Butein Ameliorates Oxidative Stress in H9c2 Cardiomyoblasts through Activation of the NRF2 Signaling Pathway

Abstract

Oxidative stress, defined as an imbalance between reactive oxygen species (ROS) production and the antioxidant defense system, contributes to the pathogenesis of many heart diseases. Therefore, oxidative stress has been highlighted as a therapeutic target for heart disease treatment. Butein, a tetrahydroxychalcone, has potential biological activities, especially antioxidant properties. However, the effect of butein on oxidative-stressed heart cells has been poorly studied. Thus, we sought to identify the antioxidant effects of butein in H9c2 cardiomyoblasts. To elucidate these antioxidant effects, various concentrations of butein were used to pretreat H9c2 cells prior to H₂O₂ treatment. Thereafter, measures of oxidative damages, such as ROS production, antioxidant expression levels, and apoptosis, were evaluated. Butein effectively increased cell viability and rescued the cells from oxidative damage through the inhibition of ROS production, apoptosis, and increased antioxidant expression. Furthermore, butein dramatically inhibited mitochondrial dysfunction and endoplasmic reticulum (ER) stress, which are the main ROS inducers. Nrf2 protein translocated from the cytosol to the nucleus and consequently activated its target genes as oxidative stress suppressors. These findings demonstrate that butein has potential antioxidant effects in H9c2 cardiomyoblasts, suggesting that it could be used as a therapeutic substance for the treatment of cardiac diseases.

Keywords: ER stress; Nrf2; butein; oxidative stress; reactive oxygen species (ROS).

Figure 1

Cytotoxic effects of butein in H9c2 cardiomyoblasts. An MTT assay was performed to measure the cell viability. The cells were treated with various concentrations of butein (0.5, 1, 3, 5, and 10 µM) for 24 (a), 48 (b), and 72 h (c). The results are shown as the mean ± SEM in triplicate. Cont; control, Bu; butein.

Figure 2

Butein increases the cell viability of H₂O₂-treated H9c2 cardiomyoblasts. The cell viability was determined by MTT assay after treating with butein and/or H₂O₂. The results are shown as the mean ± SEM in triplicate. ### p < 0.001 vs. control group. ** p < 0.01 and *** p < 0.001 vs. H₂O₂-alone group. Cont; control, Bu; butein.

Figure 3

Butein attenuates apoptotic cell death in H₂O₂-treated H9c2 cardiomyoblasts. The cells were treated with butein and/or H₂O₂ (a) Nuclear staining was performed by Hoechst staining. The arrow indicates apoptotic cells with nuclear condensation using cells that were treated with butein and/or H₂O₂. The cells in at least 10 fields (30 cells per field) were counted in each group. (b) The percentage of apoptotic cells versus total cells is shown as the apoptotic index. (c) Western blot analysis for the expression levels of apoptosis-related proteins including Bcl-2, Bax, pro- and cleaved caspase 3, and β-actin (loading control). The band densities of the ratio of Bcl-2/Bax (d) and pro- (e) and cleaved (f) caspase 3 proteins were calculated with HD imaging software. The results are shown as the mean ± SEM in triplicate. # p < 0.05 and ## p < 0.01 vs. control group. ** p < 0.01 and *** p < 0.001 vs. H₂O₂-only group. Cont; control, Bu; butein. Scale bar, 100 μm.

Figure 4

Butein attenuates oxidative stress responses in H₂O₂-treated H9c2 cardiomyoblasts. Intracellular ROS production was assessed by a DCFH-DA staining (a) and the measurement of green fluorescence intensity (b) using cells treated with butein and/or H₂O₂. The arrow indicates the cells in which ROS accumulated. (c) Western blot analysis for the expression levels of antioxidants including SOD1, SOD2, catalase, and β-actin (loading control). The band densities of SOD1 (d), SOD2 (e), and catalase (f) proteins were calculated with HD imaging software. The results are shown as the mean ± SEM in triplicate. ## p < 0.01 and ### p < 0.001 vs. control group. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. H₂O₂-only group. Cont; control, Bu; butein. Scale bar, 100 μm.

Figure 5

Butein attenuates mitochondrial ROS production and mitochondrial dysfunction in H₂O₂-treated H9c2 cardiomyoblasts. Mitochondrial ROS production was measured by MitoSox staining assay (a) and red fluorescence intensity (b) using cells treated with butein and/or H₂O₂. (c) Western blot analysis for the expression levels of mitochondrial complex II and β-actin (loading control) in butein-pretreated/H₂O₂-treated cells. (d) The band densities of the ratio of mitochondrial complex II protein were calculated with HD imaging software. The results are shown as the mean ± SEM in triplicate. ## p < 0.01 and ### p < 0.001 vs. control group. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. H₂O₂-only group. Cont; control, Bu; butein. Complex II; mitochondrial complex II. Scale bar, 100 μm.

Figure 6

Butein suppresses ER stress in oxidative-stress-induced H9c2 cardiomyoblasts. (a) Western blot analysis for the expression levels of ER stress-related proteins including PERK, eIF2α, ATF4, CHOP, GADD45α, and β-actin (loading control) using cells treated with butein and/or H₂O₂. The band densities of the ratio of phospho-PERK/total PERK (b), ratio of phopho-eIF2α/total eIF2α (c), ATF4 (d), CHOP (e), and GADD45α (f) proteins were calculated with HD imaging software. The results are shown as the mean ± SEM in triplicate. # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. control group. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. H₂O₂-only group. Cont; control, Bu; butein.

Figure 7

Butein activates the Nrf2-related signaling pathway in oxidative-stress-induced H9c2 cardiomyoblasts. (a) Western blot analysis for Nrf2 and lamin B1 (loading control) proteins using the nuclear fraction of cells treated with butein and/or H₂O₂. (b) The band density of Nrf2 protein was calculated with HD imaging software. The results are shown as the mean ± SEM in triplicate. (c) Immunofluorescence images of Nrf2 protein (red fluorescence). The nucleus (blue fluorescence) was stained with DAPI. (d) The mRNA expression of Nrf2 target genes including NQO1, HMOX1, and GCLC was performed by qRT-PCR analysis in triplicate. ## p < 0.01 and ### p < 0.001 vs. control group. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. H₂O₂-only group. Cont; control, Bu; butein. Scale bar, 100 μm.