Introduction

Morphine belongs to a large family of drugs known as narcotic analgesics. Despite the advancement in pain medicine, morphine with other opioids still remains the most powerful and widely used analgesic available [1,2,3]. Interestingly, besides its potent antinociceptive effects, morphine may apparently confer protection against anoxia/reoxygenation injury in some cells and tissues. In 1996, Schulz et al. [4] reported that morphine is able to significantly reduce tissue damage that occurs during myocardial ischemia. Since that time, a number of studies have been conducted in an attempt to delineate the molecular mechanisms underlying opioid-induced cardioprotection [5,6,7]. There are some indications that glycogen synthase kinase-3β (GSK-3β) inhibition mediated via phosphatidylinositol 3-kinase (PI3K), target of rapamycin (TOR) [8], JAK/STAT [9], or the NO/cGMP/PKG pathway [10] can play a central role in morphine-induced protection. Inactivation of GSK-3β prevents opening of the mitochondrial permeability transition pore (mPTP), thereby improving mitochondrial respiratory function and reducing the production of reactive oxygen species (ROS) [11, 12]. The p38 MAPK (mitogen-activated protein kinase) signaling cascade is another important pathway potentially involved in opioid-induced cardioprotection [13, 14]. However, there is only one study suggesting that activation of p38 MAPK may be essential to cardioprotection afforded by morphine [15].

The enhanced ROS generation, which takes place during myocardial ischemia and reperfusion, is one of the main risk factors implicated in tissue injury [16]. According to a recent study of Borges et al. [17], morphine can serve as a potent antioxidant capable of strengthening the cellular antioxidant capacity [18]. Nevertheless, as summarized previously, the impact of morphine on cellular redox balance may vary according to concrete circumstances, and it depends on multiple factors, such as species, age, sex, tissue type, dose and length of exposure, and simultaneous administration of other drugs [19]. Previous studies performed on H9c2 cells, a rat cardiomyoblast cell line, demonstrated that treatment of these cells with morphine (1 μM to 10 mM) does not lead to excessive ROS formation [20] and can diminish arsenic trioxide-induced ROS production [21]. Moreover, morphine was found to increase viability of H9c2 cells after simulated ischemia and reperfusion and to prevent oxidative stress-caused mitochondrial damage through increased ROS production [22, 23]. According to these studies, ROS play an important role in activation of the PI3K/Akt signaling pathway and may thus participate in morphine-mediated cytoprotection.

In the present study, we set out to investigate the presumed protective effect of morphine on H9c2 cell viability during oxidative stress induced by tert-butyl hydroperoxide (t-BHP). It has been shown previously that exposure of H9c2 cells to t-BHP may induce intracellular oxidative stress, which is accompanied by distinct morphological alterations, such as translocation of phosphatidylserine to the outer leaflet of the plasma membrane and membrane blebbing, one of the defined features of apoptosis [24]. In addition to that, high concentrations of t-BHP or prolonged exposure to this compound were found to lead to loss of cell membrane integrity and necrotic cell death. Besides examining the effects of different doses of morphine, in this study we also aimed to explore the role of PI3K/GSK-3β and p38 MAPK pathways in the molecular mechanism underlying the cytoprotective actions of morphine. For this purpose, cells were preincubated with selective inhibitors of these two signaling pathways prior to the addition of morphine. Finally, we examined the impact of morphine on the functional state of GSK-3β and p38 MAPK, as well as on the expression of the antioxidant enzymes catalase and superoxide dismutase. Our results indicated that morphine can quite effectively prevent the adverse consequences of t-BHP-induced oxidative stress in H9c2 cells.

Materials and Methods

Compounds and Reagents

Fetal bovine serum was purchased from Gibco (Grand Island, NY, USA), acrylamide and bis-acrylamide were from SERVA (Heidelberg, Germany), complete protease inhibitor and PhosSTOP phosphatase inhibitor cocktails were from Roche Life Science (Indianapolis, IN, USA), 2′,7′-dichlorodihydrofluorescein diacetate was from Molecular Probes (Eugene, OR, USA), Protran BA85 nitrocelulose membrane was from GE Healthcare Life Sciences (Little Chalfort, Buckinghamshire, UK), and SuperSignal West Dura chemiluminescent substrate was from Pierce Biotechnology (Rockford, IL, USA). Antibodies against Bcl-2, Bax, p38α MAPK, phospho-p38 MAPK (Thr180/Tyr182), GSK-3β, catalase, and superoxide dismutase 1 (SOD1) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-GSK-3β (pSer9) antibody was obtained from Cell Signaling Technology (Beverly, MA, USA). Annexin V Dyomics 647 was purchased from Apronex Biotechnologies (Vestec, Czech Republic). All other chemicals were from Sigma-Aldrich (St. Louis, MI, USA), and they were of the highest purity available.

