Eaton P, Li J M, Hearse D J, Shattock M J
Cardiovascular Research, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, United Kingdom.
Am J Physiol. 1999 Mar;276(3):H935-43. doi: 10.1152/ajpheart.1999.276.3.H935.
4-Hydroxy-2-nonenal (HNE) is a major lipid peroxidation product formed during oxidative stress. Because of its reactivity with nucleophilic compounds, particularly metabolites and proteins containing thiol groups, HNE is cytotoxic. The aim of this study was to assess the extent and time course for the formation of HNE-modified proteins during ischemia and ischemia plus reperfusion in isolated rat hearts. With an antibody to HNE-Cys/His/Lys and densitometry of Western blots, we quantified the amount of HNE-protein adduct in the heart. By taking biopsies from single hearts (n = 5) at various times (0, 5, 10, 15, 20, 35, and 40 min) after onset of zero-flow global ischemia, we showed a progressive, time-dependent increase (which peaked after 30 min) in HNE-mediated modification of a discrete number of proteins. In studies with individual hearts (n = 4/group), control aerobic perfusion (70 min) resulted in a very low level (296 arbitrary units) of HNE-protein adduct formation; by contrast, after 30-min ischemia HNE-adduct content increased by >50-fold (15,356 units, P < 0.05). In other studies (n = 4/group), administration of N-(2-mercaptopropionyl)glycine (MPG, 1 mM) to the heart for 5 min immediately before 30-min ischemia reduced HNE-protein adduct formation during ischemia by approximately 75%. In studies (n = 4/group) that included reperfusion of hearts after 5, 10, 15, or 30 min of ischemia, there was no further increase in the extent of HNE-protein adduct formation over that seen with ischemia alone. Similarly, in experiments with MPG, reperfusion did not significantly influence the tissue content of HNE-protein adduct. Western immunoblot results were confirmed in studies using in situ immunofluorescent localization of HNE-protein in cryosections. In conclusion, ischemia causes a major increase in HNE-protein adduct that would be expected to reflect a toxic sequence of events that might act to compromise tissue survival during ischemia and recovery on reperfusion.
4-羟基-2-壬烯醛（HNE）是氧化应激过程中形成的一种主要脂质过氧化产物。由于其与亲核化合物，特别是与含硫醇基团的代谢产物和蛋白质发生反应，HNE具有细胞毒性。本研究的目的是评估在离体大鼠心脏缺血及缺血加再灌注过程中HNE修饰蛋白的形成程度和时间进程。我们使用抗HNE-半胱氨酸/组氨酸/赖氨酸抗体并通过蛋白质印迹法进行光密度测定，对心脏中HNE-蛋白质加合物的量进行了定量。通过在零流量全心缺血开始后的不同时间点（0、5、10、15、20、35和40分钟）从单个心脏（n = 5）取活检组织，我们发现HNE介导的特定数量蛋白质的修饰呈时间依赖性逐渐增加（在30分钟后达到峰值）。在对单个心脏的研究（n = 4/组）中，对照有氧灌注（70分钟）导致HNE-蛋白质加合物形成水平非常低（296任意单位）；相比之下，30分钟缺血后HNE-加合物含量增加了50多倍（15356单位，P < 0.05）。在其他研究（n = 4/组）中，在30分钟缺血前立即向心脏给予N-（2-巯基丙酰基）甘氨酸（MPG，1 mM）5分钟，可使缺血期间HNE-蛋白质加合物的形成减少约75%。在包括缺血5、10、15或30分钟后心脏再灌注的研究（n = 4/组）中，HNE-蛋白质加合物的形成程度没有比单独缺血时进一步增加。同样，在使用MPG的实验中，再灌注对HNE-蛋白质加合物的组织含量没有显著影响。蛋白质印迹免疫分析结果在使用冷冻切片中HNE-蛋白质的原位免疫荧光定位的研究中得到了证实。总之，缺血导致HNE-蛋白质加合物大量增加，这有望反映一系列毒性事件，这些事件可能在缺血期间损害组织存活并在再灌注时影响恢复。
