藏红花素对大鼠缺氧心肌的保护作用及其机制研究

The Protective Role of Crocin in Hypoxic Cardiomyocytes With its Underlying Mechanism in Neonatal Rats

目的:研究藏红花素(Crocin)对大鼠缺氧损伤心肌细胞的保护作用及其机制。方法:采用原代培养SD大鼠心肌细胞,应用氯化钴(CoCl2)方法建立心肌细胞缺氧损伤实验模型。研究分为两部分,(1)实验分组(各组样品数n=6):空白对照组、CoCl2组(CoCl2250μmol/L)、不同浓度藏红花素[10(-7^-3)mol/L]+CoCl2组和阳性对照组(丹参酮ⅡA磺酸钠组)。采用细胞活力和细胞毒性检测法测定不同浓度藏红花素对缺氧心肌细胞活力影响。应用生物化学方法测定心肌细胞超氧化物歧化酶活性和丙二醛含量变化。(2)实验分组(各组样品数n=3):实验对照组,缺氧组(CoCl2250μmol/L)、藏红花素(10-5mol/L)组和藏红花素(10-5mol/L)+缺氧细胞组。应用蛋白质印迹法和逆转录聚合酶链式反应检测心肌细胞缺氧诱导因子-1α、血管内皮生长因子、脯氨酰羟化酶1、2、3(PHD1、2、3)蛋白和mRNA表达的变化。结果:①藏红花素10-5mol/L+CoCl2组与CoCl2组比较,心肌细胞活力增强(P<0.01),缺氧心肌细胞超氧化物歧化酶活性提高(P<0.01),丙二醛的含量降低(P<0.01)。②藏红花素(10-5mol/L)+缺氧细胞组与缺氧组比较,缺氧诱导因子-1α及其下游靶基因血管内皮生长因子蛋白表达均增高(P<0.01)。③藏红花素(10-5mol/L)+缺氧细胞组心肌细胞PHD2蛋白和mRNA表达较缺氧组均明显增加(P均<0.01),PHD3蛋白和mRNA表达则均明显减少(P均<0.01),差异均有统计学意义。结论:藏红花素对缺氧损伤心肌细胞具有明显的保护作用,其作用机制与藏红花素激活缺氧诱导因子-1介导的低氧反应通路有关,PHDs可能参与了该反应的病理生理调控过程。

Objective：To investigate the protective role of Crocin in hypoxic cardiomyocyte with its underlying mechanism in neonatal rats. Methods：We established the CoCl2-induced hypoxic injury model of cardiomyocyte in neonatal rats at primary culture. The cells were pre-treated with or without Crocin, and the cell condition was examined by Cell counting kit 8. The activity of super oxide dismutase （SOD） and the amount of malondaldehyde（MDA） in cardiomyocytes were examined with biochemical methods. The expression of hypoxia-inducible factor 1 （ HIF-1 ） and prolylhydroxylase （ PHD1,2,3 ） were detected by RT-PCR and Western blot analysis respectively. Results：Compared with CoC12 group,①the viability of cardiomyocytes was significantly increased by 24h treatment of Crocin at the concentration of 10-5 mol/L in Crocin ＋ CoC12 group, P 〈 0.01;②the activity of SOD was increased and the amount of MDA was decreased in Crocin ＋ CoCl2 group,P 〈 0. 01 respectively ;③the expression of HIF-la and PHD2 was increased, while the expression of PHD3 was decreased in Crocin ＋ CoCl2 group,P 〈 0. 01 respectively. Conclusion：Crocin could protect the cardiomyocytes from hypoxic injury in neonatal rats, which possibly related to the activation of HIF-1 mediated hypoxia response. PHDs might be involved in this patho-physiologic process.