Cell Culture and Drug Treatment

H9c2 cardiomyoblasts (ATCC, CRL-1446) from the American Type Culture Collection (Rockville, MD, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic–antimycotic solution at 37 °C in a humidified atmosphere of 5% CO2. Culture medium was replaced every 2–3 days, and cells were subcultured at 70% confluence. All experiments were performed on cells at 70–80% confluence. At least 1 h before experiment, the culture medium was replaced with starvation medium (DMEM supplemented with 1% FBS). Cells were incubated in phosphate-buffered saline (PBS) (CON) or in PBS containing morphine (MOR) for 15 min and subsequently exposed to oxidative stress induced by tert-butyl hydroperoxide (t-BHP) for 15 h. For cell viability and cytotoxicity assays, t-BHP was used over a broad range of concentrations (100–500 μM) selected on the basis of preliminary investigations. In some experiments, cells were pretreated with 10 μM SB203580 (SB), a specific inhibitor of the p38 MAPK pathway, or with 1 μM wortmannin (W), PI3K inhibitor, or with a combination of both these inhibitors (SB + W) for 1 h prior to the addition of morphine.

Cell Viability Assay

Cell viability was assessed by a rapid MTT colorimetric assay described by Mosmann [25]. The assay was performed on cells grown in 96-well tissue culture plates. After treatment with t-BHP, the culture medium was replaced by 100 μl of phenol red-free DMEM supplemented with 1% FBS and cells incubated in the presence of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) at a concentration of 0.2 mg/ml for 4 h. Thereafter, 75 μl of the medium was carefully removed from each well and the remaining MTT-formazan crystals were dissolved by adding 50 μl of DMSO. The color that developed was read at 540 nm against a blank sample (without cells) using BioTek synergy HT microplate reader.

Cell Cytotoxicity Assay

The loss of plasma membrane integrity accompanying cell death was determined by measuring the release of lactate dehydrogenase (LDH) in the culture medium. LDH activity was monitored by the reduction of a tetrazolium salt (pale yellow) to formazan (red). LDH activity was measured using a cytotoxicity detection kit (Roche Applied Science, Indianapolis, IN, USA), and the assay was conducted according to the protocol supplied by the manufacturer.

Detection of Apoptosis by Flow Cytometry

The percentage of living and dead cells was assessed by Annexin V/Hoechst 33258 staining followed by flow cytometric analysis. For this purpose, cells were seeded into six-well plates. After treatment with drugs culture media was removed, attached cells were released with trypsin and washed twice with PBS by centrifugation (200×g for 5 min at 4 °C). Approximately 1.5 × 105 cells were stained with Dyomics 647-labeled Annexin V according to the manufacturer’s protocol (Apronex Biotechnologies). Then the cells were washed twice in Annexin binding buffer, stained with Hoeschst 33258 (1:6000), and 104 events of every sample were analyzed on a BD LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The final evaluation of results was carried out with Gatelogic 400.2A software (Invai, Mentone, Australia).

Assessment of Total Antioxidant Capacity

This assay is based on the ability of antioxidants present in the sample to prevent oxidation of 2,2′-azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS) by metmyoglobin. The resulting decrease in the absorbance at 405 nm is proportional to antioxidant concentration. Total antioxidant capacity of cellular homogenates was measured using a commercially available antioxidant assay kit (Cayman Chemical Company, Ann Arbor, MI, USA), according to the supplier’s instructions. The assay was calibrated with trolox, and the results were expressed in terms of micromoles of Trolox equivalents per liter (μmol Trolox Equiv/L).

