Oxidative stress is a major cause of cellular injuries in a variety of human disorders [17,18]. Considerable efforts have been made to find natural antioxidants with protective potential against oxidative stress. Myricetin (Figure 1) is a potent flavonoid antioxidant and we have recently shown that myricetin can protect cells against H₂O₂-induced cell damage [16]. H₂O₂ has been extensively used as an inducer of oxidative stress, resulting in cell death including apoptosis [19–21]. The effect of myricetin on cell survival in H₂O₂-treated cells was measured at 24 h using the MTT test. Myricetin at 30 μM did not show cytotoxicity compared to control (Figure 2). Treatment with myricetin increased cell survival to 65% compared to 40% survival with H₂O₂ treatment. We did experiment to elucidate the up-take of myricetin into cells; cells were pre-treated with myricetin for 24 hours, removed the medium, then washed, and added new media without myricetin. After addition of H₂O₂ treatment, the cell viability was detected using MTT test after 24 hours. The cell viability results were consistent with results of continued incubation of myricetin in Figure 2 (and data not shown). These results suggest that myricetin was up-taken into cells and showed a cytoprotective effect on H₂O₂-induced cell damage.

In the present study, we found that myricetin decreased cell death induced by H₂O₂, and this result was further confirmed by apoptotic observation. To evaluate the cytoprotective effect of myricetin on apoptosis induced by H₂O₂, the nuclei of cells were stained with Hoechst 33342 and assessed by microscopy. The microscopic pictures in Figure 3A show that the control cells had intact nuclei, while the H₂O₂-treated cells exhibited significant nuclear fragmentation, which is indicative of apoptosis. However, when the cells were treated with myricetin for one hour prior to H₂O₂ treatment, reduced nuclear fragmentation was observed. These data suggest that H₂O₂-treated cells displayed typical features of apoptosis with fragmented nuclei; however, myricetin inhibited these morphological changes. In addition, H₂O₂-treated cells had increased levels of cytoplasmic histone-associated DNA fragmentation compared to the control group. However, myricetin decreased the degree of DNA fragmentation (Figure 3B). The protective effect of myricetin against apoptosis was also confirmed by an apoptotic sub-G₁ DNA analysis. As shown in Figure 3C, an analysis of the DNA content in H₂O₂-treated cells revealed a 24% increase in the apoptotic sub-G₁ DNA content. Moreover, myricetin decreased the apoptotic sub-G₁ DNA content to 10%. Mitochondrial membrane potential (Δψ_m) analysis showed that the level of Δψ_m loss was increased in H₂O₂-treated cells, as substantiated by an increase in fluorescence with the JC-1 dye, however, myricetin recovered the level of Δψ_m loss (Figure 3D). The flow cytometric data was consistent with the image analysis data; the red fluorescence of JC-1 (JC-1 aggregated form, indicative of mitochondrial polarization) was decreased in H₂O₂-treated cells, whereas the green fluorescence (JC-1 monomer form, indicative of mitochondrial depolarization) was greatly increased. Myricetin blocked the loss of Δψ_m after H₂O₂ treatment, as shown in Figure 3E. These results suggest that myricetin protects cells via inhibition of the mitochondria dependent apoptosis pathway. Mitochondria act as an important apparatus for signals during apoptosis, and the loss of mitochondrial integrity can be prompted or inhibited by many regulators of apoptosis [22]. In many cases, oxidative stress induces caspase activation through cytochrome c release from the mitochondrial inter-membrane space into the cytosol [23]. H₂O₂ treatment increased the expressions of Bax, active caspase-9, and -3, which are pro-apoptotic factors, but decreased the expression of Bcl-2, which is an anti-apoptotic factor. And H₂O₂ treatment also increased the release of cytochrome c from mitochondria to cytosol (Figure 3F). Myricetin inhibited the H₂O₂-induced release of mitochondrial cytochrome c. During the apoptotic process, Bcl-2 prevents the opening of the mitochondrial membrane pores, whereas Bax induces the opening of membrane pores [24]. Therefore, the blocked loss of Δψ_m by myricetin may be the result of Bcl-2 up-regulation, and Bax down-regulation.

Activation of PI3K and its downstream effector Akt has also been shown to suppress apoptosis and promote cell survival [25–27]. It has been shown that activation of PI3K leads to the phosphorylation and activation of Akt, which promotes cell survival by enhancing the expression of anti-apoptotic proteins and inhibiting the activity of pro-apoptotic proteins [28,29]. To further elucidate the mechanism of myrcetin-mediated cell survival, we examined the activation of Akt, a major signaling enzyme involved in cell survival against oxidative stress. Western blot analysis showed that myricetin increased Akt phosphorylation compared to the decreased Akt phosphorylation with H₂O₂ treatment (Figure 4A). In addition, LY294002 (a specific PI3K inhibitor) attenuated the protective effect of myricetin against H₂O₂-induced cytotoxicity (Figure 4B). These results suggest that PI3K/Akt is activated by myricetin and rescues cells from H₂O₂-induced apoptosis. Because p38 MAPK and JNK play important roles in modulating apoptosis, we next examined the effects of myricetin on the activation of p38 MAPK and JNK. The activation states of MAPKs were determined by measuring the expression of their phosphorylated forms. H₂O₂ treatment increased phosphorylation of p38 MAPK and JNK compared to that in control cells, however, myricetin decreased their phosphorylation (Figure 4C). In addition, SB203580 (a specific p38 MAPK inhibitor) and SP600125 (a specific JNK inhibitor) maintained the protective effect of myricetin against H₂O₂-induced cytotoxicity (Figure 4D). These results suggest that myricetin provides a cytoprotective effect against H₂O₂-induced apoptosis via inhibition of p38 MAPK and JNK.

