Simoni J, Simoni G, Martinez-Zaguilan R, Wesson D E, Lox C D, Prien S D, Kumar R V
Department of Surgery, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
ASAIO J. 1998 Sep-Oct;44(5):M356-67.
The authors have previously documented that appropriate chemical and pharmacologic modification of the hemoglobin molecule are required to attenuate certain pathophysiologic reactions of the reticuloendothelium. The current study further investigates the molecular responses of human coronary artery endothelial cells to a high concentration (0.4 mmol) of 1) unmodified bovine hemoglobin; and 2) an improved blood substitute that comprises hemoglobin cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione. In this study, the scavenging effect of hemoglobins toward nitric oxide (NO) was evaluated by the measurement of nitrite (NO2-) and nitrate (NO3-) formation. The pro-oxidant effect of hemoglobin on endothelial cells was examined by the measurement of intracellular reduced glutathione, and by monitoring the formation of lipid hydroperoxides and 8-iso prostaglandin F2alpha, a novel potent vasoconstrictor, which is produced by a noncyclooxygenase mechanism involving free radical catalyzed peroxidation of arachidonic acid. The inflammatory reactions of endothelial cells were evaluated by the expression of the adhesion molecule, intracellular adhesion molecule-1, and the activation of nuclear transcription factor, nuclear factor kappaB. In additional, endothelial cell responses were investigated by analysis of intracellular ionized calcium concentrations. Results indicate that unmodified hemoglobin in a concentration of 0.4 mmol/L can aggravate endothelial cell oxidative and inflammatory responses. This hemoglobin produced a significant (p < 0.01) depletion of reduced glutathione, acceleration of lipid peroxidation, and a greater influx of Ca2+. The formation of 8-iso prostaglandin F2alpha increased compared with the control cells (p < 0.01). Unmodified hemoglobin was found to be a potent scavenger of NO, great activator of nuclear factor kappaB, and a stimulator of intracellular adhesion molecule-1 expression. Contrarily, the improved blood substitute did not appear to induce oxidative stress nor to increase the intracellular Ca2+. The concentration of 8-iso prostaglandin F2alpha was similar to that in the control cells, whereas the formation of NO2-/NO3- was much lower (p < 0.05) than in the unmodified hemoglobin group. The effect of an improved blood substitute can be linked with the anti-inflammatory and cytoprotective properties of adenosine, which is used as a cross-linker and surface modifier, and the type of the chemical modification procedure that lowers hemoglobin pro-oxidant potential.
作者们之前已证明，血红蛋白分子需要进行适当的化学和药理学修饰，以减轻网状内皮系统的某些病理生理反应。当前研究进一步探究了人冠状动脉内皮细胞对高浓度（0.4 mmol）的以下两种物质的分子反应：1）未修饰的牛血红蛋白；2）一种改良的血液替代品，其包含分子内与o - 三磷酸腺苷交联、分子间与o - 腺苷交联并与还原型谷胱甘肽共轭的血红蛋白。在本研究中，通过测量亚硝酸盐（NO2-）和硝酸盐（NO3-）的形成来评估血红蛋白对一氧化氮（NO）的清除作用。通过测量细胞内还原型谷胱甘肽，并监测脂质氢过氧化物和8 - 异前列腺素F2α（一种新型强效血管收缩剂，由涉及花生四烯酸自由基催化过氧化的非环氧化酶机制产生）的形成，来检测血红蛋白对内皮细胞的促氧化作用。通过黏附分子细胞间黏附分子-1的表达以及核转录因子核因子κB的激活来评估内皮细胞的炎症反应。此外，通过分析细胞内游离钙浓度来研究内皮细胞反应。结果表明，浓度为0.4 mmol/L的未修饰血红蛋白可加重内皮细胞的氧化和炎症反应。这种血红蛋白使还原型谷胱甘肽显著（p < 0.01）耗竭，加速脂质过氧化，并使Ca2+大量内流。与对照细胞相比，8 - 异前列腺素F2α的形成增加（p < 0.01）。发现未修饰血红蛋白是一种强效的NO清除剂、核因子κB的强大激活剂以及细胞间黏附分子-1表达的刺激剂。相反，改良的血液替代品似乎不会诱导氧化应激，也不会增加细胞内Ca2+。8 - 异前列腺素F2α的浓度与对照细胞相似，而NO2-/NO3-的形成比未修饰血红蛋白组低得多（p < 0.05）。改良血液替代品的作用可能与用作交联剂和表面修饰剂的腺苷的抗炎和细胞保护特性以及降低血红蛋白促氧化潜力的化学修饰程序类型有关。
