丹参多酚通过减轻线粒体DNA氧化损伤抑制缺氧复氧诱导的心肌细胞凋亡

Salvianolate protects H9c2 cells from hypoxia/reoxygenation injury-induced apoptosis by attenuating mitochondrial DNA oxidative damage

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。

ObjectiveTo investigate the possible mechanism related to the protective effects of salvianolate in H9c2 cells underwent hypoxia/reoxygenation （H/R）-injury. MethodsH9c2 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. ConclusionSalvianolate 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.