NRG ≥95%, DOX, SB203580, N-acetyl-L-cysteine (NAC), Hoechst 33258, rhodamine123 (Rh123) and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were all bought from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Cell Counting kit-8 (CCK-8) was provided by Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Caspase-3, t-p38 and p-p38 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM)-F12 were supplied by Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). H9c2 cells were obtained from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China).

Embryonic rat cardiac cell line H9c2 was provided by the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China) and cultured at 37°C in DMEM-F12 that was supplemented with 10% FBS in an atmosphere comprising 5% CO₂.

Following being cultured in 96-well plates (1×10⁶), the cells received different treatments, with the viability detected by CCK-8 assay. CCK-8 solution (10 µl, diluted by 1/10) was added into each well, following which the cells were further incubated for 2 h at 37°C. The absorbance at 450 nm was read using Multiskan MK3 microplate reader (Thermo Fisher Scientific, Inc.). According to the following formula, the percentage of cell viability was calculated based on the average optical density (OD) of five wells from each group: Cell viability percentage = OD_{treatment group} / OD_{control group} × 100%. The experiments were conducted in triplicate.

The apoptosis of H9c2 cells was evaluated by Hoechst 33258 staining. Following different treatments, the cells were fixed for 10 min with 4% paraformaldehyde in phosphate-buffered saline (PBS), then washed by PBS three times, stained for 5 min by 5 mg/l Hoechst 33258, rinsed again by PBS and observed under BX50-FLA fluorescence microscope (Olympus Corporation, Tokyo, Japan). The viable cells emitted uniform blue fluorescence and presented normal nuclear sizes, but the apoptotic ones had fractured, condensed or distorted nuclei.

Intracellular ROS levels were detected by the oxidative conversion of DCFH-DA (cell-permeable) to dichlorofluorescein (DCF, fluorescent). Following being cultured in 24-well plates (7×10⁶), H9c2 cells were treated differently and washed with PBS twice, into which serum-free medium containing 10 µM DCFH-DA solution was thereafter added to incubate the cells for 60 min at 37°C. They were then washed with PBS three times, DCF fluorescence in which was detected over the whole visual field by BX50-FLA fluorescence microscope (Olympus Corporation) connected with an imaging system. In addition, the mean fluorescence intensity (MFI) of four randomly selected fields was determined with ImageJ software (version, 1.41o; National Institutes of Health, Bethesda, MD, USA). ROS levels were represented by MFI of DCF.

MMP was detected by Rh123, a cell-permeable, fluorescent cationic dye preferentially entering mitochondria on the basis of a highly negative MMP. MMP depolarization leads to Rh123 loss from mitochondria, thus decreasing intracellular fluorescence. H9c2 cells were herein cultured in 24-well plates (7×10⁶) and treated differently. Following addition of Rh123 (100 mg/l) into the culture medium, the cells were further incubated at 37°C for 45 min and observed under BX50-FLA fluorescence microscope (Olympus Corporation) connected with an imaging system. The MFI of Rh123 from four randomly selected fields, which was analyzed using ImageJ software, represented the level of MMP. The experiments were repeated three times.

Differently treated H9c2 cells were collected and lysed with ice-cold lysis solution (10 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 5 mM EDTA (pH 8.0), 1% Triton X100), and the resulting homogenate was centrifuged for 10 min at 9,500 × g and 4°C. Subsequently, total protein in the supernatant was quantified by using bicinchoninic acid assay protein assay kit (Sigma-Aldrich), resolved by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane that was then blocked by 5% fat-free milk in TBS-0.05% Tween 20 at room temperature for 1 h, incubated overnight with t-p38MAPK (Sigma-Aldrich; cat no. M8177; dilution, 1:1,000), p-p38MAPK (Sigma-Aldrich; cat no. CS0430; dilution, 1:1,000) and caspase-3 antibodies (Sigma-Aldrich; cat no. C9598; dilution, 1:1,000) or horseradish peroxidase (HRP)-conjugated GAPDH antibody (Sigma-Aldrich; cat no. SAB2108668; dilution, 1:5,000) at 4°C under gentle agitation, and further incubated with a HRP-conjugated secondary antibody (Sigma-Aldrich; cat no. A9542; dilution, 1:3,000) at room temperature for 1.5 h. Following being washed with TBS-0.05% Tween three times, the membrane was developed by enhanced chemiluminescence (Sigma-Aldrich) and thereafter exposed to X-ray films that were scanned and determined by ImageJ software to quantify protein expression.

All experimental data were expressed as mean ± standard error of the mean. Inter-group differences were analyzed with one-way analysis of variance and Fisher's Least Significant Difference by using SPSS software (version, 13.0; Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Fig. 1A demonstrated that 24 h of exposure of H9c2 cells to 5 µmol/l DOX induces obvious cytotoxicity (P<0.01) that reduces their viability, compared with the control group. The NRG concentration required to protect against DOX-induced cytotoxicity was calculated by performing a dose-response study in the presence of 0.1, 1, 10 and 20 µmol/l NRG (Fig. 1A). The cytotoxic effects of DOX were significantly attenuated after 2 h of pretreatment by using 0.1–20 µmol/l NRG. Although 1 µmol/l NRG exerted the maximum effect, it alone hardly affected cell viability (P>0.05).

To find out the optimum treatment time of NRG for DOX-induced cytotoxicity, H9c2 cells were pretreated by 1 µmol/l NRG for 30, 60, 90, 120, 150 and 180 min respectively prior to DOX exposure (Fig. 1B). Within 30–180 min after NRG pretreatment, the cell viability continuously increased and peaked at 150 min. Therefore, 150 min of pretreatment with 1 µmol/l NRG was selected for subsequent experiments.