Measurement of Intracellular ROS by Flow Cytometry

The amount of intracellular ROS was measured using the oxidant-sensing fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA). The nonpolar H2DCFH-DA can enter the cells where it becomes deacetylated to the polar derivative 2′,7′-dichlorohydrofluorescein DCFH by cellular esterases, and this nonfluorescent product is switched to highly fluorescent DCF when oxidized by ROS. Cells grown in six-well plates were loaded with 10 μM H2DCF-DA for 30 min at 37 °C and subsequently treated with morphine and t-BHP according to the treatment protocol. After incubation with drugs culture media was removed, attached cells were released with trypsin, washed twice with PBS by centrifugation (200×g for 5 min at 4 °C), and immediately analyzed on a BD LSR II flow cytometer. The final evaluation of results was carried out with Gatelogic 400.2A software.

Protein Carbonyl Formation

Carbonyl content was determined by the derivatization of protein carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) leading to the formation of stable dinitrophenylhydrazone (DNP) adducts, which can be detected spectrophotometrically [26]. The cells were lysed in lysis buffer (0.1 M Tris–HCl, 1% w/v SDS; pH 6.8) in the presence of protease inhibitors and sonicated using a Bandelin Sonopuls HD 2070 utrasonic homogenizer (GmbH & Co. KG, Berlin, Germany) for three cycles of 20 s on and 20 s off at 80% amplitude on ice. Cell debris were pelleted (10,000×g, 15 min, 4 °C), and the collected supernatant was tested for the presence of nucleic acid contamination by measuring absorbance at 260 and 280 nm [27]. The concentration of carbonyl groups was determined by the colorimetric protein carbonylation assay described earlier [28].

Analysis of Lipid Peroxidation

The amount of lipid peroxides in cell homogenates was determined by measuring so-called thiobarbituric acid reactive substances (TBARS). The determination of TBARS is a well-established method for monitoring lipid peroxidation utilizing the reaction between malondialdehyde (MDA), a product of lipid peroxidation, and thiobarbituric acid (TBA). This measurement was conducted according to the TBARS assay protocol described by Stöhr et al. [29], using 1,3,3-tetramethoxypropane as standard. The MDA–TBA product formed was measured colorimetrically at 535 nm.

Electrophoresis and Immunoblotting

Detection of the levels of selected proteins was performed by electrophoresis and immunoblotting. After treatment with drugs, cells were scraped and collected by centrifugation (510×g for 10 min at 4 °C). Pellet was resuspended in TMES buffer (20 mM Tris, 3 mM MgCl2, 1 mM EDTA, 250 mM sucrose; pH 7.4) in the presence of protease and phosphatase inhibitors and homogenized with the aid of a 1-ml tuberculin syringe with a 22-gauge needle (20 strokes). Cell homogenates were solubilized in Laemmli buffer and loaded (20 μg per line) on standard 10 or 15% polyacrylamide gels for SDS–PAGE. After electrophoresis, the resolved proteins were transferred onto a nitrocellulose membrane. After blocking in 5% nonfat dry milk or 5% bovine serum albumin (for phosphorylated forms of proteins) in TBS buffer (10 mM Tris, 150 mM NaCl; pH 8.0) containing 1% (v/v) Tween 20 for 1 h at room temperature, membranes were incubated with primary rabbit polyclonal antibodies against Bcl-2, Bax, p38α, phospho-p38 (Thr 180/Tyr 182), GSK-3β, phospho-GSK-3β (pSer9), catalase, and SOD1 on an orbital shaker at 4 °C overnight. After three 10-min washes in TBS-Tween, the secondary donkey anti-rabbit IgG labeled with horseradish peroxidase (GE Healthcare) was applied for 1 h at room temperature. After another three 10-min washes in TBS-Tween, the blots were visualized by enhanced chemiluminiscence technique according to the manufacturer´s instructions (Pierce Biotechnology, Rockford, IL, USA). The immunoblots were scanned and quantitatively analyzed by ImageQuantTM TL software (Amersham Biosciences). Ponceau staining of nitrocellulose membranes was used to check equal loading of total proteins and for data normalization.

Protein Determination

Protein concentrations were determined by the bicinchoninic acid assay [30]. Samples were incubated at 37 °C for 30 min before analysis on BioTek synergy HT microplate reader at 562 nm.

Statistical Analysis

All results were expressed as the mean ± SEM. Data for analysis were obtained from at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Student–Newman–Keuls test. For each comparison, differences were considered statistically significant at P ≤ 0.05.

