外源性一氧化碳对肢体缺血再灌注所致中性粒细胞肺内扣押作用的实验研究

Effect of exogenous carbon monoxide on sequestration of polymorphonuclear neutrophils in the lung following limb ischemia-reperfusion: an experimental study

目的观察外源性一氧化碳(CO)对肢体缺血再灌注(IR)所致中性粒细胞(PMN)在肺内扣押的作用及机制。方法应用双大腿根部止血带复制大鼠双后肢缺血及再灌注后肺损伤模型。将动物置于含或不含0·025%CO的空气中。进行同样操作只是不造成肢体缺血的动物作为对照。在含5%CO2和0·025%CO+5%CO2的空气中应用肢体IR患者血清孵育人肺微血管内皮细胞(PMVEC)和PMN,对照细胞采用健康志愿者血清孵育。观察大鼠肺组织学、肺组织中中性粒细胞(PMN)数目及肺组织细胞间黏附分子-1(ICAM-1)表达的变化。检测PMVEC的ICAM-1、PMN的整合素-CD11b的表达及PMVEC-PMN黏附率的变化。结果肢体IR后动物肺组织出现出血、水肿及PMN扣押等病理征象。与对照组相比,肺组织中PMN数目显著增加(39·3±5·7vs19·6±2·8,P<0·01)、ICAM-1表达显著增强;应用CO后每高倍镜PMN数目显著减少(28·3±3·8,P<0·01),ICAM-1表达显著降低,肺损伤减轻。与对照组相比,肢体IR患者血清使人PMVEC的ICAM-1表达、PMN整合素的表达以及PMVEC-PMN黏附率均显著增高(分别为28·3±2·6vs12·9±1·9、18·9±2·6vs9·8±1·2和30·7±2·9vs13·4±1·1,P<0·01),应用CO后均显著降低(分别为19·9±2·1、14·1±1·9和19·8±1·5,P<0·01)。结论外源性CO可抑制肢体IR所致PMN在肺内扣押,其机制与其下调黏附分子表达、抑制PMN与PMVEC粘附有关。

Objective To investigate the effect of exogenous carbon monoxide （CO） in inhibiting the sequestration of polymorphonuclear neutrophils （PMNs） in the lung following limb ischemia-reperfusion （IR） and the mechanism thereof. Methods PMNs of peripheral blood were isolated from the venous blood of a healthy volunteer. Serum was collected from a patient undergoing bilateral knee joint replacement as IR serum. Human pulmonary microvascular endothelial cells （PMVECs） were cultured and divided into 4 groups： control group （cultured under the condition of room air containing 5% CO2 for 5 h and cultured in normal human serum instead of medium during the last 4 hours of experiment）, IR group （ cultured under the condition of air containing 5% CO2 for 5h and cultured in the serum of IR patient during the last 4 hours）, IR ＋ CO group （ cultured under the condition of air containing 0. 025% CO and 5% CO2 for 5 hours and cultured in IR serum during the last 4 hours）, and control ＋ CO group （cultured under the condition of air containing and O. 025% CO and 5% CO2 for 5 hours and cultured in normal human serum during the last 4 hours）. Immunofluorescence flow cytometry was used to detect the expression of intercellular adhesion molecule （ICAM） -1 and integrin CD11b in the PMVECs. Human PMVECs were put into the wells of a 96-well plate and added with PMNs to calculate the PMVEC-PMN adhesion rate. Tourniquettes were bound at the bilateral hind thighs of 32 healthy male SD rats for 4 hours so as to establish a rate IR model. The rats were randomly divided into 4 equal groups： control group （ undergoing the same operation without causing limb ischemia and exposed to room air）, IR group （ undergoing bilateral hind limb ischemia for 4 h and reperfusion for 4 h and exposed to room air）, IR ＋ CO group （exposed to the containing O. 025% CO one hour before reperfusion till 4 hours after reperfusion）, and control ＋ CO group （ exposed to air containing 0. 025% CO at the corresponding time point as that of the IR ＋ CO group）. Then the rats were killed and their middle pulmonary lobes were taken out for microscopy and calculation of the number of PMNs in alveolar septum. Western blotting was used to examine the ICAM-1 protein expression in the lung. Results The ICAM-1 expression and integrin CDllb expression of the IR group PMVECs were significantly stronger than those of the IR ＋ CO group PMVECs （both P 〈 0. 05） and there were no significant differences in the ICAM-1 expression and CDllb expression between the control ＋ CO and control groups （both P 〉 0. 05）. The PMN-PMVEC adhesion rate of the IR group PMVECs was 30 ± 2. 9%, significantly higher than those of the IR ＋ CO group and control group PMVECs （ 19. 8 ± 1.5% and 13.4 ± 1.1% respectively, both P 〈 0. 05 ） and there was no significant difference in the PMN-PMVEC adhesion rate between the CO ＋ control group and control group （P 〉 0. 05 ）. The lung tissues of the IR group rats showed edema and hemorrhage. The number of PMNs in the alveolar septum was 60. 6 ± 1.7/10 high power fields, significantly higher than those of the IR ± CO group and control group （36.4 ± 1.6 and 22. 5 ± 1.6 respectively, both P 〈0. 05） and there was no significant difference between the latter 2 groups （P 〉 0. 05）. The ICAM-1 protein expression in the lung of the IR group was the strongest, followed by the IR ＋ CO group, control ＋ CO group, and control group. Conclusion Exogenous CO inhibits the limb/IR-induced PMN sequestration in the lung, probably by the mechanism of down-regulation of the expression of adhesion molecules and suppression of the PMN／PMVEC adhesion following IR.