Myricetin, Hoechst 33342, propidium iodide, and [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium] bromide (MTT) were purchased from Sigma Chemical Company (St. Louis, U.S.). 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) was purchased from Invitrogen Corporation (Carlsbad, U.S.). The primary anti-Bcl-2, -Bax, -cytochrome c, -caspase-9, -caspase-3, -Akt, -phospho Akt, -ERK, -phospho ERK, -JNK, -phospho JNK, -p38, and -phospho p38 antibodies were purchased from Cell Signaling Technology (Beverly, U.S.). LY294002, SB203580, and SP600125 were purchased from Calbiochem (San Diego, U.S.).

Chinese hamster lung fibroblasts (V79-4) cells from the American Type Culture Collection (Rockville, U.S.) were maintained at 37 °C in an incubator with a humidified atmosphere of 5% CO₂ and cultured in Dulbecco’s Modified Eagle’s medium containing 10% heat-inactivated fetal calf serum, streptomycin (100 μg/mL) and penicillin (100 units/mL).

The effect of myricetin on the cell viability was determined by the MTT assay [30]. The cells were seeded in a 96 well plate at a concentration of 1 × 10⁵ cells/mL, and 16 h after plating, were treated with 30 μM of myricetin, and 1 h later, 1 mM H₂O₂ was added to the plate and incubated for an additional 24 h at 37 °C. Fifty microliters of MTT stock solution (2 mg/mL) was then added to each well in a total reaction volume of 200 μL. After incubating for 4 h, the plate was centrifuged at 800 × g for 5 min and the supernatants were aspirated. The formazan crystals in each well were dissolved in 150 μL DMSO and the A₅₄₀ was read on a scanning multi-well spectrophotometer. To determine the effects of LY294002, SB203580, and SP600125 on cell viability, cells were pretreated with inhibitors for 1 h, followed by 1 h of incubation with myricetin and exposure to 1 mM H₂O₂ for 24 h, and cell viability was then measured using the MTT test.

The cells were treated with 30 μM of myricetin. After 1 h, 1 mM of H₂O₂ was added to the plate, and the mixture was incubated for 24 h. 1.5 μL of Hoechst 33342 (stock 10 mg/mL), a DNA specific fluorescent dye, was added to each well and incubated for 10 min at 37 °C. The stained cells were then observed under a fluorescent microscope, which was equipped with a CoolSNAP-Pro color digital camera to examine the degree of nuclear condensation.

Cellular DNA fragmentation was assessed by using a cytoplasmic histone-associated DNA fragmentation kit from Roche Diagnostics (Mannheim, Germany) according to the manufacturer’s instructions.

Flow cytometry was performed to determine the content of apoptotic sub G₁ hypo-diploid cells [31]. The cells were harvested, and fixed in 1 mL of 70% ethanol for 30 min at 4 °C. The cells were washed twice with phosphate buffered saline (PBS), and then incubated for 30 min in the dark at 37 °C in 1 mL of PBS containing 100 μg propidium iodide and 100 μg RNase A. Flow cytometric analysis was performed and the proportion of sub G₁ hypo-diploid cells was assessed by the histograms generated using the computer program, Cell Quest and Mod-Fit.

The cells were harvested, washed, and suspended in PBS containing JC-1 (10 μg/mL). After 15 min of incubation at 37 °C, the cells were washed, suspended in PBS and analyzed by flow cytometery [32]. For image analysis, the cells were loaded with JC-1, and incubated for 30 min at 37 °C. Cells were washed and the stained cells were mounted onto a microscope slide with mounting medium (DAKO, Carpinteria, U.S.). Microscopic images were collected using the Laser Scanning Microscope 5 PASCAL program (Carl Zeiss, Jena, Germany) on a confocal microscope.

The cells were harvested, washed twice with PBS, lysed on ice for 30 min in 100 μL of lysis buffer [120 mM NaCl, 40 mM Tris (pH 8), 0.1% NP 40] and then centrifuged at 13,000 × g for 15 min. The supernatants were collected from the lysates and the protein concentrations were determined. Aliquots of the lysates (40 μg of protein) were boiled for 5 min and electrophoresed in 10% sodium dodecylsulfate-polyacrylamide gel. The proteins in the gels were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, U.S.), which were then incubated with the primary antibodies. The membranes were subsequently incubated with the secondary immunoglobulin-G-horseradish peroxidase conjugates (Pierce, Rockford, U.S.). Protein bands were detected using an enhanced chemiluminescence Western blotting detection kit (Amersham, Little Chalfont, Buckinghamshire, U.K.), and then exposed to X-ray film.

All values were expressed as means ± standard error of the mean (SEM). The results were subjected to analysis of variance (ANOVA) using Tukey’s test to analyze differences. p < 0.05 were considered significant.