Results

Effect of Morphine and t-BHP on Cell Viability

In our initial experiments, we investigated the effect of different doses of morphine on H9c2 cells. Morphine was added to the cell culture medium at concentrations ranging from 1 μM to 1 mM. After incubation for 15 h at 37 °C, cytotoxicity and cell viability were assessed using the LDH and MTT assays, respectively. Even when used at the highest concentrations (1 mM), morphine did not negatively affect cell viability. By contrast, addition of t-BHP caused a dose-dependent decrease in cell viability (Fig. 1).

Fig. 1
figure 1

Effects of morphine and t-BHP on cell viability. H9c2 cells were either not treated (without MOR) or treated with 1 mM morphine (MOR) and subsequently incubated in the presence of increasing concentrations of t-BHP as described in Materials and Methods. Cell viability and cytotoxicity were assessed by MTT (a) and LDH (b) assays, respectively. In MTT assay, the absorbance of MTT-formazan dissolved in DMSO was read at 540 nm using a microplate reader and LDH activity was determined by spectrophotometric absorbance at 490 nm. Data were expressed as percentages of the untreated control (100%). Values are mean ± S.E.M. (n = 5); **P < 0.01 and ***P < 0.001 versus corresponding morphine-untreated cells

Full size image

Protective Effect of Morphine Against t-BHP-Induced Cell Damage

As stated above, morphine per se did not exert any significant effects on cell viability, but pretreatment of H9c2 cells with this drug markedly enhanced resilience of these cells to the harmful effect of t-BHP (Fig. 1). Interestingly, high doses of morphine were able to preserve cell viability even in the presence of very high t-BHP concentrations. Whereas only about 5% of cells remained viable after treatment with 500 μM t-BHP, addition of 1 mM morphine increased the proportion of viable cells to about 49%. Lower concentrations of morphine (below 1 × 10−5 M) did not yield such a prominent cytoprotective effect. In most subsequent experiments, t-BHP was used at a concentration of 300 μM. Higher t-BHP concentrations did not incur more severe cellular damage.

The cytoprotective effect of morphine was further corroborated by flow cytometric determination of cell apoptosis and necrosis using Annexin V/Hoechst 33258 staining (Fig. 2a). These measurements indicated that the viability of H9c2 cells cultured under normal conditions was about 88% and that treatment with 1 mM morphine did not markedly change cell viability (Fig. 2b). Moreover, addition of morphine to the culture medium considerably improved cell resilience to oxidative stress induced by t-BHP. Whereas 15 h of incubation with 300 μM t-BHP caused a 96% decrease in cell viability, pretreatment of H9c2 cells with morphine efficiently forestalled such a devastating damage (Fig. 2b). In this case, cell viability dropped only slightly to about 67%.

Fig. 2
figure 2

Ameliorating effect of morphine on t-BHP-induced apoptotic cell death. H9c2 cells were either not treated (CON) or treated with 1 mM morphine (MOR) or 300 μM t-BHP (t-BHP), or incubated in the presence of both these agents (MOR + t-BHP) as described in Materials and Methods. The proportion of alive and apoptotic/dead cells was estimated by Annexin V/Hoechst 33258 dual staining and flow cytometry. Representative dot plots show the distinct populations of viable cells (Annexin V−/Hoechst 33258−, QI), early apoptotic (Annexin V+/Hoechst 33258−, QII), late apoptotic (Hoechst 33258+/Annexin V+, QIII), and necrotic (Hoechst 33258+/Annexin V−, QIV) cells (a). Graphs show the proportion of surviving cells (expressed as a percentage of the total cell number) under different conditions. Values are mean ± S.E.M. (n = 5); ***P < 0.001 versus control, ###P < 0.001 versus t-BHP

Full size image

Effect of Morphine on Total Antioxidant Capacity and Oxidative Stress in Cells Affected by t-BHP

First, we investigated the impact of 1 mM morphine on total antioxidant capacity of H9c2 cells using the ABTS decolorization assay. Treatment of cells with morphine resulted in a marked increase (by about 64%) in total antioxidant capacity. Contrarily, 300 μM t-BHP significantly reduced (by about 35%) total antioxidant capacity. Interestingly, the observed increase in total antioxidant capacity of H9c2 cells induced by morphine treatment remained preserved even in the presence of t-BHP (Fig. 3).

