脓毒性休克早期生理盐水复苏剂量和时机对内皮多糖包被的影响

Dose and timing of normal saline resuscitation on endothelial glycocalyx in early septic shock

目的观察不同生理盐水（NS）复苏策略联合去甲肾上腺素（NE）对脓毒性休克早期兔肺内皮多糖包被的影响。方法按随机数字表法将30只雄性新西兰大白兔分为假手术（Sham）组、模型组、30 mL和60 mL及时复苏组（30 mL、60 mL及时组）及30 mL延迟复苏组（30 mL延迟组），每组6只。采用盲肠结扎穿孔术（CLP）复制脓毒性休克动物模型；Sham组只开腹探查后关腹，不进行盲肠结扎、穿孔。30 mL、60 mL及时组和30 mL延迟组于制模后即刻或1 h静脉输注NS 30 mL/kg或60 mL/kg，持续1 h，联合NE 0.02～0.05 μg·kg-1·min-1静脉泵入，维持平均动脉压（MAP）〉75 mmHg（1 mmHg＝0.133 kPa），以5 mL/h NS维持至实验结束；Sham组和模型组仅给予5 mL/h NS。观察3个液体复苏组复苏前及复苏后即刻动脉血气变化。各组分别于制模0、3、6 h取颈内静脉血，采用酶联免疫吸附试验（ELISA）检测血浆多糖包被标志物多配体蛋白聚糖-1（syndecan-1）水平；于制模6 h处死动物取肺组织，采用蛋白质免疫印迹试验（Western Blot）检测肺组织细胞间黏附分子-1（ICAM-1）、基质金属蛋白酶2（MMP-2）及syndecan-1的蛋白表达；采用免疫组化法观察肺组织syndecan-1阳性表达。结果①血气分析：与复苏前比较，3个液体复苏组复苏后血乳酸（Lac）水平均明显降低，以30 mL及时组更加显著；中心静脉血氧饱和度（ScvO2）均明显升高，以30 mL延迟组更加显著；30 mL及时或延迟复苏对氧合指数（PaO2/FiO2）均有改善作用，但60 mL及时组PaO2/FiO2反而下降。②血浆标志物：与Sham组比较，模型组血浆syndecan-1水平明显升高，并呈一定时间依赖性。采取30 mL及时或延迟复苏策略后，血浆syndecan-1水平在3 h即较模型组明显降低（ng/L：138.0±2.4、139.7±15.7比161.5±4.1，均P〈0.05），但30 mL延迟组6 h syndecan-1水平较模型组明显升高（ng/L：213.1±19.4比206.4±15.5，P〈0.05）；60 mL及时组3 h和6 h血浆syndecan-1水平反而较模型组升高（ng/L：233.0±28.9比161.5±4.1，252.3±27.2比206.4±15.5，均P〈0.05）。③肺组织蛋白表达：与Sham组比较，模型组肺组织ICAM-1、MMP-2蛋白表达明显升高，而syndecan-1蛋白表达明显降低。采取30 mL及时或延迟复苏策略后，肺组织ICAM-1、MMP-2蛋白表达明显降低，syndecan-1蛋白表达明显升高，以30 mL及时组变化最为显著，与模型组比较差异有统计学意义（ICAM-1蛋白：0.56±0.09比1.04±0.05，MMP-2蛋白：0.83±0.15比1.06±0.06，syndecan-1蛋白：2.09±0.08比0.99±0.03，均P〈0.05）；而60 mL及时组各蛋白表达变化趋势与其他两个复苏策略组相反。④免疫组化：Sham组syndecan-1阳性表达明显；模型组syndecan-1阳性表达强度明显减弱；30 mL及时或延迟复苏后syndecan-1阳性表达增强，但60 mL及时组syndecan-1阳性表达较模型组进一步减弱。结论脓毒性休克时NS复苏剂量和时机均可影响肺血管内皮多糖包被的功能，以30 mL及时复苏对多糖包被的保护作用相对更好；NS复苏不及时或复苏过量都可使多糖包被降解更加明显，导致内皮通透性增加，微循环受损，加重肺损伤。

