基于报告基因的丹参PPAR-α受体激动效应研究

Studies on the Activation Effect of Radix Salviae Miltiorrhizae on Peroxisome Proliferator-activated Receptor-alpha in Vitro Based on Report Gene

目的研究丹参不同组分及部分单一成分的PPAR-α激动效应,探讨丹参抗动脉粥样硬化的作用机理。方法设丹参提取物组:SM1、SM2、SM3、SM4,单一成分组:丹参素、丹酚酸B、丹参酮、隐丹参酮,非诺贝特阳性对照组及空白对照组,pc DNA-h PPAR-α、p GL3-PPRE-luc及pRLCMV载体共转染293T细胞6h后,各组加相应试验溶液对转染细胞进行干预,试验液终浓度,提取物组为0.1、1、10、50μg·m L^(-1),单一成分及非诺贝特组为0.1、1、10、50μmo L·L^(-1),常规培养24h后测定萤光素强度,计算相对萤光素酶表达强度。每浓度点重复5个样本,所有数据皆采用算术平均数±标准偏差(x±s)表示,不同转染组之间的差异性比较,采用t检验和方差分析,P<0.05认为有显著性差异。结果丹参素、丹酚酸B、丹参酮、隐丹参酮、SM1、SM2、SM3、SM4对报告基因的相对诱导率均小于1.06,与空白组比较无显著差异,而浓度为0.1、1、10、50μm的非诺贝特阳性组相对诱导率分别为:0.82、1.29、1.72、1.94,表现有显著差异。结论经筛药体系的体外研究,4种丹参主要活性成分丹参素、丹酚酸B、丹参酮、隐丹参酮无显著的体外PPAR-α激动活性,在确保筛药体系活性的给药浓度下丹参4种不同提取组分无显著的体外PPAR-α激动作用。

OBJECTIVE To study the activation effect of 4 extract fractions and several constituent from Radix Salviae Miltiorrhizae（ RSM） on PPAR-α,aim to approach the anti-atheroscleresis pathway of RSM. METHODS pc DNA-h PPAR-α,p GL3-PPRE-luc and pRL-CMV（ 0. 67μg each） were Co-transfected into 293 T cell seeded in 96 wells plate,fenofibrate,danshensu,salvianolic acid B,tanshinoneⅡA and cryptotanshinone standards were added into different wells after 6 hours transfection,respectively. The terminal concentration of extracts was 0,0. 1,1,10,50μmol·L- 1each,SM1,SM2,SM3 and SM4 were added at terminal concentration of 0. 1,1,10,50μg·m L- 1,5 replicates for each concentration points. Two luciferase active were determined using Dual-Glo Lucifarase Assay System after 24 hours incubation,and relative luciferase active was used to express induction activity. RESULTS There was no significant deviation for danshensu,salvianolic acid B,tanshinoneⅡA,cryptotanshinone standards and fractions of SM1,SM2,SM3,SM4. The relative luciferase inductivity of them were all lower than 1. 06,compared with vehicle group. CONCLUSION There is rare possibility that some active components in RSM can activate PPAR-α in vitro.