Fig. 3
figure 3

Effects of morphine and t-BHP on total antioxidant capacity. H9c2 cells were either not treated (CON) or treated with 1 mM morphine (MOR) or 300 μM t-BHP (t-BHP), or incubated in the presence of both these agents (MOR + t-BHP) as described in Materials and Methods. Total antioxidant capacity of cellular homogenates was then determined by the bleaching of preformed ABTS radical cations. Trolox was used as a reference standard and the results expressed as Trolox equivalent antioxidant capacity (TEAC, μmol/L Trolox). Values are presented as mean ± S.E.M. (n = 5); **P < 0.01 and ***P < 0.001 versus control, ###P < 0.001 versus t-BHP

Full size image

The generation of intracellular ROS was measured by flow cytometry using DCF as a fluorescence probe (Fig. 4a). Exposure of H9c2 cells to 300 μM t-BHP resulted in a considerable increase (by about 155%) in DCF fluorescence, indicating the presence of elevated levels of ROS. On the other hand, morphine significantly attenuated the production of ROS, which was reflected by a decrease (by about 30%) in DCF fluorescence. Furthermore, this drug turned out to be able to achieve a very high degree of reduction (by about 34%) of the elevated level of DCF fluorescence caused by 300 μM t-BHP (Fig. 4b).

Fig. 4
figure 4

Effects of morphine and t-BHP on intracellular ROS generation and levels of oxidative stress markers. H9c2 cells were either not treated (CON) or treated with 1 mM morphine (MOR) or 300 μM t-BHP (t-BHP), or incubated in the presence of both these agents (MOR + t-BHP) as described in Materials and Methods. The intracellular levels of ROS were determined by flow cytometry using H2DCFH-DA as the probe. Representative histogram of DCF fluorescence intensity (a) and bar graph showing DCF-detectable ROS levels (expressed in relative fluorescence units) in H9c2 cells under different conditions (b). The carbonyl content was calculated from the absorbance of the protein-DNT derivative at 370 nm (c) and the lipid peroxidation was monitored by colorimetric measurement of MDA at 535 nm (d). Values are presented as mean ± S.E.M. (n = 7); *P < 0.05, **P < 0.01 and ***P < 0.001 versus control, ###P < 0.001 versus t-BHP

Full size image

Next, we were interested to find out whether morphine can affect the extent of oxidative cell damage. Oxidative damage was assessed by measuring protein carbonyl content and lipid peroxidation (determination of TBARS). Treatment with 1 mM morphine significantly lowered (by 30%) the levels of protein carbonylation (Fig. 4c), as well as lipid peroxidation (Fig. 4d). Consistently with previous results, the addition of 300 μM t-BHP to H9c2 cells led to increased lipid and protein oxidation; the extent of lipid peroxidation and protein carbonylation increased by about 40 and 39%, respectively. Importantly, pretreatment of cells with morphine completely prevented the harmful impact of t-BHP.

Effect of Morphine and t-BHP on the Expression of Bcl-2 and Bax Proteins

To evaluate the effect of morphine and t-BHP on the sensitivity of H9c2 cells to apoptosis, the expression levels of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax were determined using immunoblotting (Fig. 5a, b). Whereas treatment with t-BHP markedly suppressed (by about 46%) Bcl-2 and increased (by about 87%) Bax expression, morphine did not affect Bcl-2 and decreased (by about 21%) Bax expression. In addition, morphine completely abolished the effect of t-BHP and maintained the expression of both Bcl-2 and Bax at the control level. Thus, the Bcl-2/Bax ratio substantially decreased or increased after treatment of H9c2 cells with morphine or t-BHP, respectively (Fig. 5c).

Fig. 5
figure 5

Effects of morphine and t-BHP on Bcl-2 and Bax expression. H9c2 cells were incubated in the absence (CON) or presence either of 1 mM morphine (MOR), 300 μM t-BHP (t-BHP) or both these agents (MOR + t-BHP) as described in Materials and Methods. Cells were harvested after 15 h and immunoblot analyses were performed using Bcl-2 (a) and Bax (b) specific antibodies. The blots are representatives of five independent experiments. Bcl-2 and Bax band intensities were measured by densitometry and normalized to total protein levels. The ratios of Bcl-2 to Bax obtained under different experimental conditions were plotted (c). Values are presented as mean ± S.E.M. (n = 5); **P < 0.01 and ***P < 0.001 versus control, ##P < 0.01 and ###P < 0.001 versus t-BHP

