Establishment of a model of hydrogen peroxide-induced injury in pulmonary artery endothelium cells and relevant mechanisms of oxidative stress / 中华心血管病杂志

ABSTRACT

Objective@#To establish a hydrogen peroxide (H2O2) induced injury model of pulmonary artery endothelial cells (PAECs) and explore the molecular mechanisms of oxidative stress on the structure and function of PAECs in this model.@*Methods@#Human PAECs were treated with H2O2 at different concentrations (25, 50, 100, 200, 400, 800, 1 600, 3 200, 6 400 μmol/L) for 4 and 24 h, respectively. The PAECs survival curve was obtained according to the cell viability measured by CCK-8 assay. The cell apoptosis of PAECs was detected by flow cytometry. The reactive oxygen species (ROS) generation and mitochondrial activity were measured using small molecule fluorescent probes. Proteins were extracted and the phosphorylation levels of signal molecules in PAECs were detected by Western blot assays.@*Results@#(1) The effect of H2O2 at various concentrations on cell viability of PAECs cell viability of PAECs decreased in proportion to increasing concentration of H2O2 after incubation for 4 h. The half maximal inhibitory concentration (IC50) of PAECs exposed to H2O2 for 4 and 24 h were 397.00 and 488.77 μmol/L, respectively. (2) The effect of H2O2 on cell apoptosis of PAECs After H2O2 incubation for 4 h, proportions of PAECs at late-apoptosis ((22.58±3.69) %) and necrotic stage( (11.86±4.27)%) were significantly higher than those of control PAECs at late-apoptosis stage( (3.41±1.44)%, P<0.01) and at necrotic stage ((1.94±1.15) % , P<0.05). The survival rate of PAECs post H2O2 was dramatically lower than that of control PAECs ((7.98±3.21)% vs. (48.89±8.08)%, P<0.01). However, there is no statistical difference between both groups regarding to the early apoptosis. (3) The effect of H2O2 on mitochondrial activity and ROS production of PAECs the mitochondrial activity and ROS generation of PAECs treated by H2O2 were significantly increased compared to those in control PAECs (P<0.01). (4) The effect of H2O2 on signaling molecules in PAECs there was a significant increase in phosphorylation level of Akt in PAECs incubated with H2O2 for 30 minutes compared to that in control PAECs (P<0.01), while there was no significant difference in levels of Akt between H2O2 treated PAECs and control PAECs. Phosphorylation level of JNK as well as p38 were also significantly upregulated in H2O2 treated PAECs (P<0.01).@*Conclusion@#H2O2 at the concentration of 400 μmol/L could induce human PAECs injuries via the regulation of Akt and MAPK signaling pathways.