Effects of berbamine against myocardial ischemia/reperfusion injury: Activation of the 5' adenosine monophosphate-activated protein kinase/nuclear factor erythroid 2-related factor pathway and changes in the mitochondrial state

Abstract

This study was designed to investigate whether berbamine (BA)-induced cardioprotective effects were related to 5' adenosine monophosphate-activated protein kinase (AMPK)/nuclear factor erythroid 2-related factor (Nrf2) signaling and changes in the mitochondria in myocardial ischemia/reperfusion (I/R) injury. C57/BL6 mice were exposed to BA (10 mg/kg/d), with or without administration of the AMPK specific inhibitor compound C (5 mg/kg/d) or the Nrf2 specific inhibitor ML-385 (30 mg/kg/d), and then subjected to a myocardial I/R operation. As expected, BA significantly improved post-ischemic cardiac function, reduced infarct size and apoptotic cell death, decreased oxidative stress, and improved the mitochondrial state. Furthermore, BA markedly increased AMPK activation, Nrf2 nuclear translocation, and the levels of NAD(P)H quinone dehydrogenase and heme oxygenase-1. Nevertheless, these BA-induced changes were abrogated by compound C. In addition, ML-385 also canceled the cardioprotective effects of BA but had little effect on AMPK activation. Our results demonstrate that BA alleviates myocardial I/R injury and the mitochondrial state by inhibiting apoptosis and oxidative stress via the AMPK/Nrf2 signaling pathway.

Keywords: AMPK/Nrf2; berbamine; heart; ischemia/reperfusion injury; mitochondrion; oxidative stress.

FIGURE 1

Compound C abrogated the cardioprotective effects of berbamine in myocardial ischemia/reperfusion. (A) Evans blue and TTC staining of heart slices; (B) myocardial infarct size; (C) serum CK activity; (D) serum LDH activity; (E) representative images of motion‐mode echocardiography; (F) LVEF; (G) LVFS. Data are presented as the mean ± SEM, n = 6. **p < 0.01 versus the sham group; ^## p < 0.01 versus the I/R group; ^^ p < 0.01 versus the BA+I/R group. BA, berbamine; CC, compound C; CK, creatine kinase; I/R, ischemia/reperfusion; LDH, lactate dehydrogenase; LVEF, left ventricular ejection fraction; LVFS, left ventricular fraction shortening; SEM, standard error of the mean. TTC, triphenyltetrazolium chloride

FIGURE 2

Compound C abolished the antiapoptotic effects of BA in myocardial I/R injury. (A) Representative immunoblots of cleaved caspase‐3, cytosolic cytochrome c, and GAPDH (internal control); (B) representative images of TUNEL staining of left ventricular tissue; (C,D) semiquantitative analysis of cleaved caspase‐3 and cytosolic cytochrome c; (E) caspase‐3 activity; (F) quantitative analysis of apoptotic index (magnification, ×400). Data are presented as the mean ± SEM, n = 6. **p < 0.01 versus the sham group, ^## p < 0.01 versus the I/R group, ^^ p < 0.01 versus the BA+I/R group. BA, berbamine; CC, compound C; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; I/R, ischemia/reperfusion; SEM, standard error of the mean; TUNEL, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling

FIGURE 3

Compound C obstructed the antioxidative effects of BA and the mitochondrial state in myocardial I/R injury. (A) Representative immunoblots of gp91^phox and GAPDH (internal control); (B) representative images of DHE staining of left ventricular tissue (magnification, ×400); (C) semi‐quantitative analysis of gp91^phox; (D) DHE intensity; MDA contents; (F) SOD activity in myocardial tissues; and (G) mitochondrial morphology and state. Data are presented as the mean ± standard error of the mean, n = 6. **p < 0.01 versus the sham group, ^## p < 0.01 versus the I/R group, ^^ p < 0.01 versus the BA+I/R group. BA, berbamine; CC, compound C; DHE, dihydroethidium; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; I/R, ischemia/reperfusion; MDA, malondialdehyde

