Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), streptomycin, and penicillin were purchased from WelGENE Inc. (Daegu, Republic of Korea). Morin, 3-(4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 4′,6-diamidino-2-phenylindole (DAPI), N-acetyl-L-cysteine (NAC), 6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) and zinc protoporphyrin IX (ZnPP), a specific inhibitor of heme oxygenase-1 (HO-1), were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). A SOD assay kit was obtained from Dojindo Molecular Technologies (Tokyo, Japan). 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 system was purchased from Amersham Co. (Arlington Heights, IL, USA). Primary antibodies (Table I) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), Cell Signaling Technology, Inc. (Danvers, MA, USA) and Abcam, Inc. (Cambridge, MA, USA). Horseradish (HRP)-conjugated secondary antibodies were obtained from Amersham Co. Morin was dissolved in dimethyl sulphoxide (DMSO; Sigma-Aldrich Chemical Co.) and then diluted with medium to the desired concentration prior to use. The final DMSO concentration was <0.1% in all experiments. All other chemicals were purchased from Sigma-Aldrich Chemical Co.

The SOD-like activity of morin was measured using a SOD assay kit according to the manufacturer's instructions. Briefly, 20 μl of morin stock solution was added to 200 μl of the working solution in the SOD assay kit. The mixture was incubated at 37°C for 20 min after gentle shaking and then 20 μl of the kit enzyme working solution was added. The absorbance of the mixtures was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Sunnyvale, CA, USA), and the SOD activity was calculated according to the manufacturer's instructions (33). Vitamin C was used as a positive control.

The ABTS assay was based on a previously described method (34) with slight modifications. ABTS radical cation (ABTS^•+) was produced by the reaction of a 7 mM ABTS solution with potassium persulphate (2.45 mM). The ABTS^•+ solution was diluted with ethanol to an absorbance of 0.70±0.05 at 734 nm. The mixture was stored in the dark at room temperature for 12 h before use. After the addition of 25 μl of morin solution or vitamin C as a positive control to 2 ml of diluted ABTS^•+ solution, absorbance was measured at 734 nm after exactly 6 min. Inhibition of the ABTS radical by morin was calculated using the formula: ABTS scavenging activity (%) = [1 - (absorbance of the sample/absorbance of the control)] × 100.

The Chinese hamster lung fibroblast V79-4 cell line was obtained from the American Type Culture Collection (ATCC; Manassas, MD, USA) and cultured in DMEM containing 10% heat-inactivated FCS, streptomycin (100 μg/ml) and penicillin (100 Units/ml). The cells were maintained at 37°C in an incubator with a humidified atmosphere of 5% CO₂.

For the cell viability assay, V79-4 cells were seeded at 1×10⁵ cells/ml in a 96-well plate and cultured for 24 h before being treated with the indicated concentrations of morin for 24 h in the presence or absence of 1 mM H₂O₂ with or without 1 h pretreatment of morin or NAC. After the treatments, the MTT solution (0.5 mg/ml) was added, followed by a 2-h incubation at 37°C in the dark, after which the medium was removed. The formazan precipitate was dissolved in DMSO. The absorbance of the formazan product was measured at 540 nm using an ELISA reader (35).

The cells were harvested, washed with PBS, and fixed with 3.7% paraformaldehyde for 30 min at room temperature. After washing twice with PBS, the cells were attached on glass slides using cytospin (Shandon, Pittsburgh, PA, USA) and stained with 2.5 μg/ml DAPI solution for 20 min at room temperature. The stained cells were washed twice with PBS and then analyzed using a fluorescence microscope (Carl Zeiss, Deisenhofen, Germany).

To assess the induced cell apoptosis rate quantitatively, the cells were washed with PBS and stained with 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI) in each sample according to the manufacturer's protocols. After a 15-min incubation at room temperature in the dark, the degree of apoptosis was quantified as a percentage of the Annexin V-positive and PI-negative (Annexin V⁺/PI⁻cells) cells using a flow cytometer (BD Biosciences).

The oxidation-sensitive dye DCF-DA was used to determine the formation of intracellular ROS. Briefly, the cells from each treatment were harvested, washed twice with PBS, and then re-suspended in 10 μM DCF-DA for 30 min at 37°C in the dark. The production of ROS in the cells was monitored immediately using a flow cytometer (36).

