Institute of Nutritional Sciences, Laboratory for Nutrition in Prevention and Therapy, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, Schubertstrasse 81, 35392 Giessen, Germany.
Molecular Nutrition Research, Interdisciplinary Research Center, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany.
Oxid Med Cell Longev. 2019 Nov 15;2019:6840540. doi: 10.1155/2019/6840540. eCollection 2019.
Mitochondria supply cellular energy and are key regulators of intrinsic cell death and consequently affect longevity. The nematode is frequently used for lifespan assays. Using paraquat (PQ) as a generator of reactive oxygen species, we here describe its effects on the acceleration of aging and the associated dysfunctions at the level of mitochondria.
Nematodes were incubated with various concentrations of paraquat in a heat-stress resistance assay (37°C) using nucleic staining. The most effective concentration was validated under physiological conditions, and chemotaxis was assayed. Mitochondrial membrane potential (m) was measured using rhodamine 123, and activity of respiratory chain complexes determined using a Clark-type electrode in isolated mitochondria. Energetic metabolites in the form of pyruvate, lactate, and ATP were determined using commercial kits. Mitochondrial integrity and structure was investigated using transmission electron microscopy. Live imaging after staining with fluorescent dyes was used to measure mitochondrial and cytosolic ROS. Expression of longevity- and mitogenesis-related genes were evaluated using qRT-PCR.
PQ (5 mM) significantly increased ROS formation in nematodes and reduced the chemotaxis, the physiological lifespan, and the survival in assays for heat-stress resistance. The number of fragmented mitochondria significantly increased. The ∆m, the activities of complexes I-IV of the mitochondrial respiratory chain, and the levels of pyruvate and lactate were significantly reduced, whereas ATP production was not affected. Transcript levels of genetic marker genes, , , , and , were significantly upregulated after PQ incubation, which implicates a close connection between mitochondrial dysfunction and oxidative stress response. Expression levels of and were unchanged.
Using paraquat as a stressor, we here describe the association of oxidative stress, restricted energy metabolism, and reduced stress resistance and longevity in the nematode making it a readily accessible model for mitochondrial dysfunction.
线粒体为细胞提供能量，是细胞内在死亡的关键调节剂，因此影响寿命。秀丽隐杆线虫常用于寿命测定。本文使用百草枯（PQ）作为活性氧（ROS）的产生剂，描述其对加速衰老的影响以及与线粒体水平相关的功能障碍。
使用核酸染色，在热应激抗性测定（37°C）中用不同浓度的百草枯孵育线虫。在生理条件下验证最有效的浓度，并测定趋药性。使用罗丹明 123 测量线粒体膜电位（m），并使用Clark 型电极在分离的线粒体中测定呼吸链复合物的活性。使用商业试剂盒测定丙酮酸、乳酸和 ATP 的形式的能量代谢物。使用透射电子显微镜研究线粒体的完整性和结构。使用荧光染料染色后的实时成像测量线粒体和胞质 ROS。使用 qRT-PCR 评估与寿命和有丝分裂相关的基因的表达。
PQ（5 mM）显著增加线虫中的 ROS 形成，并降低趋药性、生理寿命以及热应激抗性测定中的存活率。碎片化线粒体的数量显著增加。∆m、线粒体呼吸链复合物 I-IV 的活性以及丙酮酸和乳酸的水平显著降低，而 ATP 产生不受影响。PQ 孵育后，遗传标记基因 、 、 、 和 的转录水平显著上调，这表明线粒体功能障碍与氧化应激反应之间存在密切联系。 和 的表达水平没有变化。
本文使用百草枯作为应激源，描述了氧化应激、限制能量代谢以及线虫应激抵抗和寿命降低之间的关联，使其成为一种易于获得的线粒体功能障碍模型。
