Sargassum horneri methanol extract rescues C2C12 murine skeletal muscle cells from oxidative stress-induced cytotoxicity through Nrf2-mediated upregulation of heme oxygenase-1

Abstract

Background: Sargassum horneri, an edible marine brown alga, is typically distributed along the coastal seas of Korea and Japan. Although several studies have demonstrated the anti-oxidative activity of this alga, the regulatory mechanisms have not yet been defined. The aim of the present study was to examine the cytoprotective effects of S. horneri against oxidative stress-induced cell damage in C2C12 myoblasts.

Methods: We demonstrated the anti-oxidative effects of a methanol extract of S. horneri (SHME) in a hydrogen peroxide (H2O2)-stimulated C2C12 myoblast model. Cytotoxicity was determined using the 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl-tetrazolium assay and mode of cell death by cell cycle analysis. DNA damage was measured using a comet assay and expression of phospho-histone γH2A.X (p-γH2A.X). Levels of cellular oxidative stress as reactive oxygen species (ROS) accumulation were measured using 2',7'-dichlorofluorescein diacetate. The involvement of selected genes in the oxidative stress-mediated signaling pathway was explored using Western blot analysis.

Results: SHME attenuated H2O2-induced growth inhibition and exhibited scavenging activity against intracellular ROS that were induced by H2O2. The SHME also inhibited comet tail formation, p-γH2A.X expression, and the number of sub-G1 hypodiploid cells, suggesting that it prevents H2O2-induced cellular DNA damage and apoptotic cell death. Furthermore, the SHME significantly enhanced the expression of heme oxygenase-1 (HO-1) associated with induction of nuclear factor-erythroid 2 related factor 2 (Nrf2) in a time- and concentration-dependent manner. Moreover, the protective effect of the SHME on H2O2-induced C2C12 cell damage was significantly abolished by zinc protoporphyrin IX, a HO-1 competitive inhibitor, in C2C12 cells.

Conclusions: These findings suggest that the SHME augments cellular antioxidant defense capacity through both intrinsic free radical scavenging activity and activation of the Nrf2/HO-1 pathway, protecting C2C12 cells from H2O2-induced oxidative cytotoxicity.

Figure 1

Effect of SHME on viability of C2C12 cells. C2C12 cells were incubated for 24 h with various concentrations of SHME for 24 h. Cell viability was estimated by the MTT assay. Data are presented as the mean ± SEM obtained from three independent experiments (*P < 0.05, compared with the control group).

Figure 2

Effect of SHME on H ₂ O ₂ -induced growth inhibition and morphological changes in C2C12 cells. C2C12 cells were pretreated with 300 μg/ml SHME for 1 h and then incubated with or without 1 mM H₂O₂ for 6 h. Then, cell viability (A) and changes in cell morphology (B) were measured. Data are presented as the mean ± SEM obtained from three independent experiments (*P < 0.05, compared with the control group; ^# P < 0.05, compared with the H₂O₂-treated group).

Figure 3

SHME attenuates H ₂ O ₂ -induced apoptosis and ROS generation in C2C12 cells. C2C12 cells were pretreated with 300 μg/ml SHME or 5 mM NAC for 1 h and then stimulated with and without 1 mM H₂O₂ for 6 h. (A) To quantify the degree of apoptosis, media were discarded and the cells were evaluated for sub-G1 DNA content using a flow cytometer. (B) To monitor ROS production, the cells were incubated at 37°C in the dark for 20 min with new culture media containing 10 μM DCF-DA. ROS generation was measured using a flow cytometer. Data are presented as the mean ± SEM obtained from three independent experiments (*P < 0.05, compared with the control group; ^# P < 0.05, compared with the H₂O₂-treated group).

Figure 4

SHME protects against H ₂ O ₂ -induced DNA damage in C2C12 cells. C2C12 cells were pretreated with 300 μg/ml SHME for 1 h and then incubated with and without 1 mM H₂O₂ for 6 h. (A) To detect cellular DNA damage, the comet assay was performed and representative pictures of the comets were taken using a fluorescence microscope at × 200 original magnification. (B) To quantify the degree of apoptosis, the cells were stained with Annexin V, and the percentages of apoptotic cells were then analyzed using flow cytometric analysis. Each point represents the means of two independent experiments. (C) Whole-cell lysates were prepared and subjected to Western blot analysis with a specific antibody against phospho-histone γH2A.X. Actin was used as the loading control. A representative blot from three independent experiments is shown. The numbers represent the average densitometric analyses as compared with actin in, at a minimum, two or three different experiments.

Figure 5

Induction of HO-1 and Nrf2 expression by SHME in C2C12 cells. Cells were incubated with various concentrations of the SHME for 6 h (A) or for the indicated periods with 300 μg/mL SHME (B). The levels of HO-1 and Nrf2 proteins were determined by Western blot analyses, and representative blots of three independent experiments are shown. Actin was used as a loading control. The numbers represent the average densitometric analyses as compared with actin in, at a minimum, two or three different experiments.

Figure 6

Effects of an inhibitor of HO-1 on SHME-mediated protection of DNA damage by H ₂ O ₂ in C2C12 cells. C2C12 cells were pretreated for 1 h with 300 μg/ml SHME and then treated for 6 h with or without 1 mM H₂O₂ in the absence or presence of 10 μM ZnPP. (A) The comet assay was performed and representative pictures of the comets were taken using a fluorescence microscope at × 200 original magnification. (B) Cell lysates were prepared and subjected to Western blot analysis with a specific antibody against phospho-histone γH2A.X. Actin was used as a loading control. A representative blot from three independent experiments is shown. The numbers represent the average densitometric analyses as compared with actin in, at a minimum, two or three different experiments.

Figure 7

Effects of an inhibitor of HO-1 on SHME-mediated attenuation of ROS formation and apoptosis induction by H ₂ O ₂ in C2C12 cells. (A) Cells grown under the same conditions as those in Figure 6 were assayed for ROS generation by DCF fluorescence. (B) The degree of apoptosis was evaluated by sub-G1 DNA content using a flow cytometer. (C) Cell viability was estimated by the MTT assay. Data are presented as the mean ± SEM, obtained from three independent experiments (*P < 0.05, compared with the control group; ^# P < 0.05, compared with the H₂O₂-treated group; ^$ P < 0.05, compared with the H₂O₂ and SHME-treated group).