Full size image

Analysis of the p38 MAPK and GSK-3β Pathways

To explore the effect of morphine and t-BHP on the p38 MAPK and GSK-3β pathways, total expression of p38 and GSK-3β, as well as changes in their phosphorylation state were monitored by immunoblotting (Fig. 6). The results were expressed as the ratio of phosphorylated p38 to total p38 (phosho-p38/p38) and phosphorylated GSK-3β to total GSK-3β (phospho-GSK-3β/GSK-3β). Morphine treatment of H9c2 cells did not affect total expression of either p38 or GSK-3β, but increased phosphorylation of p38 (Thr 180, Tyr 182) by about 128%. Phosphorylation of GSK-3β (Ser 9) was not changed under these conditions. Although incubation of cells in the presence of t-BHP substantially diminished phosphorylation of both p38 and GSK-3β, treatment of cells with morphine prior to the addition of t-BHP reversed the phosphorylation state of these two proteins. In other words, the morphine-induced increase in phospho-p38 level was not affected by t-BHP (Fig. 6a) and, intriguingly, morphine in combination with t-BHP markedly enhanced (by about 21%) the level of pospho-GSK-3β (Fig. 6b).

Fig. 6
figure 6

Effects of morphine and t-BHP on p38 MAPK and GSK-3β expression. H9c2 cells were incubated in the absence (CON) or presence either of 1 mM morphine (MOR), 300 μM t-BHP (t-BHP) or both these agents (MOR + t-BHP) as described in Materials and Methods. Cells were harvested after 15 h and immunoblot analyses were performed using p38 and phosho-p38 (a) or GSK-3β and phospho-GSK-3β (b) specific antibodies. The blots are representatives of four independent experiments. Intensities of the bands corresponding to the phoshorylated and nonphoshorylated forms of p38 and GSK-3β were measured by densitometry and normalized to total protein levels. Graphs show the ratios of phosho-p38 to total p38 and phospho-GSK-3β to GSK-3β obtained under different experimental conditions. Values are presented as mean ± S.E.M. (n = 4); *P < 0.05, **P < 0.01 and ***P < 0.001 versus control, ###P < 0.001 versus t-BHP

Full size image

Finally, we wanted to test whether the protective effect of morphine is mediated through the p38 MAPK and/or PI3K/GSK-3β pathways. In these experiments, H9c2 cells were preincubated either with the p38 MAPK inhibitor SB203580 (10 μM), or with the PI3K inhibitor wortmannin (1 μM), or with a combination of both these inhibitors for 1 h before the addition of morphine and t-BHP. While treatment with SB203580, wortmannin, or both these inhibitors together has no toxic effect on cultured H9c2 cell in terms of cell viability (data not shown), preincubation with these compounds, either individually or in combination, markedly attenuated (by about 45–74%) the protective effect of morphine against oxidative damage caused t-BHP (Fig. 7). Importantly, the combined application of SB203580 and wortmannin resulted in a significantly greater attenuation of morphine-induced cytoprotection than that observed when these inhibitors were added individually.

Fig. 7
figure 7

Inhibitory effects of p38 MAPK and PI3K blockade on the morphine-mediated protection against t-BHP-induced cell damage. Before incubating with 300 μM t-BHP (t-BHP), H9c2 cells were either not treated or treated with 1 mM morphine (MOR). In some cases, cells were pretreated with 10 μM SB203580 (SB) or 1 μM wortmannin (W) or both these inhibitors as described in Materials and Methods. Treatment with both these inhibitors either separately or in combination did not affect cell survival (data not shown). The proportion of alive and apoptotic/dead cells was estimated by Annexin V/Hoechst 33258 dual staining and flow cytometry. Representative dot plots show the distinct populations of viable cells (Annexin V−/Hoechst 33258−, QI), early apoptotic (Annexin V+/Hoechst 33258−, QII), late apoptotic (Hoechst 33258+/Annexin V+, QIII) and necrotic (Hoechst 33258+/Annexin V−, QIV) cells (a). Graphs show the proportion of surviving cells (expressed as a percentage of the total cell number) under different conditions. Values are mean ± S.E.M. (n = 5); ***P < 0.001 versus t-BHP, ###P < 0.001 versus MOR + t-BHP

Full size image

Effect of Morphine of on Selected Antioxidant Enzymes

Expression levels of the key antioxidant enzymes, namely catalase and superoxide dismutase 1 (SOD1), in control H9c2 cells and in cells treated with morphine were determined by immunoblotting (Fig. 8). Whereas exposure to morphine resulted in a marked upregulation of catalase (by about 40%), the expression level of SOD1 was not significantly changed.

