Zeng Jianying, Yang Guo-Yuan, Ying Weihai, Kelly Mark, Hirai Kiyoshi, James Thomas L, Swanson Raymond A, Litt Lawrence
Department of Anesthesia, University of California at San Francisco, 94143-0648, USA.
J Cereb Blood Flow Metab. 2007 Feb;27(2):304-15. doi: 10.1038/sj.jcbfm.9600335. Epub 2006 May 24.
Previous neuron and glial cell culture studies of excessive poly (ADP-ribose) polymerase (PARP-1) activation found NAD(+) depletion, glycolytic arrest, and cell death that could be avoided by exogenous tricarboxylic acid cycle (TCA) metabolites, especially pyruvate (pyr). Pyruvate neuroprotection has been attributed to cytosolic NAD(+) replenishment, TCA metabolism, and antioxidant activity. We investigated the first two mechanisms in respiring cerebrocortical slices after a 1-h H(2)O(2) exposure to activate PARP-1. H(2)O(2) was followed by a 4-h recovery with oxy-artificial cerebrospinal fluid superfusion having either: (1) no glucose (glc) or pyruvate; (2) 10 mmol/L glc only; (3) 10 mmol/L pyruvate only; (4) both 10 mmol/L glc and 10 mmol/L pyruvate. Poly-ADP-ribosylation was quantified from Western blots and immunohistochemistry. Perchloric acid extracts were quantified with 14.1 T (31)P nuclear magnetic resonance spectroscopy. Just after H(2)O(2) exposure, ATP and NAD(+) decreased by approximately 50%, PCr decreased by 75%, and the ADP/ATP ratio approximately doubled. ATP and NAD(+) changes, but not PCr changes, were nearly eliminated if PARP inhibitors accompanied the H(2)O(2). Recovery with both pyruvate and glc was better than with glc alone, having higher ATP (0.161 versus 0.075, P<0.01) and PCr levels (0.144 versus 0.078, P<0.01), and higher viable cell counts in TUNEL and Fluoro-Jade B staining. Two-dimensional [(1)H-(13)C] HSQC spectra showed metabolism during recovery of (13)C glc or pyr. Pyruvate metabolism was primarily via pyruvate dehydrogenase, with some via pyruvate carboxylation. Pyruvate superfusion of PARP-injured brain slices helps replenish NAD(+) while providing metabolic fuel. Although this augments recovery, a strong antioxidant role for pyruvate has not been ruled out.
先前对过度激活的聚（ADP - 核糖）聚合酶（PARP - 1）进行的神经元和神经胶质细胞培养研究发现，烟酰胺腺嘌呤二核苷酸（NAD(+)）耗竭、糖酵解停滞以及细胞死亡，而外源性三羧酸循环（TCA）代谢产物，尤其是丙酮酸（pyr）可以避免这些情况。丙酮酸的神经保护作用归因于胞质NAD(+)的补充、TCA代谢以及抗氧化活性。我们研究了在暴露于1小时过氧化氢（H(2)O(2)）以激活PARP - 1后，正在进行呼吸的大脑皮质切片中的前两种机制。在H(2)O(2)处理后，用含氧人工脑脊液灌注进行4小时恢复，灌注液分别为：（1）不含葡萄糖（glc）或丙酮酸；（2）仅含10 mmol/L葡萄糖；（3）仅含10 mmol/L丙酮酸；（4）同时含10 mmol/L葡萄糖和10 mmol/L丙酮酸。通过蛋白质免疫印迹和免疫组织化学对多聚ADP - 核糖基化进行定量。用14.1 T（31）P核磁共振波谱对高氯酸提取物进行定量。在H(2)O(2)暴露后，三磷酸腺苷（ATP）和NAD(+)下降约50%，磷酸肌酸（PCr）下降75%，ADP/ATP比值约增加一倍。如果在H(2)O(2)处理时同时使用PARP抑制剂，ATP和NAD(+)的变化，但不是PCr的变化，几乎可以消除。丙酮酸和葡萄糖同时灌注的恢复效果优于单独使用葡萄糖，ATP水平更高（0.161对0.075，P<0.01），PCr水平更高（0.144对0.078，P<0.01），并且在TUNEL和Fluoro - Jade B染色中活细胞计数更高。二维[(1)H-(13)C] HSQC谱显示了(13)C葡萄糖或丙酮酸恢复过程中的代谢情况。丙酮酸代谢主要通过丙酮酸脱氢酶，部分通过丙酮酸羧化。对PARP损伤的脑切片进行丙酮酸灌注有助于补充NAD(+)，同时提供代谢燃料。尽管这增强了恢复效果，但丙酮酸的强大抗氧化作用尚未排除。
