Attenuation of doxorubicin-induced cardiotoxicity by esculetin through modulation of Bmi-1 expression

Abstract

The protective effects and mechanisms of esculetin on doxorubicin (DOX)-induced injury of H9c2 cells were investigated. H9c2 cells were cultured and the logarithmic growth phase of the cells was divided into a control group, a DOX group and an esculetin + DOX group. Cell viability was detected by MTT assay. Annexin V-PI (AV-PI) double staining flow cytometry was carried out to detect cell apoptosis. Intracellular reactive oxygen species (ROS) were detected by flow cytometry. Transmission electron microscope (TEM) was used to evaluate cell ultrastructure. Cleaved caspase-3, cleaved PARP, Bcl-2, Bid and Bmi-1 proteins levels were investigated by western blot analysis. Bmi-1 siRNA was used to detect the role of Bmi-1 in the protective effects of esculetin against DOX-induced toxicity in H9c2 cells. The MTT and AV-PI double staining results showed that esculetin significantly increased H9c2 cell viability. Compared with the control group, the levels of cleaved caspase-3, cleaved PARP, Bid and ROS levels were significantly decreased, but the expression of Bcl-2 and Bmi-1 were significantly increased in the esculetin + DOX group. TEM showed that the cell structure of the mitochondria was protected by esculetin. The results of Bmi-1 siRNA showed that esculetin could protect DOX-induced cardiotoxicity by modulating Bmi-1 expression. Esculetin can protect DOX-induced cardiotoxicity and the effects may be attributable to modulation of Bmi-1 expression, provoking intracellular ROS accumulation, protecting the structure of mitochondria and reducing cell apoptosis.

Keywords: Bmi-1; H9c2 cells; doxorubicin; esculetin.

Figure 1.

Effects of esculetin against DOX-induced apoptosis of H9c2 cells. (A) MTT assay cell survival after treatment with different concentrations of DOX. (B) Flow cytometry of apoptosis among different treatment groups. (C) Western blot analysis of the expression of apoptosis proteins, cleaved caspase-3 and cleaved PARP, when compared with the DOX group alone (*P<0.05, **P<0.01). DOX, doxorubicin.

Figure 2.

Effects of esculetin on DOX-induced H9c2 mitochondrial function. (A) Mitochondrial morphology of the three groups was observed by transmission electron microscopy. (B) Western blot analysis of mitochondrial pathway associated proteins, Bcl-2 and Bid, compared with DOX alone (*P<0.01). DOX, doxorubicin.

Figure 3.

Flow cytometry detection of total content of intercellular ROS species, compared with the DOX group (**P<0.01). DOX, doxorubicin; ROS, reactive oxygen species.

Figure 4.

Effects of esculetin against DOX-induced Bmi-1 expression in H9c2 cells. (A) Effects of esculetin on Bmi-1 expression in DOX-induced H9c2 cells, compared with the DOX (**P<0.01). (B) Effects of Bmi-1 siRNA on Bmi-1 expression in each group. (C) Effects of Bmi-1 siRNA on cell apoptosis in each group. (D) Effects of Bmi-1 siRNA on ROS levels in each group, compared with the blank siRNA group (**P<0.01). DOX, doxorubicin; ROS, reactive oxygen species.