C2C12 myoblasts obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) were grown in Dulbecco's modified Eagle's medium (DMEM; WelGENE Inc., Daegu, Korea), supplemented with 10% fetal bovine serum (FBS) and 100 µg/ml penicillin/streptomycin antibiotics (WelGENE Inc.) in a humidified 5% CO₂ atmosphere at 37°C. For the preparation of the crude garlic saponins, an improved method was used for saponin extraction based on a previous study (22). Garlic was collected around Namhae city (Gyeongsangnam-do, Korea); the bulbs were peeled, washed and chopped before being stored at −20°C. The frozen samples were lyophilized and homogenized using a grinder before extraction. The samples were extracted twice with methanol by refluxing at 80°C for 2 h. The methanol extract was then suspended in water and partitioned sequentially with n-hexane, chloroform, ethyl acetate and n-butanol. Subsequently, the water-saturated n-butanol fraction was evaporated to dryness in a vacuum. The crude saponins recovered in this process were loaded onto a Diaion^® HP-20 MCI gel (Sigma-Aldrich Chemical Co., St. Louis, MO, USA). The sugar residues were then removed with 40% CH₃OH. The fractions were eluted with 60–80% CH₃OH, collected, and then dried to obtain the garlic saponins. The saponins were then dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich Chemical Co.) and adjusted to final concentrations using complete DMEM prior to use.

Cell viability was measured based on the formation of blue formazan, which was metabolized from colorless 3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bromide (MTT; Sigma-Aldrich Chemical Co.) by mitochondrial dehydrogenases. These are active only in live cells. Briefly, the C2C12 cells were seeded in 6-well plates at a density of 1×10⁵ cells per well. After 24 h of incubation, the cells were treated with the specified concentrations of garlic saponins in the absence or presence of H₂O₂ and/or zinc protoporphyrin IX (ZnPP; Sigma-Aldrich Chemical Co.) and N-acetyl-L-cysteine (NAC; Sigma-Aldrich Chemical Co.) for the specified duration. MTT working solution was then added to the culture plates following by continuous incubation at 37°C. Three hours later, the supernatant was removed, and the formation of formazan was measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (Dynatech Laboratories, Chantilly, VA, USA). Control cells were supplemented with complete medium containing 0.05% DMSO (vehicle control). The inhibitory effect on cell growth was assessed as the percentage of cell viability, where the vehicle-treated cells were considered 100% viable.

The intracellular accumulation of ROS was determined using the fluorescent probes, 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Molecular Probes, Eugene, OR, USA). In order to monitor ROS generation, the cells were incubated with 10 µM H₂DCFDA for 20 min at room temperature in the dark. ROS production in the cells was monitored using a flow cytometer (Becton Dickinson, San Jose, CA, USA) using CellQuest Pro software, as previously described (23).

Comet assay, a sensitive and rapid technique for detection of DNA damage in individual cells, was performed as previously described (24). Briefly, harvested individual cells were mixed with molten low melt agarose and spread on a fully-frosted microscopic slide pre-coated with 1% normal melting agarose. The embedded cells were then lysed using lysis solution and treated with alkaline solution to relax and denature the DNA. Subsequently, electrophoresis of the samples was carried out under alkaline condition at 25 V and 300 mA for 20 min. Following electrophoresis, the slides were washed, stained with 20 µg/ml propidium iodide (PI; Sigma-Aldrich Chemical Co.), and were then examined under a fluorescence microscope (Carl Zeiss, Jena, Germany).

