导痰汤对人脐静脉内皮细胞细胞间黏附分子1和p53表达的影响

Effect of Daotan Decoction on Intercellular Adhesion Molecule-1 mRNA and p53 Expression in Human Umbilical Vein Endothelial Cells

目的:通过研究导痰汤干预细胞间黏附分子1(ICAM-1)与p53的表达,分析导痰汤治疗动脉粥样硬化的机制。方法:人脐静脉内皮细胞(HUVEC),传代培养1～3代用于实验。含药血清的制备为将导痰汤按照0.9 g.kg-1.d-1剂量给SD大鼠ig 10 d后,经腹主动脉采血,分离血清。实验共分7组:正常HUVEC为空白对照组,不含药血清处理HUVEC为空白血清对照组,肿瘤坏死因子α(TNF-α,200 U.mL-1)预处理HUVEC为TNF-α诱导组,先采用5%(0.015 g.mL-1),10%(0.03 g.mL-1),20%(0.06 g.mL-1)含药血清预处理HUVEC,再与TNF-α共培养为5%,10%,20%导痰汤组,使用p53特异性阻滞剂PFT-α(0.009 g.mL-1)处理HUVEC为PFT-α阻滞组。通过PCR和Western blot等方法,观察导痰汤对TNF-α刺激脐静脉内皮细胞培养内皮细胞ICAM-1 mRNA的表达和p53表达的影响。结果:①TNF-α诱导组ICAM-1 mRNA和p53 mRNA的表达显著高于正常对照组和导痰汤对照组(P<0.01);使用导痰汤含药血清或PFT-α处理后,ICAM-1和p53 mRNA的表达显著下降(P<0.05);②TNF-α诱导组ICAM-与p53蛋白显著高于正常对照组和导痰汤对照组(P<0.01)。使用导痰汤含药血清或PFT-α处理后,ICAM-1与p53表达显著下降(P<0.05);③p53 mRNA与ICAM-1 mRNA表达水平呈显著正相关(r=0.981,P<0.01);p53活性与ICAM-1水平呈显著正相关(r=0.854,P<0.01)。结论:导痰汤可以通过调节p53表达而抑制TNF-α刺激所致的脐静脉内皮细胞ICAM-1 mRNA的表达,故能起到治疗动脉粥样硬化的作用。

Objective： To investigate intervention of D aotan decoction （DTD） in of intercellular adhesion molecule-1 （ 1CAM-1 ） mRNA expression through the regulation of p53 in order to reveal the mechanisms of DTD in the treatment of atherosclerosis. Method ： Neonatal umbilical cords were used to isolate endothelial cells, and they were subcultured for 1 to 3 generations in experiment. According to a dose of 0.9 g·kg-l-d-1, DTD was given to SD rats by gavage. Blood samples were obtained via the abdominal aorta after 10 days and the serum was separated. The experiment i, asdivided into 7 groups： blank control group, blank serum control group （HUVECs were treated with serum without DTD）, tumor necrosis factor-α（TNF-α） induced group （200 U ·mL-I TNF-α, 12 h）, 5% DTD group （5% DTD serum, 6 h, and then 200 U -mL-1 TNF-α, 12 h）, 10% DTD group （10% DTD serum, 6 h, and then 200 U ·mL-1 TNF-α, 12 h） , 20% DTD group （20% DTD serum, 6 h, and then 200 U -mL-1 TNF-α, 12 h） and PFT-α group （p53 blocker） blocking groups （PFT-α 0. 009 g -mL-1, 30 min, and then 200 U·mL-1 TNF-α, 12 h）. The impact of DTD on ICAM-I mRNA and p53 expression in human umbilical vein endothelial cells stimulated by TNF-α were observed with the PCR and Western blot. Result： （1）The expression of ICAM-1 mRNA and p53 in the TNF-α induced group was significantly higher than the normal control and DTD control groups. The difference was statistically significant （ P 〈 O. 01 ） ; after being treated with DTD serum or PFT-α, the expressions of ICAM-1 and p53 mRNA were reduced （P 〈 0.05）. （1）The protein expressions of ICAM-I and p53 in the TNF-αinduced group were significantly higher than the normal control and DTD control groups. The difference was statistically significant （P 〈 0.01 ） ; after being treated with DTD serum or PFT-α, the protein expressions of ICAM-1 and p53 were reduced （P 〈 0. 05）. （1）There was a significant positive correlation between the p53 and ICAM-1 mRNA level （ r = 0. 981, P 〈 0.01 ）, and protein level （ r = 0. 854, P 〈0. 01 ）. Conclusion： DTD inhibits TNF-α stimulation caused ICAM-1 mRNA expression in umbilical vein endothelial cells through the regulation of p53 expression, therefore, atherosclerosis. it may play a therapeutic role in the treatment of