Szabados E, Fischer G M, Gallyas F, Kispal G, Sumegi B
Department of Biochemistry, University Medical School Pecs, Hungary.
Free Radic Biol Med. 1999 Nov;27(9-10):1103-13. doi: 10.1016/s0891-5849(99)00151-3.
Poly-ADP-ribose polymerase (PARP) is considered to play an important role in oxidative cell damage. We assumed that ischemia-reperfusion resulting from the increasing reactive oxygen species (ROS) can lead to the activation of endogenous mono- and poly-ADP-ribosylation reactions and that the reduction of ROS level by lipoamide, a less known antioxidant, can reverse these unfavorable processes. Experiments were performed on isolated Langendorff hearts subjected to 60-min ischemia followed by reperfusion. ROS, malondialdehyde, deoxyribonucleic acid (DNA) breaks, and NAD+ content were assayed in the hearts, and the ADP-ribosylation of cytoplasmic and nuclear proteins were determined by Western blot assay. Ischemia-reperfusion caused a moderate (30.2 +/- 8%) increase in ROS production determined by the dihydrorhodamine 123 method and significantly increased the malondialdehyde production (from < 1 to 23 +/- 2.7 nmol/ml), DNA damage (undamaged DNA decreased from 71 +/- 7% to 23.1 +/- 5%), and NAD+ catabolism. In addition, ischemia-reperfusion activated the mono-ADP-ribosylation of GRP78 and the self-ADP-ribosylation of the nuclear PARP. The perfusion of hearts with lipoamide significantly decreased the ischemia-reperfusion-induced cell membrane damage determined by enzyme release (LDH, CK, and GOT), decreased the ROS production, reduced the malondialdehyde production to 5.5 +/- 2.4 nmol/ml, abolished DNA damage, and reduced NAD+ catabolism. The ischemia-reperfusion-induced activation of poly- and mono-ADP-ribosylation reactions were also reverted by lipoamide. In isolated rat heart mitochondria, dihydrolipoamide was found to be a better antioxidant than dihydrolipoic acid. Ischemia-reperfusion by ROS overproduction and increasing DNA breaks activates PARP leading to accelerated NAD+ catabolism, impaired energy metabolism, and cell damage. Lipoamide by reducing ROS levels halts PARP activation and membrane damage and improves the recovery of postischemic myocardium.
聚 ADP - 核糖聚合酶（PARP）被认为在氧化性细胞损伤中起重要作用。我们推测，由活性氧（ROS）增加导致的缺血再灌注可引发内源性单 ADP - 核糖基化和多 ADP - 核糖基化反应的激活，而一种鲜为人知的抗氧化剂硫辛酸通过降低 ROS 水平可逆转这些不利过程。实验在离体 Langendorff 心脏上进行，先使其经历 60 分钟缺血，随后再灌注。检测心脏中的 ROS、丙二醛、脱氧核糖核酸（DNA）断裂情况以及 NAD⁺含量，并通过蛋白质印迹法测定细胞质和细胞核蛋白的 ADP - 核糖基化。缺血再灌注导致通过二氢罗丹明 123 法测定的 ROS 生成适度增加（30.2±8%），并显著增加丙二醛生成（从<1 增加至 23±2.7 nmol/ml）、DNA 损伤（未损伤 DNA 从 71±7%降至 23.1±5%）以及 NAD⁺分解代谢。此外，缺血再灌注激活了 GRP78 的单 ADP - 核糖基化以及细胞核 PARP 的自身 ADP - 核糖基化。用硫辛酸灌注心脏显著降低了通过酶释放（乳酸脱氢酶、肌酸激酶和谷草转氨酶）测定的缺血再灌注诱导的细胞膜损伤，降低了 ROS 生成，将丙二醛生成降至 5.5±2.4 nmol/ml，消除了 DNA 损伤，并减少了 NAD⁺分解代谢。硫辛酸还逆转了缺血再灌注诱导的多 ADP - 核糖基化和单 ADP - 核糖基化反应的激活。在离体大鼠心脏线粒体中，发现二氢硫辛酸比二氢硫辛酸是更好的抗氧化剂。ROS 过量生成和 DNA 断裂增加所导致的缺血再灌注激活 PARP，导致 NAD⁺分解代谢加速、能量代谢受损以及细胞损伤。硫辛酸通过降低 ROS 水平可阻止 PARP 激活和膜损伤，并改善缺血后心肌的恢复。
