Meroterpenoid-Rich Fraction of the Ethanolic Extract from Sargassum serratifolium Suppressed Oxidative Stress Induced by Tert-Butyl Hydroperoxide in HepG2 Cells

Abstract

Sargassum species have been reported to be a source of phytochemicals, with a wide range of biological activities. In this study, we evaluated the hepatoprotective effect of a meroterpenoid-rich fraction of the ethanolic extract from Sargassum serratifolium (MES) against tert-butyl hydroperoxide (t-BHP)-treated HepG2 cells. Treatment with MES recovered the cell viability from the t-BHP-induced oxidative damage in a dose-dependent manner. It suppressed the reactive oxygen species production, lipid peroxidation, and glutathione depletion in the t-BHP-treated HepG2 cells. The activity of the antioxidants induced by t-BHP, including superoxide dismutase (SOD) and catalase, was reduced by the MES treatment. Moreover, it increased the nuclear translocation of nuclear factor erythroid 2-related factor 2, leading to the enhanced activity of glutathione S transferase, and the increased production of heme oxygenase-1 and NAD(P)H:quinine oxidoreductase 1 in t-BHP-treated HepG2 cells. These results demonstrate that the antioxidant activity of MES substituted the activity of the SOD and catalase, and induced the production of detoxifying enzymes, indicating that MES might be used as a hepatoprotectant against t-BHP-induced oxidative stress.

Keywords: Nrf2; Sargassum serratifolium; antioxidant; oxidative stress; tert-butyl hydroperoxide.

Figure 1

Hepatoprotective effect of MES on t-BHP-stimulated oxidative stress. (A) The cell viability of MES was measured by using Cell Proliferation Assay Kit. (B) The hepatoprotective effect was determined with various concentrations of MES in t-BHP-treated HepG2 cells. The data are the means ± SD of three independent experiments. # p < 0.05 indicates significant differences from the control group. * p < 0.05 indicates significant differences from the t-BHP treatment group.

Figure 2

Inhibitory effect of MES on the reactive oxygen species generation, lipid peroxidation, and GSH depletion in t-BHP-treated HepG2 cells. HepG2 cells were treated with MES (0.25~1.0 μg/mL) and t-BHP. (A) ROS were measured by a fluorescent probe, DCFH-DA. (B) Lipid peroxidation was measured by using a TBARS assay. (C) The GSH level was measured by fluorescent probe, CMF-DA. The data are means ± SD of three independent experiments. # p < 0.05 indicates significant differences from the control group. * p < 0.05 indicates significant differences from the t-BHP treatment group.

Figure 2

Inhibitory effect of MES on the reactive oxygen species generation, lipid peroxidation, and GSH depletion in t-BHP-treated HepG2 cells. HepG2 cells were treated with MES (0.25~1.0 μg/mL) and t-BHP. (A) ROS were measured by a fluorescent probe, DCFH-DA. (B) Lipid peroxidation was measured by using a TBARS assay. (C) The GSH level was measured by fluorescent probe, CMF-DA. The data are means ± SD of three independent experiments. # p < 0.05 indicates significant differences from the control group. * p < 0.05 indicates significant differences from the t-BHP treatment group.

Figure 3

Effect of MES on antioxidant enzyme activities in t-BHP-treated HepG2 cells. Cells were treated with MES and t-BHP for 18 h. The cell lysates were prepared and used for superoxide dismutase (A) and catalase (B) activities. The values are the means ± SD of three independent experiments. # p < 0.05 indicates significant differences from the control group. * p < 0.05 indicates significant differences from the t-BHP treatment group.

Figure 4

Effect of MES on the activity of GST and the productions of HO-1 and NQO1 in t-BHP-treated HepG2 cells. Cells were treated with MES and t-BHP for 18 h. (A) The GST activity was measured with a glutathione S-transferase assay kit. (B) The expressions of HO-1 and NQO1 were analyzed by Western blot. The data are means ± SD of three independent experiments. # p < 0.05 indicates significant differences from the control group. * p < 0.05 indicates significant differences from the t-BHP treatment group.

Figure 5

Effect of MES on the activation and translocation of Nrf2. (A) Cells were treated with indicated concentrations of MES for 1 h, and the nuclear fraction was analyzed with Western blot. (B) The cells were treated with MES and t-BHP for 1 h and separated cytosolic and nuclear fraction were analyzed with Western blot. (C) Cells were fixed and immunostained with anti-Nrf2 antibody (Alexa Fluor 586) and DAPI. The images were captured by confocal microscopy. The data are means ± SD of three independent experiments. * p < 0.05 indicates significant differences from the t-BHP treatment group.