木犀草素經由誘發第一型血基質氧化酶抑制硫酸吲哚酚誘導 EA.hy926內皮細胞發炎機制之探討

Inductionof Heme Oxygenase 1 and Inhibition of Indoxyl Sulate-Induced Inflammation by Luteolin in EA.hy926 Cells

莊雅祺(YA-CHI CHUANG)

指導教授 ： 李健群

中山醫學大學/健康管理學院/營養學系碩士班/碩士(2017年)

摘要

心血管疾病(cardiovascular disease, CVD)為慢性腎臟病(chronic kidney disease, CKD)患者常見之併發症，研究指出CKD患者體內累積之尿毒素-硫酸吲哚酚(indoxyl sulfate)是導致腎臟損傷進而加速腎病惡化主因之一。此外，硫酸吲哚酚可透過誘導血管內皮細胞發炎反應與氧化壓力促進CKD患者心血管疾病發生。細胞間黏著分子1 (ICAM-1)、血管細胞黏著分子1 (VCAM-1)和單核球趨化蛋白1 (MCP-1)為發炎反應指標蛋白，與白血球細胞黏附至內皮細胞作用有關。木犀草素(luteolin)屬於黃酮類植化素，又稱3’,4’,5,7-四氫黃酮。文獻顯示其具有抗氧化與抗發炎特性。本實驗主要探討木犀草素對indoxyl sulfate誘導EA.hy926血管內皮細胞ICAM-1、VCAM-1、MCP-1表現及其相關作用機制。結果顯示，隨著indoxyl sulfate濃度增加顯著誘發ROS生成和ICAM-1、VCAM-1、MCP-1蛋白質表現，而預處理2 µM luteolin可抑制indoxyl sulfate誘發ROS生成和ICAM-1、VCAM-1、MCP-1蛋白質表現與HL-60白血球細胞黏附。此外，luteolin減少indoxyl sulfate誘導細胞核內NF-kB p65與c-jun蛋白質累積；先前研究指出第一型血基質氧化酶(HO-1)在血管內皮組織對抗氧化壓力扮演重要角色，本研究另一重點在探討luteolin是否透過誘發HO-1來抑制indoxyl sulfate誘導ICAM-1、VCAM-1與MCP-1蛋白質表現。結果顯示，隨著luteolin濃度增加顯著誘發HO-1蛋白質表現，將HO-1基因knockdown後則明顯減少luteolin對indoxyl sulfate之抗發炎作用。綜合以上結果得知，在EA.hy926細胞中，indoxyl sulfate可能藉由誘發ROS生成活化NF-kB與AP-1訊號路徑進而誘發ICAM-1、VCAM-1與MCP-1蛋白質表現。luteolin可藉由抑制NF-kB與c-jun訊號路徑或透過增加HO-1蛋白質表現來下調indoxyl sulfate所誘導的ICAM-1、VCAM-1和MCP-1表現；本研究結果支持luteolin具有保護因indoxyl sulfate誘發的心血管發炎性疾病。

關鍵字

木犀草素；硫酸吲哚酚；第一型血基質氧化酶；細胞黏著分子；心血管疾病；慢性腎臟病

並列摘要

Cardiovascular disease (CVD) is an inflammatory vascular disease frequently associated with chronic kidney disease (CKD). Indoxyl sufate, one of the uremic toxins increased in CKD patients, has been shown to accelerate kidney injury and progression of CKD. In addition, indoxyl sulfate may also be responsible for vascular disease by inducing inflammation in endothelial cells. Intercellular adhesion molecule (ICAM-1)、vascular cell adhesion molecule,(VCAM-1) and monocyte chemoattractant protein (MCP-1), inflammatory biomarkers, play pivotal roles in CVD progression. Luteolin, also known as 3’,4’,5,7-hydroxyl flavones, has been reported to have anti-oxidative and anti-inflammatory properties. The aim of our study was to investigate the effect of luteolin on indoxyl sulfate-mediated ROS production as well as ICAM-1, VCAM-1, and MCP-1 protein expression in EA.hy926 cells and the possible mechanisms involved. The results showed that indoxyl sulfate markedly induced ROS production as well as ICAM-1, VCAM-1 and MCP-1 protein expression, whereas pretreatment with 2 µM luteolin significantly suppressed indoxyl sulfate-induced ROS production, ICAM-1, VCAM-1 and MCP-1 protein expression as well as HL-60 cell adhesion. Moreover, luteolin attenuated indoxyl sulfate-induced nuclear accumulation of p65 and c-jun. Previous study indicated that heme oxygenase-1 (HO-1) plays an important role in anti-oxidative stress in vascular endothelial tissue. The other aim of this study will examine whether induction of HO-1 involved in the inhibition of luteolin on indoxyl sulfate-induced ICAM-1, VCAM-1 and MCP-1 protein expression. In the study, luteolin dose-dependently increased HO-1 expression, whereas knockdown of HO-1 attenuated the luteolin-mediated suppression of ICAM-1, VCAM-1, and MCP-1 expression by indoxyl sulfate in EA. hy926 cells. In conclusion, our results suggested that indoxyl sulfate upregulates the expression of ICAM-1, VCAM-1 and MCP-1 possibly through induction of ROS and NF-kB and AP-1 signaling pathway; luteolin inhibits indoxyl sulfate-induced ICAM-1, VCAM-1 and MCP-1 expression at least partially through suppression of NF-kB and AP-1 signaling pathway and induction of HO-1 expression in endothelial cells. The anti-inflammatory effects of luteolin may implicate its CVD-protective potential.
Keywords: Luteolin; Indoxyl sulfate; Heme oxygenase-1; Cell adhesion molecule; Cardiovascular disease; Chronic kidney disease

並列關鍵字

Luteolin ； Indoxyl sulfate ； Heme oxygenase-1 ； Cell adhesion molecule ； Cardiovascular disease ； Chronic kidney disease

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