Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin antibiotics were purchased from WelGENE Inc. (Daegu, Republic of Korea). Morin, 3-[4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-acetyl-L- cysteine (NAC), 4,6-diamidino-2-phenyllindile (DAPI), and PD98059 (2′-amino-3′-methoxyflavone) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (DCF-DA) and a fluorescein-conjugated Annexin V (Annexin V-FITC) staining assay kit were purchased from Molecular Probes (Eugene, OR, USA) and BD Biosciences (San Jose, CA, USA), respectively. An enhanced chemiluminescence (ECL) detection kit and FITC-conjugated donkey anti-rabbit IgG were purchased from Amersham Co. (Arlington Heights, IL, USA) and Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA), respectively. Various primary antibodies (Table I) for western blot analysis were obtained from Cell Signaling Technology, Inc. (Boston, MA, USA), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and Abcam, Inc. (Cambridge, MA, USA). Horseradish peroxidase-conjugated anti-rabbit (SC-2004), anti-mouse (SC-2005) and anti-goat (SC-2350) antibodies were used as the secondary antibodies, which were obtained from Santa Cruz Biotechnology, Inc. All other chemicals were purchased from Sigma-Aldrich Chemical Co.

C2C12 myoblasts obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were grown in DMEM supplemented with 10% FBS and 100 μg/ml penicillin/streptomycin antibiotics in a humidified 5% CO₂ atmosphere at 37°C. Morin was dissolved in dimethyl sulfoxide (DMSO) and adjusted to final concentrations using complete DMEM prior to use; the final DMSO concentration was <0.1% in all experiments. In order to evaluate the degree to which a single morin treatment affects C2C12 cell viability, the C2C12 cells were seeded at a density of 1×10⁴ cells/well in a 96-well plate, incubated at 37°C for 24 h, and treated with morin at different concentrations (100–10,000 μM). Additional cell cultivation occurred for 6 h in media where H₂O₂ and NAC were simultaneously administered, singularly administered, or were not administered. For the cell viability assay using a colorimetric MTT assay, the medium was discarded, MTT solution was added to each well, and the cells were further incubated for 3 h at 37°C. The medium was discarded and DMSO was added to dissolve the formazan product. The optical density was then read at 450 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (Dynatech MR-7000; Dynatech Laboratories, Chantilly, VA, USA). Relative cell cytotoxicity was evaluated according to the quantity of MTT converted to insoluble formazan salt.

In order to monitor ROS generation, the cells were incubated with 10 μM DCF-DA for 20 min at room temperature in the dark. The ROS production in the cells was monitored with a flow cytometer (Becton-Dickinson, San Jose, CA, USA) using CellQuest Pro software (25).

To quantitatively assess the induced cell apoptosis rate, an Annexin V-FITC staining assay was performed as previously described (26). The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and stained with Annexin V-FITC and propidium iodide (PI) in each sample for 15 min at room temperature in the dark. The degree of apoptosis was quantified as a percentage of Annexin V-positive and PI-negative (Annexin V⁺/PI⁻ cells) cells using a flow cytometer.

To assess oxidative DNA damage, the cell suspension was mixed with 0.5% low melting agarose (LMA) at 37°C, and the mixture was spread on a fully frosted microscopic slide precoated with 1% normal melting agarose. After the solidification of the agarose, the slide was covered with 0.5% LMA and then immersed in a lysis solution (2.5 M NaCl, 100 mM Na-ethylenediaminetetraacetic acid (Na-EDTA), 10 mM Tris, 1% Triton X-100, and 10% DMSO, pH 10.0) for 1 h at 4°C. The slides were then placed in a gel electrophoresis apparatus containing 300 mM NaOH and 10 mM Na-EDTA (pH 13.0) for 40 min to allow for DNA unwinding and the expression of alkali-labile damage. Next, an electrical field was applied (300 mA, 25 V) for 20 min at 4°C to draw the negatively charged DNA toward the anode. After electrophoresis, the slides were washed three times for 5 min at 4°C in a neutralizing buffer (0.4 M Tris, pH 7.5), followed by staining with 20 μg/ml PI. The cells were washed twice with PBS, and images were then captured using a fluorescence microscope (Carl Zeiss, Jena, Germany) (27).

