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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 study the mechanism of Guntan Decoction on the treatment of atherosclerosis by studying the expression of intercellular adhesion molecule-1 (ICAM-1) and p53 in Guanyu decoction. Methods: Human umbilical vein endothelial cells (HUVEC) were subcultured for 1-3 passages for experiment. The drug-containing serum was prepared by inducing sputum soup to SD rats at a dose of 0.9 g.kg-1.d-1 for 10 days and then blood was collected from the abdominal aorta and the serum was separated. The experiment was divided into 7 groups: normal HUVEC was blank control group, without serum treatment HUVEC as blank serum control group, tumor necrosis factor α (TNF-α, 200 U.mL-1) pretreated HUVEC as TNF-α induction group HUVECs were pretreated with 5% (0.015 g.mL-1), 10% (0.03 g.mL-1) and 20% (0.06 g.mL-1) 5%, 10%, 20% Dantan Decoction group, PFT-α block group was treated with p53-specific blocker PFT-α (0.009 g.mL-1) The effects of Toutan Decoction on the expression of ICAM-1 mRNA and p53 protein in cultured endothelial cells of umbilical vein endothelial cells stimulated by TNF-α were observed by PCR and Western blot. Results: ① The expression of ICAM-1 mRNA and p53 mRNA in TNF-αinduced group was significantly higher than that in normal control group and Gundan Decoction group (P <0.01). After treated with Guotan Decoction-containing serum or PFT-α, ICAM- (P <0.05). ② The levels of ICAM- and p53 in TNF-α-induced group were significantly higher than those in normal control group and Dantan decoction group (P <0.01). The expression of ICAM-1 and p53 were significantly decreased (P <0.05) after treated with the drug-containing serum of Nao-Tang group or PFT-α treatment; ③ The expression of p53 mRNA and ICAM-1 mRNA was positively correlated There was a significant positive correlation between p53 activity and ICAM-1 level (r = 0.854, P <0.01). Conclusion: Dantan Decoction can inhibit the expression of ICAM-1 mRNA induced by TNF-α in human umbilical vein endothelial cells by regulating the expression of p53, so it can treat atherosclerosis.