Abstract:

Objective To discuss the effect of chrysophanol on hydrogen peroxide （H₂O₂）-induced oxidative damage of the EA.hy926 cells， and to clarify its therapeutic role in bronchopulmonary dysplasia （BPD） and related mechanism. Methods The EA.hy926 cells were induced with 25， 50， 100， 200， 400， 800， and 1 600 μmol·L^-1 H₂O₂， and 8， 16， 32， 64， 128， and 256 μmol·L^-1 chrysophanol. CCK-8 method was used to detect the viabilities of the EA.hy926 cells treated with different concentrations of H₂O₂ and chrysophanol. The cells were divided into control group， model group （200 μmol·L^-1 H₂O₂）， low dose of chrysophanol group （8 μmol·L^-1 chrysophanol and 200 μmol·L^-1 H₂O₂）， and high dose of emodin group （256 μmol·L^-1 chrysophanol and 200 μmol·L^-1 H₂O₂）. Western blotting method was used to detect the expression levels of apoptosis-inducing factor （AIF） protein in the cytoplasm and nucleus in various groups； immunofluorescence staining was used to detect the AIF nuclear translocation in the cells in various groups； kits were used to detect the activities of superoxide dismutase （SOD） and the levels of malondialdehyde （MDA）， cysteinyl aspartate specific proteinase（Caspase）-8， and Caspase-9 in the cells in various groups. Results Under different concentrations of H₂O₂， the viabilities of EA.hy926 cells showed an inverted S-shaped curve， with good cell viability， and the half-maximal inhibitory concentration （IC₅₀） was 261.52 μmol·L^-1. The cell model was induced by 200 μmol·L^-1 H₂O₂ for 24 h. As the increaseing of concentration of chrysophanol， there was no significant change of the viability in the EA.hy926 cells （P>0.05）， and interventions were performed using 8 and 256 μmol·L^-1 chrysophanol. The Western blotting results showed that compared with control group， the expression level of AIF protein in the nucleus in model group was significantly increased （P<0.05）， and the expression level of AIF protein in the cytoplasm was significantly decreased （P<0.05）. Compared with model group， the expression levels of AIF protein in the nucleus in both low and high doses of chrysophanol groups were significantly decreased （P<0.05）， and the expression level of AIF protein in the cytoplasm was significantly increased（P<0.05）. The immunofluorescence staining results showed that AIF was less localized in the nucleus in the cells in control group. Compared with control group， the positive value of AIF nuclear translocation in model group was significantly increased （P<0.05）； compared with model group， the positive values of AIF nuclear translocation in both low and high doses of chrysophanol groups were significantly decreased （P<0.05）. Compared with control group， the activity of SOD in the cells in model group was significantly decreased（P<0.05）， and the level of MDA was significantly increased（P<0.01）. Compared with model group， the activities of SOD in the cells in low and high doses of chrysophanol groups were significantly increased（P<0.05）， and the level of MDA was significantly decreased（P<0.05 or P<0.01）. There were no significant differences in the levels of Caspase-8 and Caspase-9 in the cells among various groups（P>0.05）. Conclusion Chrysophanol improves the H₂O₂-induced apoptosis of the EA.hy926 cells by inhibiting the oxidative stress and AIF nuclear translocation， which may be beneficial for the treatment of BPD.

Key words: Chrysophanol, Bronchopulmonary dysplasia, Apoptosis-inducing factor, Nuclear translocation, Apoptosis, Oxidative stress