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To study the effects of particulate matter on
the inflammation of high glucose-treated endothelial cells through mitophagy
Particulate matter,Diabetes,Endothelial dysfunction,ROS,mitophagy,Inflammation,Vitamin D3,
|Publication Year :||2019|
|Abstract:||近年來，空氣污染已逐漸成為全世界關注的公共衛生問題。空氣中的懸浮微粒 (Particulate matter, PM2.5)直徑小於2.5 μm，容易進入呼吸道深處並參與全身血液循環，使得生物體內各種不良反應的產生。因此，PM2.5對人體健康的影響已成為一個重要的議題。流行病學研究顯示，PM2.5會導致心血管、肺癌等疾病的發生，且可能誘導氧化壓力的產生、發炎反應，內皮細胞功能障礙等。空氣汙染是糖尿病發生率增加的原因之一，但仍需更多研究加以闡明。在本實驗中，我們以30 mM葡萄糖與50 μg/ml的PM2.5處理人類臍靜脈內皮細胞 (Human umbilical vein endothelial cells, HUVECs )模擬高糖與空氣污染之環境。實驗結果顯示，PM2.5會促使高糖處理的細胞存活率顯著性的下降，也會使細胞凋亡的現象增加。在此處理下還可觀察到活性氧化物 (reactive oxygen species, ROS)的含量上升、清除ROS的酵素- SOD1 (superoxide dismutase 1)的蛋白表現量下降。另外，也會使粒線體膜電位下降、自噬小體的形成、粒線體自噬相關蛋白p62、LC3B (microtubule-associated protein 1 light chain 3 beta)、Bnip3 (BCL2 Interacting Protein 3)及透過MAPKs (mitogen-activated protein kinases)這條訊息傳遞路徑使發炎相關蛋白ICAM-1 (intracellular adhesion molecule-1)及VCAM-1 (vascular cell adhesion protein 1)的表現上升。而加入維他命D處理後，可以透過降低ROS生成而使細胞存活率回升、降低粒線體自噬與發炎現象而達到保護內皮的效果。動物實驗部分，我們以STZ (streptozotocin)誘導小鼠使其產生高血糖，以氣管內注射(intratracheal instillation) PM模擬暴露空氣污染之環境。結果顯示，暴露PM2.5的糖尿病小鼠其胸主動脈之ROS含量有增加的現象，而VCAM-1, p62及Bnip3的蛋白表現量也有上升的趨勢；而給予維他命D處理則可以減少此現象。透過本篇實驗，我們了解懸浮微粒在高糖環境下增加細胞內氧化壓力、粒線體自噬及發炎現象，而維他命D可以透過降低ROS生成而達到保護內皮細胞之效果。|
Environmental particulate matter 2.5 (PM2.5) can penetrate deep into the respiratory tract, reach the alveolar region, participate in blood circulation, and is associated with the development of lung cancer and cardiovascular diseases. It may also promote inflammation, oxidative stress and endothelial dysfunction. Air pollution has been found to be one of the risk factors for diabetes, but evidence is limited. Here, we treated human umbilical vein endothelial cells (HUVECs) with 30 mM glucose and 50 μg/ml PM (HG + PM) to mimic endothelial cells under hyperglycemia and air pollution. First, we demonstrated that HUVECs exposed to PM in a high glucose (HG) environment showed a significant increase in cell damage and apoptosis. In addition, PM significantly increased reactive oxygen species (ROS) production in HG-treated HUVECs. The expression of superoxide dismutase 1 (SOD1), an enzyme of ROS scavenger, was also reduced. Using the JC-1 assay and MitoStatus staining to examine mitochondrial membrane potential, we demonstrated that mitochondrial membrane potential tends to collapse in the PM and HG + PM groups. We also showed that high glucose and PM can induce the formation of autolysosomes in HUVECs by acridine orange (AO) staining. Proteins associated with mitochondrial autophagy, p62, microtubule-associated protein 1 light chain 3β (LC3B) and BCL2 interacting protein 3 (Bnip3), were increased in the PM and HG + PM group compared to the control and HG group. In addition, autophagosome-like structures were observed in the HG + PM group by transmission electron microscopy. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were also increased through JNK/p38 signaling pathway in the HG + PM group. Furthermore, we investigated whether vitamin D protects HUVECs under high glucose and PM conditions. Endothelial cells pretreated with vitamin D significantly increased cell viability and reduced mitochondrial ROS production under high glucose and PM conditions. Vitamin D also reduced the formation of autolysosomes using AO staining. The levels of p62, LC3B, Bnip3, ICAM-1 and VCAM-1 expression were decreased after vitamin D treatment. In addition, we used streptozotocin (STZ)-induced diabetic mice as an animal model. Mice were treated with PM by intratracheal instillation. The aortas of the HG + PM group showed increased ROS production by DHE staining and increased levels of VCAM-1, p62, and Bnip3 by Western blot; while vitamin D could reverse these phenomena. Taking together, these results suggested that PM exerted the enhancement of cell injury under high glucose environment via ROS production, mitophagy and inflammation, and vitamin D could protect HUVECs against these phenomena by suppressing oxidative stress.
|Appears in Collections:||解剖學暨細胞生物學科所|
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