BNC Protects H9c2 Cardiomyoblasts from H 2 O 2 -Induced Oxidative Injury through ERK1/2 Signaling Pathway

Abstract

Buchang naoxintong capsule (BNC) is a traditional Chinese medicine approved for the treatment of cerebrovascular and cardiovascular diseases. However, little is known about the specific protective function or mechanism by which BNC protects against myocardial injury. This research was designed to investigate the cardioprotective effects of BNC in vitro model of hydrogen peroxide (H2O2)-induced H9c2 rat cardiomyoblasts. BNC intestinal absorption liquid was used in this study instead of drug-containing serum or extracting solution. Our study revealed that BNC preconditioning enhanced antioxidant function by increasing the activities of total-antioxygen capacity, total-superoxide dismutase, and catalase and by decreasing the production of reactive oxygen species and malondialdehyde. BNC preconditioning also activated extracellular signal-regulated kinases (ERK1/2) and inhibited apoptosis-related proteins such as poly ADP-ribose polymerase (PARP) and caspase-3. Additionally, preincubation with BNC reduced intracellular Ca(2+) concentration, improved mitochondrial membrane potential, and decreased the apoptosis rate of H9c2 cells in a dose-dependent manner. These data demonstrated that BNC protects H9c2 cardiomyoblasts from H2O2-induced oxidative injury by increasing antioxidant abilities, activating ERK1/2, and blocking Ca(2+)-dependent and mitochondria-mediated apoptosis. Based on our results, the potency of BNC for protecting H9c2 cells from oxidative damage is comparable to that of trimetazidine.

Figure 1

UPLC of Control (a), BNC extraction solution (b), and BNC intestinal absorption liquid (c). Chromatograms numbered from 1 to 11 represent paeoniflorin, protocatechualdehyde, calycosin, salvianolic acid B, senkyunolide A, ligustilide, tanshinone I, caffeic acid, ferulic acid, rosmarinic acid, and hydroxysafflor yellow A.

Figure 2

Determination of H₂O₂ working conditions. H9c2 cells were cultured with different concentrations of H₂O₂ for 1 h (a) or with 100 μM for different incubation times (b). Cell viability was detected by MTT assay. Values are expressed as mean ± SD from three independent experiments. **P < 0.01 versus control; *P < 0.05 versus control.

Figure 3

BNC rescued H₂O₂-induced loss of cell viability. H9c2 cells were pretreated with different concentrations of BNC (0, 7.81, 15.63, 31.25, 62.50, 125, or 250 μg/mL) for 24 h and then treated with 100 μM H₂O₂ for 1 h (a). H9c2 cells were preconditioned with different concentrations of BNC (7.81, 15.63, 31.25, or 62.50 μg/mL) for 24 h (b). Cell viability was measured by MTT assay. Values are expressed as mean ± SD from three independent experiments. **P < 0.01 versus control; ^## P < 0.01 versus model.

Figure 4

Changes in H9c2 cell morphology in response to H₂O₂ and/or BNC. (a) H9c2 cells without BNC or H₂O₂ (control); (b) H9c2 cells exposed to 100 μM H₂O₂ (model); ((c)–(h)) H9c2 cells pretreated with 0, 7.81, 15.63, 31.25, 62.50 μg/mL BNC or with 10 μM TMZ followed by treatment with 100 μM H₂O₂. Magnification: 200x.

Figure 5

Protective effects of BNC via enhancing antioxidant function by increasing the activities of T-AOC, T-SOD, and CAT and decreasing the production of MDA detected by the T-AOC, T-SOD, CAT, and MDA assay kits. Values are expressed as mean ± SD from three independent experiments. **P < 0.01 versus control; *P < 0.05 versus control; ^## P < 0.01 versus model; ^# P < 0.05 versus model.

Figure 6

Protective effects of BNC due to reduced H₂O₂-induced generation of intracellular ROS measured by the DCFDA assay. Values are expressed as mean ± SD from three independent experiments. **P < 0.01 versus control; *P < 0.05 versus control; ^## P < 0.01 versus model; ^# P < 0.05 versus model.

Figure 7

BNC protected H9c2 cells from H₂O₂-induced apoptosis, as detected by Annexin V-FITC and PI staining with FCM analysis. (a) H9c2 cells without BNC or H₂O₂ (control); (b) H9c2 cells exposed to 100 μM H₂O₂ (model); ((c)–(h)) H9c2 cells pretreated with 0, 7.81, 15.63, 31.25, 62.50 μg/mL BNC, or 10 μM TMZ followed by the treatment of 100 μM H₂O₂. Values are expressed as mean ± SD from three independent experiments. **P < 0.01 versus control; *P < 0.05 versus control; ^## P < 0.01 versus model; ^# P < 0.05 versus model.

Figure 8

BNC reduced H₂O₂-induced cytosolic Ca²⁺ concentrations in H9c2 cells, as observed by Fluo-3/AM staining with FCM analysis. (a) H9c2 cells without BNC or H₂O₂ (control); (b) H9c2 cells exposed to 100 μM H₂O₂ (model); ((c)–(h)) H9c2 cells pretreated with 0, 7.81, 15.63, 31.25, 62.50 μg/mL BNC, or 10 μM TMZ followed by the treatment with 100 μM H₂O₂. Values are expressed as mean ± SD from three independent experiments. **P < 0.01 versus control; *P < 0.05 versus control; ^## P < 0.01 versus model; ^# P < 0.05 versus model.

Figure 9

BNC attenuated H₂O₂-induced mitochondrial membrane potential loss in H9c2 cells, as assessed by JC-1 staining with FCM analysis. (a) H9c2 cells without BNC or H₂O₂ (control); (b) H9c2 cells exposed to 100 μM H₂O₂ (model); ((c)–(h)) H9c2 cells pretreated with 0, 7.81, 15.63, 31.25, 62.50 μg/mL BNC or 10 μM TMZ followed by treatment of 100 μM H₂O₂. Values are expressed as mean ± SD from three independent experiments. **P < 0.01 versus control; *P < 0.05 versus control; ^## P < 0.01 versus model; ^# P < 0.05 versus model.

Figure 10

Effects of BNC on expression of ERK1/2, p-ERK1/2, PARP, and caspase-3 in H9c2 cells treated with H₂O₂ (a) and effects of BNC on expression of ERK1/2 and p-ERK1/2 in the presence of ERK1/2 inhibitor, PD98059 (10 μM) (b). Protein expression was detected by western blot analysis. The ratio of p-ERK1/2/ERK1/2 significantly increased in response to BNC pretreatment (c). Values are expressed as mean ± SD from three independent experiments. **P < 0.01 versus control; *P < 0.05 versus control; ^## P < 0.01 versus model.

Figure 11

Schematic representation of BNC-mediated protection from H₂O₂-induced oxidative injury in H9c2 cells.