谷氨酰胺对脓毒症大鼠肺血管内皮细胞的作用

The role of glutamine in pulmonary vascular endothelial cells of sepsis

目的观察脓毒症大鼠肺血管内皮细胞（vascular endothelial cell，VEC）、细胞间黏附分子-1（intercellular adhesion molecule-1,ICAM-1）和E-选择素的变化特点并探讨谷氨酰胺对其的作用。方法将Sprague-Dawlev（SD）大鼠56只随机分成对照组、脓毒症组和治疗组。实验地点在中山大学北校区动物实验中心。脓毒症组注射脂多糖4mg／kg制备大鼠脓毒症模型，治疗组注射脂多糖4mg／kg和谷氨酰胺0．3g／kg，对照组不注射脂多糖。造模成功后，在6，12，24b时间点分离大鼠肺标本，采用逆转录聚合酶链反应和免疫组织化学方法研究肺ICAM-1和E-选择素的表达；用Hoechest染色评价肺VEC凋亡；用电子显微镜观察肺VEC。采取SPSS 13．0软件包进行统计学处理，统计分析方法采用方差分析法。结果脓毒症组大鼠肺ICAM-1和E-选择素的表达与治疗组和对照组比较明显增加（P〈0．01），治疗组ICAM-1蛋白和E-选择素的表达明显高于对照组（P〈0．05）。脓毒症组和治疗组ICAM-1的表达24h达到高峰，E-选择素的表达6h达高峰。脓毒症组大鼠肺VEC凋亡明显多于治疗组（P〈0．05）和对照组（P〈0．01）；治疗组大鼠VEC凋亡明显多于对照组（P〈0．01）。电子显微镜观察也得到证实。结论脓毒症大鼠肺ICAM-1和E-选择素的表达明显增加，导致肺VEC的坏死和凋亡以及急性肺损伤的发生。谷氨酰胺对其有保护作用。

Objective To explore the effect of glutamine on changes in the expression of intercellular adhesion molecule-1 （ICAM-1） and E-selectin and the characteristics of pulmonary vascular endothelial ceils （VECs） in sepsis rats. Method Totally 56 Sprague-Dawley rats were randomly divided into three groups： a control group, a sepsis group and a treatment group. All experiments were performed at the animal research center at Sun Yat-sen University in Guangzhou. Sepsis was induced by injecting 4 mg/kg lipopolysaccharide （LPS）. The treatment group was injected with 4 mg/kg LPS and 0.3 g/kg glutamine. The control group was not injected with either LPS or glutamine. The rats were killed at 6, 12 or 24 h after treatment and pulmonary tissue samples were obtained. The expression of ICAM-1 and E-selectin in VECs was detected by reverse transcription-polymerase chain reaction and immunohistochemistry. Apoptosis of VECs lung tissue was analyzed by Hoechest-33258 staining. The ultramicrostructure of VECs was observed under an electron microscope. Data were analyzed using analysis of variance using SPSS 13.0. Results At 6, 12 and 24 h, the expression of ICAM-1 and E-selectin was significantly higher in the sepsis group （relative expression; ICAM- 1 ： 0. 0864 ± 0. 0101, 0. 141 ± 0. 0147 and 0. 1677 ± 0. 0127, respectively; E-selectin： 0. 1535 ± 0.0180, 0.0811 ± 0.0107 and 0.0505 ± 0.0031, respectively） compared with the control group （ICAM-1 ： 0.021 ± 0.0032, 0.0228 ± 0.0042 and 0.0204 ± 0.0059, respectively; E-selectin： 0.0423±0.0108, 0.0412±0.0066 and 0.0418±0.0092, respectively） （all： P 〈0.01）. Glutamine treatment significantly decreased （ P 〈 0.01） the expression of ICAM-1 （0.0646 ± 0.0136, 0. 1202 ± 0.0143 and 0. 1378 ±0.0085, respectively） and E-seleetin （0.1071 ± 0.0189, 0.0628± 0.0088 and 0.0463 ± 0.0049, respectively） at all time points compared with the sepsis group. However, the expression of ICAM-1 and E-selectin remained significantly higher than that in the control group （all： P 〈 0.05）. There were similar changes in the expression of pulmonary ICAM-1, E-selectin mRNA and the results of VEC apoptosis. Electron microscopy confirmed these find- ings. Conclusions The expression of ICAM-1 and E-selectin was significantly increased in sepsis rats, leading to necrosis and apoptosis of VECs, and the onset of acute lung injury. Glulamine had a protective effect in VECs against lipopolysaccharide-induced sepsis.