Whole-cell protein extracts from C2C12 myoblasts were prepared with cell lysis buffer [25 mM of Tris-Cl (pH 7.5), 250 mM of NaCl, 5 mM of EDTA, 1% Nonidet P-40, 0.1 mM of sodium orthovanadate, 2 μg/ml of leupeptin, and 100 μg/ml of phenylmethylsulfonyl flouride] containing protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany) for 30 min. In a parallel experiment, nuclear and cytosolic proteins were prepared using nuclear extraction reagents (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer's protocol. After cell debris was discarded following centrifugation at 13,000 × g for 15 min, the protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). For western blot analysis, equal amounts of protein extracts were subjected to electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred onto nitrocellulose membranes (Schleicher and Schuell Bioscience, Inc., Keene, NH, USA) by electroblotting. The blots were probed with the desired primary antibodies for 1 h, incubated with the diluted enzyme-linked secondary antibodies, and visualized using an ECL method according to the recommended procedure.

Nrf2 siRNA and control siRNA were purchased from Santa Cruz Biotechnology, Inc. The siRNAs were transfected into cells according to the manufacturer's instructions using Lipofectamine 2000 Transfection Reagent (Life Technologies, Carlsbad, CA, USA). For transfection, the cells were seeded in 6-well culture plates and incubated with the control siRNA or Nrf2 siRNA at 50 nM for 6 h in serum-free Opti-MEM media (Life Technologies). After incubation, the transfected cells were subjected to treatment, as previously described (28).

C2C12 cells were seeded on glass coverslips in 6-well plates for 24 h, and the cells were treated with morin for 6 h. Next, the cells were rinsed twice with PBS and fixed with 3.7% paraformaldehyde in PBS for 10 min at 4°C. The cells were incubated with 0.4% Triton X-100 for 10 min and then blocked with 5% bovine serum albumin for 1 h, followed by probing with the anti-Nrf2 antibody overnight at 4°C and incubation with FITC-conjugated donkey anti-rabbit IgG for 2 h at room temperature. After washing with PBS, nuclei were counterstained with DAPI solution (1 mg/ml) for 15 min in the dark. Images were collected using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

Data are expressed as mean ± standard deviation (SD) from at least three independent experiments. Statistical comparisons between different groups were performed using one-way ANOVA, followed by Student's t-tests after comparing each treated group to the negative control. Probability values P<0.01 were considered to indicate statistically significant differences.

To evaluate the protective effect of morin on H₂O₂-induced cytotoxicity, the cells were treated with various concentrations of morin for 24 h, and the cell viability was evaluated for primary dose selection by determining the percentage of MTT reduction. Morin alone at 100–500 μM showed no cytotoxic effects, but significant cytotoxicity was noted at 1,000 μM morin (Fig. 1A). Thus, 500 μM morin was chosen as the optimal dose for studying the cytoprotective effect of this flavonoid.

The viability of the C2C12 cells when exposed to H₂O₂ at the concentration of 1 mM for 6 h was significantly decreased as compared with the control group, and the survival rate was ~60% that of the control group. When the C2C12 cells were pretreated with 500 μM morin or 10 mM NAC, an ROS scavenger that was used as positive control, for 1 h and exposed to H₂O₂ for an additional 6 h, the cell viability was significantly increased compared to the H₂O₂ group, and the survival rate was 81.00 and 99.08% that of the control group, respectively (Fig. 1B).

As the cytotoxicity of H₂O₂ is mainly mediated by oxidative stress, we investigated the effect of morin on H₂O₂-induced ROS accumulation using DCF-DA reagent. Compared with the non-treated control cells, the level of intracellular ROS was markedly increased in the C2C12 cells treated with 1 mM H₂O₂ for 6 h, as indicated by the increase in the DCF-liberated fluorescent signal (Fig. 2A). However, when the C2C12 cells were pretreated with morin or NAC, the ROS formation was significantly decreased, suggesting that morin pretreatment induced a cellular antioxidative response.