ObjectiveTo observe the effect of different doses and timing of normal saline （NS） resuscitation combined with norepinephrine （NE） on endothelial glycocalyx in rabbits with early septic shock.MethodsThirty New Zealand male rabbits were randomly divided into sham group, model group, 30 mL and 60 mL timely resuscitation groups （30 mL and 60 mL timely group）, and 30 mL delayed resuscitation group （30 mL delayed group） with 6 rabbits in each group. The rabbit model of septic shock was reproduced by cecal ligation and puncture （CLP）. The rabbits in sham group were only received abdominal cavity open without cecal and ligation. The rabbits in 30 mL and 60 mL timely groups and 30 mL delayed group were intravenous infused with 30 mL/kg or 60 mL/kg NS immediately or 1 hour after model reproduction for 1 hour, and the mean arterial pressure （MAP） was maintained over 75 mmHg （1 mmHg = 0.133 kPa） compared with intravenous pumping of 0.02-0.05 μg·kg-1·min-1 NE followed by 5 mL/h NS infusion till the end of the experiment. The rabbits in sham and model groups were only given 5 mL/h NS. The changes in arterial blood gas before and immediately after resuscitation were observed in three fluid resuscitation groups. The internal jugular vein blood was collected at 0, 3, 6 hours after model reproduction. The levels of syndecan-1 （polysaccharide envelope marker） in plasma were determined by enzyme linked immunosorbent assay （ELISA）. The rabbits were sacrificed at 6 hours after model reproduction, and the lung tissue was harvested. Western Blot was used to determine the protein expressions of intercellular adhesion molecule-1 （ICAM-1）, matrix metalloproteinase 2 （MMP-2） and syndecan-1. The positive expression of syndecan-1 in lung tissue was observed by immunohistochemical method.Results① Blood gas analysis： compared with the results before resuscitation, the levels of lactic acid （Lac） after resuscitation in three fluid resuscitation groups were significantly decreased, especially in 30 mL timely group; the central venous blood oxygen saturation （ScvO2） was significantly increased, especially in 30 mL delayed group. Oxygenation index （PaO2/FiO2） was improved in 30 mL timely and 30 mL delayed resuscitation groups, which was decreased in 60 mL delayed group. ② Plasma marker： compared with sham group, plasma syndecan-1 level in model group was significantly increased with a time-dependent manner. Plasma syndecan-1 levels at 3 hours in 30 mL timely and 30 mL delayed groups were significantly decreased as compared with those of model group （ng/L： 138.0±2.4, 139.7±15.7 vs. 161.5±4.1, both P 〈 0.05）, but it was significantly increased at 6 hours in 30 mL delayed group （ng/L： 213.1±19.4 vs. 206.4±15.5, P 〈 0.05）. The plasma syndecan-1 levels at 3 hours and 6 hours in 60 mL timely group were significantly higher than those in model group （ng/L： 233.0±28.9 vs. 161.5±4.1, 252.3±27.2 vs. 206.4±15.5, both P 〈 0.05）. ③ Protein expression in lung tissue： compared with sham group, the protein expressions of ICAM-1 and MMP-2 in lung tissue of model group were significantly increased, and syndecan-1 protein expression was significantly decreased. After 30 mL timely or 30 mL delayed resuscitation, the protein expressions of ICAM-1 and MMP-2 in lung tissue were significantly decreased, and syndecan-1 protein expression was significantly increased, especially in 30 mL timely group, which showed statistical differences as compared with those of model group （ICAM-1 protein： 0.56±0.09 vs. 1.04±0.05, MMP-2 protein： 0.83±0.15 vs. 1.06±0.06, syndecan-1 protein： 2.09±0.08 vs. 0.99±0.03, all P 〈 0.05）. The change tendency of protein expressions in 60 mL timely group was opposite to the other two resuscitation groups. ④ Immunohistochemistry： the positive expression of syndecan-1 in lung tissues was significant in the sham group, and it was lowered in model group. The positive expression of syndecan-1 was increased after 30 mL timely or 30 mL delayed resuscitation, but further weakened in 60 mL timely group.ConclusionsThe dose and timing of resuscitation with NS in septic shock can affect pulmonary vascular endothelial glycocalyx function. The timely resuscitation with 30 mL NS in combination with NE plays a protective effect on endothelial cell and glycocalyx. However, NS resuscitation which was not timely or excessive infusion can make the glycocalyx degradation more obvious, resulting in increased endothelial permeability, microcirculation damaged, thus aggravate lung injury.