Inhibition of autophagy by berberine enhances the survival of H9C2 myocytes following hypoxia - PubMed (original) (raw)

Inhibition of autophagy by berberine enhances the survival of H9C2 myocytes following hypoxia

Zhuyin Jia et al. Mol Med Rep. 2017 Aug.

Abstract

Hypoxia may induce apoptosis and autophagy to promote cardiomyocyte injury. The present study investigated the effect of berberine, a natural extract of Rhizoma Coptidis, on hypoxia‑induced autophagy and apoptosis in the H9c2 rat myocardial cell line. Expression levels of apoptosis and autophagy markers were upregulated in H9c2 myocytes during hypoxia and cell viability was reduced. However, berberine significantly reduced hypoxia‑induced autophagy in H9c2 myocytes, as demonstrated by the ratio of microtubule‑associated proteins 1A/1B light chain 3 I/II and the expression levels of B‑cell lymphoma 2 (Bcl‑2)/adenovirus E1B 19 kDa protein‑interacting protein 3, and promoted cell viability. In addition, expression levels of the Bcl‑2 anti‑apoptotic protein were significantly downregulated, and expression levels of pro‑apoptotic proteins Bcl‑2‑associated X protein and cleaved caspase‑3 were upregulated during hypoxia injury in cardiac myocytes. This was reversed by treatment with berberine or the autophagy inhibitor 3‑methyladenine, whereas the autophagy agonist rapamycin had the opposite effects, suggesting that berberine reduces myocyte cell death via inhibition of autophagy and apoptosis during hypoxia. In addition, Compound C, a 5' adenosine monophosphate‑activated protein kinase (AMPK) inhibitor, reduced apoptosis and autophagy in hypoxic myocytes, suggesting that the activation of the AMPK signaling pathway may be involved in this process. These findings suggested that berberine protects cells from hypoxia‑induced apoptosis via inhibition of autophagy and suppression of AMPK activation. Therefore, berberine may be a potential therapeutic agent for the treatment of patients with cardiac myocyte injury and ischemia.

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Figures

Figure 1.

Figure 1.

Expression levels of autophagy-associated proteins following hypoxia in H9c2 myocytes. Following 0–12 h hypoxia, whole cell lysates were collected for western blot analysis. (A) Representative blots of LC3-I, LC3-II and β-actin. (B) Densitometry of LC3-II/LC3-I signals. The protein expression level of the control (0 h) group was arbitrarily set as 1 in each blot, and the target protein signals on the same blot were normalized to the control to generate relative densities. Data are expressed as the mean ± standard deviation and were obtained from three independent experiments. **P<0.01 vs. control. LC3; microtubule-associated proteins 1A/1B light chain 3.

Figure 2.

Figure 2.

Effect of berberine on cell viability and apoptosis in H9c2 cells exposed to hypoxia. Cells were treated with 0, 5, 10 or 25 µM berberine, prior to hypoxia. (A) Cell viability of H9c2 cells, as assessed by an MTT assay. (B) Induction of apoptosis in H9c2 cells was measured by Annexin-V/PI double-staining followed by flow cytometric analysis. The upper right and lower right areas, which represented the percentage of late apoptosis and early apoptosis, were analyzed. Data are expressed as the mean ± standard deviation and were obtained from three independent experiments. **P<0.01 vs. control group; #P<0.05 and ##P<0.01 vs. hypoxia group. Ber, berberine; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 3.

Figure 3.

Berberine inhibits the hypoxia-induced increase in the expression levels of autophagy- and apoptotic-associated proteins. (A) Protein expression levels of Bax, cleaved caspase-3, BNIP3, LC3 and β-actin were measured by western blot analysis. (B) Quantification of bands by densitometry. β-actin served as the loading control. Data are expressed as the mean ± standard deviation and were obtained from three independent experiments. **P<0.01 vs. control group; #P<0.01 vs. hypoxia group. Bax, B-cell lymphoma 2-associated X protein; BNIP3, B-cell lymphoma 2/adenovirus E1B 19 kDa protein-interacting protein 3; LC3, microtubule-associated proteins 1A/1B light chain 3; Ber, berberine.

Figure 4.

Figure 4.

Effect of 3-MA or rapamycin on cell viability and apoptosis in hypoxic H9c2 cells. Cells were subjected to glucose starvation with the exception of the control group, which was cultured in complete Dulbecco's modified Eagle's medium. Subsequently, cells were treated with 3-MA (an autophagy inhibitor) or rapamycin (an autophagy inducer) prior to hypoxia exposure. (A) Viability of H9c2 cells. (B) Induction of apoptosis in H9c2 cells was measured by Annexin-V/PI double-staining followed by flow cytometric analysis. The apoptotic proportion of H9c2 cells was calculated according to the flow cytometry results. The upper right and lower right areas, which represented the percentage of late apoptosis and early apoptosis, were analyzed. Data are expressed as the mean ± standard deviation and were obtained from three independent experiments. **P<0.01 vs. control group; #P<0.05 and ##P<0.01 vs. hypoxia group. 3-MA, 3-methyladenine; FITC, fluorescein isothiocyanate; PI, propidium iodide; Rapa, rapamycin.

Figure 5.

Figure 5.

Inhibition of autophagy reduces apoptosis in hypoxia-induced myocytes. (A) Protein expression levels of Bax, Bcl-2, cleaved caspase-3, BNIP3, LC3 and β-actin were measured by western blot analysis. Quantification of (B) Bcl-2, Bax and cleaved caspase-3, and (C) LC3-II/LC3-I and BNIP3 protein expression levels by densitometry. β-actin served as the loading control. Data are expressed as the mean ± standard deviation and were obtained from three independent experiments. **P<0.01 vs. control group; ##P<0.01 vs. hypoxia group. Bax, B-cell lymphoma 2-associated X protein; Bcl-2, B-cell lymphoma 2; BNIP3, B-cell lymphoma 2/adenovirus E1B 19 kDa protein-interacting protein 3; LC3, microtubule-associated proteins 1A/1B light chain 3; 3-MA, 3-methyladenine.

Figure 6.

Figure 6.

Activation of the AMPK signaling pathway may be involved in the regulation of apoptosis by berberine in hypoxic H9c2 cells. Cells were subjected to glucose starvation with the exception of the control group, which was cultured in complete Dulbecco's modified Eagle's medium. Cells were treated with berberine, Compound C (an AMPK inhibitor) or a combination of the two, prior to hypoxia exposure. Induction of apoptosis in H9c2 cells was measured by Annexin-V/PI double-staining followed by flow cytometric analysis. The apoptotic proportion of H9c2 cells was calculated according to the flow cytometry results. The upper right and lower right areas, which represented the percentage of late apoptosis and early apoptosis, were analyzed. Data are expressed as the mean ± standard deviation and were obtained from three independent experiments. **P<0.01 vs. control group; #P<0.05 vs. hypoxia group. AMPK, 5′ adenosine monophosphate-activated protein kinase; FITC, fluorescein isothiocyanate; PI, propidium iodide; Ber, berberine.

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