背景：血红蛋白类氧载体（hemoglobin-based oxygen carriers，HBOCs）具有携释氧功能，在失血性休克复苏、肿瘤放化疗增敏、器官保存等方面均有疗效，但在其制备过程中仍存在诸多问题。血红蛋白容易被氧化生成高铁血红蛋白而失去携释氧能力，目前主要通过全程通惰性气体、维持低温和加入抗氧化剂等方式抑制氧化过程。一氧化碳（carbon monoxide，CO）是机体重要的信号传递分子，与血红蛋白的结合位点同氧气一致。生理条件下，CO对血红蛋白亲和力约为氧气的250倍，可以保护血红蛋白不易被氧化，而低浓度外源性CO可用于与炎症、氧化应激等相关的疾病治疗。本课题在前期研究的基础上，制备聚合碳氧血红蛋白（poly-Carboxyhemoglobin，poly-COHb）并对其在急性失血性休克中的作用进行研究。

目的：以提高聚合度、控制超大分子含量为目的，在常温、无需隔氧条件下制备poly-COHb；研究poly-COHb在急性失血性休克大鼠模型中的复苏效果及其对休克复苏后大鼠结肠组织的作用。

方法：选择血红蛋白浓度、戊二醛与碳氧血红蛋白摩尔比、反应时间和反应温度四个因素进行正交试验，确定最佳匹配条件，制备poly-COHb并检测其理化性质。体内试验中，制备50%急性失血性休克模型，将大鼠随机分成3组：对照组为羟乙基淀粉组（HES组）、聚合血红蛋白组（poly-Hb组）；实验组为聚合碳氧血红蛋白组（poly-COHb组）。各组大鼠进行等容液体复苏，动态监测平均动脉压、心率、动脉血气等生理指标并记录72h内存活情况。另取SD大鼠随机分为两组：poly-Hb组和poly-COHb组，复苏后6h取样，检测结肠组织渗透性、炎症因子、氧化应激等指标，并观察结肠组织病理形态变化。

结果：制备poly-COHb的最佳匹配条件为：[Hb]=70g/L；戊二醛与COHb摩尔比=11:1；反应时间=60min；反应温度=25℃。poly-Hb与poly-COHb比较，聚合度（%）与平均分子量（kDa）分别为54.16±5.12 vs 84.25±0.99，97.62±6.57 vs 235.27±13.50（P<0. 05），其余理化指标无统计学差异。体内有效性研究结果显示，复苏后，与HES组比较，poly-Hb和poly-COHb组平均动脉压、总血红蛋白及氧合血红蛋白含量均显著增加；其中，复苏0h，与HES组比较，其余两组动脉血氧分压、氧合血红蛋白百分比下降；与poly-Hb组比较，poly-COHb组平均动脉压下降（P＜0.05）。复苏1h，与HES组比较，poly-COHb组心率加快，乳酸水平降低（P＜0.05）。复苏72h，poly-Hb和poly-COHb组较HES组生存率显著提高（P＜0.05）。研究poly-COHb对休克复苏后结肠组织的作用结果显示，复苏后6h，与poly-Hb组比较，poly-COHb组结肠组织中超氧化物歧化酶活力增加，丙二醛、髓过氧化物酶、活性氧含量下降，核转录因子E2相关因子2（nuclear factor erythroid2-related factor 2，Nrf2）、血红素加氧酶-1（heme oxygenase-1，HO-1）表达水平上升（P＜0.05），病理HE染色显示结肠组织病变减轻。

结论：常温、无需隔氧条件能制备poly-COHb，与poly-Hb相比，poly-COHb可以提高聚合度，简化制备方法，达到实验预期目标。poly-COHb具有携氧扩容作用，可用于急性失血性休克复苏，改善大鼠休克状态，提高大鼠生存率，其可能通过调节Nrf2/HO-1信号通路降低大鼠结肠组织氧化应激水平，对结肠组织具有保护作用。

