo with or without tumor necrosis factor α (TNF-α) treatment. After TNF-α stimulation, ICAM-1 expression, mitochondrial ROS (mtROS), mitochondrial fission and mitophagy were significantly increased in A549 cells. In this study, Vit. D prevents TNF-α-induced pulmonary inflammation by the regulation of mitochondrial function. Vit. D inhibits TNF-α-induced mitochondrial fission and mitophagy by the inhibition of the expression of DRP1, Mff and Bnip3 in A549 cells. This effect is mediated through the AKT/NF-κB pathway. Due to these findings, Vit. D should be considered as a new and novel therapeutic agent for the targeting epithelial activation in lung inflammation. In addition, we use particulate matter in air pollutants as an inflammatory stimulant to study the damage and mechanism of lung epithelial cells. Particulate matters (PMs) can easily enter the human circulatory system through the respiratory tract, causing systemic inflammation and immune response. However, the role of PMs in epithelial-mesenchymal transition (EMT) and pulmonary fibrosis needs to be clarified. In the part II study, we aimed to investigate the effect of PMs on the formation of EMT, the development of pulmonary fibrosis and the related mechanisms in pulmonary epithelial cells by in vitro and in vivo studies. In this study, we found that EMT, fibronectin, cell migration and EMT-related transcription factors (NF-κB p65 and ETS-1) were increased in A549 cells treated with PMs. In addition, the EMT formation and the expression of fibronectin and ETS-1 were increased in lung tissues of mice for 7 and 14 days after PMs exposure. The results of human lung fibrosis tissue microarray showed that ETS-1 expression was closely related to the fibronectin expression. Therefore, PMs induced EMT and pulmonary fibrosis via the activation of ETS-1. Based on the above findings, these data provide a mechanism for the damage of respiratory diseases caused by inflammatory stimulants. In addition, we also provide a new and practical treatment for this injury.

並列關鍵字

inflammation ； fibrosis ； TNF-α ； particulate matters ； mitochondria ； EMT

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