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背景:导致血管内皮细胞损伤的因素中,肾素-血管紧张素系统、尤其是局部肾素-血管紧张素系统所产生的血管紧张素Ⅱ起着重要的病理生理作用。目的:观察丹参酮ⅡA对血管紧张素Ⅱ作用下的血管内皮细胞分泌一氧化氮及其内皮型一氧化氮合酶基因表达的影响和细胞内游离钙离子浓度水平的改变,探讨丹参酮ⅡA对血管内皮细胞的保护作用。设计:观察对比实验。单位:华中科技大学同济医学院附属同济医院急诊内科。材料:实验于2006-03/10在华中科技大学同济医学院基础医学实验中心完成。实验用猪主动脉由同济医学院病理生理教研室提供。方法:采用硝酸还原法、逆转录聚合酶链反应,分别检测不同浓度(10-8～10-6mol/L)作用不同时间(1,6,24h)的血管紧张素Ⅱ对培养的猪主动脉内皮细胞产生一氧化氮及其内皮型一氧化氮合酶mRNA表达的影响;然后比较在10-6mol/L的血管紧张素Ⅱ作用的不同点(0,6h)加入50mg/L浓度的丹参酮ⅡA,分别检测作用1,6,24h后内皮细胞的一氧化氮生成和内皮型一氧化氮合酶的基因表达变化。用激光共聚焦扫描显像系统检测内皮细胞内游离钙离子浓度水平的变化。主要观察指标:①一氧化氮含量。②内皮型一氧化氮合酶mRNA表达。③游离钙离子浓度。结果:①随着血管紧张素Ⅱ浓度的增加、作用时间的延长,内皮细胞一氧化氮的产生及内皮型一氧化氮合酶mRNA的表达呈顺序下降(P<0.01)。②丹参酮ⅡA各组一氧化氮的产生及内皮型一氧化氮合酶mRNA的表达明显高于血管紧张素Ⅱ组,在丹参酮ⅡA作用1,6h,血管紧张素Ⅱ+丹参酮ⅡA组一氧化氮的产生及内皮型一氧化氮合酶mRNA的表达明显高于血管紧张素Ⅱ6h+丹参酮ⅡA组(P<0.05);随着作用时间延长至24h,两组间差异无显著性(P>0.05)。③主动脉内皮细胞游离钙离子浓度:血管紧张素Ⅱ组显著高于空白对照组(P<0.01),丹参酮+血管紧张素Ⅱ组显著低于血管紧张素Ⅱ组(P<0.05)。结论:丹参酮ⅡA可抑制血管紧张素Ⅱ对血管内皮细胞分泌一氧化氮以及细胞内皮型一氧化氮合酶基因表达的负性作用,可能通过多种途径对血管内皮细胞及其功能起到保护作用。
Background: Among factors that cause damage to vascular endothelial cells, angiotensin II produced by the renin-angiotensin system, especially the local renin-angiotensin system, plays an important pathophysiological role. Objective: To observe the effect of tanshinone IIA on the secretion of nitric oxide and endothelial nitric oxide synthase gene expression by vascular endothelial cells induced by angiotensin II and the changes of intracellular free calcium ion levels to investigate the effect of tanshinone II A on vascular endothelium Cell protection. Design: Observe the contrast experiment. Unit: Emergency Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: The experiment was performed at the Basic Medical Experimental Center of Tongji Medical College, Huazhong University of Science and Technology from March to October 2006. Experimental porcine aorta was provided by the Department of Pathophysiology, Tongji Medical College. METHODS: Nitro-reduction and reverse transcription polymerase chain reaction were used to detect angiotensin II in cultured porcine aorta at different concentrations (10-8 to 10-6 mol/L) for different time (1, 6, and 24 hours). Effects of endothelial cells on the expression of nitric oxide and endothelial nitric oxide synthase mRNA; comparison of tanshinone IIA at 50 mg/L at different points (0,6 h) of 10-6 mol/L angiotensin II , Nitric oxide production and endothelial nitric oxide synthase gene expression changes were detected in endothelial cells at 1, 6, and 24 h respectively. Confocal laser scanning imaging system was used to detect the change of intracellular free calcium concentration in endothelial cells. MAIN OUTCOME MEASURES: 1 Nitric oxide content. 2 Endothelial nitric oxide synthase mRNA expression. 3 free calcium ion concentration. Results:1With the increase of angiotensin II concentration and the prolongation of action time, the production of nitric oxide and the expression of endothelial nitric oxide synthase mRNA in endothelial cells decreased in a sequential manner (P<0.01). 2 The production of nitric oxide and the expression of endothelial nitric oxide synthase mRNA were significantly higher in the tanshinone II A groups than in the angiotensin II group. The effect of tanshinone II A was 1,6 h, and the nitric oxide was found in the angiotensin II+tanshinone II A group. The production of endothelial nitric oxide synthase mRNA was significantly higher than that of angiotensin II 6h+Tanshinone IIA group (P<0.05). With the prolongation of the action time to 24h, there was no significant difference between the two groups (P>0.05). 3Intracellular free calcium concentration of aortic endothelial cells: Angiotensin II group was significantly higher than that of blank control group (P<0.01), and tanshinone + angiotensin II group was significantly lower than that of angiotensin II group (P<0.05). Conclusion: Tanshinone II A can inhibit the negative effect of angiotensin II on the secretion of nitric oxide and endothelial nitric oxide synthase gene expression in vascular endothelial cells, and may protect endothelial cells and their functions through various ways. .