关键词：聚合碳氧血红蛋白；聚合血红蛋白；氧化应激；一氧化碳；失血性休克

Background:Hemoglobin-based oxygen carriers (HBOCs) have been shown to have abilities to carry and release oxygen. Curative effects of HBOCs have been reported in hemorrhagic shock, tumor chemoradiotherapy sensitization, and organ preservation. Nonetheless, many problems still exist in the preparation process. Hemoglobin is easy to be oxidized to produce methemoglobin and lose the ability to carry and release oxygen.At present, the oxidation process is mainly inhibited by passing inert gas through the whole process, maintaining low temperature and adding antioxidants. Carbon monoxide (CO), sharing same binding site on hemoglobin with oxygen, is an important signal transduction molecule in the body.Under physiological conditions, the affinity of CO to hemoglobin is about 250 times that of oxygen, so CO can prevent hemoglobin from being oxidized.In addition, low concentration exogenous CO can be used to treat diseases related to inflammation and oxidative stress. Based on the previous research, we prepared poly-Carboxyhemoglobin (poly-COHb) and studied its role in acute hemorrhagic shock.

Objective:In order to improve the degree of polymerization and control the super molecular content, we prepared poly-COHb at room temperature without oxygen isolation. To study the resuscitation effect of poly-COHb in acute hemorrhagic shock and its effect on colon tissue of rats after shock resuscitation.

Methods:Hemoglobin concentration, molar ratio of glutaraldehyde to carboxyhemoglobin (COHb), reaction time and reaction temperature were selected for orthogonal test.The best matching conditions were obtained to prepare poly-COHb, and its physical and chemical properties were characterized. In the in vivo experiment, 50% acute hemorrhagic shock model was prepared. The rats were randomly divided into three groups:the control group were hydroxyethyl starch (HES) group and poly-hemoglobin (poly-Hb) group,the experimental group was poly-Carboxyhemoglobin (poly-COHb) group.The rats were resuscitated with isovolumetric fluid. The indexes such as average arterial pressure, heart rate and arterial blood gas were dynamically monitored, and the survival of rats was recorded at 72 hours following resuscitation. In addition, SD rats were randomly divided into two groups: poly-Hb group and poly-COHb group. Colon tissues were taken 6 hours after resuscitation to detect the permeability, inflammatory factors,oxidative stress and other indicators.The pathological changes of colon tissue were observed.

Results:The best matching condition for the preparation of poly-COHb was: [Hb] = 70g/L; molar ratio of glutaraldehyde to COHb = 11:1; reaction time = 60min; reaction temperature = 25 ℃. Comparison of poly-Hb and poly-COHb, the degree of polymerization (%) and average molecular weight (kDa) were 54.16 ± 5.12 vs 84.25 ± 0.99, 97.62 ± 6.57 vs 235.27 ± 13.50 (P < 0.05), and there was no significant difference in other physical and chemical indexes.The results of in vivo efficacy study showed that after resuscitation, compared with HES group, the mean arterial pressure, total hemoglobin and oxygenated hemoglobin in both poly-Hb and poly-COHb groups were increased significantly (P < 0.05). At 0 h after resuscitation, compared with HES group, the arterial partial pressure of oxygen and the percentage of oxygenated hemoglobin in poly-Hb and poly-COHb groups were decreased (P < 0.05). Compared with poly-Hb group, the mean arterial pressure in poly-COHb group was decreased (P < 0.05). After 1 hour of resuscitation, compared with HES group, the heart rate in poly-COHb group increased while the lactic acid content decreased (P < 0.05). After 72 hours of resuscitation, the survival rate of poly-Hb and poly-COHb groups was significantly higher than that of HES group (P < 0.05). The results from the effect study of poly-COHb on colonic tissues showed that 6 hours after resuscitation, compared with poly-Hb group, the expression levels of malondialdehyde, myeloperoxidase and reactive oxygen species in colonic tissues were decreased while the activity of superoxide dismutase and the expression of nuclear factor erythroid2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)were increased  in poly-COHb group(P < 0.05). Furthermore, HE staining showed that the tissue lesion was alleviated.

Conclusion:Poly-COHb can be prepared at room temperature without oxygen isolation.Compared with poly-Hb, poly-COHb can improve the degree of polymerization, simplify the preparation method and achieve the expected goal of the experiment. Poly-COHb has the function of oxygen carrying and expanding capacity. It can be used for resuscitation of acute hemorrhagic shock, relieve the shock state, increase the survival rate of rats and protect colon tissue. One of the potential mechanisms may be related to the reduced level of oxidative stress in rat colon by regulating Nrf2 / HO-1 signal pathway.

Keywords:poly-Carboxyhemoglobin; poly-hemoglobin; oxidative stress; carbon monoxide；hemorrhagic shock