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

Abstract:

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+H2O2 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 tri-gas incubator to generate a longer hypoxia. One hour later added EUK134（10 μmol/L） and H2O2（100 μmol/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 ac－tivities（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 hy－poxic 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 hypoxic preconditioning could be counteracted by high dose H2O2. Conclusion：Hypoxic preconditioning could promote cell viability and eNOS activity probably through reducing oxidative stress and eNOS glutathionylation，thus increasing the production of NO.