Objective: To investigate the possible mechanism related to the protective effects of salvianolate in H9c2 cells underwent hypoxia/reoxygenation (H/R)-injury. Methods: H9c2 cells were divided into four groups: control group, salvianolate group (S group), H/R group, and salvianolate+ H/R group(S+ H/R group), in which the H9c2 cells were pretreated with salvianolate before H/R-treatment.Apoptotic cells were detected by Tunel assays and AnnexinⅤ-FITC apoptosis detection kit.The intracellular ATP level, the change of mitochondrial membrane potential and the mitochondrial DNA oxidative damage were also determined in these groups. Results: (1) The apoptosis rate of H/R group(26.36±5.14)% was significantly higher compared to control group(2.71±1.66)%(P=0.000 4), which could be significantly reduced in S+ H/R group(17.28±4.75)%(P=0.012 8 vs. H/R group , P=0.003 9 vs. control group). The ratio of AnnexinⅤ and PI double positive cells in H/R group(28.23±6.73)% was significantly higher compared to control group(3.53±2.83)%(P=0.001 1), which was significantly reduced in S+ H/R group(18.10±4.56)%(P=0.037 2 vs. H/R group, P=0.038 3 vs. control group). (2)The ATP level of H9c2 cells in H/R group(49.05±10.12)% was significantly lower than in control group 100%(P=0.000 5), which was significantly increased in S+ H/R group(68.67±13.32)%(P=0.019 9 vs. H/R group). Confocal microscope showed that red fluorescence was dominant in the control group, red fluorescence was significantly reduced, while green fluorescence was significantly increased in H9c2 cells of H/R group and the fluorescence ratio of red to green in H/R group((37.13±8.47)%) was significantly decreased compared to control group (100%, P=0.000 1), fluorescence ratio of red to green was significantly increased in S+ H/R group((63.77±12.32)% vs. H/R group, P=0.007 3). (3)The mitochondrial DNA oxidative damage in different groups: there was only few 8-hydroxyguanine (8-OHdG) expression, which marked as green, in control group, and 8-OHdG expression was significantly upregulated in H/R group, moreover, the 8-OHdG was co-localized with mitochondria.The expression of 8-OHdG was significantly lower in S+ H/R group compared to H/R group. Conclusion: Salvianolate can reduce mitochondrial DNA oxidative damage, and protect mitochondrial function, thus inhibit myocardial cell apoptosis and eventually reduce the myocardial H/R-injury in H9c2 cells.
目的： 探讨丹参多酚减轻心肌细胞缺氧复氧损伤的机制。 方法： H9c2心肌细胞株来源于中国科学院细胞库。将H9c2心肌细胞分为4组，即对照组(细胞在普通培养基中正常培养)、丹参多酚组[先用含有0.5 g/L丹参多酚的培养基预处理4 h(37 ℃)后换普通培养基正常培养]、H/R组(进行缺氧复氧处理)和丹参多酚＋H/R组(先用含有0.5 g/L丹参多酚的培养基预处理4 h后再进行缺氧复氧处理)。检测各组心肌细胞的凋亡率、细胞内三磷酸腺苷(ATP)水平、线粒体膜电位的变化以及线粒体DNA氧化损伤情况。 结果： (1)各组心肌细胞凋亡情况：H/R组Tunel阳性细胞率为(26.36±5.14)%明显高于对照组的(2.71±1.66)%(P＝0.000 4)，丹参多酚＋H/R组Tunel阳性细胞率为(17.28±4.75)%，虽高于对照组(P＝0.003 9)，却明显低于H/R组(P＝0.012 8)。H/R组AnnexinⅤ及PI双阳性的细胞比率为(28.23±6.73)%明显高于对照组的(3.53±2.83)%(P＝0.001 1)，丹参多酚＋H/R组AnnexinⅤ及PI双阳性的细胞比率为(18.10±4.56)%虽高于对照组(P＝0.038 3)，却明显低于H/R组(P＝0.037 2)。(2)各组心肌细胞内ATP水平及线粒体膜电位：H/R组心肌细胞内ATP水平为(49.05±10.12)%明显低于对照组(100%)(P＝0.000 5)，而丹参多酚＋H/R组心肌细胞内ATP水平为(68.67±13.32)%明显高于H/R组(P＝0.019 9)。激光共聚焦显微镜下可见H/R组心肌细胞的红色荧光较对照组弱，绿色荧光较对照组强，酶标仪检测结果示H/R组心肌细胞中线粒体膜电位为(37.13±8.47)%明显低于对照组(100%)(P＝0.000 1)。而丹参多酚＋H/R组心肌细胞的红色荧光较H/R组强，绿色荧光较H/R组弱，酶标仪检测结果示丹参多酚＋H/R组心肌细胞中线粒体膜电位为(63.77±12.32)%明显高于H/R组的(37.13±8.47)%(P＝0.007 3)。(3)各组心肌细胞线粒体DNA氧化损伤情况：对照组及丹参多酚组心肌细胞中几乎未见绿色的8-OHdG表达，H/R组心肌细胞中绿色的8-OHdG表达则较多，而且与代表线粒体的红色的MitoTracker Red重合。丹参多酚＋H/R组心肌细胞线粒体内绿色的8-OHdG表达较H/R组少。 结论： 丹参多酚可能是通过减轻线粒体DNA氧化损伤，保护线粒体功能，从而抑制心肌细胞凋亡，最终减轻心肌细胞缺氧复氧损伤。.
Keywords: Apoptosis; Mitochondria; Myocytes, cardiac; Reperfusion injury; Salvia miltiorrhiza.