eNOS谷胱甘肽化在缺氧预适应保护人脐静脉内皮细胞缺氧复氧损伤中的作用及机制

Role of eNOS glutathionylaiton in hypoxic preconditioning protecting human umbilical vein endothelial cells from hypoxia and reoxygenation injury

目的:研究缺氧预适应(hypoxic preconditioning,HPC)保护人脐静脉内皮细胞(human umbilical veinous endothelial cells,HUVECs)缺氧/复氧损伤的可能机制。方法:将培养的HUVECs分为5组:对照(Control)组、缺氧复氧(hypoxia/reoxygenation,HR)组、HPC组、HR+自由基清除组(HR+EUK134组)、HPC组+自由基处理组(HPC+H_2O_2组)。先将Control组和HR组、HR+EUK134组放在常规孵育箱中,而HPC组和HPC+H_2O_2组放入三气低氧箱中进行3个10 min的缺氧/复氧小循环,即缺氧预适应,再将HR组、HR+EUK134组与HPC组、HPC+H_2O_2组一起放入三气低氧箱中进行1 h大缺氧。1 h后,向HPC+H_2O_2组加入H_2O_2(100 mmol/L)、HR+EUK134组加入EUK134(10 mmol/L),然后将4组同时放入常规孵育箱复氧2 h。复氧后检测细胞存活率,测定细胞内还原型谷胱甘肽(reductive glutathione,GSH)和氧化型谷胱甘肽(oxidative glutathione,GSSG)内皮型一氧化氮合成酶(endothelial nitric oxide synthetase,eNOS)谷胱甘肽化水平、超氧化物阴离子(superoxide anion,O2.)和一氧化氮(nitric oxide,NO)水平以及抗氧化酶过氧化氢酶(catalase,CAT)和超氧化物歧化酶(superoxide dismutase,SOD)的活性。结果:与对照组相比,HR组O2.增加(P=0.004),抗氧化酶活性降低(P=0.000),细胞内氧化型谷胱甘肽/还原型谷胱甘肽比例(GSSG/GSH)和eNOS谷胱甘肽化水平(P=0.000)均增加,而NO生成减少(P=0.003)。自由基清除剂EUK134和HPC(P=0.028)均可减少O2.生成,增加SOD(P=0.002)和CAT活性(P=0.003),降低GSSG/GSH和eNOS谷胱甘肽化水平(P=0.001),NO生成增加(P=0.042),高浓度H_2O_2可消除HPC的作用。结论:HPC可提高细胞存活率,恢复内皮细胞eNOS功能活性,其机制可能是通过减轻氧化应激,降低细胞内GSSG/GSH和eNOS谷胱甘肽化水平,促进NO生成而实现。

Objective：To explore the effect of hypoxic preconditioning （HPC） on human umbilical vein endothelial cells （HUVECs） undergoing hypoxia and reoxygenation. Methods：Cultured HUVECs were divided into 5 groups,control, hypoxia/reoxygenation（HR）, hypoxic preconditioning（HPC）, HR＋EUK134 and HPC＋H2O2. The hypoxia with tri-gas incubator and glucose-free and serum-free DMEM culture medium was stimulated and reoxygenation was generated using normal incubator and high glucose DMEM culture medium containing 10% fetal bovine serum. First, group control, HR and HR＋EUK134 were put into normal incubator, and groups HPC and HPC＋HzO2 into tri-gas incubator to exert hypoxic preconditioning with three 10 minutes hypoxia/reoxygenation cycles. Then together with group HR and HR＋EUK134,the two groups underwent one-hour hypoxia in trigas incubator to generate a longer hypoxia. One hour later added EUK134（10μmol/L） and H2O2（100μmol/L） were added into group HR＋EUK134 and HPC＋H2O2, and all the five groups were oxygenated in normal incubator for two hours. After reoxygenation,the cells were harvested to detect cell viability, oxidative and reductive glutathione （GSSG and GSH）, and endothelial nitric oxide synthetase （eNOS） glutathionylation,superoxide anion （O2.） and nitric oxide （NO） levels,also the activities of superoxide dismutase（SOD） and catalase（CAT）. Results：Compared with control,group HR showed an increase in O2 level （P=0.004） and a decrease in SOD and CAT activities（P=0.000）, and had a higher GSSG/GSH ratio and eNOS glutathionylation（P=0.000）,thus a lower production of NO（P=0.003）. The free radical scavenger EUK134 and hypoxic preconditioning （P=0.028） both could ameliorate O2. level and increase activities of SOD （P=0.002） and CAT（P=0.003）, resulting lower GSSG/GSH ratio and eNOS glutathionylation （P=0.001 ）, thus a higher production of NO （P=0.042）, but the effect of hypoxie preconditioning could be counteracted by high dose H202. Conclusion：Hypoxie preconditioning could promote cell viability and eNOS activity probably through reducing oxidative stress and eNOS glutathionylation, thus increasing the production of NO.