Zholobenko Aleksey V, Mouithys-Mickalad Ange, Dostal Zdenek, Serteyn Didier, Modriansky Martin
Department of Medical Chemistry & Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic.
Centre for Oxygen, R&D (CORD), Institut de Chimie, Sart-Tilman, Université de Liège, Liège, Belgium.
PLoS One. 2017 Oct 4;12(10):e0185691. doi: 10.1371/journal.pone.0185691. eCollection 2017.
Quercetin and dehydrosilybin are polyphenols which are known to behave like uncouplers of respiration in isolated mitochondria. Here we investigated whether the effect is conserved in whole cells. Following short term incubation, neither compound uncouples mitochondrial respiration in whole H9c2 cells below 50μM. However, following hypoxia, or long term incubation, leak (state IV with oligomycin) oxygen consumption is increased by quercetin. Both compounds partially protected complex I respiration, but not complex II in H9c2 cells following hypoxia. In a permeabilised H9c2 cell model, the increase in leak respiration caused by quercetin is lowered by increased [ADP] and is increased by adenine nucleotide transporter inhibitor, atractyloside, but not bongkrekic acid. Both quercetin and dehydrosilybin dissipate mitochondrial membrane potential in whole cells. In the case of quercetin, the effect is potentiated post hypoxia. Genetically encoded Ca++ sensors, targeted to the mitochondria, enabled the use of fluorescence microscopy to show that quercetin decreased mitochondrial [Ca++] while dehydrosilybin did not. Likewise, quercetin decreases accumulation of [Ca++] in mitochondria following hypoxia. Fluorescent probes were used to show that both compounds decrease plasma membrane potential and increase cytosolic [Ca++]. We conclude that the uncoupler-like effects of these polyphenols are attenuated in whole cells compared to isolated mitochondria, but downstream effects are nevertheless apparent. Results suggest that the effect of quercetin observed in whole and permeabilised cells may originate in the mitochondria, while the mechanism of action of cardioprotection by dehydrosilybin may be less dependent on mitochondrial uncoupling than originally thought. Rather, protective effects may originate due to interactions at the plasma membrane.
槲皮素和脱氢水飞蓟宾是多酚类物质，已知它们在分离的线粒体中表现得像呼吸解偶联剂。在此，我们研究了这种效应在完整细胞中是否也存在。短期孵育后，在浓度低于50μM时，这两种化合物均不会使完整的H9c2细胞中的线粒体呼吸解偶联。然而，在缺氧或长期孵育后，槲皮素会增加渗漏（用寡霉素处理后的状态IV）氧消耗。在缺氧后的H9c2细胞中，这两种化合物都能部分保护复合体I的呼吸，但对复合体II无保护作用。在透化的H9c2细胞模型中，槲皮素引起的渗漏呼吸增加会因[ADP]增加而降低，因腺嘌呤核苷酸转运体抑制剂苍术苷而增加，但不会因邦克雷酸而增加。槲皮素和脱氢水飞蓟宾都会使完整细胞中的线粒体膜电位耗散。就槲皮素而言，缺氧后这种效应会增强。靶向线粒体的基因编码Ca++传感器使我们能够利用荧光显微镜观察到，槲皮素会降低线粒体[Ca++]，而脱氢水飞蓟宾则不会。同样，槲皮素会减少缺氧后线粒体中[Ca++]的积累。荧光探针显示，这两种化合物都会降低质膜电位并增加胞质[Ca++]。我们得出结论，与分离的线粒体相比，这些多酚类物质的解偶联样效应在完整细胞中有所减弱，但下游效应仍然明显。结果表明，在完整细胞和透化细胞中观察到的槲皮素效应可能起源于线粒体，而脱氢水飞蓟宾对心脏的保护作用机制可能比最初认为的更少依赖于线粒体解偶联。相反，保护作用可能源于质膜上的相互作用。
