Abstract:

Objective To study the effect of different stimulated duration of 200 μmol/L H2O2 on the senescence of human umbilical vein endothelial cells（HUVEC），soas to provide reference for oxidative stress⁃ induced senescence cell model. Methods The HUVEC were divided into control group and stimulated group which was subdivided into 8 groups according to the different stimulated duration of 200 μmol/L H2O2 including 0.5，1，3，6，12，24，48，96 h. MTT assays were performed to assess cell proliferation in each group；senescence ⁃associated β⁃galactosidase（SA ⁃β）staining was performed to measure the senescent cells；ELISA was used to measure the content of endothelin⁃1（ET⁃1）and nitric oxide（NO）；colorimetry assays were performed to assess the content of malondialdehyde（MDA），super oxidase dismutase（SOD）and glutathionperoxidase（GSH⁃px）and western blot was performed to measure the expressions of Bax，Bcl⁃2 and p21. Results Compared with that in the control group，cell proliferation in H2O2 stimulated groups with a short⁃duration（＜3 h）was significantly increased accom⁃ panied with very low positive rate of SA⁃β staining and increased secretion of ET⁃1 and NO. With the prolonging of the stimulated duration of H2O2（3 ~ 24 h），the cell proliferation decreased but the positive rate of SA⁃β staining increased；the secretion of ET⁃1 increased but NO decreased，accompanied with increased expression of p21 but unchanged Bax and Bcl⁃2. In the 48 ~ 96 h，both the cell proliferation and the secretion of ET⁃1 and NO kept decreasing while there was no change of SA⁃ β staining. However，the expression of Bax increased but the Bcl⁃2 decreased while there was no change of the p21. What′s more，compared with that in the control，MDA kept increasing but SOD and GSH⁃px decreasing in the stimulated groups. Conclusion The HUVEC stimulated with 200 μmol/L H2O2 for 24 h may lead to significant senescence while over 24 h stimulation may lead to increased apoptosis. 

Objective To study the effect of different stimulated duration of 200 μmol/L H2O2 on the senescence of human umbilical vein endothelial cells（HUVEC），soas to provide reference for oxidative stress⁃ induced senescence cell model. Methods The HUVEC were divided into control group and stimulated group which was subdivided into 8 groups according to the different stimulated duration of 200 μmol/L H2O2 including 0.5，1，3，6，12，24，48，96 h. MTT assays were performed to assess cell proliferation in each group；senescence ⁃associated β⁃galactosidase（SA ⁃β）staining was performed to measure the senescent cells；ELISA was used to measure the content of endothelin⁃1（ET⁃1）and nitric oxide（NO）；colorimetry assays were performed to assess the content of malondialdehyde（MDA），super oxidase dismutase（SOD）and glutathionperoxidase（GSH⁃px）and western blot was performed to measure the expressions of Bax，Bcl⁃2 and p21. Results Compared with that in the control group，cell proliferation in H2O2 stimulated groups with a short⁃duration（＜3 h）was significantly increased accom⁃ panied with very low positive rate of SA⁃β staining and increased secretion of ET⁃1 and NO. With the prolonging of the stimulated duration of H2O2（3 ~ 24 h），the cell proliferation decreased but the positive rate of SA⁃β staining increased；the secretion of ET⁃1 increased but NO decreased，accompanied with increased expression of p21 but unchanged Bax and Bcl⁃2. In the 48 ~ 96 h，both the cell proliferation and the secretion of ET⁃1 and NO kept decreasing while there was no change of SA⁃ β staining. However，the expression of Bax increased but the Bcl⁃2 decreased while there was no change of the p21. What′s more，compared with that in the control，MDA kept increasing but SOD and GSH⁃px decreasing in the stimulated groups. Conclusion The HUVEC stimulated with 200 μmol/L H2O2 for 24 h may lead to significant senescence while over 24 h stimulation may lead to increased apoptosis. 

Key words:

different duration, hydrogen peroxide, HUVEC, senescence