Western blot analysis and protein extraction were performed as previously described (24). In brief, the cells were lysed, and then equal amounts of cell lysates were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto nitrocellulose membranes (Schleicher & Schuell Bioscience, Inc., Keene, NH, USA). The membranes were probed with specific antibodies for 1 h and incubated with the diluted enzyme-linked secondary antibodies (Amersham Co., Arlington Heights, IL, USA). The proteins were visualized using an enhanced chemiluminescence (ECL) detection system (Amersham Co.) according to the manufacturer's instructions. The primary antibodies used in this study were as follows: γH2AX (1:500, CS #2577; rabbit polyclonal, Cell Signaling Technology, Inc., Danvers MA, USA), p-γH2AX (1:500, CS #9718S; rabbit polyclonal, Cell Signaling Technology, Inc.), Nrf2 (1:500, SC-13032; rabbit polyclonal, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), p-Nrf2 (1:500, ab76026; rabbit monoclonal, Abcam, Inc., Cambridge, UK), HO-1 (1:500, SC-136960; mouse monoclonal, Santa Cruz Biotechnology, Inc.), Keap1 (1:1,000, SC-33569; rabbit polyclonal, Santa Cruz Biotechnology, Inc.), NQO-1 (1:1,000, SC-16464; goat polyclonal, Santa Cruz Biotechnology, Inc.), TrxR1 (1:1,000, SC-28321; mouse monoclonal, Santa Cruz Biotechnology, Inc.), ERK (1:1,000, SC-154; rabbit polyclonal, Santa Cruz Biotechnology, Inc.), p-ERK (1:500, #9106S; mouse monoclonal, Cell Signaling Technology, Inc.), p38 (1:1,000, SC-535; rabbit polyclonal, Santa Cruz Biotechnology, Inc.), p-p38 (1:500, #9211S; rabbit polyclonal, Cell Signaling Technology, Inc.), JNK (1:1,000, #9252S; rabbit polyclonal, Cell Signaling Technology, Inc.), p-JNK (1:500, #9255S; mouse monoclonal, Cell Signaling Technology, Inc.) and actin (1:1,000, SC-1616; goat polyclonal, Santa Cruz Biotechnology, Inc.). Actin and lamin B were used as the internal controls for cytosolic and nuclear fractions, respectively. In order to examine the effects of MAPK signaling pathway on the activation of Nrf2 and the induction of HO-1 by garlic saponins, specific inhibitors of MAPKs such as PD98059 (an ERK inhibitor, Cell Signaling Technology, Inc.), SP600125 (a JNK inhibitor, Sigma-Aldrich Chemical Co.) and SB203580 (a p38 MAPK inhibitor, Cell Signaling Technology, Inc.) were applied.

siRNA targeting Nrf2 (Nrf2 siRNA) and control siRNA were purchased from Santa Cruz Biotechnology. The siRNA was transfected into the cells following 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 control siRNA or Nrf2 siRNA at 50 nM for 6 h in serum-free OPTI-MEM medium (Life Technologies). Following transfection, the cells were treated with garlic saponins (500 µg/ml) for 6 h or pre-treated with garlic saponins (500 µg/ml) for 1 h and then stimulated with or without 1 mM H₂O₂ (1 mM) in the presence of garlic saponins for a further 6 h. The cells were then lysed and equal amounts of cell lysates were subjected to western blot analysis.

Data are expressed as the means ± standard deviation (SD) values. One-way analysis of variance (ANOVA) was used for comparisons in the experiments with multiple time points and concentrations. When ANOVA indicated statistical significance, Duncan's multiple range test was used to determine which means were significantly different. A probability value of P<0.05 was used as the criterion for statistical significance.

We first examined the effects of garlic saponins on the viability of C2C12 cells by MTT assay. As shown in Fig. 1, the results revealed that treatment with garlic saponins (10–1,000 µg/ml) alone had no obvious effect on C2C12 cell viability. To examine the protective effects of garlic saponins against oxidative stress-induced cytotoxicity in C2C12 cells, the cells were pre-treated with garlic saponins for 1 h and exposed to H₂O₂ for an additional 6 h. The results revealed that treatment of the C2C12 cells with 1 mM of H₂O₂ for 6 h resulted in approximately a 40% loss of cellular viability, as compared with the control cells. However, the H₂O₂-induced reduction in cell viability was significantly reversed by pre-treatment with garlic saponins in a concentration-dependent manner (Fig. 2A). These results indicate that garlic saponins have properties that protect C2C12 cells against oxidative stress.

We then measured the intracellular ROS levels in order to investigate whether garlic saponins had any effect on intracellular ROS generation induced by stimulation with H₂O₂. As expected, exposure of the C2C12 cells to H₂O₂ for 6 h induced an increase in intracellular ROS levels (Fig. 2B). However, pretreatment of the cells with garlic saponins (500 µg/ml for 1 h) significantly reduced the H₂O₂-induced ROS production. As a positive control, the ROS scavenger, NAC, was used, and we noted that this also reduced H₂O₂-induced ROS generation. Moreover, we noted that the garlic saponins themselves did not contribute to ROS generation, suggesting that pre-treatment with garlic saponins induced a cellular antioxidant response.

