Paeonol protects rat vascular endothelial cells from ox-LDL-induced injury in vitro via downregulating microRNA-21 expression and TNF-α release
Paeonol protects rat vascular endothelial cells from ox-LDL-induced injury in vitro via downregulating microRNA-21 expression and TNF-α release
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DOI：
10.1038/aps.2013.190
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发表时间：
2014-04-01
影响因子：
8.2
通讯作者：
Dai, Min
中科院分区：
文献类型：
--
作者：
Liu, Ya-rong;Chen, Jun-jun;Dai, Min
目的：丹皮中的丹皮酚(2‘-羟基-4’-甲氧基苯乙酮)是一种潜在的抗动脉粥样硬化药物。本研究旨在探讨丹皮酚对体外培养的大鼠血管内皮细胞(VECs)的抗炎作用机制。用丹皮酚处理细胞24小时，再用氧化低密度脂蛋白刺激细胞24小时，用定量逆转录聚合酶链式反应分析微RNA-21(miR-21)和PTEN在血管内皮细胞中的表达。Western blotting检测PTEN蛋白的表达。结果：氧化型低密度脂蛋白可抑制血管内皮细胞的生长，呈剂量和时间依赖性(24 h的IC50值约为20 mg/L)。此外，氧化低密度脂蛋白(20 mg/L)可显著上调血管内皮细胞miR-21的表达，抑制miR-21下游靶基因PTEN的表达。此外，氧化型低密度脂蛋白(20 mg/L)可显著增加血管内皮细胞释放肿瘤坏死因子-α。丹皮酚可剂量和时间依赖性地提高ox-LDL处理的血管内皮细胞存活率。丹皮酚(120mU/L)可拮抗氧化型低密度脂蛋白诱导的miR-21表达和肿瘤坏死因子-α的释放，以及氧化型低密度脂蛋白对PTEN表达的抑制。双荧光素酶报告实验表明miR-21直接与PTEN的3‘-UTR结合，从而抑制PTEN的表达。在氧化型低密度脂蛋白处理的血管内皮细胞中，miR-21模拟表达miR-21，抑制PTEN的表达，并减弱丹皮酚的保护作用，而miR-21抑制剂的转染显著降低miR-21的表达，增加PTEN的表达，从而增强丹皮酚的保护作用。结论：MIR-21是丹皮酚的重要靶点，对氧化-低密度脂蛋白诱导的血管内皮细胞损伤具有保护作用，可能在动脉粥样硬化的发生发展中起重要作用。
Aim: Paeonol (2 '-hydroxy-4 '-methoxyacetophenone) from Cortex moutan root is a potential therapeutic agent for atherosclerosis. This study sought to investigate the mechanisms underlying anti-inflammatory effects of paeonol in rat vascular endothelial cells (VECs) in vitro.Methods: VECs were isolated from rat thoracic aortas. The cells were pretreated with paeonol for 24 h, and then stimulated with ox-LDL for another 24 h. The expression of microRNA-21 (miR-21) and PTEN in VECs was analyzed using qRT-PCR. The expression of PTEN protein was detected by Western blotting. TNF-alpha release by VECs was measured by ELISA.Results: Ox-LDL treatment inhibited VEC growth in dose-and time-dependent manners (the value of IC50 was about 20 mg/L at 24 h). Furthermore, ox-LDL (20 mg/L) significantly increased miR-21 expression and inhibited the expression of PTEN, one of downstream target genes of miR-21 in VECs. In addition, ox-LDL (20 mg/L) significantly increased the release of TNF-a from VECs. Pretreatment with paeonol increased the survival rate of ox-LDL-treated VECs in dose-and time-dependent manners. Moreover, paeonol (120 mu mol/ L) prevented ox-LDL-induced increases in miR-21 expression and TNF-a release, and ox-LDL-induced inhibition in PTEN expression. A dual-luciferase reporter assay showed that miR-21 bound directly to PTEN's 3 '-UTR, thus inhibiting PTEN expression. In ox-LDL treated VECs, transfection with a miR-21 mimic significantly increased miR-21 expression and inhibited PTEN expression, and attenuated the protective effects of paeonol pretreatment, whereas transfection with an miR-21 inhibitor significantly decreased miR-21 expression and increased PTEN expression, thus enhanced the protective effects of paeonol pretreatment.Conclusion: miR-21 is an important target of paeonol for its protective effects against ox-LDL-induced VEC injury, which may play critical roles in development of atherosclerosis.