Fig. 8
figure 8

Effect of morphine on catalase and superoxide dismutase 1 (SOD1) expression. After incubation for 15 h in the absence (CON) or presence of 1 mM morphine (MOR), H9c2 cells were harvested and immunoblot analyses were performed using catalase (a) or SOD1 (b) specific antibodies. The blots are representatives of four independent experiments. Intensities of the bands corresponding to catalase and SOD1 were measured by densitometry and normalized to total protein levels. Values are presented as mean ± S.E.M. (n = 4); ***P < 0.001 versus control

Full size image

Discussion

It was reported earlier that treatment of H9c2 cells with t-BHP may lead to increased oxidative stress and result in apoptotic and necrotic cell death [24]. It was also found that opioids including morphine may confer protection against oxidative injury in some cells and tissues [6, 7]. However, it is currently not known whether morphine is able to protect against oxidative stress elicited by t-BHP and what kind of molecular mechanisms may possibly underlie its action. Therefore, the present study aimed to explore these issues. We observed that exposure of H9c2 cells to increasing concentrations (100–500 μM) of t-BHP resulted in a dose-dependent decrease in cell viability and a concomitant dose-dependent increase in cell death. Morphine (even at 1 mM concentration) did not negatively impact cell viability, but it markedly reduced the adverse effect of t-BHP. Furthermore, this drug was found to act as a strong antioxidant agent. Morphine markedly enhanced total antioxidant capacity of H9c2 cells and prevented the increase in ROS generation, protein carbonylation as well as lipid peroxidation that would have otherwise occurred in the presence of t-BHP.

We tested different concentrations (ranging from 1 μM to 1 mM) of morphine for their cytoprotective potency. Although there are some studies showing that high doses of morphine can induce oxidative stress and apoptosis in different cell types [31,32,33], the highest morphine concentration (1 mM) proved to be the most effective in providing cytoprotection in our experimental conditions. In essence, these data agree well with previous findings of Hargrave et al. [20]. These authors reported that incubation of H9c2 cells with morphine (1 μM–10 mM) did not lead to increased ROS production. Likewise, it has been observed that treatment of isolated rat liver cells with 1 mM morphine decreased H2O2 formation and lipid peroxidation [34]. Hence, studies dealing with the relationship between morphine and cellular oxidative state provide rather contradictory results. Morphine cannot be easily considered as a pro-oxidant or antioxidant compound because different factors, such as dosage, exposure time, tissue type and species, may affect the outcome of morphine treatment [19]. Interestingly, heroin (a closely related morphine derivative) increased the production of ROS and oxidative damage of proteins and lipids in the brain and liver but not in the heart, indicating better tolerance of opioids by heart tissue [35]. These findings are supported by results of proteomic analysis of the rat heart after prolonged morphine exposure which did not reveal any significant alternations in the levels of several proteins involved in the control of cellular redox state (superoxide dismutase 1 and 2 (SOD1 and SOD2), glutathione-peroxidase 4 isoform A precursor, peroxiredoxins Prx 2, Prx 3, Prx 5 and Prx 6, catalase, and some others) [36]. On the other hand, prolonged morphine exposure decreased the expression of SOD1, SOD2, and glutathione in the rat brain [37, 38] and the level of glutathione in the mouse liver [39]. By contrast, Arabian et al. [40] recently described increased catalase activity in hippocampal CA1 neurons after chronic morphine preconditioning. Similarly, here we observed that treatment of H9c2 cells by morphine resulted in increased rather than decreased expression of catalase. Concomitantly, the expression of SOD1 was not significantly changed. These data provide further support to the notion that the effects of morphine are highly dependent on tissue and other experimental conditions.