FIGURE 4

Compound C prevented BA‐induced AMPK activation and Nrf2 nuclear translocation in myocardial I/R injury. (A) Representative immunoblots of p‐AMPK, AMPK, p‐ACC, ACC, and GAPDH (internal control); (B,C) semiquantitative analysis of p‐AMPK and p‐ACC; (D) semiquantitative analysis of nuc‐Nrf2; (E) representative immunoblots of nuc‐Nrf2, histone H3 (internal control), cyto‐Nrf2, NQO‐1, HO‐1, and GAPDH (internal control); (F–H) semiquantitative analysis of cyto‐Nrf2, NQO‐1, and HO‐1. Data are presented as the mean ± standard error of the mean, n = 6. **p < 0.01 versus the sham group, ^## p < 0.01 versus the I/R group, ^^ p < 0.01 versus the BA+I/R group. BA, berbamine; CC, compound C; cyto‐Nrf2, cytoplasmic Nrf2; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; I/R, ischemia/reperfusion; nuc‐Nrf2, intranuclear Nrf2

FIGURE 5

ML‐385 abolished the antiapoptotic effects of BA in myocardial I/R injury. (A) Representative immunoblots of cleaved caspase‐3, cytosolic cytochrome c, and GAPDH (internal control); (B,C) semiquantitative analysis of cleaved caspase‐3 and cytosolic cytochrome c; (D) representative images of TUNEL staining of left ventricular tissue; (E) quantitative analysis of apoptotic index (magnification, ×400). Data are presented as the mean ± standard error of the mean, n = 6. **p < 0.01 versus the sham group, ^## p < 0.01 versus the I/R group, ^^ p < 0.01 versus the BA+I/R group. BA, berbamine; I/R, ischemia/reperfusion; ML, ML‐385; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; TUNEL, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling

FIGURE 6

ML‐385 blunted the antioxidative effects of BA in myocardial I/R injury. (A) Representative immunoblots of gp91^phox and GAPDH (internal control); (B) semiquantitative analysis of gp91^phox; (C) representative images of DHE staining of left ventricular tissue (magnification, ×400Χ); (D) DHE intensity; (E) MDA contents; and (F) SOD activity in myocardial tissues. Data are presented as the mean ± standard error of the mean, n = 6. **p < 0.01 versus the sham group. ^## P < 0.01 versus the I/R group; ^^ P < 0.01 versus the BA+I/R group. BA, berbamine; I/R, ischemia/reperfusion; DHE, dihydroethidium; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; MDA, malondialdehyde; ML, ML‐385

FIGURE 7

ML‐385 prevented the cardioprotective effects of BA in myocardial I/R. (A) Evans blue and TTC staining of heart slices; (B) myocardial infarct size; (C) serum CK activity; (D) serum LDH activity; (E) representative images of motion‐mode echocardiography; (F) LVEF; (G) LVFS; and (H) mitochondrial morphology and state. Data are presented as the mean ± standard error of the mean, n = 6. **p < 0.01 versus the sham group, ^## p < 0.01 versus the I/R group, ^^ p < 0.01 versus the BA+I/R group. BA, berbamine; CK, creatine kinase; LVEF, left ventricular ejection fraction; LVFS, left ventricular fraction shortening; I/R, ischemia/reperfusion; ML, ML‐385; TTC, triphenyltetrazolium chloride

FIGURE 8

ML‐385 retarded BA‐induced Nrf2 nuclear translocation but had little effect on AMPK activation in myocardial I/R injury. (A) Representative immunoblots of p‐AMPK, AMPK, p‐ACC, ACC, and GAPDH (internal control); (B,C) semi‐quantitative analysis of p‐AMPK and p‐ACC; (D) semi‐quantitative analysis of nuc‐Nrf2; (E) representative immunoblots of nuc‐Nrf2, histone H3 (internal control), cyto‐Nrf2, NQO‐1, HO‐1, and GAPDH (internal control); (F–H) semi‐quantitative analysis of cyto‐Nrf2, NQO‐1, and HO‐1. Data are presented as the mean ± standard error of the mean, n = 6. **p < 0.01 versus the sham group; ^## p < 0.01 versus the I/R group; ^^ p < 0.01 versus the BA+I/R group. BA, berbamine; cyto‐Nrf2, cytoplasmic Nrf2; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; I/R, ischemia/reperfusion; ML, ML‐385; nuc‐Nrf2, intranuclear Nrf2