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目的观察丹参酮ⅡA磺酸钠(sodium tanshinoneⅡA sulfonate,STS)对血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)诱导的心肌细胞氧化应激反应的影响及其机制。方法培养新生大鼠心肌细胞,建立AngⅡ诱导的心肌细胞氧化应激反应模型。用活性氧(reactive oxygen species,ROS)敏感的二氮荧光黄双乙酸盐(DCFH-DA)探针测定胞内ROS水平。酶联免疫分析法测定细胞培养物上清中8-羟脱氧鸟苷(8-OHdG)含量。以细胞存活率、细胞内超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量、培养上清液乳酸脱氢酶(LDH)活性作为反应心肌细胞损伤的指标。比色法测定还原型辅酶Ⅱ(NADPH)氧化酶(NADPH oxi-dase,NOX)活性。蛋白质印迹法(Western-blot)测定NOX亚单位p47 phox表达。结果 Ang Ⅱ能显著提高心肌细胞内DCF荧光信号强度,降低细胞存活率及SOD活性,同时上调培养上清液LDH活性及MDA含量。以上氧化应激相关指标能被各浓度STS抑制。进一步研究发现,STS能抑制Ang Ⅱ上调的NADPH氧化酶活性及亚单位p47 phox表达。结论 STS可以抑制Ang Ⅱ诱导的心肌细胞氧化应激损伤反应,机制可能与下调NADPH氧化酶活性及亚单位p47 phox表达有关。
Objective To investigate the effect and mechanism of sodium tanshinone Ⅱ A sulfonate (STS) on oxidative stress in cardiomyocytes induced by angiotensin Ⅱ (Ang Ⅱ). Methods The neonatal rat cardiomyocytes were cultured and AngⅡ-induced cardiomyocyte oxidative stress response model was established. Intracellular ROS levels were measured by reactive oxygen species (ROS) -dependent fluorescent dye diazepin yellow diacetate (DCFH-DA). Enzyme-linked immunosorbent assay was used to determine the content of 8-OHdG in the cell culture supernatant. Cell viability, intracellular superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and lactate dehydrogenase (LDH) activity in culture supernatant were used as indicators of cardiomyocyte injury. The activity of NADPH oxi-dase (NOX) was measured by colorimetry. The NOX subunit p47 phox expression was determined by Western blotting. Results Ang Ⅱ could significantly increase the fluorescence intensity of DCF, decrease cell viability and SOD activity, and increase LDH activity and MDA content in culture supernatant. Above indicators of oxidative stress can be inhibited by various concentrations of STS. Further study found that STS inhibited Ang Ⅱ upregulated NADPH oxidase activity and subunit p47 phox expression. Conclusion STS can inhibit Ang Ⅱ-induced oxidative stress injury in cardiomyocytes, which may be related to down-regulation of NADPH oxidase activity and expression of p47 phox.