心理应激大鼠血浆NO水平及内皮细胞NO调控蛋白表达的变化

Psychological stress reduces the plasma NO level and NO-regulating protein expression in the vascular endothelial cells of rats

目的心理应激对血管内皮细胞(vascular endothelial cells,VEC)损伤的确切机制尚未清楚。文中拟建立空瓶刺激心理应激大鼠血管内皮损伤模型,探讨心理应激所致VEC损伤大鼠血浆中NO水平的改变及NO相关调控蛋白表达的变化。方法将20只雄性SD大鼠按随机数字表法分为对照组和模型组,各10只。对照组:群居饲养,早晚9点规律饮水10min;模型组:孤独饲养,前7 d固定早晚9点饮水10 min,第8天开始造模,早晚9点按随机表给予饮水或空瓶刺激10 min。造模时间为期90 d。2组均按体重腹腔注射等渗盐水(0.2 m L/100 g),2次/d。用旷场实验观察大鼠行为学变化;酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测血浆中皮质醇、肾上腺素、内皮素-1含量,硝酸还原酶法检测血浆NO含量,LC-ESI-MS分析VEC NO调控相关蛋白的表达。结果对照组垂直得分(7.00±0.70)分、水平得分(37.60±4.50)分、总得分(44.50±5.50)分,模型组分别为(11.28±1.79)分、(56.25±1.71)分、(67.25±2.98)分,2组差异均有统计学意义(P<0.01);对照组血浆皮质醇、肾上腺素[(65.28±2.21)ng/m L、(3.85±0.35)pg/m L]较模型组[(83.85±1.95)ng/m L、(15.14±3.97)pg/m L]明显降低(P<0.01);对照组NO/内皮素-1(1.04±0.19)较模型组(0.64±0.08)明显升高(P<0.01)。LC-ESI-MS分析得知,与对照组比较,模型组差异蛋白表达数为238,其中上调134,下调104;与对照组比较,模型组与NO调控相关的蛋白表达均下降。透射电镜显示,模型组VEC的线粒体嵴模糊,部分线粒体出现空泡;对照组VEC线粒体结构完整,嵴清晰、排列紧密。结论空瓶刺激心理应激引起NO水平降低及其相关调控蛋白的表达下降,是造成VEC功能和结构损伤的重要机制之一。

Objective The mechanism of psychological stressi-nduced damage to vascular endothelial cells （VEC） is not yet clear.This study was to investigate the impact of psychological stress on the NO level and NO-related regulatory protein expression in the plasma of rat models of psychological stress-induced VEC injury. Methods Twenty male SD rats were equally randomized into a con-trol group and a model group, the former raised together, with water regularly fed at 9：00 and 21：00 hours for 10 minutes a day, while the latter raised alone, with water regularly fed at the same hours for 10 minutes a day for the first 7 days and then with water or an empty bottle at the same hours for 10 minutes a day according to the random table.Ninety days later,all the rats were intraperitoneally injected with normal saline at 0.2 ml per 100 g body weight twice a day.Then the behaviors of the animals were observed by open field tests, the contents of cortisol （COR）,adrenaline （Adr）, and endothelin-1 （ET-1） in the plasma measured by ELISA, the NO level detected by the nitrate reductase method, and the expression of NO-related regulatory proteins in VECs determined by LC-ESI-MS. Results Com-pared with the control group, the model rats showed significant increases in the vertical score （7.00 ±0.70 vs 11.28 ±1.79）, horizontal score （37.60 ±4.500 vs 56.25 ±1.71）, and total score （44.50 ±5.50 vs 67.25 ±2.98） （P〈0.01） as well as in the levels of COR （[65.28 ±2.21] vs [83.85 ±1.95] ng/mL ） and Adr （[3.85 ±0.35] vs [15.14 ±3.97] pg/mL） （P〈0.01）, but a remarkable de-with vacuoles in the VECs in the model group and structural integrity, ridge clarity, and tight pack in the VEC mitochondria in the con-trol. Conclusion Psychological stress induced by empty bottle stimulation can reduce the NO level and the expression of NO-related regulatory proteins in the plasma, which may cause both functional and structural damages to VECs.