Recently, it has been reported that morphine, when used at common therapeutic (analgesic) concentrations (~ 1 μM), may also contribute to minimalizing oxidative stress and cell death in glioma cells [41, 42] and H9c2 cells [21], as well as in HL-1 cells (mouse atrial cardiomyocyte cell line) and isolated rat cardiomyocytes [43]. However, cell injury induced by oxidative stress in all these experiments was significantly less prominent than that in our experimental conditions where treatment with t-BHP decreased the number of viable cells in a population by about 97%. It can be assumed that lower morphine concentrations may be sufficient to prevent the adverse consequences of mild oxidative stress in H9c2 cells, but cell damage caused by harsh oxidative stress conditions can be efficiently ameliorated only by higher morphine doses.

There is some evidence to suggest that activation of the p38 MAPK signaling pathway can be implicated in the development of a cardioprotective phenotype upon opioid-mediated preconditioning [13, 14]. In the present study, we decided to evaluate the potential role of this signaling pathway in morphine-induced cellular resistance to oxidative stress elicited by t-BHP. Treatment of H9c2 cells with morphine markedly enhanced phosphorylation of p38 MAPK and abolished the suppressive effect of t-BHP on its activity, indicating the ability of morphine to upregulate this signaling pathway. This conclusion has been further corroborated by the finding that morphine’s protective effect on cell survival could be abrogated in the presence of the p38 MAPK inhibitor SB203580. These observations are consistent with data obtained by Zhang et al. [15], who identified the p38 MAPK pathway as an important mediator of morphine-induced preconditioning in vivo. It is interesting to note that the consequences of p38 MAPK activation on cell viability can be time dependent. Whereas p38 MAPK activation before ischemia was found to be protective [13,14,15], activation of this enzyme during a period of reperfusion resulted in increased tissue damage [44].

There are some indications that PI3K-mediated inactivation of GSK-3β can also participate in the molecular mechanism of morphine-induced cardioprotection [8, 11]. GSK-3β is a crucial regulator of cellular function as it plays a key role in pro-survival signaling. Inhibition of GSK-3β is important for the prevention of mPTP opening that could otherwise lead to the impairment of mitochondrial respiratory function and excessive ROS production [11, 12]. Here we observed that incubation of H9c2 cells in the presence of both morphine and t-BHP resulted in increased phosphorylation of GSK-3β on Ser 9, thereby inhibiting kinase activity, and maintaining high cell viability. In line with this, the addition of the PI3K inhibitor wortmannin to the culture medium significantly diminished the protective effect of morphine against t-BHP-induced cell damage. Intriguingly, concomitant application of SB203580 and wortmannin further reduced the ability of morphine to confer cytoprotection, when compared to the individual inhibitory effects of these two compounds. These data imply that both the p38 MAPK and PI3K/GSK-3β signaling pathways play a role in morphine-induced cytoprotection. In this context, it is also worth mentioning that activation of p38 MAPK in MEF cells (mouse embryonic fibroblasts) increased catalase and SOD levels, diminished ROS accumulation and promoted cell survival [45]. Interestingly, these data fit well with our current findings.

Apoptosis regulator proteins Bcl-2 and Bax seem to exert distinct effects on the mPTP. Whereas the pro-apoptotic Bax protein facilitates outer membrane permeability of the mPTP, thereby leading to necrotic cell death [46], interaction of the anti-apoptotic Bcl-2 protein with the mPTP renders the pore more resistant to opening [47]. Therefore, preservation of a favorable ratio of Bcl-2 and Bax is critically important for controlling cell survival. Here we found that treatment of H9c2 cells with t-BHP decreased Bcl-2 and increased Bax expression. Morphine treatment abolished the detrimental effect of t-BHP and maintained the Bcl-2/Bax ratio similar to that in control untreated cells. Similar results were recently reported by Amini-Khoei et al. [21], who demonstrated that morphine was able to substantially restore the levels of Bcl-2 and Bax that were found to be significantly altered upon treatment of H9c2 cells with arsenic trioxide.

Collectively, our data support previous studies that highlight the importance of opioid preconditioning in cellular functions. We have shown that morphine can confer a strong protection against oxidative stress caused by t-BHP in H9c2 cells and that this protection is at least partially mediated through the p38 MAPK and PI3K/GSK-3β cascades. Our findings suggest that activation of these signaling pathways is required to trigger the defense reaction antagonizing oxidative stress by mechanisms involving the upregulation of catalase, as well as the modulation of Bcl-2 family proteins. However, the complete molecular mechanism that coordinates these events remains to be determined.