We further examined the effects of garlic saponins on DNA damage induced by H₂O₂ using single-cell gel electrophoresis (comet assay) and western blot analysis. As shown in Fig. 3A, stimulation with H₂O₂ alone significantly increased the number of DNA breaks, resulting in an increase in fluorescence intensity in the tails of the comet-like structures in C2C12 cells. These adverse effects were markedly reduced by pre-treatment with garlic saponins. In addition, stimulation of the C2C12 cells with H₂O₂ alone resulted in the upregulation of the level of the phosphorylated histone variant H2AX at serine 139 (p-γH2AX), a sensitive marker for DNA double-strand breaks (25) (Fig. 3B). By contrast, pre-treatment with garlic saponins resulted in a decreased p-γH2AX expression, which again indicates that garlic saponins exert a protective effect against H₂O₂-induced DNA damage.

The fact that Nrf2 signaling regulates the cellular antioxidant response by promoting ARE-dependent gene expression has been well documented (3,7,26). As a result, we wished to determine whether garlic saponins protect cells from intracellular oxidative stress by activating the Nrf2 signaling pathway. As shown in Fig. 4A and B, treatment of the C2C12 cells with garlic saponins induced Nrf2 expression and the phosphorylation of Nrf2 at Ser40 in a duration- and dose-dependent manner and was associated with the induction of HO-1. However, NQO1 and Keap1 were relatively unaffected by treatment with garlic saponins. We then examined the effect of garlic saponins on the intracellular localization of Nrf2 and found that there was an increased nuclear translocation of phosphorylated Nrf2 proteins following treatment with garlic saponins (Fig. 4C and D).

We then developed an Nrf2 gene knockdown model using siRNA transfection to demonstrate the contribution of Nrf2 signaling to the counteractive effects of garlic saponins on H₂O₂-induced cytotoxicity. Western blot analysis revealed that Nrf2 siRNA reduced the expression of Nrf2 and the phosphorylation of Nrf2 induced by treatment with garlic saponins. The expression of HO-1 which was induced by treatment with garlic saponins was also blocked following transfection of the cells with Nrf2 siRNA (Fig. 5A), which is evidence that the augmentation of HO-1 expression is mediated by Nrf2. To confirm the involvement of Nrf2, the protective effects of garlic saponins against the H₂O₂-induced reduction in cell viability were determined in cells in which Nrf2 was knocked down. As shown in Fig. 5B, transfection with Nrf2 siRNA cancelled out the cytoprotective effects of garlic saponins when compared with the control siRNA-transfected cells, providing evidence that garlic saponins initiate the cellular antioxidant defense system through the activation of the Nrf2/HO-1 signaling pathway.

To provide further confirmation that the antioxidant and cytoprotective activities of garlic saponins against oxidative stress in C2C12 cells are mediated through the activation of the Nrf2/HO-1 signaling pathway, the C2C12 cells were pre-incubated with or without ZnPP, a specific inhibitor of HO-1. The ROS levels and cell viability were also assessed. As shown in Fig. 6, ZnPP nullified the protective effect of garlic saponins on the H₂O₂-induced production of ROS and the reduction in cell viability. These data suggest that garlic saponins exert their protective effects by activating the cellular defense mechanisms against oxidative stress through the Nrf2-related cytoprotective pathway. The subsequent upregulation of HO-1 thus plays a crucial role in the protective effects of saponins in C2C12 cells.

Previous studies have demonstrated that multiple phosphorylation cascades participate in regulating the translocation of Nrf2 and Nrf2-mediated HO-1 gene expression (27–29). To identify the upstream signaling events involved in the activation of Nrf2 and the induction of HO-1 by garlic saponin, the potential involvement of MAPKs was explored. MAPKs are classified into three major subgroups, namely ERK, c-Jun N-terminal kinase (JNK) and p38 MAPK. Although garlic saponins induced the phosphorylation of JNK to a certain extent, it was found that their effect was only significant on the phosphorylation of ERK in a duration-dependent manner. There were no significant changes observed in the levels of phosphorylated p38 MAPK compared with the controls (Fig. 7A). To determine whether garlic saponins induce Nrf2 expression and phosphorylation, and HO-1 expression through the activation of ERK, the cells were pre-treated with garlic saponins for 1 h and then incubated with MAPK inhibitors. As shown in Fig. 7B, when the cells were incubated with a selective inhibitor of ERK (PD98059), the induction and phosphorylation of Nrf2 were blocked; HO-1 induction was diminished accordingly. However, the p38 MAPK inhibitor (SB203580) and JNK inhibitor (SP600125) were unable to reduce Nrf2 and HO-1 expression and Nrf2 phosphorylation induced by garlic saponins. Taken together, these observations indicate that the way in which garlic saponins activate the Nrf2/HO-1 signaling pathway involves the ERK pathway.