高盐激活氧化应激-p38丝裂原活化蛋白激酶-肌动蛋白骨架重组诱导血管内皮细胞损伤  

High sodium induced endothelial cells impairment by activating oxidative stress-p38-MAPK-F-actin reorganization

作　　者：张玉婕[1] 朱男[1] 张效林[1] 闫承慧[1] 赵昕[1] 韩雅玲[1] 

机构地区：[1]沈阳军区总医院全军心血管病研究所,辽宁沈阳110840

出　　处：《中华高血压杂志》2014年第8期742-748,共7页Chinese Journal of Hypertension

基　　金：国家973课题资助项目(2012CB517800);国家自然科学基金资助项目(81130072;81070097);辽宁省科技攻关课题资助项目(2009225009-9)

摘　　要：目的探讨高盐对血管内皮细胞损伤的生物学作用及其机制。方法用含10%胎牛血清、0.45nmol/L醛固酮的杜尔伯科改良伊格尔培养基（DMEM）培养人主动脉内皮细胞3d,应用不同浓度NaCl（135、145、155mmol/L）及NaCl（135mmol/L）＋一氧化氮合酶抑制剂——左旋硝基精氨酸甲酯（l-NAME,1μmol/L）刺激内皮细胞3h。Rhodamin-Phalloidin检测内皮细胞纤维型肌动蛋白骨架（F-actin）分布;硝酸还原酶法/酶联免疫吸附试验（ELISA）分析法检测内皮细胞上清中一氧化氮及过氧化亚硝基阴离子（ONOO-）含量的变化;通过Western blot检测内皮细胞中内皮型一氧化氮合酶（eNOS）、还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的亚基gp91、磷酸化p38（P-p38）、磷酸化HSP27（P-HSP27）的蛋白表达量。应用磷酸化p38丝裂原活化蛋白激酶（P-p38 MAPK）特异性抑制剂SB203580（25μmol/L,1h）或氧化应激清除剂tempol（1mmol/L,1h）预处理内皮细胞后,检测盐负荷诱导内皮细胞骨架、细胞上清及蛋白表达的改变情况。结果当细胞外钠离子浓度为145-155mmol/L时,较135mmol/L时内皮细胞骨架F-actin增多,细胞上清中一氧化氮含量降低、ONOO-含量增加,且内皮细胞中磷酸化eNOS（P-eNOS）表达量降低,gp91、P-p38、P-HSP27表达量增加。预先给予氧化应激清除剂tempol后,可以缓解由盐负荷引起的内皮细胞损伤。预先给予SB203580后,可以部分缓解由盐负荷引起的内皮细胞损伤。结论高盐通过激活氧化应激及p38通路诱导F-actin重组,进而引起血管内皮细胞eNOS表达降低及一氧化氮释放减少。Objective To investigate the biological function and its mechanism of high sodium on vascular endothelial cells impairment. Methods The human endothelial cells were incubated for 3 days in a culture medium containing 10 % fetal bovine serum and aldosterone at a physiological concentration （0.45 nmol/L）, then salt concentration （135, 145, 155 mmol/L） and salt concentration （ 135 mmol/L） ＋ Nω-nitro-L-arginine-methyl-ester （/-NAME, 1 μmol/L） were used to stimulate endothelial cells for 3 hours. The Rhodamine-Phalloidin was used to observe living endothelial cells distribution of F-actin. The content change of cell supernatant NO and ONOO- were measured by Nitrate reductase method/enzyme linked immunosorhent assay （ELISA）. Protein expression of P-eNOS, NADPH, gp91, P-p38 and P-HSP27 were measured by Western blot. After using SB203580 （25 μmol/L, 1 h1 or tempol （1 mmol/L, 1 h） to preprocess the endothelial cells, the change of high sodium on vascular endothelial cells skeleton, supernatant and the expression of protein were detected. Results When the extracellular sodium concentration from 145 to 155 mmol/L, F-actin was higher than endothelial cells skeleton in 135 mmol/L, nitric oxide level in cell-supernatant was lower, ONOO- level increased and expression of P-eNOS in endothelial cells decreased, while gp91, P-p38 and P-HSP27 increased. Being pre-exposed to tempol and SB203580, the damage of endothelial cells caused by high sodium could be alleviated. Conclusion The results suggest that changes in sodium concentration may reduce eNOS expression and release of NO by activating oxidative stress, p38 and F-actin reorganization in endothelial cells in vitro.

关 键 词：血管内皮细胞 盐 氧化应激 内皮型一氧化氮合酶 纤维型肌动蛋白 

分 类 号：R544.1[医药卫生—心血管疾病]