口山酮抑制ox-LDL诱导的内皮细胞组织因子表达及机制研究

Inhibition of Oxidative Low-density Lipoprotein Induced-tissue Factor Expression in Endothelial cells by Demethylbellidifolin and Its Mechanism

【目的】研究口山酮化合物去甲基雏菊叶龙胆酮(DMB)对氧化型低密度脂蛋白(ox-LDL)诱导的内皮细胞组织因子(TF)表达的影响及机制。【方法】培养人脐静脉内皮细胞,加入ox-LDL(0.2mg/mL)孵育24h诱导TF表达,一期凝固法和反转录PCR分别检测TF的活性和mRNA表达,用荧光试剂H2DCF-DA检测细胞内活性氧(ROS)的水平,EMSA检测核因子(NF)-κB活性。【结果】孵育ox-LDL(0.2mg/mL)24h能显著增加内皮细胞TF的活性和上调其mRNA表达,同时增加细胞内ROS生成和激活NF-κB。在加入ox-LDL前1h分别加入1、3或10μmol/LDMB能呈浓度依赖性地抑制ox-LDL诱导的TF活性和mRNA水平的增加。而且,DMB能显著降低ox-LDL诱导的细胞内ROS的生成增加和NF-κB的激活。【结论】DMB能抑制ox-LDL诱导的内皮细胞TF表达,其作用与抑制细胞内ROS生成和NF-κB激活有关。

[Objective] To investigate the effect of demethylbellidifolin （DMB）, a xanthone compound, on oxidative low-density lipoprotein （ox-LDL）-induced tissue factor （TF） expression in endothelial cells and its underlying mechanism. [Methods] Human umbilical vein endothelial cells （HUVECs） were cultured and treated with ox-LDL （0.2 mg/mL） for 24h to induce TF expression. The activity and mRNA expression of TF were determined by one-stage clotting assay and reverse transcription PCR, respectively. The level of intracellular reactive oxygen species （ROS） was determined by H2DCF, an oxidative sensitive fluorescent indicator. The DNA-binding activity of nuclear factor （NF）-κB in nuclear extracts was analyzed by electrophoretic mobility shift assay （EMSA）, [Results] Treatment with ox-LDL could induced TF mRNA expression and increase its activity. The increase in intracellular ROS production and the activation of NF-κB were observed in HU- VECs treated with ox-LDL. However, the addition of different concentrations of DMB （1, 3 and 10 μmol/L） lh before ox-LDL stimulation could markedly inhibit ox-LDL-induced increases in TF activity and mRNA level in a concentration-dependent manner. Also, the elevation of intracellular ROS production and the activation of NF-κB elicited by ox-LDL were significantly suppressed by pretreatment with DMB. [Conclusion] DMB inhibits ox-LDL-induced TF expression in endothelial cells, and such effects of DMB may be related to inhibiting intracellular ROS production and NF-κB activation.