羟基红花黄色素A对缺氧/复氧心肌细胞增殖与凋亡的影响

Effects of hydroxysafflower yellow A on the proliferation and apoptosis of hypoxic/reoxygenated cardiomyocytes

目的研究羟基红花黄色素A(HSYA)调控微小RNA-499(miR-499)表达对缺氧/复氧心肌细胞增殖与凋亡的影响。方法将大鼠H9c2心肌细胞分为空白组(正常培养)、模型组(缺氧/复氧处理)和实验组(20μmol HSYA+缺氧/复氧处理)。干预48 h后于光学倒置显微镜下观察细胞形态,以活细胞计数试剂盒法检测细胞存活率,以DCF-DA荧光染色检测细胞内活性氧(ROS)水平,以试剂盒检测细胞损伤指标含量,以蛋白质印迹法检测心肌细胞凋亡相关蛋白及miR-499表达。结果干预48 h后,空白组、模型组及实验组H9c2心肌细胞存活率分别为(99.26±0.15)%,(45.12±3.62)%,(78.69±2.15)%,细胞上清液中肌酸激酶(CK)分别为(2.63±0.14),(6.25±0.29),(4.63±0.21)U·L-1,乳酸脱氢酶(LDH)分别为(22.63±3.15),(46.85±2.96),(32.16±2.86)U·L-1,细胞凋亡率分别为(2.63±0.24)%,(36.25±4.92)%,(13.29±4.15)%。细胞中Bax相对表达量分别为0.24±0.03,0.69±0.03,0.42±0.02,Caspase-3相对表达量分别为0.28±0.04,2.56±0.21,1.85±0.03,miR-499相对表达量分别为1.63±0.02,0.56±0.05,1.16±0.02,差异均有统计学意义(P<0.05)。结论 HSYA通过调控miR-499蛋白表达,抑制缺氧/复氧心肌细胞氧化应激反应,抑制细胞凋亡,促进细胞增殖,减轻心肌损伤。

Objective To investigate the effect of hydroxysafflower yellow A(HSYA) on the proliferation and apoptosis of hypoxic/reoxygenated cardiomyocytes by regulating the expression of microRNA-499(miR-499). Methods Rat H9c2 myocardial cells were divided into blank group(normal culture), model group(hypoxia/reoxygenation treatment) and test group(20 μmol HSYA + hypoxia/reoxygenation treatment). After 24 h of intervention, the cell morphology was observed under optical inverted microscope, the survival rate was detected by living cell counting kit, the level of reactive oxygen species(ROS) was detected by DCF-DA fluorescence staining, the content of cell damage index was detected by kit, and the expression of apoptosis-related proteins and miR-499 was detected by Western blot. Results After 48 hours of intervention, the survival rates of H9c2 cardiomyocytes in the blank group,the model group and the test group were(99. 26 ± 0. 15) %,(45. 12 ± 3. 62) %,(78. 69 ± 2. 15) %,the creatine kinase(CK) in cell supernatant were(2. 63 ± 0. 14),(6. 25 ± 0. 29),(4. 63 ± 0. 21) U·L-1,lactate dehydrogenase(LDH) were(22. 63 ± 3. 15),(46. 85 ± 2. 96),(32. 16 ± 2. 86) U·L-1,the apoptosis rates were(2. 63 ± 0. 24) %,(36. 25 ± 4. 92) %,(13. 29 ± 4. 15) %. The relative expression of Bax in H9c2 cardiomyocytes were 0. 24 ± 0. 03,0. 69 ± 0. 03,0. 42 ± 0. 02,Caspase-3 were 0. 28 ± 0. 04,2. 56 ± 0. 21,1. 85 ± 0. 03,miR-499 were 1. 63 ± 0. 02,0. 56 ± 0. 05,1. 16 ± 0. 02,all with significant difference(all P < 0. 05). Conclusion HSYA can inhibit oxidative stress response, inhibit apoptosis, promote cell proliferation and alleviate myocardial injury by regulating the expression of microRNA-499.