The effects of morin on C2C12 cell apoptosis induced by H₂O₂ were observed using an Annexin V-FITC/PI assay. As illustrated in Fig. 2B, the percentage of apoptotic cells treated with 1 mM H₂O₂ was ~49.54%; however, morin or NAC pretreatment effectively decreased cell apoptosis induced by H₂O₂ to 7.99 and 7.86%, respectively. The results indicate that the H₂O₂-induced apoptosis was mediated by ROS generation and that morin exerted a potent ROS scavenging effect, preventing H₂O₂-induced apoptosis.

We next examined the effects of morin on H₂O₂-mediated DNA damage in the C2C12 cells using the comet assay (single cell gel electrophoresis) and western blot analysis. Exposure to H₂O₂ alone induced significant DNA breaks, resulting in an increase in fluorescence intensity in the tails of the comet-like structures (Fig. 3A); however, this adverse effect was markedly reduced by pretreatment with morin or NAC. In addition, the exposure of the C2C12 cells to H₂O₂ resulted in upregulation in the level of the phosphorylated histone variant H2AX (p-γH2AX) at serine 139, a sensitive marker of DNA double strand breaks (29); however, pretreatment with morin or NAC resulted in a significant decrease in p-γH2AX expression.

As Nrf2 signaling regulates cellular antioxidant response (20,22), we examined whether morin protects cells from oxidative stress by activating the Nrf2 signaling pathway. Our immunoblotting results indicated that treatment of C2C12 cells with morin induced the expression of HO-1 protein in a time-dependent manner, but other antioxidant enzymes, NADPH-quinone oxidoreductase 1 (NQO1) and thioredoxin reductase 1 (TrxR1), were unaffected by morin treatment, which was associated with the upregulation of Nrf2 expression and the downregulation of Keap1 (Fig. 4A).

Since the phosphorylation of Nrf2 at Ser40 by several kinases is also a critical process in its stabilization and nuclear translocation (20,23,24), we examined the phosphorylation of Nrf2 under morin treatment to further confirm the Nrf2 activating property of morin and observed that the treatment of cells with morin time-dependently increased the expression levels of phosphorylated Nrf2 (Fig. 4A). Additionally, western blot analysis was carried out using the nuclear and cytosolic fractions of the C2C12 cells. The results shown in Fig. 5A and B indicate that the amounts of total and phosphorylated Nrf2 proteins in the nucleus were markedly increased following treatment with morin. The immunofluorescence images also revealed that the nuclear localization and accumulation of Nrf2 in the C2C12 cells was significantly increased after stimulation with morin (Fig. 5C).

In order to provide evidence for the involvement of Nrf2 in the induction of HO-1 by morin, we transiently transfected C2C12 cells with Nrf2 siRNA. Western blot analysis results revealed that the silencing of Nrf2 using specific siRNA abrogated the morin-induced increase and phosphorylation in Nrf2 expression (Fig. 4B). Therefore, we investigated whether Nrf2 siRNA transfection attenuated morin-induced upregulation of HO-1 and downregulation of Keap1 and found that Nrf2 siRNA reversed these effects, which is evidence that the augmentation of HO-1 by morin was mediated by Nrf2.

To identify the upstream signaling events involved in morin-mediated Nrf2-dependent HO-1 induction, the potential involvement of the PI3K/Akt and MAPK signaling pathways was investigated. The total and phosphorylated levels of PI3K and Akt, a downstream target of PI3K, did not show a notable change in the morin-treated C2C12 cells as compared with levels in the untreated control cells (Fig. 6). Subsequently, although there were no notable changes observed in the phosphorylated levels of c-Jun N-terminal kinase (JNK) and p38 MAPK, the activation of extracellular signal-regulated kinase (ERK) was noted as early as 30 min after morin treatment, and it lasted at least for 6 h. However, treatment with a selective inhibitor of ERK, PD98059, blocked the morin-triggered phosphorylation of Nrf2, and HO-1 induction was accordingly diminished without the blockade of Nrf2 induction (Fig. 7). By contrast, when an inhibitor of ERK was utilized, the induction of the total protein level of Nrf2 in the morin-treated C2C12 cells was not attenuated.