The expression of p-p38MAPK was detected by western blot analysis. As presented in Fig. 2, the expression level of p-p38MAPK, which is significantly elevated by 60 min of exposure of H9c2 cells to DOX at 5 µM (P<0.01) compared with the control group, can be reduced by pretreatment with 1 µM NRG for 150 min (P<0.01 compared with the control group). However, 1 µM NRG alone barely affected the basal expression of p-p38MAPK (P>0.05).

To elucidate whether NRG exerted inhibitory effects on DOX-induced increase in p-p38MAPK expression through antioxidative action, H9c2 cells were pretreated for 60 min with NAC (a ROS scavenger) at 1,000 µM prior to 5 µM DOX exposure. As presented in Fig. 3, pretreating the cells with NAC, similar to that of using NRG, significantly depresses DOX-induced upregulation of p-p38MAPK expression (P<0.01). Nevertheless, NAC alone hardly affected basal p-p38MAPK expression (P>0.05). Accordingly, NRG inhibited the DOX-induced increase in p-p38MAPK expression by resisting oxidation.

The viability of H9c2 cells was analyzed by detecting the reduction in percentage with CCK-8 assay. Such viability, which was decreased by ~50% following 24 h of exposure to 5 µM DOX, was significantly increased by preconditioning with 1 µM NRG for 150 min (P<0.01 compared with the control group; Fig. 4). Moreover, preconditioning of the cells for 150 min by using SB203580 (a selective inhibitor of p38MAPK) at 3 µM had similar cytoprotective effects to those of NRG, as suggested by the increase in cell viability. Either NRG or SB203580 alone failed to significantly affect the viability of H9c2 cells (P>0.05). Therefore, the activation of p38MAPK was involved in the cytotoxicity against H9c2 cells induced by DOX.

The morphological changes during H9c2 cell apoptosis were observed by Hoechst 203580 staining (Fig. 5). To evaluate the effects of NRG and SB203580 on the apoptosis induced by DOX, H9c2 cells were pretreated for 150 min with 1 µM NRG or for 60 min with 3 µM SB203580 prior to DOX exposure. As presented in Fig. 5B, 24 h of preconditioning with 1 µM DOX induces typical apoptosis characteristics including chromatin condensation and nuclear shrinkage. The apoptotic cells with these characteristics decreased by preconditioning with NRG (Fig. 5C). In contrast, NRG alone almost had no visible effect on cell apoptosis (Fig. 5E). Furthermore, western blotting (Fig. 6A) indicated that 12 h of treatment by using 5 µM DOX significantly upregulated the expression of cleaved caspase-3, an effector protease degrading most cellular targets that result in apoptotic cell death, with respect to the control group. However, the upregulation was markedly suppressed by preconditioning with 1 µM NRG for 150 min. Individual NRG at 1 µM did not affect the expression of cleaved caspase-3 (P>0.05). These results indicated that H9c2 cells were protected by NRG against apoptosis induced by DOX.

In order to examine whether p38MAPK activation was implicated in the apoptosis of H9c2 cells induced by DOX, they were pretreated for 60 min by 3 µM SB203580 prior to 24 or 12 h of exposure to DOX at 5 µM (to test the expression of cleaved caspase-3). As indicated in Figs. 5D and 6B, SB203580 pretreatment decreases both the number of apoptotic cells and the expression of cleaved caspse-3 induced by DOX, suggesting that the activation of p38MAPK participated in DOX-induced cytotoxicity. Individual SB203580 hardly affected cell apoptosis or caspase-3 activation (Figs. 5F and 6B).

All above findings suggested that the p38MAPK pathway was involved in the apoptosis of H9c2 cells induced by DOX. Notably, NRG was able to protect H9c2 cells against this kind of apoptosis by inhibiting the p38MAPK signaling pathway.

Following this, the authors assessed the antioxidative effects of NRG and whether p38MAPK activation contributed to the ROS overproduction induced by DOX through detecting the levels of intracellular ROS based on DCFH-DA staining. Following being treated for 24 h by using 5 µM DOX, the levels of intracellular ROS in H9c2 cells significantly increased (P<0.01; Fig. 7). Interestingly, 150 min of pretreatment by 1 µM NRG or 60 min of pretreatment by 3 µM SB203580 significantly reduced the levels of intracellular ROS, revealing the antioxidative effects of NRG and the involvement of the p38MAPK pathway in the oxidative stress induced by DOX. Similar to the control group, the cells treated with 1 µM NRG or 3 µM SB203580 individually emitted weak DCF fluorescence.

It is well-documented that (8,9,25) disruption of MMP was involved in the cardiotoxicity induced by DOX, so the authors examined the effects of NRG and a p38MAPK inhibitor on DOX-disrupted MMP. As presented in Fig. 8B, following being treated for 24 h by 5 µM DOX, the mitochondrial number of H9c2 cells is evidently decreased, which reduced the uptake of Rh123, thereby verifying the loss of MMP. However, such loss was relieved by 150 h of preconditioning by 1 µM NRG (Fig. 8C), suggesting that NRG managed to protect the cells against the mitochondrial damage induced by DOX. Similarly, pretreatment with SB203580 significantly depressed the loss of MMP induced by DOX (P<0.05; Fig. 8D), suggesting that NRG provided mitochondrial protection by inhibiting p38MAPK expression. Alone, neither NRG nor SB203580 had any effect on MMP (both P>0.05).