阿司匹林对血管紧张素Ⅱ诱导平滑肌细胞增殖的影响及其机制

The Role and Mechanism of Aspirin in Ang Ⅱ -Induced Proliferation of Vascular Smooth Muscle Cells

目的:观察阿司匹林对血管紧张素Ⅱ(AngⅡ)诱导的人脐动脉平滑肌细胞增殖的影响及其机制。方法:采用组织贴块法培养人脐动脉平滑肌细胞,选3-9代细胞用于实验。①阿司匹林细胞毒性检测:实验分对照组和阿司匹林5 mmol/L组。两组培养24 h后,取上清液,用乳酸脱氢酶活性测定法,观察阿司匹林加入对细胞有无毒性。②阿司匹林抗增殖效果:实验分5组,对照组、AngⅡ组、AngⅡ+阿司匹林0.5 mmol/L组、AngⅡ+阿司匹林1 mmol/L组和AngⅡ+阿司匹林2 mmol/L组。以上各组分别培养1 d、3 d、5 d后,采用四唑盐比色法测各孔的吸光值。③NO含量检测:实验分4组,对照组、阿司匹林2 mmol/L组、AngⅡ组和AngⅡ+阿司匹林2 mmol/L组。以上各组分别培养24 h后,收集细胞培养上清液按试剂盒说明书检测NO含量。④细胞周期检测:实验分3组,对照组、AngⅡ组和AngⅡ+阿司匹林2 mmol/L组。以上各组分别培养24 h后,收集细胞,用流式细胞仪检测细胞周期。结果:①与对照组比较,阿司匹林5 mmol/L组细胞上清液中乳酸脱氢酶活性无明显升高(P>0.05)。②阿司匹林(0.5、1、2 mmol/L)呈剂量依赖性地抑制脐动脉平滑肌细胞的增殖,抑制的最大效果在本实验内显示为2 mmol/L浓度的阿司匹林在培养第5天时,与AngⅡ组比较有极显著性差异(0.27±0.01 vs 0.74±0.03,P<0.01)。③AngⅡ刺激脐动脉平滑肌细胞24 h后,可降低细胞培养上清液中NO的含量,与对照组比较有显著差异[(54.08±5.69)μmol/L vs (70.27±3.99)μmol/L,P<0.05];而加入阿司匹林2 mmol/L干预后,可升高上清液中NO的含量,与AngⅡ组比较有极显著差异[(63.31±5.41)μmol/L vs(54.08±5.69)μmol/L,P<0.01]。④与AngⅡ组比较,阿司匹林(2 mmol/L)可使细胞静止期/DNA合成前期(G0/G1期)的脐动脉平滑肌细胞所占比例显著升高[(62.05±1.55)%vs(48.72±2.31)%,P<0.05],而DNA合成期(S期)细胞所占比例显著降低[(24.77±2.82)%vs(35.77±2.13)%,[<0.05]。结论:阿司匹林能呈剂量依赖性地抑制AngⅡ诱导的脐动脉平滑肌细胞的增殖,抑制细胞在G0/G1期,促进脐动脉平滑肌细胞NO的产生。

Objective： To explore the role and mechanism of aspirin in Ang Ⅱ-induced proliferation of human umbilical arterial smooth muscle cells （ HUASM Cs）. Methods ： The primary culture of HUASMC was performed using tissue explant method. Cells at passage 3 to 9 were used for the experiment. ①Assessment of cytotoxicity：There were two groups, control group and aspirin（5 mmol/L） group . Lactate dehydrogenase（LDH） levels were measured to assess cyctotoxicity after 24 h incubation. ②The anti-proliferative effect of aspirin： There were five groups, control group, Ang 11 （10^-6 mol/L） group, Ang Ⅱ ＋ aspirin 0.5mmol/L group, Ang Ⅱ ＋ aspirin 1 mmol/L group and Ang Ⅱ ＋ aspirin 2 mmol/L group. Cell proliferation was measured by MTT colorimetric assay at 1,3,5 days after treatment with or without aspirin at different concentration. ③Measurements of nitric oxide（NO） levels in supernatant medium： The cells were divided into four group, control group,aspirin（2 mmol/L） group, Ang Ⅱ （ 10^-6 mol/L） group and Ang Ⅱ ＋aspirin（2 mmol/L）. Concentration of NO in supernatant medium was measured by enzyme-method at 24 h after treatment with or without aspirin. ④Cell cycle experiment： The cells were divided into three groups control group, Ang Ⅱ （10^-6mol/L） group and Ang Ⅱ ＋ aspirin（2 mmol/L）. After 24 h incubation, cell cycle was measured by flow cytometry（FCM）. Results：①Compared with the control group, concentrations of 5 mmol/L aspirin did not increase LDH levels significantly in supernatant medium. ②Compared with the Ang Ⅱ group, aspirin with（0. 5,1,2 mmol/L） dose-dependently inhibited proliferation of HUASMCs with no eytotoxic effect, with the maximum effect of inhibition taking place at 5 days of cuhure（0. 27 ±0. 01 vs 0. 74 ±0. 03, P 〈0. 01 ）. ③Compared with the Ang 1I group, aspirin（2 mmol/L） could increased NO production of HUASMC（ [63.31 ± 5.41 ] μmol/L vs [ 54. 08 ± 5.69 ] μmol/L, P 〈 0. 01 ] ）. ④Compared with the Ang Ⅱ group, flow cytometric DNA analysis revealed that aspirin（2 mmol/L） induced significantly an increase in the number of HUASMC in G0/G1 （[62. 05 ± 1.55]% vs [48.72 ±2.31 ]%, P 〈0. 05] ） and a decrease in the S phase（ [24. 77±2. 82]% vs [35.77 ±2. 131%, P 〈0. 05] ）. Conclusions： Aspirin（0. 5,1,2 mmol/L） dose-dependently inhibited proliferation of HUASMCs, restrained cells in G0/G1 phase with no cytotoxic effect and increased NO production of HUASMCs.