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| 題名: | 親蛋白質尿毒素—硫酸吲哚酚及硫酸對甲酚引發內皮功能障礙之分子機轉研究
Mechanistic study of protein-bound uremic toxins, indoxyl sulfate and p-cresyl sulfate, induced endothelial dysfunction |
| 作者: | 潘思翰
Pan, Szu-Han |
| 貢獻者: | 許準榕 |
| 關鍵詞: | 硫酸吲;哚;酚;硫酸對甲酚;神經醯胺;內皮功能障礙
indoxyl sulfate;p-cresyl sulfate;ceramide;endothelial dysfunction |
| 日期: | 2015-07-21 |
| 上傳時間: | 2020-08-28 15:09:48 (UTC+8) |
| 摘要: | 親蛋白質尿毒素硫酸吲哚酚（indoxyl sulfate, IS）及硫酸對甲酚（p-cresyl sulfate, PCS），分別為色氨酸與酪氨酸的代謝產物，當患者腎功能下降時會過度蓄積對人體造成影響。之前的臨床研究發現，進行透析之患者因心血管疾病而死亡之比例高於常人5倍之多，且此現象與尿毒素之血中濃度呈正相關性。動脈粥狀硬化是心血管疾病的主要起因之一，IS已被發現可能經由誘導血管內皮細胞功能障礙而加劇動脈粥狀硬化，進而引發心血管疾病；然而，PCS對血管內皮細胞的影響及親蛋白質尿毒素影響血管內皮細胞之分子機轉尚待釐清。本實驗室先前已利用人類臍靜脈內皮細胞（human umbilical vein endothelial cells, HUVECs）評估IS（100-1000 M）及PCS（100-1000 M）對血管內皮細胞的影響，發現IS較PCS更明顯地降低血管內皮細胞存活率、誘發血管內皮細胞衰老、抑制細胞中一氧化氮合成酶（eNOS）的磷酸化且抑制血管內皮細胞血管新生的能力。而這些作用可能都與尿毒素刺激過多的活性氧自由基（reactive oxygen species, ROS）生成有關。本篇研究進一步的發現，較長時間地（24 小時）給予血管內皮細胞IS或PCS可經由活化芳香烴受體（aryl hydrocarbon receptor, AhR）而增加NADPH oxidase 4 (NOX4)的蛋白質表現，進而造成ROS的產生及氧化傷害的增加。另一方面，給予血管內皮細胞IS或PCS於短時間內（5-30 分鐘）即可經由刺激p47phox 磷酸化及粒線體活化而造成ROS產生。而事先給予神經醯胺(ceramide)合成酶抑制劑3-OMe-SM，可明顯地減少IS或PCS所刺激的p47 phox磷酸化及粒線體的活化；免疫螢光染色的結果亦顯示給予IS及PCS會刺激血管內皮細胞中ceramide的生成。本篇研究結果顯示，親蛋白質尿毒素IS或PCS所引發的過量ROS產生及對血管內皮細胞造成的氧化傷害，可能經由活化AhR及ceramide路徑而來。此深入的分子作用機轉釐清，可為臨床改善尿毒素誘發心血管疾病病程進行提供可能的新治療標的。
Protein-bound uremic toxins: indoxyl sulfate (IS) and p-cresyl sulfate (PCS) which accumulate in patients with declining renal function are the metabolites of tryptophan and tyrosine, respectively. Previous clinical studies indicated that the mortality of cardiovascular disease in dialysis patients was five times higher than general population, and this event is positive correlated with the blood level of protein-bound uremic toxins. IS was known to exacerbate atherosclerosis, a major cause of cardiovascular diseases, by inducing endothelial cell dysfunction. However, the effect of PCS on endothelial cells and the underlying mechanisms of protein bound uremic toxins-induced endothelial cell dysfunction are still unclear. In our previous study, human umbilical vein endothelial cells (HUVECs) were used to determine the effects of IS (100-1000 M) or PCS (100-1000 M) on endothelial cells. We have found that IS was more potent than PCS in reducing cell viability, inducing cell senescence, inhibiting endothelial nitric oxide synthase phosphorylation, and suppressing tube formation in HUVECs. These phenomena may associate with excessive reactive oxygen species (ROS) production and subsequent oxidative damage in uremic toxin-treated endothelial cells. In the present study, we have found that the long term treatment of IS or PCS induced ROS production and oxidative damage may be due to the activation of aryl hydrocarbon receptor (AhR) and increased NADPH oxidase 4 (NOX4) in HUVECs. On the other hand, IS and PCS could produce ROS within 5-30 min by stimulating p47phox phosphorylation and mitochondrial activation. Furthermore, the pretreatment of 3-O-methyl-sphingomyeline (3-OMe-SM), a ceramide synthase inhibitor, could abolish IS or PCS-induced p47phox phosphorylation and mitochondrial activation, and the formation of ceramide was directly observed in uremic toxin-treated HUVECs by confocal microscopy. The results collectively indicated that protein-bound uremic toxins, IS and PCS, induced excessive ROS production and subsequent oxidative damage by AhR and ceramide signaling pathways in endothelial cells. This study may provide new potential therapeutic targets for treating uremic toxin-induced or exacerbated cardiovascular diseases. |
| 描述: | 碩士
指導教授:許準榕
委員:許銘仁
委員:黃德富
委員:林冠宏 |
| 資料類型: | thesis |
| 顯示於類別: | [醫學科學研究所] 博碩士論文
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