Berberine protects human and rat cardiomyocytes from hypoxia/reoxygenation-triggered apoptosis

Abstract

Berberine (BBR) confers potential cardioprotective effects. However, the relevant mechanisms underlying its regulation of cardiomyocyte survival following hypoxia/reoxygenation (H/R) treatment remain unknown. The present study investigated whether BBR could protect H/R by suppressing apoptosis and explored how TGF-β/Smad4 signaling pathway influenced H/R in vitro. Two cardiomyocyte cell lines-AC16 and H9c2- were treated with H/R and BBR. The survival and apoptosis of these two cell lines were assessed using the MTT and BrdU assays and western blotting (WB) and flow cytometry. Mitochondrial reactive oxygen species (ROS) and caspase (Cas)-3, Cas-8, and Cas-9 activation were evaluated using enzyme-linked immunosorbent assay as well as WB. Compared to the control group, H/R resulted in notable cell apoptosis, whereas BBR treatment evidently counteracted the process. BBR also markedly suppressed H/R-triggered excessive mitochondrial ROS generation and inhibited Smad4 expression. Overexpressing Smad4 in BBR-treated H/R-exposed cardiomyocytes reversed the effect of BBR treatment on apoptosis. Therefore, BBR protects H/R-treated cardiomyocytes from apoptosis by inhibiting the TGF-β/Smad4 signaling pathway.

Keywords: BBR; TGF-β/smad4 pathway; apoptosis; cardiomyocytes; hypoxia/reoxygenation.

Figure 1

Effect of BBR treatment on cardiomyocyte viability following exposure to H/R. Human and rat cardiomyocytes were pretreated with various concentrations of BBR (0, 10, 33, and 100 μM) and then subjected to H/R treatment. A, B. MTT assay was performed to assess cell survival following different treatments. C, D. BrdU assay was performed to examine cell proliferation following different treatments. E, F. Colony formation assay was performed to assess cell growth following different treatments. G. Non-proliferating cardiomyocytes from 4 day-old Wistar rats were isolated, cultured, pretreated with 100 μM BBR, and subjected to H/R treatment. MTT assay was performed to assess cell survival following the treatments. Bars indicate mean (from three parallel experiments) ± SD. *P < 0.05, **P < 0.01 vs. the control group; ^#P < 0.05, ^##P < 0.01 vs. the H/R group.

Figure 2

Effect of BBR treatment on ROS production. Human and rat cardiomyocytes were pretreated with various concentrations of BBR (0, 10, 33, and 100 μM) and then subjected to H/R treatment. A, B. ROS levels in AC16 and H9c2 cells were measured via DCFH-DA for 20 min following BBR incubation and H/R exposure. C, D. Mitochondrial ROS exhibited MitoSOX RED fluorescence, as determined using a microplate reader. Bars indicate mean ± SD. *P < 0.05, **P < 0.01 vs. the control group; ^#P < 0.05, ^##P < 0.01 vs. the H/R group.

Figure 3

Effects of BBR treatment on H/R-triggered cardiomyocyte apoptosis. Human and rat cardiomyocytes were pretreated with BBR (100 μM) and then subjected to H/R treatment. (A, C) Flow cytometry was performed to determine the extent of apoptosis in human and rat cardiomyocytes following different treatments. (B, D) Histograms show the level of cardiomyocyte apoptosis following different treatments. (E, F) WB was performed to assess the expression levels of Bcl-2, Bax, and Cas-3 and its cleaved form. (G, H) Bcl-2 and Bax bands were quantified to measure the Bcl-2/Bax ratio. (I, J) WB was performed to assess the expression level of Gal3. (K) Quantification of WB bands of (G-J). Bars indicate mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the H/R group.

Figure 4

Effects of BBR treatment on the TGF-β/Smad4 signaling pathway. Human and rat cardiomyocytes were pretreated with BBR (100 μM) and then subjected to H/R treatment. A-F. RT-qPCR and WB were performed to determine mRNA and protein expression, respectively, of TGF-β and Smad4 in AC16 and H9c2 cells. G, H. IFA was performed to observe the cellular localization of Smad4 in AC16 and H9c2 cells. I, J. Cellular fractionation was performed to show the Smad4 distribution in each cellular fraction. N, nuclear; C, cytoplasmic. Bars indicate mean ± SD. *P < 0.05 vs. the control group; ^#P < 0.05 vs. the H/R group.

Figure 5

Smad4 overexpression in H/R-triggered BBR-treated cardiomyocytes. Human and rat cardiomyocytes were pretreated with BBR (100 μM) and then subjected to H/R treatment. Human and rat cardiomyocytes were pretreated with BBR and then transfected with pcDNA3-Smad4/NC following H/R exposure. A-D. RT-qPCR and WB were performed to determine the mRNA and protein expression levels, respectively, of TGF-β and Smad4 in AC16 and H9c2 cells. E, F. IFA was performed to observe the cellular localization of Smad4 in AC16 and H9c2 cells. Bars indicate mean ± SD. *P < 0.05, **P < 0.01 vs. the control group; ^#P < 0.05, ^##P < 0.01 vs. the H/R group.

Figure 6

Effects of Smad4 overexpression on cardiomyocyte apoptosis and viability. Human and rat cardiomyocytes were pretreated with BBR (100 μM) and then transfected with pcDNA3-Smad4/NC following H/R exposure. A-D. Flow cytometry was performed to assess the extent of apoptosis in human and rat cardiomyocytes following different treatments. E, F. MTT assay was performed to assess cell survival following different treatments. Bars indicate mean ± SD. *P < 0.05 vs. the control group; ^#P < 0.05 vs. the H/R group.