The MMP values were determined using the dual-emission potential-sensitive probe JC-1. Briefly, the cells were collected and incubated with 10 μM of JC-1 for 20 min at 37°C in the dark. After the JC-1 was removed, the cells were washed with PBS to remove the unbound dye, and the amount of JC-1 retained by 10,000 cells per sample was measured by using a flow cytometer (37).

After each treatment, the cells were washed with PBS, and the cell suspension was mixed with 0.5% low melting agarose (LMA) at 37°C before adding it to the slides precoated with 1.0% normal melting agarose. After the agarose was solidified, the slides were covered with another 0.5% LMA and then immersed in lysis buffer [2.5 M NaCl, 500 mM Na-ethylenediaminetetraacetic acid (EDTA), 1 M Tris buffer, 1% sodium lauroyl sarcosinate and 1% Triton X-100] for 1 h at 4°C. The slides were later transferred into an unwinding buffer for another 20 min for DNA unwinding. The slides were then placed in an electrophoresis tank containing 300 mM NaOH and 1 mM Na-EDTA (pH 13.0). An electrical field was then applied (300 mA, 25 V) for 20 min at 25°C to draw the negatively charged DNA toward the anode. The slides were washed three times for 5 min at 25°C in a neutral-izing buffer (0.4 M Tris, pH 7.5), and then stained with 20 μg/ml PI. The slides were examined under a fluorescence microscope.

All cell lysates were lysed in an extraction buffer [25 mM Tris-Cl (pH 7.5), 250 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.1 mM sodium orthovanadate, 2 μg/ml leupeptin, and 100 μg/ml phenylmethylsulfonyl fluoride]. The protein concentration in the cell lysate was determined using a detergent-compatible protein assay from Bio-Rad (Hercules, CA, USA). In the Western blot analysis, equal amounts of protein (30–50 μg) were separated by 8–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then were electro-transferred to polyvinylidene fluoride (PVDF) membranes (Schleicher & Schuell, Keene, NH, USA). The membranes were blocked with 5% skim milk for 1 h and then subjected to immunoblot analysis with the appropriate antibodies. Using an ECL detection system, immunoreactive bands were detected and exposed to X-ray film.

HO-1 enzyme activity was measured as previously described (38). In brief, lysates of the cells were prepared, and homogenates containing biliverdin reductase were obtained from rat liver. After quantifying the protein concentration, the cell lysates and homogenates were incubated with nicotinamide adenine dinucleotide phosphate (NADPH) and hemin for 1 h, whereas the blank samples were incubated with hemin only. The concentration of bilirubin, which was the product of degradation by HO-1, was determined as the difference in absorbance at 464 and 530 nm using an ELISA plate reader. HO-1 activity was expressed as picomoles of bilirubin per milligram of protein.

All experiments were conducted in triplicate (n=3), and a one-way ANOVA (SPSS 11.5 statistical software; SPSS, Inc., Chicago, IL, USA) was used to compare the mean values of each treatment. Significant differences were determined using Duncan's test. P<0.05 was considered to indicate a statistically significant difference.

To determine the antioxidant activity of morin, the SOD-like enzyme and ABTS radical scavenging activities were evaluated. As shown in Fig. 1A, the SOD-like enzyme activity of morin was highly increased in a concentration-dependent manner to the given concentration. For example, the percentage of SOD-like enzyme activity was ~72% at a concentration of 100 μM morin, which was very similar after treatment with the same concentration of vitamin C used as a positive control. Similar to SOD-like enzyme activity, at different concentrations (10–1,000 μM), morin was also found to effectively scavenge ABTS^•+ (Fig. 1B).

To exclude the cytotoxicity caused by morin treatment in V79-4 fibroblasts, the cells were treated with different concentrations (100–1,000 μM) of morin for 24 h. The MTT assay indicated that the treatments did not result in any cytotoxic effect at the concentration of 500 μM, and cell viability was significantly decreased at concentrations higher than 1,000 μM (Fig. 2A). Therefore, 500 μM morin was selected for use in the subsequent examination of the protective effect of morin on H₂O₂-induced cytotoxicity. A further MTT assay revealed that treatment with 1 mM H₂O₂ significantly reduced cell viability. However, the H₂O₂-induced reduction in cell viability was effectively protected by pretreatment with both 500 μM morin and 10 mM NAC (Fig. 2B).

To elucidate the cytoprotective effect of morin against the H₂O₂-induced reduction in V79-4 cell viability, we investigated the effects of morin on H₂O₂-mediated apoptosis. The results of the DAPI staining showed that treatment with H₂O₂ alone significantly increased the number of cells with condensed or blebbing nuclei. In contrast, when these cells were pretreated with morin or NAC, these phenomena were markedly reduced (Fig. 3A). The results of the flow cytometry consistently indicated that the H₂O₂ treatment enhanced the population of Annexin V⁺/PI⁻ apoptotic cells. However, the pretreatment of cells with morin or NAC prior to exposure to H₂O₂ effectively protected the V79-4 cells against apoptosis (Fig. 3B).

In a further experiment to study the mechanisms underlying the protective effect of morin, the intracellular ROS levels were determined. The results of the flow cytometric analysis using DCF-DA as a fluorescence probe demonstrated that the intensity of the DCF-liberated fluorescent signal from the H₂O₂-treated cells was markedly increased. However, the signal was effectively attenuated in the presence of morin as well as NAC (Fig. 4A).

The mitochondrial-mediated intrinsic apoptosis pathway is initiated by the loss of mitochondrial membrane potential (MMP, Δψm) and the subsequent release of pro-apoptotic proteins, which leads to the activation of caspase-9 and -3 (39,40). We therefore investigated the effects of morin on the H₂O₂-induced loss of MMP and the activation of caspase-3. The results indicated that the loss of MMP in H₂O₂-treated V79-4 cells increased 6.4-fold relative to the untreated control; however, the reduction in MMP was significantly inhibited by pretreatment with morin (Fig. 4B). The immunoblotting results indicated a marked increase in the level of activated caspase-3 expression in the H₂O₂-treated cells compared with the control. A subsequent increase in cleaved poly(ADP-ribose) polymerase (PARP), a well-known substrate of caspase-3 (41), was also observed. However, the results clearly showed that H₂O₂-induced caspase-3 activation and PARP degradation were completely abrogated by pretreatment with morin (Fig. 4C).

We then assessed the protective effects of morin against H₂O₂-induced DNA damage by applying the alkaline comet assay and Western blot analysis. The comet assay measures single- and double-strand breaks that are caused either directly by the DNA-damaging system or indirectly by the repair mechanism (42). As shown in Fig. 5A, the exposure of cells to H₂O₂ increased DNA breaks, resulting in an increase in fluorescence intensity in the tails of the comet-like structures. However, pretreatment with morin led to a marked decrease in damage to DNA (Fig. 5A). Furthermore, as expected, H₂O₂ enhanced the phosphorylation of histone λH2AX on serine 139, which was rapidly increased after the induction of DNA double-strand breaks (43), whereas pretreatment with morin prevented the increase in the phosphorylation of histone λH2AX (Fig. 5B).

The transcription factor Nrf2 regulates the expression of antioxidant responsive element (ARE)-driven antioxidant and cytoprotective genes, including HO-1 and quinone oxidoreductase-1 (NQO-1), to control the cellular defense against oxidative stress (44,45). Thus, we examined the effects of morin on the levels of Nrf2, HO-1 and NQO-1 expression, and found that treatment with morin gradually increased Nrf2 expression and its phosphorylation, but not NQO-1 expression, in a time-dependent manner. It concomitantly decreased the level of Kelch-like ECH-associated protein 1 (Keap1), which is a negative regulator of Nrf2 (Fig. 6A). Morin also enhanced HO-1 expression as well as HO-1 activity in a time-dependent manner (Fig. 6B).

To determine whether the morin-induced antioxidant and cytoprotective activities against oxidative stress were mediated through the activation of the Nrf2/HO-1 pathway, V79-4 cells were pretreated with or without a specific inhibitor of HO-1, ZnPP, and the levels of ROS and cell viability were assessed. As shown in Fig. 7, ZnPP abrogated the protective effect of morin on the H₂O₂-induced production of ROS and the reduction in cell viability.